The Toxicology of Radioactive Substances. Volume 3.59

THE TOXICOLOGY OF RADIOACTIVE SUBSTANCES

VOLUME 3

Iron-59

Edited by

A.A.LETAVET and

E. B. KURLYANDSKAYA

Translated by

R.E. TRAVERS

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Library of Congress Catalog Card No. 61-9783

This is a translation of the original Russian Toksikologiya radioaktivnykh veshchestv published in 1962 by Medgiz, Moscow

2691/67

TOXICOLOGY OF RADIOACTIVE IRON-59

E. B. KURLYANDSKAYA

THIS volume contains experimental results obtained by the Radiotoxicology Laboratory of the Institute of Occupational Hygiene and Disease, Academy of Medical Sciences, U.S.S.R., in studying the toxicology of radioactive iron.

In accordance with the general direction of the Laboratory's work the data presented here stem from the further development of investigations chiefly concerned with the long-term effect of small doses of radioactive substances close to permitted maxima.

We chose to study the toxicology of 59Fe for two reasons. Firstly 59Fe has been used in various branches of science and technology. Thus, in the metal industry, it is used to study the movement of metal and distribution of alloy elements in the open-hearth furnace and pouring ladle (N. G. Bogdanova, 1958). It is also used to study the movement of charging materials in blast furnaces and open-hearth furnaces, the blending of pig iron in the furnace (M.T.Bul'skii et al., 1958), the hydrodynamics and crystallization of ingots of melted and cold steel (A. A. Zborovskii and L. K. Strelkov, 1958), and so on. , ■ ■ ■

In engineering 59Fe is used to study friction mechanisms, wear of machine parts, metal fatigue, etc.

In biology it is employed in the study of haemoglobin metabolism in the red cells, bone marrow, etc.

The above techniques far from exhaust all the possible uses of 59Fe in scientific investigation. But its employment in industry and the laboratory entails the possibility of intake by workers of very small quantities of 59Fe close to the maximum permissible. Moreover, the existing maximum permissible concentrations of radioactive iron in air and water are based solely on calculation and require biological verification. This was shown convincingly in our previous work on other radioisotopes and is one of the practical purposes of the present investigations (see The Toxicology of Radioactive Substances, vol. 1, 1957; vol. 2, 1960).

But the study of the long-term effect of 59Fe is also of theoretical importance. In recent years evidence has accumulated in the laboratory which indicates the great significance of the chemical and biological properties of radioactive elements in the picture of chronic radiation sickness produced by intake of these substances. This applies especially to those elements whose stable analogues are essential to normal body function (45Ca, 60Co, 59Fe,

1

2 Toxicology of Radioactive Substances 65Zn, and others). It is suggested that the direction of the pathological process and the early signs of radiation sickness produced by these radioéléments will differ from the effect of other radioéléments in accordance with the functional background which these elements create as chemical substances, as in fact was shown by our earlier work on 60Co (vol. 2, 1960).

Consequently, it might be expected that 59Fe, which plays an important role in haemopoiesis and in the activity of the respiratory enzymes, should also manifest its own characteristic effects. Study of these peculiarities of the effect of 59Fe was the second aspect of our investigations.

There is a fair volume of literature on the behaviour of 59Fe in the normal and anaemic animals following the administration of trace amounts (Aus-toni and Greenberg, 1940; Copp and Greenberg, 1946; Hahn, 1948; and others). The effect of small doses of 59Fe (single dose) has been studied by R. E. Kavetskii, L.B.Stolyarova, R. D. Nikitenko and P.M.Amdurskaya. These authors have established that the body reacts even to trace quantities of 59Fe. But the literature which has reached us contains no references to the long-term effect of 59Fe, administered in different ways and in amounts close to the maximum permissible.

Not wishing to overburden the reader with information of a general nature we shall summarize the physical, chemical and biological properties of iron briefly.

59Fe has a half-life (Γ±) of 45^ days and a complex β- (Εβ = 0-26 and 0-46 MeV) and y-spectrum (1-025 y-quanta on decay; Ey = 1-1-1-3 MeV).

In the human and mammalian body stable iron is an essential component of haemoglobin and the respiratory enzymes. The human body normally absorbs, daily, according to some data, from 6 to 8 mg iron (L.A.Klyu-charev, 1953; Ts.D.Savve, 1954; and others) and according to other results, 8-12 mg (Granick and Hahn, 1944). In iron deficiency, the absorption may increase greatly. On entering the blood, iron passes to the cells and takes part in the metabolic processes and is also utilised by the bone marrow cells for formation of haemoglobin. All these aspects were taken into consideration when interpreting our results.

Our investigations into the long term effect of 59Fe consisted of two main series of experiments.

The first series was carried out on 103 rabbits divided into 4 groups. In the first group (20 rabbits), iron was administered orally at a dose rate of 1 μο ferric chloride (59FeCl3) per kg weight per day, the radioactivity of which exceeded by 10 times the international maximum permissible concentration for water. The animals of the second group (42 rabbits) received 10 μc/kg, the third group (24 rabbits) received stable iron in an amount equivalent to that administered to the second group, from 1 to 4 mg/kg, and the fourth group (17 rabbits) constituted a physiological control.

In the second series of experiments, 45 rats were used. In some, 0-03 μο of 59Fe oxide (the maximum permissible dose for a single administration) was injected intratracheally on three occasions. The others received a single intratracheal injection of 106, 3-36 and 27-5 [LC per rat, which are respectively

Toxicology of Radioactive Iron-59 3

10, 30 and 250 times the maximum permissible dose for a single administration. Nine rats were given a single dose of 20 Resoluble 59Fe citrate per rat, which exceeded by 100 times the maximum permissible concentration for soluble iron compounds. This series of experiments was made to examine the effect of soluble and insoluble compounds of radioactive iron, the significance of local doses of insoluble compounds in the development of tumours, and also to verify the maximum permissible concentrations of soluble and insoluble compounds. The animals were maintained on a constant standard diet.

In the first series of experiments on the long-term effects of 59Fe the following factors were studied :

(1) metabolism of orally administered 59Fe in the body (absorption, distribution and excretion) ;

(2) tissue doses in individual organs and in the whole body; (3) the electrical activity of the brain as shown by encephalograms; (4) the electrical activity of the heart under normal conditions and against

a background of pharmacological stresses ; (5) haemopoiesis under normal conditions and during functional stresses

(parturition, bloodloss) ; (6) certain biochemical changes (proteins and protein fractions, sugar un

der normal conditions and with sugar loading); (7) morphological changes in organs and tissues at different intervals dur

ing administration; (8) means of stimulating excretion of 59Fe from the body.

In the second series (intratracheal injection) the following factors were investigated:

(1) effect of 59Fe on the central nervous system (stimulation threshold, summation of subliminal impulses) ;

(2) peripheral blood; (3) long-term effects of intratracheal injection of soluble and insoluble

59Fe compounds with calculation of tissue doses in the lungs and whole body (morphological investigation).

Such complex investigations on the same animals over a period of up to 21 months enabled the detection of peculiarities in the behaviour and biological effect of 59Fe in the long-term experiment, distinguishing it from the effects of other isotopes studied by us (cf. E.B.Kurlyandskaya: Toxicology of Radioactive Substances, vol. 1, 1957 and vol. 2, 1960) and also the formulation of maximum permissible concentrations of 59Fe based on biological results.

In studies on the metabolism of 59Fe after oral administration (G. A. Avru-nina) it was established that on a normal diet, without iron deficiency in the body, only about 5 per cent of soluble ferric chloride is absorbed by the gastro-intestinal tract. The unabsorbed portion is almost entirely excreted with the faeces in the course of a week. At the same time, however, the half-

4 Toxicology of Radioactive Substances

life of iron in the blood is fairly long, about 200 days, and is due to its fixation in the bone marrow and utilization in haemoglobin synthesis.

In the earlier published work of the Laboratory, it was shown that with daily administration of radioisotopes (89Sr, 134Cs, 60Co) an equilibrium level of body content is reached. The time taken to reach this equilibrium and the level of activity attained depend on the character of the isotope, the amount of carrier, the animal's diet and other factors. Similar principles were found to apply also to 59Fe (G.A. Avrunina). After 45-65 days, according to the amount of carrier, a fairly constant level of 59Fe is maintained in the blood, bone marrow, liver and other organs, and varies insignificantly throughout 21 months. However, it is significant that no such constancy obtains in the gastro-intestinal tract of animals dying at different stages, and the amount of 59Fe found there is from 5 to 8 times the amount of the daily administered dose. As a result, fairly heavy doses of irradiation may occur in the abdominal cavity, and can be detected by systematic in vivo measurement of external y-radiation from the animals submitted to chronic 59Fe intoxication. This factor is not sufficiently considered by physicists in calculating maximum permissible concentrations, and it confirms the necessity for their experimental verification.

Tissue doses were calculated by G.A.Avrunina from the results of measurement in particular organs and also in the whole body, obtained by systematic in vivo measurement of external y-radiation of the animals. Doses of ß- and y-radiation in the liver in rabbits of the first and second groups were 0-133 and 1-35 rads per day, in the spleen 0-173 and 1-56 rads, in the bone marrow 0-109 and 0-55 rads, and in the blood 0-145 and 0-91 rads per day respectively. In the remaining organs doses were considerably lower (cf. Avrunina's paper in the present volume).

The mean whole body dose (mean of the in vivo measurements) was in the first group about 0-040 rads per day, and in the second about 0-3 rads per day. Thus, mean doses under our experimental conditions were either close to the maximum permissible or, when the administered amounts were increased by 10 and 100 times, exceeded maximum permissible by 15 times, which again underlines the absence of a complete correlation between calculated results and the data obtained experimentally.

The picture of chronic radiation sickness produced by administration of 59Fe bore peculiarities characteristic for iron.

Three to five weeks after beginning daily administration of a dose of 10 μc per kg weight to rabbits of the second group changes appeared in the response of the brain currents to rhythmic photostimulation (D. A. Ginsburg), manifested by the appearance of evoked rhythm at higher stimulation frequencies. In most animals trace rhythms were detected in the motor-sensory regions which were not found in the control animals.

It must be emphasized that resting E.E.G. activity was unchanged and current disturbance was manifested only on application of functional stress, such as rhythmic photostimulation. This indicates a functional rather than an organic impairment of the brain at these stages.


Earlier, even in the first month of administration of 10 [/.c/kg 59Fe, changes in the haemopoiesis occurs (N.L.Beloborodova, V. L. Ponomareva, E.K. Red'kina). In our previous volume (The Toxicology of Radioactive Substances, vol. 2, 1960) detailed results concerning the chronic effect of 60Co on haemopoiesis were given. The specific effect of 60Co on red blood formation, connected with the role of the stable isotope of cobalt in these processes, was dealt with. Taking into consideration the importance of iron in erythropoie-sis, and also differences in the mechanisms of action of Co and Fe, a comparison was made between the effects of these two elements on the red blood. The number, form, diameter and osmotic fragility of the red cells were investigated, along with the size and character of the red cell precursors in the bone marrow (V.L.Ponomareva).

It transpired that the very early changes in erythropoiesis caused by administration of 60Co and 59Fe were quite different.With prolonged oral administration of 60Co the main change was an initial increase, followed by a reduction in the number of red cells. After 16 months of administration pronounced anaemia had developed in the rabbits. The colour index was virtually unchanged.

With administration of 59Fe, the haemoglobin level begins to rise from the first month, reaching its highest point by the 6th month (1-7 g per cent). There was no significant change in the number of red cells, and consequently the colour index was considerably increased. This increase became statistically significant after the first month of administration of 59Fe (data from 62 experimental and 35 control animals). The question arises as to whether the rise in the haemoglobin level was associated with the administration of iron as a component part of haemoglobin. And indeed, by the end of the first month a statistically significant short-term increase in the haemoglobin level and colour index was detected in rabbits receiving an equivalent amount of stable iron. Thus, there is a basis for supposing that, as in the 60Co experiments, micro-quantities of the element iron change the functional condition of the haemopoietic system, giving specific changes in the pathological process in the haemopoietic organs.

In view of the particularly important role of iron in erythropoiesis changes in size and shape of the red cells were studied (V.L.Ponomareva). It was shown that administration of 59Fe produces an increased mean red cell diameter, which becomes statistically significant from the 3rd, and reaches a maximum by the 6th, month of administration. It should be emphasized that the number of reticulocytes in the peripheral blood, which normally have a larger diameter, did not increase. The diameter distribution curves of both peripheral blood red cells and of their nuclear precursors in the bone marrow (erythroblasts and normoblasts) were shifted to the right, i.e. towards larger diameters. Red cell volume was also increased, but to a lesser degree than diameter. In this connection increased numbers of leptocytes were observed in the experimental rabbits, earlier in the second group (ΙΟμ/kg) and somewhat later in animals of the first group. It may be supposed that the increased diameter of the red cell precursors in the bone marrow is linked with developmental abnormalities.


These results show that the appearance in the peripheral blood of macro-cytic leptocytes with increased haemoglobin content, due to increased volume of the red cells, must be considered one of the early reactions of the haemo-poietic organs to administration of 59Fe. Increased haemoglobin and red cell volume is a peculiarity of the effect of 59Fe, whereas leptocytosis was also observed with administration of 60Co.

Qualitative and quantitative analysis of the morphological composition of the bone marrow showed that, in the experimental animals, the total number of nucleated bone marrow cells increases from the first months of administration, and, in most, also the number of cells of the red series, especially in animals of the first group (1 μό).

Analysis of the experimental results shows that during the first year of administration of 59Fe definite changes in erythropoiesis occur, apparently of a compensatory character. It may be supposed that one of the causes of erythroid hyperplasia, during daily administration of small amounts of 59Fe, is concealed anoxia, associated with, on the one hand, formation of physiologically sub-standard haemoglobin, and on the other, with reduced efficiency of the respiratory enzymes, the molecules of which include radioactive iron. Evidence for this supposition is provided by the fact that, despite the raised level of haemoglobin, when a significant stress is placed upon the haemopoietic organs, such as bloodloss, the haemoglobin level is restored more slowly in the experimental animals than in the controls. It would seem that this can be considered a symptom of defective haemoglobin synthesis, and is shown by the condition of the haemopoietic processes in pregnant animals after prolonged administration of 59Fe (N.L.Beloborodova, V.L. Ponomareva, E.K.Red'kina). As the results of these authors in the present volume show, anaemia, developed earlier during pregnancy in the experimental animals than in the controls, and was accompanied by signs of pathological erythropoiesis. Post-partum anaemia in the former was marked by its duration and by the formation of defective erythrocytes.

It is of interest to note that with all the specificity of the effect of 59Fe on red blood formation, changes in leuco- and lymphopoiesis in no way differed from those observed with other isotopes, especially 60Co. With administration of both isotopes, transitory lymphopenia was observed from the first month, together with a fall in the absolute number of lymphocytes. Further investigation showed that the fall in the absolute lymphocyte number was a consequence of impaired lymphopoiesis, which during the first 12-14 months was of a functional character (A. S. Kaplanskii, E.S.Gaidova) without the morphological changes which occurred at later periods.

In analysing the results obtained on the effect of 59Fe on the haemopoietic processes, the suggestion can be made that changes in erythropoiesis during prolonged administration of this isotope are the result of the combined radiation and chemical effect of the element, which plays a very important part in erythropoiesis.

Although 59Fe has specific effects on haemopoiesis, changes in cardiac responses, as shown by electrocardiograms, differ little from those produced


by 60Co (A.O.Saitanov). As in the latter case, daily administration of 59Fe in an amount of 10 and 1 μc per kg body weight produced a change in the terminal part of the ventricular complex, chiefly of the T wave, which appeared during the 10th—11th month and was especially pronounced from the 14th to the 20th months of administration, i.e. during the period when parallel histological investigations were revealing dystrophic lesions in the cardiac muscle. At approximately the same period changes in certain biochemical processes occur. Thus, serial estimations of the fractional composition of the serum proteins by R. L. Orlyanskaya revealed a reduction of albumin, detected by paper electrophoresis, and an increase of y-globulin in the serum of animals of the first and second groups, which may be associated with a disturbance of the liver's synthesizing function. This is also shown by changes in the sugar curve in experimental animals following oral administration of glucose.

Observations on the condition of rabbits subjected to prolonged administration of radioisotopes showed great variation in individual sensitivity both to 59Fe and other isotopes. The most sensitive animals die during the early stages of the experiment (chiefly during the first few months), mainly from pneumonia. Examination of animals dying at this stage revealed a diffuse suppurative process with leucocytic reaction in the lungs, and foci of myeloid haemopoiesis were found. The animals which survived for a fairly long period (up to 11-12 months) were in good condition and gained weight. At this stage, no gross morphological lesions were found (E. S. Gaidova). The latter arise chiefly during the period from 10 to 17 months and are most pronounced in second group animals. These rabbits show adiposis, sluggish inflammatory reactions, diffuse and focal sclerosis of the pulmonary tissue, and atypical bronchial epithelium; areas of adenomatosis appear, blood vessel walls become softened and oedematous and the amount of lymphoid tissue in the spleen decreases. Dystrophic and sclerotic lesions are found in the liver and kidneys. In some animals, the thyroid gland showed a reduction in follicle size, and a reduction and even complete disappearance of colloid. The gonads showed atrophie lesions (E. S. Gaidova). These morphological features are in complete agreement with the functional disturbances in the different systems and organs. It must be emphasized that the lesions observed were little different from those previously found with other isotopes.

Analysis of the results obtained showed that even with a ten-fold increase of the maximum permissible concentration recommended by ICRP* for 59Fe and with a relatively small (2-3 times) excess of the radiation dose in the body and "critical" organs, the changes characteristic of radioactive iron occur. This led us to question the level of the existing international maximum permissible concentrations of 59Fe for water (4 x 10"6 c/1). The permissible concentrations proposed by us (according to our results they should not exceed 1 x 10~8 c/1) have been incorporated into the U.S.S.R.

* Recommendation of the International Commission on Radiological Protection. Report of Committee II on Permissible Dose for Internal Radiation, p. 45. Pergamon Press, 1960.


Rules of Hygiene for work with radioactive substances and sources of ionizing radiation (No. 333-66 of 26 June 1960).

Further work in the field of soluble and insoluble radioactive compounds included investigation into the effects of intratracheal injection of a soluble iron citrate compound and insoluble iron oxide (N. D. Sagaidak). By this method of administration virtually all the insoluble iron compound remains in the lungs for a prolonged period, only negligible amounts being found in other organs. Distribution of iron citrate after intratracheal injection is similar to that obtained in G.A.Avrunina's experiments (cf. the present volume. Body dose in rabbits receiving daily oral administration of 59FeCl3, and some data on accumulation and excretion of 59Fe). A peculiarity of the behaviour of iron citrate in the lungs is of interest. Although most is absorbed from the lungs during the first day after injection, nevertheless by the end of a month after a single injection about 9 per cent of the injected quantity has become fixed in the lungs for a prolonged period, probably in the form of a compound of 59Fe with proteins. The possibility of formation in the lungs of insoluble radioactive compounds, which may create a rather large total radiation dose, our lack of knowledge concerning conversion of soluble compounds in the lungs, and the gravity of the possible consequences, lead us to suggest that work on radioactive aerosols for inhalation must be carried out under the most strict conditions, i.e. as though for insoluble compounds, which would make human contact with radioactive aerosols safe. The necessity for this is dictated by the following facts. N.D. Sagaidak, studying the long-term effects of intratracheal injection of 59Fe oxide in amounts exceeding the maximum permissible concentrations by 10 and 30 times, found bronchial cancer in 6 of 25 rats (25 per cent). Total radiation dose in the lungs during the 15 months of the experiment was 484-1296 rads, and on the first day 8*5-26 rads. With injection of permissible quantities of iron oxide for 15 months no cancer was observed. It is noteworthy that the threshold for the carcinogenic effect with iron was significantly lower than with the other insoluble isotope compounds we have studied (32P and 198Au). This has enabled us to suggest that there is apparently no single threshold for the carcinogenic action of radioactive isotopes, and the value of the threshold may be affected by many factors, including the chemical and biological properties of the element, in this case 59Fe. It can also be assumed that for isotopes with short half-lives, the amount of radiation and the total energy absorbed are the main factors, which for these isotopes (24Na, 198Au, 32P) must be large (thousands of rads), as T. A. Kochetkova and G.A.Avrunina's investigations have shown. The time factor is of primary importance for isotopes with long half-lives, i.e. the duration of the prolonged effect of small doses. This proposition requires further experimental verification and in fact forms one of this laboratory's current research problems.

But the data already available are enough to indicate the necessity for the strict standardization of radioactive aerosols, the prolonged inhalation of which either creates large local doses at the sites of deposition or has an irritant effect in these areas, leading to the occurrence of neoplasms in the

Toxicology of Radioactive Ir on-5 9 9

lungs (T.A.Kochetkova dnd G.A.Avrunina, N.D.Sagaidak, Cember, and others).

In accordance with the work programme of this laboratory and the scope of the Institute of Occupational Hygiene and Disease AMN U.S.S.R., each investigation on the chronic effect of a radioactive isotope must conclude by suggesting not only biologically based standards, but also means of stimulating excretion. A. A. Rubanovskaya has studied the effect of the calcium di-sodium salt of cyclohexane-diaminotetra-acetic acid (CDTA) and the same salt of ethylene-diaminotetra-acetic acid (EDTA) on distribution and excretion of 59Fe. Both complexes had a similar effect when administered shortly after the dose of the 59Fe under these circumstances, the excretion of 59Fe in the urine increased by 2-3 times as compared with the controls, while content in the liver was reduced by 35 to 67-5 per cent, and in the kidneys by 39 to 54 per cent of the control. But whereas administration of the complexes may be effective and justifiable in the case of acute 59Fe injury, where the chronic effect of small doses is concerned these substances, apart from stimulating excretion of extracellular iron, may lead to loss of other elements, as shown by Teisinger and others. Therefore these complexes cannot be recommended for prophylactic administration as a means of controlling the chronic effect of 59Fe. A. A. Rubanovskaya has shown that pectin can be used successfully in prophylaxis. When given with small quantities of stable iron, pectin limits the absorption of 59Fe from the gastro-intestinal tract and reduces its concentration in the organs. In this respect polyvinyl-pyrrolidone proved quite ineffective in similar experiments. Thus, on the basis of A.A.Rubanovskaya's work, it can be stated that pectin and compounds of CDTA and EDTA are effective means of stimulating excretion of 59Fe from the body in the early stages of intoxication.

This brief review far from exhausts all the problems in the toxicology of 59Fe which are discussed in the articles which follow. The aim was to draw a few general conclusions and to direct attention to a series of principles important for an understanding of the toxicology of 59Fe.

REFERENCES

AUSTONI M.E. and G R E E N B E R G D . M . , / . Biol Chem., 134, 27-41 (1940). BOGDANOVA N.G. , ByulleterC TsentraV nogo in-ta informat sii chernoi metallurgii, 14 (346),

15-18 (1958). BOGDANOVA N.G. , G R U Z I N P . L . , ERMOLAYEVG.I . and NIKULINSKII I .D. , The use of

radioisotopes in investigating metallurgical processes (Primeneniye radioaktivnykh izo-topv dlya issledovaniya metallurgicheskikh protsessov). Transactions of the 2nd International Conference on the Peaceful Uses of Atomic Energy, Geneva (1958).

BUL'SKII M.T., SKREBTSOV A. M., VAL'TER AA.etal., Byull. Tsent. in-ta informat sii chernoi metallurgii, 4 (346), 18-21 (1957).

C O P P D . H . and G R E E N B E R G D . M . , / . Biol Chem., 164, 389-401 (1946). GRANICK S. and HAHN P., J. Biol. Chem., 155 (2), 661 (1944). GREENBERG D.M. and COPP D.H. , / . Biol. Chem., 164, 377-387 (1946). HAHN P., Advances in Biological and Medical Physics, 1 (7), 288-^81 (1948).


KAVETSKIIR.E., STOLYAROVAL.B., NIKITENKOR.D. and AMDURSKAYAP.M., The effect of small doses of radioactive substances on the morphological and biochemical composition of the blood (Vliyaniye malykh doz radioaktivnykh veshchestv na morfologiches-kii i biokhimicheskii sostav krovi). Paper read at the All-Union Conference on the uses of radioactive and stable isotopes and radiations in the economy, Moscow (1957).

KLYUCHAREV L. A., Iron metabolism in bloodloss and shock (Obmen zhelezy pri krovopo-teryakh i shoke), Dissertation (1953).

KURLYANDSKAYA E.B., The Toxicology of Radioactive Substances (Materialy po toksiko-logii radioaktivnykh veshchestv), vol. 1, Moscow (1957); vol. 2 (1960).

ZBOROVSKII A.A. et al, StaV, 1, 24-30 (1957).

DISTRIBUTION AND EXCRETION OF DIFFERENT RADIOACTIVE IRON (59Fe)

COMPOUNDS AFTER INTRATRACHEAL INJECTION IN WHITE RATS

N. D. S A G A I D A K

E.B.KURLYANDSKAYA and co-workers, D.I.Zakutinskii and co-workers, Yu. I. Moskalev and others, have shown that the body's reaction to internal administration of small quantities of radioactive substances depends, to a considerable extent, on the physico-chemical properties of these substances and the means of administration. The solubility of the substance in the biological media of the body is of particular significance.

In this connection the distribution and excretion of different 59Fe compounds after entering the respiratory organs is of interest.

There are many references in the literature concerning iron metabolism. According to Granick the human body contains about 5 g iron, 60-70 per cent of which is in haemoglobin. The presence of iron-containing haem in the haemoglobin molecule is responsible for its respiratory activity. The muscles contain 3-5 per cent of total iron in the form of myoglobin, which creates some oxygen reserve. In addition, iron is contained in many enzymes (catalase, peroxidase, cytochrome, cytochromoxidase), the properties of which, as biological catalysts of the oxidative processes, are linked with the presence of iron in the molecule. These substances include the iron-containing porphyrin—haem, from which they have received the name of haem compounds.

The liver and spleen contain 15-22 per cent of total body iron. It is contained in non-haem compounds—ferritin and haemosiderin, which have a storage function.

Iron metabolism in the body can be described as follows : The iron in food is absorbed from the gastro-intestinal tract, enters the blood and is then transferred in considerable amounts to the liver, which is the main storage depot of iron. As it is required, iron enters the bone marrow from the depot or directly from the plasma, and is there used in haemoglobin synthesis. The iron liberated from destroyed red cells is re-utilized, firstly, in the construction of new red cells; thus the body's iron requirement is limited. The human adult's daily requirement of iron is 6-12 mg. In children during the growth period, in pregnant women, and after bloodloss, the iron requirement increases. TRS 2

11


Hahn (1948), and Copp and Greenberg (1946) in experiments on animals have shown that when 59Fe is administered orally absorption takes place along the entire length of the gastro-intestinal tract from the stomach to the caecum. Iron assimilation from food depends on the body's total iron stores and increases when the internal reserves are depleted.

Iron absorbed into the blood enters all the organs and tissues of the body. The greatest accumulation is found in the liver, bone marrow and blood. As Kheveshi (1950) indicates, accumulation of labelled iron in organs and tissues depends on the means of administration and the condition of the experimental animals.

According to Austoni and Greenberg (1940), and Copp and Greenberg (1946), the greatest accumulation of labelled iron in the liver, up,to 30-40 per cent of the administered amount, is found after intravenous and intra-peritoneal injection and the smallest (3-5 per cent of administered) after oral administration. As Hahn (1948) has shown, direct exchange between the iron of the plasma and mature red cells in the peripheral blood does not occur. The iron content of the bone marrow, and then of the liver falls when haemopoiesis is stimulated.

Hahn and co-workers have established that with uptake of iron from food in approximately physiological amounts, negligible excretion in the faeces is observed. Under normal conditions iron is not excreted in the urine. When large quantities of labelled iron are administered orally the body regulates its intake by limiting absorption. The unabsorbed iron is excreted predominantly in the faeces.

With parenteral administration of radioactive iron in large quantities excretion in the urine is observed on the first day after injection. However, only a small amount of iron is voided in the urine, 2-5 per cent of the injected amount, and as McCance has shown, there is no mechanism in the body by which it can excrete completely radioactive iron injected parenterally.

We have found no references in the literature concerning the fate of different 59Fe compounds absorbed from the respiratory tract.

M E T H O D S

The experiments were performed on adult white rats. A finely-dispersed 59Fe oxide powder was used as an insoluble compound, and a neutral solution of iron citrate as a soluble compound. The bloodless intratracheal method was used with visual insertion of the needle into the trachea.

With intratracheal injection, some of the injected material does not penetrate into the lungs but is swallowed or coughed up by the animal, so that the amount of material actually entering the lungs is somewhat different from that calculated. This difference is especially marked when a suspension in which the suspended particles are unevenly dispersed is injected.

To determine the actual amount of 59Fe entering the lungs a preliminary investigation was made. Ten rats were given intratracheal injections of a

Distribution and Excretion of Different Radioactive Iron (59Fe) 13

certain amount of 5 9 Fe 2 0 3 suspension under conditions identical with those of the main experiment (order of injections, weight of powder, degree of dispersion). The rats were killed 10-15 min after injection and the radioactivity in the lungs measured. A sample of the suspension, taken with the same syringe and needle as were used for injection, was used as a standard of radioactivity.

The results showed that from 60 to 102 per cent of the 59Fe oxide injected, mean 76 + 4 per cent, is found in the lungs immediately after injection. A similar experiment with 59Fe citrate solution showed that an average 88 ± 2 per cent of the injected quantity, with variations from 74 to 95 per cent in particular cases, is found in the lungs immediately after intratracheal injection.

These results enabled us to evaluate more accurately those of the main experiment.

In order to investigate distribution and excretion of 59Fe one group of rats (22 animals) was injected with a neutral solution of 59Fe citrate in an amount of 1 ml per rat with activity of 18-20 μο. The animals of the second group (18 rats) received a 10-mg suspension of a virtually insoluble finely-dispersed 59Fe oxide powder in 1 ml physiological saline giving a dose of 15-22 μο per rat. The animals were housed in replaceable cells enabling separate collection of urine and faeces. For 16 days the y-radiation from the urine and faeces was measured daily. The animals were killed from 1 hr to 20 days after injection, 2-3 rats at a time.

Activity of tissues and excretions was measured on a B-l apparatus using a cylindrical y-counter. The activity of 1 g wet tissue was calculated in microcuries and expressed as a percentage of the amount injected.

The samples were treated by the usual method. To calculate total 59Fe content of those organs and tissues which cannot be directly put into suspension the following ratios were used :

muscles 40 per cent of body weight blood 7-5 per cent of body weight bone marrow 2 per cent of body weight intestine 10 per cent of body weight

D I S T R I B U T I O N OF 59Fe IN RATS

Content of 59Fe in tissues and organs after intratracheal injection of radioactive iron citrate is given in Table 1.

It can be seen from Table 1 that the 59Fe content of the lungs falls sharply during the first 2 days. After 48 hr only 12-8 per cent of the amount injected remains in the lungs. During the following days, removal of 59Fe from the lungs proceeds much more slowly, and on the 30th day 9-2 per cent still remains at the injection site. The cause of this prolonged retention of some iron citrate in the lungs is apparently the formation of complex compounds of iron with tissue proteins, the absorption of which is impeded.


The largest amount of iron is found in the liver, where on the 2nd day 24-1 per cent of the injected quantity was detected. Also, a considerable accumulation of 59Fe was found in the bone marrow (16-36 per cent).

The selective accumulation of 59Fe in the liver and bone marrow is easily seen when the specific activity of organs and tissues is determined (Table 2, Fig.l).

After 24, 48 and 72 hours the specific activity of the bone marrow is higher than of any other organ, but later a comparatively rapid removal of 59Fe is

TABLE 1. Content of59Fe in Organs and Tissues as a Percentage of the Amount of Iron Citrate Injected

Arithmetical Mean Values from 2-3 Animals

Organ

Lungs Liver Spleen Kidneys Intestine

and contents Heart Muscles Blood Bone marrow

Body total

Interval after injection

hour

1

61-9 0-96 0-02 0-21

12-96 0-015 1-2 3-1 0-48

80-84

days

1

22-2 12-23 0-21 1-72

11-61 0-06 2-76 1-35

11-88

64-02

2

12-8 24-1 0-25 3-1

7-9 0-15 2-16 4-48

16-30

71-24

3

11-0 18-9 0-22 2-18

5-61 0-13 3-0 8-77

13-37

62-18

4

10-1 21-5 0-33 2-3

1-65 0-12 4-2

10-9 6-12

57-12

10

15-0 17-1 0-32 1-71

1-35 0-13 2-4

11-27 3-72

50-0

16

8-6 11-7 0-34 1-2

1-8 0-2 2-4

11-25 2-88

40-37

20

8-6 8-3 0-15 0-83

0-6 0-09 1-8

10-01 2-22

32-6

30

9-2 9-2 009 0-35

0-39 0-08 0-60 7-65 1-08

28-64

TABLE 2. Specific Activity of Organs and Tissues in Microcuries per 1 g after a Single Intratracheal Injection of 20 ßc Iron Citrate


Organ


and contents Heart Muscles Blood Bone marrow


hour

1

5-630 0-018 0-004 0-018

0-086 0-004 0-002 0-028 0-016

days

1

1-970 0-188 0-048 0-163

0-057 0-011 0-005 0-012 0-396

2

1-260 0-462 0-059 0-285

0-053 0-028 0-004 0-039 0-574

3

1-060 0-452 0-054 0-218

0-037 0-025 0-005 0-078 0-486

7

1-010 0-398 0-073 0-248

0011 0-028 0-007 0-097 0-204

10

1-360 0-332 0-079 0-138

0-009 0-028 0004 0-101 0-124

16

0-900 0-234 0-082 0-0134

0-012 0-044 0-004 0-100 0096

20

1-03 0-195 0036 0-071

0-004 0-02 0-003 0-089 0-074

30

0-960 0-198 0-020 0-030

0-002 0-022 0002 0-068 0-036


observed, and on the 30th day only 1-08 per cent of the injected isotope is found in the bone marrow (0036 μο^).

Only an hour after injection 59Fe appears in the blood, but it accumulates slowly, reaching its highest level during the 7th-10th days (11-27 per cent of the injected quantity). The 59Fe content of the blood remains at a high level for a considerable time, and 7-65 per cent still remains on the 30th day. No selective accumulation of iron in the spleen was observed. The specific activity of the spleen did not exceed 0-059 μο/g. A considerable amount of 59Fe

FIG. 1. Accumulation of 59Fe in rat organs and tissues after a single injection of 59Fe citrate.

1—liver; 2—blood; 3—bone marrow; 4—kidneys

was found in the kidneys, especially during the first days after injection. This indicates that the kidneys play a role in its excretion.

These results show that when 59Fe is injected into the lungs in the form of a citrate solution, significant quantities are withdrawn from the lungs and distributed to all the organs and tissues of the body, with accumulation chiefly in the liver, bone marrow and blood. It should be noted that iron accumulates in the bone marrow in advance of its accumulation in the blood and kidneys. But later the marrow content falls rapidly, whereas it continues to accumulate in the blood. A peculiar redistribution of 59Fe occurs, indicating that the iron in the bone marrow is being used for the formation of new red cells which subsequently enter the peripheral blood.

Rather prolonged retention of 59Fe is observed in the liver, accumulating there in the form of ferritin.

A quite different picture of 59Fe distribution is observed when an insoluble oxide in suspension is injected into the lungs (Table 3).

At 30 days after injection 61-2 per cent of the injected amount was found in the lungs, whereas 75-3 per cent was present immediately after injection. Thus, in 30 days the amount of 59Fe in the lungs declined by only 15 per cent. A significant amount of 59Fe was found in the gastro-intestinal tract on the first day, the result of swallowing a certain quantity during intratracheal injection. In the other organs and tissues a very small quantity of 59Fe was


found on certain days. After 30 days of observation, the isotope was no longer detected in the animal's organs, with the exception of the gastrointestinal tract, where a small quantity appeared from time to time.

TABLE 3. Content of59Fe in Rat Organs, Tissues and Excreta as a Percentage of the 59Fe Oxide Injected


Organ


and contents Muscles Blood Bone marrow Urine Faeces


hour

1

75-3

. 1-67

days

1

87-1 0-04 0-01

0-95

0-03

0-015 1-84

2

76-5 0-06

003

0-63

0 0 2

0-01 0-56

3

73-2 0-01 0-02 001

0-17

0-48

7

75-1

005

0-01 0-01

0-06

10

66-8

001 -

0-02

16

69-7 0-02

0-01

0-05

20

64-4 001

0-01

30

61-2

004

EXCRETION OF 59Fe FROM THE B O D Y

After intratracheal injection of 59Fe in the form of a citrate the main mass of iron is excreted during the first 7-10 days (Table 4).

The course of excretion of 59Fe from the body is shown in Fig. 2.

\ A yv

2 4 6 8 10 12 14 16 Days after injection

FIG. 2. Excretion of 59Fe in the urine and faeces as a percentage of the amount of iron citrate injected.

1—excretion in urine; 2—excretion in faeces

As can be seen from the figures, daily excretion of 59Fe falls sharply: on the 1st day 11-3 per cent of injected amount is excreted, on the 8th day 2-8 per cent, and on the 16th day 0-3 percent. In total, during the 16 days, 54-8 per

.tz c:

-» ΟΓ

co o

<*-o co

o c α> o

δ 7 6 ς 4 3 2 1


cent of the injected quantity was excreted. The biological half-excretion period of 59Fe is 10 days.

The considerable activity of the kidneys in the excretion of 59Fe is a striking feature of our experiments, whereas the role of the kidneys is hardly recognized in the literature. According to our results 23 per cent of the activity injected into the lungs was excreted in the urine during the 16 days. The high 59Fe content of the urine, especially during the first few days after injection, is clearly connected with the massive uptake of iron citrate from the lungs into the plasma, from which it cannot all be utilized by the liver and bone marrow and is removed through the kidneys.

Removal from the lungs of insoluble 59Fe oxide particles is mainly by phagocytosis. The phagocytosed particles are removed either through the respiratory tracts with the sputum or by lymphatics to the regional lymph

TABLE 4. Daily Excretion of59Fe in the Urine and Faeces as a Percentage of the Intratracheally Injected Iron Citrate

Arithmetical Mean Values from 5 Animals


(days)

1 2 3 4 5

6 and 7 8 9

10 11 12

13 and 14 15 16

Total

Excreted in urine

8-1 5-3 1-7 2-5 0-6 1-6 0-5 0-8 0-3 0-5 0-6 0-2 0-3 0 1

231

Excreted in faeces

3-2 4-6 5-9 3-2 2-9 3-0 2-3 2-0 1-6 11 0-6 0-7 0-4 0-2

31-7

Total excreted

each day aggregate

total

ί 11-3 | 9-9 21-2 7-6 28-8 5-7 j 34-5 3-5 4-6 2-8

38-0 42-6 45-4

2-8 48-2 1-9 1-6 1-2 0-8

50-1 51-7 52-9 53-8

0-7 > 54-5 0-3 j 54-8

1

54-8 | 54-8

Retained in the body

88-7 78-8 71-2 65-6 62-0 57-4 54-6 51-8 49-9 48-3 47-1 46-2 45-5 45-2

45-2

nodes. This is confirmed by the almost constant discovery of 59Fe in the gastro-intestinal tract, where it arrives in swallowed sputum containing 59Fe, and by the increased radioactivity of the regional lymph nodes in the later stages after injection.

However, this mechanism results in only partial removal of dust from the lungs, most remaining there for a considerable period. Solution and absorption obviously play little part in this case.


C O N C L U S I O N S

1. After intratracheal injection of rats with soluble 59Fe citrate and insoluble 59Fe oxide substantial differences in distribution of the two compounds in the body are detected.

2. Most of the 59Fe citrate is absorbed from the lungs during the first few days after injection and is distributed in all the organs and tissues, accumulating predominantly in the liver, bone marrow and blood. At the end of a month about 9 per cent of the quantity injected remains at the injection site.

3. When 59Fe oxide is injected intratracheally almost all is retained in the lungs, being excreted in small amounts mainly in the sputum. In a month 15 per cent of the 59Fe injected is removed from the lungs.

REFERENCES

A U S T O N I M . E . and GREENBERG D.M. , / . Biol. Chem., 134, 27-41 (1940). C O P P D . H . and GREENBERG D.M. , J. Biol. Chem., 164, 377-389 (1946). GRANICK S. and H A H N P.S., / . Biol. Chem., 155 (2), 661 (1944). GREENBERG D.M. , C O P P D . H . and CUTHBERTSONE.M. , / . Biol. Chem., 147, 749 (1943). HAHN P.F. , Advances in Biological and Medical Physics, 1, 288^84 (1948). KHEVESHI G., Radioactive Indicators (Radioaktivnye indikatory), Moscow (1950). KURLYANDSKAYA E.B., The Toxicology of Radioactive Substances (Materialy po toksikolo-

gii radioaktivnykh veshchestv), vol. 1, pp. 3-13, Medgiz (1957). MCCANCE R. A. and WIDDOWSON E.M., / . PhysioL, 94, 148-154 (1938). ZAKUTINSKII D. I., Problems in the Toxicology of Radioactive Substances (Voprosy toksiko-

logii radioaktivnykh veshchestv), Moscow (1959).

BODY RADIATION DOSE IN RABBITS PRODUCED

BY DAILY ORAL ADMINISTRATION OF 59FeCl3, AND SOME DATA

ON ACCUMULATION AND EXCRETION OF 59Fe

G. A . AVRUNINA

THE EVALUATION of the radiation dose from an isotope administered internally requires, as is known, a study of distribution and excretion of the isotope from the body. Iron metabolism and, especially, the mechanism of its uptake by the body, distribution and excretion have been studied in detail by many writers (for a review of these references cf. the article by N. D. Sagaidak in the present volume).

It is known that iron entering the blood stream is excreted through the kidneys, bile tracts and intestine in negligible amounts. The level of iron in the body is regulated solely by an increase or decrease in the intake of iron from the food by the intestine. Normally, absorption occurs in the duodenal part of the small intestine, but with increasing iron deficiency the area of absorption extends towards the distal parts of the small intestine (Wack and Wyatt, 1959).

References in the literature on the magnitude and speed of uptake of iron administered orally are somewhat imprecise and contradictory. This is because, apart from the body's requirement of iron and the state of erythro-poiesis, other factors are important, such as species of animal, amount and compound of iron, pH of the solution, type of food, speed of evacuation of the intestine, and so on.

The distribution of iron in the organs and tissues as shown with 59Fe or 55Fe, corresponds with its physiological functions. During the first few hours after a single administration, iron accumulates in the bone marrow, liver, spleen, blood (mainly in the plasma) and to a lesser extent in the muscles. By the end of the 1st or 2nd day the radioactivity of these tissues has fallen considerably, whereas the radioactivity of the blood, and to a somewhat less degree, the spleen has steadily increased, until the 5th day (Copp and Greenberg, 1946); K. S. Zamychkina and R. A. Durinyan, 1958), when the maximum level is reached. After 4-5 days the greater part of the blood's activity is in the red cells.

19


In the literature which has reached us, we have found no precise data on the course of radioactivity in the body during daily administration of 59Fe to a normal animal.

The present work is concerned with accumulation and excretion of 59Fe in animals receiving daily, over a long period, a solution of 59FeCl3 orally, and the radiation doses received in the organs separately and in the body as a whole. For comparison, some data on absorption and excretion of 59Fe following a single administration, intravenously or orally, were obtained. The connection between accumulation of 59Fe in the body and amount of carrier in the solution was also investigated.

MEASUREMENT AND C A L C U L A T I O N

The technique of measurement was the same as that described in our earlier papers (G.A.Avrunina, 1960), with the sole difference that the faeces were not incinerated but measured whole. The total amount collected from a single rabbit was transferred to a flask, made up to a volume to 1 litre with water and measured in the standard position using a y-probe. The probe for this purpose was calibrated with a solution of 59Fe of the same volume and known activity.

FIG. 1. Arrangement of apparatus for in vivo measurement of radioactivity in the body of rabbits and in liquid samples of high activity (10-100 μο).

y-probe in lead cover with aperture, box for rabbits, flask of liquid. On the table are outlined the standard positions for the box and flask.

The method of in vivo determination of total body radioactivity by measurement of external radiation described previously (G.A.Avrunina, 1960) was used extensively both in the experiments in which the animals received a single dose and in those which received many daily doses. Figure 1 shows apparatus B which was used for this purpose. This arrangement was also used to measure the radioactivity in flasks containing samples with high

Body Radiation Dose in Rabbits 21

activity including those with excreta. Calibration of the probe for in vivo measurement of whole-body radioactivity was based on the radioactivity, as measured by external radiation, in the corpses of 23 rabbits. This value was calculated according to the specific radioactivity of separate tissues and their distribution by weight in the body, as found for 29 rabbits. This distribution can be found in one of the columns of Table 4. The value of the impulse for measurement in the selected standard positions was found to be 25-9 x 10~3 fjic impulse.

TABLE 1. Doses of γ-Radiation in the Body According to Body Weight Specific Activity 1 μc|g

Weight (g)

300 350 400 450 500 550

3000 3500 4000 4500 5000 5500

Strength of dose

(rad/hr for 1 μο/g)

0-24 0-252 0-263 0-273 0-283 0-294 0-462 0-483 0-504 0-525 0-546 0-567

Daily dose (rad/day for

i με/g)

5-75 6-05 6-3 6-55 6-8 6-05

11-1 11-6 121 12-6 13-1 13-6

The activity of the gastro-intestinal tract (AGl) was calculated from in vivo measurements as follows. From Table 4 it is seen that, on average, the blood in the body accounted for 40 per cent of total body activity, without the gastro-intestinal tract (AB). Knowing that the weight of blood is 5 per cent of body weight, it is not difficult to calculate that :

AB = 0T25 x specific activity of blood ^c/g) x weight of rabbit (g)

The activity in the body AB thus obtained was subtracted from the whole activity measured by external radiation and including the gastro-intestinal tract (ABGl) and AGl obtained. The principles of dose calculation from internally administered isotopes and an evaluation of their accuracy have also been discussed by us in the above-mentioned papers (G. A. Avrunina, 1960).

For 59Fe, having mean energy of/3-particles 0T20 MeV, the daily dose of ß-radiation is 6T5 x C rads/day, where C is the specific activity of the tissue under examination.

In Table 1 are set out the y-radiation doses from 59Fe (ionization constant = 6-55) for a specific tissue activity of 1 μο/g according to weight of organ or whole body. To obtain the dose value required the figures in the table must be multiplied by the mean specific activity of the body.


SINGLE A D M I N I S T R A T I O N OF 59FeCl3 TO RABBITS

Two groups of 4 rabbits each received a solution of 59FeCl3 either orally or intravenously. Using a tube the animals of the first group were each administered 650 μc 59Fe in a volume of 2 ml, containing 3-3 mg/ml stable

FIG. 2. Total excretion of 59Fe after a single administration of 59FeCl3 to rabbits. 1—oral; 2—intravenous

— - . · - · . .

· - · - · · — · · — · _ · 10 15 20 25 30

Days 35 40 45

FIG. 3. Excretion of 59Fe from the body after single administration of 59FeCl3 to rabbits (in vivo measurement according to external y-radiation).

1—oral: 2—intravenous

iron. The animals of the second group were injected via the auricular vein with 2 ml of the same solution diluted 10 times with distilled water, i.e. about 65 [AC The ratio of activities of 10:1 was chosen in order to obtain


comparable values of activity of blood and tissues, based on the assumption that absorption in the intestine does not exceed 10 per cent. Immediately after administration, the total body radioactivity of the animals was measured after which they were placed in changeable cells. Blood was taken after 1^, 3 and 6 hr for measurement of radioactivity. Urine and faeces were collected for the first 24 hr. Subsequently, radioactivity of the blood and total body

c/co c/co

•^•-••"

1-5

0-5

10 15 20 25 30 35 40 Days

C/CO Ί-0

OS

0-6

-10-4

0-2

8 12 16 Hours

20 24

FIG. 4 a. Blood content of 59Fe after single administration to rabbits of 59FeCl3 commencing from the second day.

C/CO—ratio of Fe concentration per give of blood of the average amount administered per 1 g weight of animal.

1—oral (right-hand scale); 2—intravenous (left-hand scale). FIG. 4b.—the same during the first day.

radioactivity, as well as urine and faeces, were measured at intervals of 1-2 days. On the first and second days urine and faeces were measured separately, but then in view of the negligible excretion of iron in the urine in both groups total excreted activity was measured. After 10 days, 2 rabbits in each group were killed and the radioactivity remaining in the gastro-intestinal tract and carcass, and the specific radioactivity of certain organs and


tissues, were measured. Two rabbits in each group were left for more prolonged observation of the decline in radioactivity of the blood and whole body. Collection of excreta was terminated after 10 days since by the end of a week excretion had fallen to insignificant levels.

Figure 2 shows total excretion of 59Fe by rabbits after a single intravenous or oral administration of 59FeCl3. Only values for total excretion are given because excretion in the urine in both cases did not reach even 1 per cent per day and was not suitable for indication on the scale chosen. It can be seen

TABLE 2. Distribution of59Fe in Rabbit Organs 10 Days after a Single Administration of59FeCl3 Orally or Intravenously (μο^)

Organ

Liver Spleen Kidneys Lungs Blood Muscle Bone Suprarenals Average for

whole body

Activity ^c /g )

Intravenous injection

10-3 ± 1-44 3-3 ± 0-66 2-3 ± 0-50 1-1 ± 0 - 3 3 7-8 ± 0-78 0-04 ± 0-01 0-5 ± 0-13 0-9 ± 0-24

0-9

Oral administration

0-13 ± 0-003 0067 ± 0-016 0-075 ± 0-010 0-072 ± 0-005 0-375 ± 0-06 0-002 ± 0-0004

0-037 ± 0-001

0-031

Ratios of activities per 1 g

tissue oral to intravenous

0-013 0-02 0-032 0-065 0-048 0-05

0-041

0-034

that after intravenous injection, virtually all the radioactivity administered is retained in the body. With oral administration most of the 59FeCl3, being unabsorbed, is excreted from the intestine during the first 2-4 days. At this period the biological half-life Tb is 1-3 days. Absorption of 59Fe from the intestine can be estimated by the radioactivity found in the body of killed rabbits (excluding the contents of the gastro-intestinal tract), the radioactivity of the blood or by measurement of whole-body radioactivity of the animal (Fig. 3) after most of the isotope has been excreted. Errors caused by excretion of iron already absorbed can be discounted by comparing results for both groups. According to our results about 5 per cent of the quantity administered is absorbed.

The blood content of 59Fe is shown in Fig. 4. It can be seen that up to the 6th-8th day the radioactivity increases, thereafter remaining at a constant level for 25-30 days after which it slowly falls.

From Figs. 3 and 4 it is seen that, regardless of the means of administration, 59Fe entering the blood stream is excreted with a very long Tb. As the curves indicate, the Tb for iron in this case is not less than 100 days.

Distribution of 59Fe in the organs on the 10th day after a single administration of 59FeCl3, when the blood radioactivity has reached a maximum, is shown in Table 2.


The concentration of 59Fe is highest in the liver, followed by the spleen and kidneys. By virtue of the spleen's physiological function it might be expected that, at later periods, this order would change and greatest radioactivity (not counting the blood) would be found in the spleen. In support of this is the fact that with prolonged oral administration of 59Fe the radioactivity of the spleen is always higher than that of the liver.

A C C U M U L A T I O N AND E X C R E T I O N OF 59Fe IN R A B B I T S D U R I N G DAILY O R A L A D M I N I S T R A T I O N

In this experiment the animals received daily, by mouth, 59FeCl3 solution in doses either of 10 or 1 μ^1<£ body weight.

To investigate the course of accumulation and excretion, some rabbits from both groups were housed in interchangeable cells for 1-2 weeks from the beginning of the experiment and again at later periods. Studies were also made of whole body activity by external radiation during the period of daily administration and after (for this purpose some animals were kept under observation for a month after administration ceased). At the same time the radioactivity of the blood was measured.

100

VÌI -σ

o OJ

20

Γ r "~ T - 1

I ! i I i I

in Ì1 L i i '

J L_J I I L_

29 Weeks

FIG. 5. Total excretion of 59Fe during a week by rabbits receiving oral 59FeCl3 daily.

The broken line indicates daily excretion during the first week.

In rabbits dying at different intervals after the beginning of the experiment (from 3 to 21 months), the specific radioactivity of the tissues, the whole of the gastro-intestinal tract and the total radioactivity of the carcass were measured by external radiation.

Iron administered daily by mouth is excreted very rapidly and unevenly (because most of it is not absorbed by the intestine). Significant variations occur in different rabbits and in the same rabbit on different days. Therefore, Fig. 5 shows mean excretion for a week. Since no differences were observed


in percentage excretion of either group receiving (10 and 1 μc/kgperday) the results for the two groups have been combined. In order to illustrate variations in daily excretion, the broken line shows the daily excretion during the first week. The diagram shows that by the second week, and possibly even by the end of the first, excretion has virtually reached a maximum. This,

FIG. 6. Increase in total body radioactivity of rabbits receiving oral 5 9FeCl3 daily. 1 — 1 μο/kg (left-hand scale); 2—10 μο/kg (right-hand scale)

:=->

1 1 0 0 1 'X3 -Q

.*tr o o a. to

O

yS

^ ̂ I 1 I 1 I I I 1 1 I

o

•

1 C

2 ~i

μί/ηιι

0-1

Ü-01

IQ 20 30 40 50 60 70 Days

90 100 110 120 130 140

FIG. 7. Increase in the specific radioactivity of the blood in rabbits receiving oral 5 9FeCl3 daily.

1 — 1 μ^1<£ (left-hand scale); 2— l ( ^ c / k g (right-hand scale)

however, does not indicate that maximum level of body radioactivity has been reached, because at such low levels of absorption the level of body activity is masked by the error in measurement of excreted activity. Total body activity increases with large variations for 2-3 weeks, linked with the variation in daily excretion (Fig. 6). During this time the activity of the gastro-intestinal tract contents probably increases. As can be seen from Fig. 7, maximum blood radioactivity is not reached for some time—45-65 days. This slow increase is caused by redistribution of 59Fe in the body and partly by the daily uptake of supplementary small amounts of the isotope


from the intestine. For the reason indicated above this is not markedly shown in the general level of radioactivity G4BGI)·

The plateau reached by the radioactivity curves for the blood and whole body (cf. Figs. 6 and 7) indicates an equilibrium level of activity for 59Fe, as has already been shown and demonstrated theoretically for many isotopes (E. B. Kurlyandskaya et ai, 1957; A.A.Rubanovskaya andUshakova, 1957; L.N.Burykina, 1957; G.A.Avrunina, 1960; and others). Confirmation of this can be seen in Table 3, which shows that the level of body activity does not depend on duration of administration.

Speed of excretion and, correspondingly, the rapidity with which the equilibrium level of 59Fe in the whole body (including the gastro-intestinal tract) is established, depend greatly on the type and volume of food. Thus, in several rabbits receiving beet only for the first week instead of the usual diet (oats or bread, hay, milk beet), mean excretion during this week was only 15 per cent of the radioactivity administered, but increased to the expected level (101 per cent) during the following week, when the rabbits were transferred to their normal diet. Correspondingly, the equilibrium level of total radioactivity was reached only 4 days after commencement of administration and was very high.

TABLE 3. Radioactivity in the Body without the Gastro-Intestinal Tract (AB) of Rabbits Dying

at Various Intervals from the Beginning of the Experiment

Percentage of the Amount Administered Daily

No. cf rabbit

110 111 59 60 102 57 24 35 11 54 4 12 1 65 66

Life-span in months

2 2 3 3 7± H 12 151 16i 17 17 21 21 21 21

ΛΒ

177 206 50 44 100 131 115 320 117 160 69 54 105 112 169

Distribution of radioactivity, after reaching equilibrium level, in the period from 3 to 24 months is shown in Table 4. The highest specific radioactivity, as to be expected, is found in the blood and spleen, followed usually by the liver, then the lungs, kidneys and other parenchymatous organs. Muscle TRS 3

TABL

E 4.

Spe

cific

and

Tot

al R

adio

activ

ity o

f Ti

ssue

Fou

nd in

Rab

bits

Kill

ed o

r D

ying

whi

ch h

ad R

ecei

ved

Ora

l 59 F

e in

an

Am

ount

of

1 μc

|kg

(17

Ani

mal

s) a

nd 1

0 ßc

jkg

(36

Ani

mal

s) p

er D

ay.

Ave

rage

Wei

ght o

f Rab

bits

3-6

kg

Tis

sue

or o

rgan

Blo

od

Liv

er

Sple

en

Lun

gs

Kid

neys

H

eart

Su

prar

enal

s U

teru

s \

Ova

ries

T

este

s B

one

mar

row

O

ther

par

ench

ymat

ous

orga

ns

Mus

cles

B

ones

B

rain

Fa

t, fu

r, s

kin

and

othe

r tis

sues

sim

ilar

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cle

in a

ctiv

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>

Ave

rage

for

who

le b

ody

with

ou

t the

gas

tro-

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cont

ents

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of t

otal

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ound

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^1<£

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ific

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(μ

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/kg

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164

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177

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ws:

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ai

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he a

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m.



radioactivity is a tenth of that of the above organs. The radioactivity of the bones and brain is almost identical and of the same order as that of the muscles. The specific radioactivity of the bile is lower than that of the liver. Considerable radioactivity is not infrequently found in the lungs, caused by aspiration of part of the solution during administration. In the gastro-intestinal tract most radioactivity is found in the caecum and other sections of the large intestine. The total radioactivity of the gastro-intestinal tract contents varies within very wide limits, from a part of the amount administered daily

-o 100

5 10 15 20 25 30 35 40 Days

FIG. 8. Excretion of 59Fe from the blood and body of rabbits after cessation of daily administration of 59FeCl3. Data are expressed as a percentage of the values

obtaining on the day administration ceased. 1—blood radioactivity of rabbits receiving 1 and 10 μΰ/kg 59FeCl3 (curve common to both groups); 2—radioactivity in the body of rabbits receiving 1 μc/kg; 3—radio

activity in the body of rabbits receiving 10 μΰ/kg

to 5-8 times this amount. The spread of results in the group receiving 10 μΰ/kg per day is so great as to give the impression that some of the animals of this group were closer in total body radioactivity to those of the group receiving 1 μc/kg per day. In fact, this is only an expression of the wide variations in radioactivity of the gastro-intestinal tract correlated with large fluctuations in excretion.

In Fig. 8 the decline in radioactivity of the blood and body after cessation of daily administration is shown. The data are presented as a percentage of radioactivity on the first day the animals received no 59FeCl3 solution; the curve therefore shows excretion of 59Fe from the body. During the first 5 days radioactivity in the body declines to 60-70 per cent of its initial level. Clearly, during this time almost all the radioactivity retained in the intestine is excreted. After this, the rate of decline falls and becomes similar to that following a single intravenous injection.

Mean values of the percentage radioactivity remaining in the body of animals in the group receiving 10 μο/kg were somewhat lower than in the group receiving 1 μο/kg. Although the difference between these values is not statis-


tically significant it is maintained all along and, as will be seen later, is capable of explanation. Radioactivity of the blood during the first 20-30 days falls very little.

Doses of/3-radiation received by the tissues of rabbits in the daily experiment, and the y-dose to the body, excluding the gastro-intestinal tract (DyB), calculated on the basis of the specific radioactivity of tissues in rabbits which died (cf. Table 4) and the calculated radioactivity in the body (AB), are given in Table 5. The mean dose of/^-radiation received by the body was also calculated.

TABLE 5. Doses of ß-Radiation in Tissues of Rabbits Receiving Oral 59FeCl3 in an Amount of 10 and 1 με/kg Daily

Organ or tissue

Blood Muscles Liver Spleen Kidneys Lungs Heart Bone marrow Brain Bone Uterus Ovaries Testes Suprarenals Mean /?-dose for whole body, excluding

gastro-intestinal tract Mean y-dose for whole body, excluding

gastro-intestinal tract Mean total ß- and y-dose jn body

without gastro-intestinal tract

Dose of ß-radiation (rads/day)

group receiving ΙΟμφζ

0-596 ± 0-166 0-025 ± 0010 0-375 ± 0-160 0-700 ± 0-260 0-197 ± 0-080 0-215 ± 0-098 0-154 ± 0-049 0-234 ±0 -140 0-027 ± 0-009 0026 ± 0-010 0105 ± 0-060 0-180 ± 0-080 0-135 ± 0-060 0-180 ± 0 - 0 6 0

0-076

0-142

0-218

group receiving 1 μο/kg

0-105 ± 0-031 0-006 ± 0-002 0-062 ± 0-030 0-102 ± 0-054 0040 ± 0-015 0-059 ± 0-027 0-028 ± 0-010 0-068 ± 0-018 0-012 ± 0-006 0-012 ± 0-006 0-021 ± 0-007 0-033 ± 0-030 0-024 ± 0-008 0-031 ± 0009

0-016

0030

0-046

In order to take into account variations in the total dose connected with variations and other factors left out of consideration, for 15 months the radioactivity in the body was measured systematically by external radiation (usually once a week and not less than once a month), and further determinations of the specific radioactivity of the blood was made. Ten rabbits in the two groups receiving 10 and 1 μc/kg were kept under observation. For each rabbit mean radioactivity in the body (ABGi) throughout the observation period was determined, together with the specific radioactivity of the blood and mean weight of the animal. On the basis of these data AB and AQI were calculated and the corresponding doses of y-radiation—DyB, DyBGl and DyGl.

In Table 6 are given the values for each rabbit, averaged over time, and the mean values for the whole group.


Mea

n

Rec

eivi

ng

1 w/k

g

Mea

n

No.

of

rabb

it

I00

101

102

105

1 06

107

108 88

96

117

Wei

ght

(ks)

__

~~

~

3.75

4.

4 3.

9 3.

25

4 3.95

5.

1 3.

75

4 ~

3.7

54

55

56

65

66

67

69

90

91

92

4 4.65

3.95

3.

85

4.65

4.1

~ _-

1

44

Spec

ific r

adic

- ac

tivity

of

the

blo

od

(149

~~

~~

~

0.07

9 00

77

0.08

00

89

0074

0.

087

0.08

0.

091

0.10

4 0.

113

0087

0.01

45

0.01

42

0022

5 0.

0181

0.

0166

0.

01 19

00

125

0019

4 00

17

0.02

03

~~

~

0.0

I67

~-

~

Rad

ioac

tivity

in

the

bcd

y,

excl

udin

g ga

stro

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tinal

trac

t (A

B),

(w)

37

42.3

39

36

.2

37

43

51

42.7

52

52

.3

43.3

-~

-

8.45

8.

3 11

.2

8.95

9.

95

6.9

7-6

9.95

9

65

9-

9

9.08

.-___ ~

87.4

13

9.2

1 08 88

.8

110

I I7

I63 70

.5

58.5

79

101 10

.2

20.6

17

.4

13.4

14

.7

11.1

13

.2

15

15.9

12

-4

iadi

oact

ivi t

) in

who

le

bcdy

(ABG

I).

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14.4

50.4

98

-9

69

52.6

73

74

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6.

5 26

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57.3

I .75

I ;-3

6.

2 4.

45

4.75

4.

2 5-

6 5.

05

6-25

2.

5

__

__

_

Zad

ioac

tivit)

n

the

gast

ro.

inte

stin

al

trac

t (AG

I).

~

5.32

Bod

y do

se,

excl

udin

g ga

stro

-inte

s-

tinal

trac

t

(rad

s/da

y)

(DY

B),

0.12

7 0.

1 3

0.13

0.

138

0.12

1 0-

142

0.14

9 0.

147

0.17

0.

I76

0- I4

3

0.02

47

0.02

46

0.03

68

0.03

0.

0288

0.

0203

0.

02 19

0.

032

0.02

92

0.03

3 I

0,02

8 I

Who

le b

ody

dose

(D

~B

GI)

, (r

ads/

day)

~~~ -

0.28

5 0.

385

0-33

4 0.

339

0.36

9 0.

36

0.44

4 02

34

0.17

9 0.

249

~~

0.3 1

8

0.02

7 0.

056

0-05

3 0.

041

0.03

9 0.

03

0.03

6 0.

43

0049

0.

039

0.04

I -

__

__

D

ose

in

gast

ro-

inte

stin

al

trac

t (D

yG

lA

( rad

s/da

y)

0.77

1.

36

1.04

0.

9 1 a

7 -

1 -09

0.43

1.

41

0.09

0.02

4 0.

164

0.09

2 0.

066

0.06

3 0.

056

0.07

3 0.

074

0.08

3 0.

038


Figure 9 shows the movement of the DyBGl value averaged for each group. The coefficient of variation for each point varied from 15 to 35 per cent. As we see, variations in the total dose of y-radiation are quite considerable.

Comparing the data in Tables 4 and 6 it can be seen that the mean values of specific radioactivity of the blood (AB and Z>yB) are very similar. This indicates, in our opinion, the relative constancy of these values in time and

σ to ■σ σ ce

05

0-4

0-3

0-2

0-1

· · -· \ •

] I I . ! . .

•

I i I i

2

1

0-06 Ü-05

-g 0-04

ë 0-02

0Ό1

V VI

__ 1959

• - \

·-*

'

VII Vili IX

ft y\

I I I

X

/ —

î

XI XII Months

·

I I

I II

1960

• A •

I _ !

Ill IV V

J I I I V VI VII Vili IX X XI XII I II III IV V

Months 1959 1960

FIG. 9. Daily y-doses (/>VBGI) in rabbits receiving oral 59FeCl3 daily. 1 — 1 μο/kg; 2—10 μο/kg (mean values for 10 rabbits.

Coefficient of variation for the separate points 15-35 per cent).

confirms the suitability of the method of calculation used. However, the considerable divergence of the data on radioactivity in the gastro-intestinal tract, which on death is 2-3 times greater, attracts attention. This may be explained by the fact that radioactivity in the intestine prior to death is markedly increased. This is also confirmed by in vivo measurement — total activity in the body before death invariably increases. Apparently, at this time the rabbits cease eating and therefore little 59Fe is excreted. Less frequently, when death is accompanied by diarrhoea, negligible radioactivity may remain in the intestine. Hence the large value of C for AGl in Table 4. Attention should be drawn to the relatively high radioactivity of the intestinal contents in the 10 μο/1<£ group, which is associated with the relatively power level of tissue radioactivity. This is possibly the explanation of the fact that, after 59Fe administration is discontinued in this group, total body activity falls to a lower level than in the 1 μ^1<^ group.

We consider it more accurate to calculate DGl and D BGi on the basis of in vivo measurements of radioactivity and here is shown the value of this method.


As has already been more than once emphasized (G. A. Avrunina, 1960) in calculating doses, especially of y-radiation, so many simplifying asssump-tions and averages have to be made that the values arrived at should not be considered in any way absolute. They must be considered as pointers, giving a greater or lesser approximation to the true values, and allowing comparison with other values arrived at by similar means and under similar conditions.

Possibly a somewhat closer approximation may be obtained if the doses of /?- and y-radiation from the radioactive substance distributed in the body (excluding the contents of the gastro-intestinal tract) DyB are considered as the general background, on which the y-dose from the radioactive substance in the gastro-intestinal tract is superimposed. For the abdominal cavity such a total dose is generally close to our calculated DyGl. For the organs of the thoracic cavity and head the dose may be calculated by considering the radioactive substance in the intestine as a point source of radioactivity at the centre of the abdominal cavity (Busch et al.). The dose given by such a source at distances corresponding to the situation of the organs concerned relative to the abdominal cavity, is calculated by the known formula:

Dy = Jy —- roentgens, or 0-93 Jy — rads r2 r2

where

Jy —ionization constant; for 59Fe it is 6*55; a —activity of AGÌ in me; t —duration of irradiation in hr ; /· —distance from centre of abdominal cavity to the organ in cm.

Taking, in the rabbit, r as being: spleen 3-4 cm, liver 4-6 cm, organs of the thoracic cavity 7-12 cm and head 15-20 cm, mean doses of y-radiation additional to DyB were found. They are presented in Table 7.

TABLE 7. Radiation Doses in Organs in Different Regions of the Body from 59Fe Situated in the Gastro-intestinal Tract (AG1)

Organs

Liver Spleen Thoracic cavity Head

Distance from centre of

abdominal cavity (cm)

4-6 3-4 7-12

15-20

y-dose

group receiving ΙΟμο/kg

0-53-0-24 0-95-0-53 0-17-0-06 0-04-0-02

in rads

group receiving 1 μc/kg

0-05-0-02 0-09-0-05 0-016-0-005 0-003-0-002

If the values in Table 7 are added to DyB from Table 6 and compared with DyBGl and DyGl from the same Table, it can be seen that for the organs of the abdominal cavity the total dose is close to DyGU as we already believed;


for the organs of the thoracic cavity this dose is similar to D BG1. For the blood, muscles and bone marrow which are distributed throughout the body the dose approaches DyGl. For head organs the radiation dose from 59Fe situated in the intestine is of no appreciable importance and the dose is more precisely determined by the magnitude of DyB.

Mean doses of y-radiation and of /5-radiation received by tissues containing 59Fe are of the same order. Therefore, in evaluating tissue doses they should be aggregated.

Table 8 sets out total doses in tissues and organs, both daily and throughout the life of the animals. In these calculations the doses of β- and y-radiation given in Tables 5 and 6 were used.

TABLE 8. Total ß- andy-doses in Organs of Rabbits Receiving Oval 59FeCI3 in an Amount of 10 and 1 μο^ Daily

Organ or tissue

Liver Spleen Kidneys Suprarenals Ovaries Testes Blood Bone marrow Muscles Lungs Heart Brain

Total β-

group receiving 10 μο/kg

daily (rads/day)

1-235 1-56 1-06 1-04 1-04 1-0 0-91 0-55 0-34 0-53 0-47 0-17

total over 3-21 months (rads)

112-780 140-985 96-670 94-655 94-655 90-630 82-575 50-350 31-215 48-335 42-295 15-107

and y-doses

group receiving 1 μο/1<^

daily (rads/day)

0-133 0-173 0-111 0-102 0-104 0-095 0-146 0-109 0-047 0-1 0-069 0-04

total over 3-21 months (rads)

12-84 15-6-109

10-70 9-2-65 9-4-65-5 8-6-60

13-1-92 9-8-69 4-2-29-5

9-63 6-2-43-5 3-6-25

A C C U M U L A T I O N OF 59Fe D U R I N G DAILY O R A L A D M I N I S T R A T I O N AND THE A M O U N T OF STABLE I R O N

IN THE S O L U T I O N

As has been shown earlier, the level of 60Co in animals during prolonged oral administration of 60CoCl2 varies inversely with the amount by weight of stable carrier in the solution administered (G.A.Avrunina, 1960). A similar connection was detected in the case of iron.

Thus, the mean specific activity of the blood in the first group (1 (xc/kg) was 0Ό185 ± 0-0054 pc/ml, and in the second group (10 μ ΰ ^ ) 0Ό9 ± 0-023 (Ac/ml.

Therefore, when the weight of 59Fe was increased 10 times (with increase of activity the amount of carrier increases correspondingly) accumulation in


the blood only increased 5 times. A similar result was observed in the other tissues, as is shown in the last column of Table 4.

Accumulation of 59Fe in the whole body (including intestinal contents) falls by 1*3, since retention in the intestine, as was the case with 60Co, depends less on the amount of carrier in solution and for the second group (10 μc/kg) was relatively even slightly higher. To confirm this correlation found in rabbits an experiment was carried out with rats. Four groups of rats each received daily for a month 1 ml solution of 59FeCl3 of similar activity

001 0-1 075 5 amount of carrier (mg/ml)

FIG. 10. Level of radioactivity in the blood and carcasses of rats after daily oral administration of 59FeCl3 according to the weight of carrier in the solution. The level of radioactivity in animals receiving the lowest quantity of iron was taken as

100 per cent. 1—carcasses; 2—blood

but with stable iron content of O01 ; 01 ; 0-75 and 5 mg/ml. At the end of the month all the animals were killed and the blood, liver and lungs taken for measurement. Also, the carcasses were measured and the gastro-intestinal tract with contents extracted and measured separately. It was found that comparatively little 59Fe remains in the gastro-intestinal tract, on average one third of the daily administration, and no correlation between the radioactivity remaining and the concentration of stable iron in the solution was detected.

The radioactivity of the blood and carcasses displayed an almost identical inverse relationship with the concentration of the carrier iron solution. The radioactivity of the liver and lungs also falls with increase in concentration of carrier iron, but not identically. Figure 10 shows the data obtained. The relationship obtained for 60Co was different from that found here, but it should be borne in mind that the absolute values of the concentrations used in the two cases were quite different ; moreover, the characteristics of iron and cobalt metabolism are different. It is possible also that with low concentrations such as 001 mg/ml the period necessary to reach the equilibrium level exceeds the duration of our experiment and after a greater interval the


initial point of the curve in Fig. 10 would be higher and the entire curve straighter. Nevertheless, there remains no doubt as to the presence of an inverse relationship between the content of stable iron in the solution and the level of accumulation of 59Fe in the body during prolonged oral administration of 59FeCl3.


1. Following a single oral administration to rabbits of 2 ml solution of 59FeCl3, containing 3-3 ml stable iron, about 5 per cent of the quantity administered is absorbed. The unabsorbed portion is almost entirely excreted during the first week.

2. Radioactive iron absorbed into the bloodstream is excreted from the body very slowly; not more than half the iron is excreted by the 100th day.

3. With daily oral administration to rabbits of 2 ml solution of 59FeCl3 containing from 3 to 7 or from 0-3 to 0-7 mg/ml stable iron the maximum level of excretion is virtually attained in the course of the first week. The maximum level in the blood is established much later, after 45-65 days.

4. When administration of 59FeCl3 is terminated the 59Fe accumulated in the intestine is rapidly excreted, but the activity of other parts of the body falls as slowly as after a single parenteral injection.

5. Two to three months after commencement of administration an equilibrium level of activity in the body is established.

The greatest radioactivity is observed in the blood and spleen. Then follow the liver, lungs and other parenchymatous organs. The radioactivity of the muscles, bones and brain was similar and is 10 times less than the radioactivity of the other organs. Total blood radioactivity accounts for 40 per cent of radioactivity in the body, excluding the gastro-intestinal tract.

6. According to post-mortem measurement the amount of 59Fe contained in the gastro-intestinal tract is approximately 3 times the amount administered daily, the variation coefficient reaching 79percent. The radioactivity of the gastro-intestinal contents calculated from in vivo measurements, is 2-2\ times lower and varies less.

7. The method of in vivo measurement of body activity by external radiation, in combination with repeated measurement of blood radioactivity, enabled the detection, from time to time, of changes in the y-radiation dose in the body and the more precise evaluation of the dose received throughout the experiment.

8. The y-radiation doses in the different body regions are characterized as follows :

a) in the abdominal cavity—the mean dose from the radioactivity accumulated in the gastro-intestinal tract, calculated according to the volume of the abdominal cavity;

b) in the thoracic cavity—the mean dose from radioactivity in the whole body, including the gastro-intestinal tract, calculated for the volume of the whole body.


c) in the head organs—the mean dose from radioactivity in the body without the gastro-intestinal tract, calculated according to its volume.

9. The greatest total dose of ß- and y-radiation is found in the liver and spleen: 1*235—1-560 rads/day for rabbits receiving \0 \kcjkg, and 0-133-0-173 rads/day for rabbits receiving 1 \LCJkg.

10. As may be seen by comparing radioactivities in the blood, tissues and bodies of rabbits of both groups, with prolonged oral administration of 59Fe the equilibrium level of radioactivity in the body (excluding the gastrointestinal tract) is higher, the lower the amount of stable carrier iron in the administered solution. A similar correlation is observed in rats.

R E F E R E N C E S

AVRUNINA G.A., Toxicology of Radioactive Substances (Materialy po toksikologii radio-aktivnykh veshchestv), vol. 2, pp. 14-26 Medgiz, (1960).

AVRUNINA G. A., Toxicology of Radioactive Substances (Materialy po toksikologii radio-aktivnykh veshchestv), vol. 2, pp. 27-38 Medgiz, (1960).

B A B A D Z H A N O V F . N . , Med. zh. Uzbekistan., 62-64 (1957). B U R Y K I N A L . N . , Toxicology of Radioactive Substances (Materialy po toksikologii radio-

aktivnykh veshchestv), vol. 1, pp. 41-52 Medgiz, (1957). BUSTAD L.K. et al, Radiation Research, 6 (3), 380-413 (1957). C O P P H . and G R E E N B E R G D . M . , J. Biol. Chem., 164 (1), 337-387 (1946). C O P P H . and G R E E N B E R G D . M . , / . Biol. Chem., 164 (1), 389-401 (1946). KURLYANDSKAYAE.B. , BELOBORODOVAN.L. and B A R A N O V A E . F . , Toxicology of Radio

active Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 1, pp. 16-22, 31-40 Medgiz, (1957).

RUBANO VSKAYA A.A. and USHAKOVA V.F. , Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 1, pp. 13-15 Medgiz, (1957).

WACK J. and WYATT J., Arch. Path., 67 (3), 237-242 (1959). ZAMYCHKINA K.S. and DURINYAN R. A., Byull. eksper. biol. med., 45 (3), 51-55 (1958).

THE EFFECT OF PROLONGED ADMINISTRATION OF RADIOACTIVE IRON

ON THE ELECTRICAL ACTIVITY OF THE CEREBRAL CORTEX OF RABBITS

D. A. GlNSBURG

MUCH experimental evidence is now available which indicates that profound changes occur in the electrical activity of the central nervous system as a result of irradiation. More than 240 references are given in one of the recent reviews on this subject (V. S.Miklashevskii, 1958). As recent investigations have shown (Yu.K.Kudritskii, 1957; N.A.Rokotova and I.M.Gorbunova, 1958; I.V.Mamonova, 1958; T.A.Korol'kova, 1959; L.S.Kupalov, 1959), the central nervous system reacts to very small doses of ionizing radiation, so that theories about the special "radioresistance" of nervous tissue, prevalent until recently in the foreign literature, have proved to be basically unfounded. On the contrary, the nervous system has, apparently, the greatest radio-sensitivity, and changes in its functional state (primary or secondary in origin) occur at the earliest stages of radiation exposure (M. N.Livanov and I. N. Kondrat'yeva, 1959). Investigations have shown that even such minimal doses of external radiation as 01 r elicit certain shifts in conditioned-reflex activity, whereas no changes can be detected at this time in any other system of the body.

Similar results have been obtained in studying the effect of prolonged internal radiation by incorporated radioactive substances. Thus, Airikyan, Gaske and Serkov and co-workers (1958) have observed in dogs early changes in central nervous system activity following oral and intravenous administration of radiophosphorus (40-80 μc per kg weight). L.N.Burykina (1957) has detected changes in conditioned-reflex activity in white rats after a single subcutaneous injection of the radioactive isotopes of ruthenium, caesium and strontium (doses 8, 20 and 40 μc per 1 g weight). Along with certain differences in the effect of these three isotopes the authors noted the variable character of the changes in cortical chemical activity, when a phase of heightened excitability is replaced by a phase of suppression of conditioned-reflex activity. According to V.M.Zakharov (1959), small doses of radioactive sodium (5-10 μc per 1 kg weight) produced in rats certain changes in the closing function of the cortex even after the first injection.

In many experiments electrophysiological methods have been used successfully to detect changes in the functional state of the peripheral and central

38

Effect of Prolonged Administration of Radioactive Iron 39

nervous systems following exposure to radiation. Electrophysiologists, chiefly from the school of M.N.Livanov, have succeeded in showing that after external irradiation of rabbits with X- and y-rays changes occur in the character of the impulses from receptor organs (M.N.Livanov and N. S. Delitsyna, 1956;N.S.Delitsyna, 1957; M.N.Livanov and N.I.Kaburneyeva, 1958), the excitability of the spinal and truncal centres (Yu. K„ Kudritskii, 1957; Z.M. Gvozdikova, 1956; M.N.Livanov, 1959), and of the autonomie centres of the pars intermedia (I.N.Kondrat'yeva, 1958; T.M.Efpemova, 1959), and also the electrical activity of the medulla oblongata (M.N.Livanov, 1956), the hypothalamus (G.V.Bavro, 1957; T.M.Efpemova, 1959) and the cortex of the cerebral hemispheres (M.N.Livanov, 1956; Z.A.Yanson, 1966; T.A. Korol'kova, 1959; A.B.Tsypin and Yu. M. Grigor'yev, 1960).

According to M. N. Livanov, Z. A.Yanson, Caster, Redgate and Armstrong (1958) changes in the electrocorticogram occurring after heavy total body irradiation of the animal (doses 500-1000 r) are phasic in character. Brief increase of the background electrical activity is replaced by a phase of depression. The subsequent restoration of normal electrical activity takes place slowly over several days. In T.A.Korol'kova's work single small doses of ionizing radiation (5, 10, 25 r) produced an increase in voltage of the cortical potentials, of excitability and response to light stimuli. It is interesting that when the dose was 25 r this reaction occurred on the day of exposure, but on the following day with doses of 5 and 10 r. Repeated irradiation led, after 3-4 exposures, to the appearance of signs of suppression of electrical activity, and with further increase of the total dose the depression phase began to predominate.

Most of these electrophysiological studies were carried out on animals suffering from acute radiation injury. They provide a basis for estimating the changes in electrical activity during and after external irradiation with comparatively large doses. The literature contains no similar investigation concerned with chronic experimental radiation sickness produced by prolonged administration of small doses.

The present article gives the results of a systematic electroencephalographic investigation of rabbits which for 3 months received 10 μc per kg weight of oral 5 9FeCl3 per day.The work was carried out on 12 adult animals. The experimental group comprised 7 rabbits while the other 5 animals were used as controls.

The electrodes were inserted using D.A.Teplii's method. The electrode arrangement suggested by him for recording the potentials of the cortex of the cerebral hemispheres was a Plexiglas rod with a head, to which the metal electrodes were firmly fixed. The head of the rod was inserted through a trephined aperture and situated epidurally; the electrode block was held by a coupling which was fixed to the rod, and clamped the bone of the skull to the rod head. This electrode arrangement enabled reliable recording of cortical potentials over a prolonged period (6-7 months). The electrodes were stainless steel needles 1-5 mm in diameter. The distance between electrodes was 2-2-5 mm. The electrode block was inserted over the sensori-motor and occipital-sincipital regions of one hemisphere.


The cortical potentials were registered with a 4-channel symmetrical amplifier with a rheostat-capacitor coupling and an oscillograph (produced by the experimental workshop of the AMN SSSR). The animal was not immobilized during the experiment, being merely housed in a plexiglas box, which limited its freedom of movement. The box containing the rabbit was placed in a darkened and screened room. As functional stress, rhythmic photic stimulation of varying frequency was used (from 2 to 20 flashes per sec). The source of light was an IS-50 gas-discharge impulse lamp. The duration of flash was 30 msec, light energy 2 lumen/sec. The light source was placed 10 cm from the rabbit's head. At the same time as the electroencephalogram, the respiratory movements were recorded using an airflow resistance consisting of a thin rubber tube filled with coal dust.

In all, 172 experiments were carried out. Recordings were first done a week after the operation and repeated every 5 to 7 days. Before the radioactive isotopes were administered, recordings were made of background activity and the responses to photic stimulation throughout the frequency range and repeated 4-5 times. The experimental group (7 rabbits) received 59Fe orally in doses of 10 μο per kg per day. This amount of 59Fe, after the equilibrium level had been reached, gave a mean dose to the body in y-radiation equivalent to 0*3 rads/day, which is 15 times the maximum permissible dose (G.A.Avrunina's data). Three rabbits of the control group received stable iron in doses corresponding to the quantity of carrier in the radioactive solution. Two control rabbits received no iron. In all animals the peripheral blood picture and the state of the haemopoietic organs were systematically investigated (N. L. Beloborodova, E.K.Red'kina and V. L. Viktorova). Two of the experimental rabbits died during the 9th week of administration from diseases unconnected with the experiment and the others were killed after 3 months. Pathological examination of organs was carried out by E. S. Gai-dova.

In analysing the background records, the voltage and frequency of wave forms, as well as the general nature of the electrical activity of the anterior and posterior quadrants of the brain, were taken into account. As is known, rabbits at the alert or in a state of great excitement, in addition to increased frequency of breathing, display a characteristic synchronized rhythm (4-7 per sec) in the electroencephalogram of the occipital-sincipital regions of the cortex and desynchronization in the electroencephalogram of the sensori -motor regions (L.A.Novikova and D. A. Färber, 1959; M. M. Bantsekina, 1960). When the animal is quiet the electroencephalogram of the occipital-sincipital regions characteristically display high-voltage slow waves of varying duration (from 200 to 350 msec), which, as a rule, do not add up to a definite stable rhythm. In the sensori-motor regions at this time against a background of high-frequency low-voltage wave forms, and isolated slow waves of low amplitude, outbursts of spindles occur. The rhythm of the spindle waves is 14-20 per sec (Petsche, Marko and Monnier, 1955; L.N.Smolin, 1959).

In our experiments with rabbits, acclimatized to the experimental conditions, this "peaceful" electrical activity was established relatively quickly and


maintained, as a rule, throughout the experiment. Systematic tracings of brain potentials in animals receiving 59Fe for 3 months showed that the "spontaneous" electrocorticograms are virtually unchanged, both in general character and in the frequency spectrum and voltage.

In determining how the cortical rhythms were affected by rhythmic photic stimulation, a note was taken of the lower and upper limits of stimulus frequency, at which following rhythm advanced, and also the amplitude, index and area of manifestation of the following. The index was measured as a percentage of the waves in the following rhythm to the total number of stimuli.

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FIG. 1. Changes in the upper limit of evoked rhythm. 1—rabbit receiving 59Fe; 2—rabbit receiving stable iron.

Ordinate axis—maximum light frequency eliciting rearrangements of cortical rhythm; abscissal axis—duration of administration. Left of broken line—before administration; right—after commencement of administration. The small circles denote experiments in which evoked rhythms appeared in the sensori-motor regions

of the cortex. ·

The maximal index was usually observed at a stimulation frequency of from 3 to 6 flashes per sec (optimal range of following). The upper limit of following, i.e. the maximal frequency of stimulation which produces rearrangement of cortical rhythms corresponding in frequency, both in the control group and in the experimental animals prior to 59Fe administration, did not usually exceed 7-8 (rarely 9) per sec. Wave amplitude and index of following in one and the same rabbit in different experiments could change significantly. But the upper limit at which following occurred was fairly stable and in the control animals varied so little over a period of 3-4 months that we felt justified in taking this value as a confirmed characteristic. In Fig. 1 the broken line (2) shows variations in the upper limit of following over 3 months in rabbit No. 17, which received stable iron. As can be seen, the value of the maximum reproducible frequency varies very little, not exceeding 8 per sec. In some of the control animals the upper limit was situated still lower—in the region of 6-7 flashes per sec. It should be emphasized that in all 5 control animals (three of which received stable iron and two served as


a physiological control) the frequency range of following due to rhythmic photic stimulation was unchanged over a prolonged period. This is illustrated by Fig. 2, where the values for the control rabbits are represented by the black circles.

For rabbits in the experimental group (white circles), beginning from the 3rd week of 59Fe administration, the range of following extended towards higher frequencies and the upper limit reached 12-14 per sec. Rhythm evoked

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at low frequencies was still maintained. This shift of the upper limit was observed in all 7 rabbits of the experimental group during the first 5 weeks of administration. In 3 animals it began in the 3rd week; in 2, in the 4th; and in the other 2, in the 5th week.

The appearance of evolved rhythms of high frequency was erratic. In each of the rabbits their occurrence alternated with temporary returns to almost the original values. The duration and character of these alternations is, apparently, individual. In any case, no regular pattern could be detected in our experiments.

In Fig. 1 the circles indicate animals in which the cortical rhythms of the sensori-motor regions were rearranged in response to interrupted photic stimulation. Such specially evoked rhythms were observed in 5 of 7 rabbits of the experimental group. As can be seen from the diagram, it usually occurred episodically, at times when high frequency rhythms were evoked. No evoked rhythms in the sensori-motor regions were observed in control or experimental animals before commencement of administration of 59Fe or in the early stages of intoxication.

Figure 3 gives the electroencephalogram of rabbit No. 14 before 59Fe administration; and Fig.4, after 2 weeks of administration. In both cases interrupted photic stimulation produces following only in the occipital-


sincipital regions. In the sensori-motor regions some depression of background electrical activity (cf. Fig. 3) or maintenance of the initial character of the tracing (cf. Fig. 4) is observed. The other tracings (Fig. 5 a, b and c) illustrate the occurrence of evoked rhythms in the sensori-motor region at different light frequencies (5th week of 59Fe administration). It is interesting to note that at a moderate stimulation frequency (5 flashes per sec) high-amplitude superimposed waves are clearly seen in the electroencephalogram of

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the occipital-sincipital region. The index at which superimposition occurred in this case is 75 per cent. In the electroencephalogram of the sensori-motor region the evoked waves are of considerably lower amplitude. The index is 34 per cent. Increasing the number of flashes to 8 per sec (cf. Fig. 5 b) leads to a rise of the index in the sensori-motor region to 88 per cent. At still greater frequencies (11 flashes per sec, cf. Fig. 5 c) the evoked rhythm begins to be more marked in the anterior, rather than the posterior quadrants of the TRS 4


brain. Wave amplitude in the electroencephalogram of the sensori-motor region increases to 150-200 \JN (index 90 per cent), whereas in the occipital-sincipital region signs of rhythm transformation appear—the "following" waves are evoked in response to each second or even each fourth stimulus.

In the tracing from another rabbit (Fig. 6), the evoked waves in the sensori-motor region are extremely well shown (index 100 per cent) (5th week of 59Fe administration). At this relatively high frequency of flashes (11 per sec) there is almost no following rhythm in the electrogram of the occipital-sincipital

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regions—waves are evoked in response to each second, or to each fourth flash.

Following in the sensori-motor region was observed in 5 of 7 experimental rabbits beginning from the 3rd-5th week of administration. In 3 animals following in the sensori-motor regions took place predominantly at high stimulation frequencies (more than 7 per sec). In 2 rabbits no such dissociation was observed: following in the anterior quadrants with a high index occurred also at moderate (4-7 per sec), and even at low frequencies

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(Fig. 7 a, b and c). A record of the respiratory movements was taken in each experiment. Analysis of the curves indicated that with prolonged administration of 59Fe and development of chronic radiation sickness, at any rate in its early stages, no regular changes in respiratory rhythms were detected. In the first experiments after the operation, before the animals had become accustomed to the experimental conditions, the respiratory rate was usually increased (up to 200-250 per min). Moreover, in the first 3-4 experiments, even when breathing was normal, its variability was increased by flashing light.

Figure 8 shows a case (control rabbit, third trace after the operation) where the respiratory rhythm was at times as it were attuned to the photic stimulation rhythm. A similar situation is seen also in Fig. 3 (fourth trace after the operation). This "tuning" of respiration was not observed in all animals and only in the first 2-4 experiments ; in subsequent experiments over 4 months both in the experimental and in the control animals respiration, during light stimulation was usually unaltered, the background frequency not exceeding 40-90 per min.

In a series of papers (M.N.Livanov, 1956; Z.A.Yanson, 1956; L.G.Ter-ekhova, 1958; R. M. Rabinovich, 1958) it has been shown that in acute radiation sickness the rhythm, amplitude and form of the respiratory movements of rabbits undergo significant changes. Thus, R. M. Rabinovich showed that after external irradiation (single dose of from 25 to 450 r) respiration,


frequency fell by 2-5 times with a simultaneous increase in depth. In the articles of Z.A.Yanson and M.N.Livanova results are given which indicate that the respiration of rabbits is sharply changed both after total single irradiation (dose 500 r) and chronic local application of 60Co to various regions of the brain (dose 36 r per min) or subcutaneous injection of polonium (210Po) in an amount of 0-2 μο/kg. Whereas before irradiation, respiration frequency reached 180 per min and became attuned to rhythmic light stimulation, with the elaboration of a conditioned defensive reflex, after irradiation, this phenomenon disappeared and the background frequency fell sharply.

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E # k t o j Prolonged Administrotion of Radioactive Iron 47


In our experiments increased respiratory variability, expressed in the subordination of frequency to the rhythm of the light stimulus or simply in sharp oscillations, was observed only in the first experiments after the operation. Incidentally, as can be seen from Figs. 3 and 8, respiratory subordination appeared just at the moment when high-voltage following occurred in the occipital regions—a fact, it seems to us, of singular interest.

The establishment of a normal respiratory rhythm and the absence of frequency changes in response to light stimulation in the later experiments cannot be connected with the development of chronic radiation sickness, since similar respiratory changes occurred in the controls. It may be linked with the habituation of the animals to the experimental conditions and the fact that the stimulus ceases to provoke an orientational reaction. This is confirmed by the fact that tachypnoea and respiratory reaction to rhythmic photic stimulation again appeared in the control and experimental rabbits when the series of experiments was interrupted for 2-3 weeks.

As is seen from our results, with the type of irradiation used in these experiments, no marked and regular changes in background electrical activity were observed, at any rate during the first 3 months of administration. The results obtained in the literature, referred to at the beginning of this article, relate to animals, which were subjected to external irradiation in relatively large doses, i.e. animals suffering from acute radiation sickness. Prolonged internal administration of small doses of 59Fe, giving rise to a slow development of chronic radiation sickness, does not, during the first four months, produce significant changes in the spontaneous electrical activity of the cerebral cortex.

The reaction of the electrocorticogram to rhythmic photic stimulation in the form in which it is used in these investigations has not been examined in any of the literature with which we are familiar, even in acute radiation sickness. As is seen in the articles already quoted by M.N.Livanov's colleagues (Z.A.Yanson and T.A.Korol'kova), interrupted light stimulation was used by them but, in the first place, only one frequency was employed (4 flashes per sec), and secondly, this stimulus served as the signal for the conditioned defence reflex, while isorhythmic electrical stimulation of a paw constituted an unconditioned stimulus. The use of a single (relatively slow) rhythm of light stimulation and its combination with a painful stimulus rules out the possibility of drawing a parallel with our results.

Rhythmic light stimulation over a wide range of frequencies constitutes, as the results show, an adequate form of functional stress and enabled the detection of changes in the higher centres of the central nervous system occurring in chronic experimental radiation sickness caused by oral administration of 10 μΰ/kg 59Fe. It should be emphasized that changes in the character of following, manifested by spreading of the evoked rhythms and the appearance of evoked potentials in the anterior quadrants of the brain, occurs only in the 4th-6th weeks of administration and has an unstable wavelike character. Investigation of conditioned reflexes in chronic radiation sickness produced by administration of radioactive isotopes (V.M.Zakharov, 1959;


L. N. Burykina, 1957; E.N.Klimova, 1958) has shown that in these cases also, changes in cortical activity are variable and periods of increased conditioned-reflex activity quickly give way to more prolonged periods of reduced activity.

Data obtained by V. L. Ponomareva (cf. the article in the present volume) indicate that, in the initial period of 59Fe administration, (beginning from the first month) an increase in the haemoglobin occurs without significant changes in the red cell number. These changes in the blood, and also in the morphological composition of the bone marrow together with the results obtained when stresses are placed on the haemopoietic organs, lead E. B. Kur-lyandskaya to suggest that even in the early stages of 59Fe intoxication a condition of concealed hypoxia develops, which may also be the cause of the functional changes in central nervous system activity which our electro-physiological results would seem to indicate.

The greatest interest from our point of view is aroused by the appearance of evoked rhythms in the anterior quadrants of the brain in 5 of the 7 rabbits receiving 59Fe. As G.D.Smirnov and co-authors (1960) established, under normal conditions the region of spread of evoked rhythms in rabbits coincides, in the main, with the zone of primary response to light stimulation. These investigators demonstrated that stimulation of the reticular formation of the brain stem, in the early stages of anoxia and narcosis facilitates more spreading of evoked rhythms through the cortex. Other data can be found in the literature to indicate that the spread of responses due to rhythmic light stimulation to regions of the cortex lying outside the visual areas is possible when the functional condition of the brain is affected. Thus, Gastout (1950) obtained frontal and precentrai responses to intermittent light stimuli in patients with diencephalic injuries. Friedlender (1959) has observed evoked rhythms in the frontal and temporal regions in some patients with acute vascular lesions in the brain-stem region. E.S.Tolmasskaya and M.A.Ti-tayeva (1960) present data on the appearance of evoked rhythms in the frontal and pre-sincipital leads in patients with schizophrenia. Yoshii, Pruvot and Gastout (1957) indicate that evoked rhythms occur in the frontal-parietal cortex in cats with experimental neurosis.

Further experimental analysis is required to elucidate the mechanism of this limited spread of evoked rhythms. It should however be noted that the appearance of high-frequency evoked rhythms in the electroencephalogram of the sensori-motor regions with the simultaneous recording of double and quadruple transformed rhythms in the occipital regions of the cortex is evidence against a transcortical spread mechanism to the anterior sections of the brain. It is more probable that transmission to the anterior areas of the cortex takes place directly from the subcortical association neurones of the optic tract.


CONCLUSIONS

1. Investigations were made of the background electrical activity and response to rhythmic photic stimulation (frequency 2-20 per sec) of the sensori-motor and occipital-sincipital regions of the cortex of rabbits during prolonged (3 months) oral administration of 59Fe (10 \Lclkg). The control animals received stable iron.

2. No significant changes in background electrical activity were observed in either the experimental or the control animals. Changes in response to rhythmic light stimuli occurred in rabbits receiving 59Fe after 3-5 weeks. They took the form of a spread of the range of evoked rhythms to the right (appearance of evoked rhythms at higher stimulation frequencies—up to 13-15 per sec) and the appearance of following in the electroencephalogram of the sensori-motor regions of the cortex. These changes in evoked rhythms were inconstant, being replaced from time to time by periods of a normal electroencephalographic response to a given stress.

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TSYPIN A.B. and GRIGOR'YEV Y U . M . , Byull. eksper. biol. i med., 49 (1), 26 (1960). Y O S H I I N . , PRUVOT P. and G A S T O U T H . , E.E.G.a. clin, neurophys., 9, (4), (1957). ZAKHAROV V. M., Changes in central nervous activity and arterial pressure following inter

nal administration of small doses of 2 4 Na (Ob imenenii vysshei nervnoi deyateFnosti i arterial'nogo davleniya pri deistvii na organizm malykh doz vnutrennego oblucheniya natrium-24). Dissertation, Moscow (1959).

THE EFFECT OF PROLONGED 59Fe ADMINISTRATION ON HAEMOPOIESIS

N. L. B E L O B O R O D O V A , V. L. P O N O M A R E V A and E. K. R E D ' K I N A

STUDY of the effect on haemopoiesis of incorporation of certain radioactive isotopes (N. L. Beloborodova and E. F. Baranova, 1957; N. L. Beloborodova, 1960) has shown that not only the amount of isotope administered but also, to a significant degree, its chemical properties have great importance in determining the amount and course of damage to the different elements in haemopoiesis. Thus, the effect on haemopoiesis of 59Fe, the stable analogue of which is an extremely important bioelement, is of particular interest. No work on this problem has so far appeared in the literature.

The purpose of the present paper was to study haemopoiesis during prolonged daily internal administration of small doses of 59Fe.

The experiments were performed on rabbits, comprising two experimental and two control groups. The animals of the first experimental group (20 animals) received daily 1 μο 59Fe per kg weight, rabbits of the second experimental group (42 animals)—10 μc 59Fe per kg weight, in the form of a solution of 59FeCl3.

In one of the two control groups, the animals (24 rabbits) received daily stable iron in the same quantity and form (FeCl3) as the rabbits of the second experimental group. The other group (17 rabbits) constituted an untreated control. Each month the peripheral blood of every animal was examined, including, apart from the usual analysis, a reticulocyte and platelet count. Cytological changes in the leucocytes were also examined using Prof. A. P. Egorov's classification.

The morphological composition of the bone marrow was investigated. Marrow was obtained from the ilium before the experiment and 1-2, 3-4, 6-8,10-12 months after. Apart from counting the myelogram and the number of reticulocytes, the total number of nucleated cells per mm3 of bone marrow aspirate was determined. It is well known that with existing methods of in vivo bone marrow puncture the aspirate always contains a certain admixture of blood. Control experiments with repeated aspiration of marrow from the same animal showed that in most cases the number of nucleated cells per mm3 of aspirate varied from 150,000 to 250,000, and only in isolated rabbits did we obtain either very low (80,000-90,000) or very high figures (500,000-600,000). Since in a single rabbit the difference in numbers of nucleated cells in repeated aspirations did not exceed 40,000-50,000 per mm3 we felt justified in using this parameter as an experimental index.

52

Effect of Prolonged 59Fe on Haemopoiesis * 53

In consideration of the specific role of iron in erythropoiesis it was thought necessary to use all available methods for its investigation. Thus, in addition, measurements were made of changes in red cell diameter, volume and form and the degree of anisocytosis was determined. Osmotic fragility to hypo-tonic saline was also studied. In some cases the diameter of the different types of erythroblasts in the bone marrow was determined. The results of these investigations, carried out by V. L. Ponomareva, are of interest and are presented in a separate article.

R E S U L T S

Erythropoiesis. Changes in erythropoiesis in all the experimental groups, taking into account the qualitative characteristics of the red cells, are dealt with in detail in V. L. Ponomareva's article. Here we shall only touch briefly on the main factors vital to an understanding of the effect of 59Fe on haemopoiesis.

In the control group the red cell, haemoglobin and reticulocyte counts in the peripheral blood showed negligible changes throughout 15 months of

-2L I 1 1 L

1-4 6-8 12-14 Months

FIG. 1. Changes in the number of cells of the red series per 100 cells of the white series in the bone marrow.

1—control; 2—administration of stable iron; 3—administration of 10 μο/kg 59Fe; 4—administration 1 μΰ/kg 59Fe

observation. The mean red cell count during \\ years varied around 5,000,000 and the haemoglobin around 13 g per 100 ml. Changes in the absolute and relative number of cells of the erythroblastic series in the bone marrow were also insignificant, as also were changes in the ratio of these to the white cells (Fig. 1)*. Nor were any marked changes in the reticulocyte count in the bone marrow, or in the erythroblast maturation index (ratio of erythroblasts containing haemoglobin to those without) observed.

In the group of animals receiving stable iron, an increase in red cell size and quantity of haemoglobin without an increased red cell count was observed, mainly in the first 4 months, connected, apparently, with the effect of the iron. Continuing observation of marrow aspirate showed after 8-10 months an increase in the red cell count (up to 40-9 ± 2) compared with the controls

* In the figures each point represents a group mean calculated for the period shown on the abscissa.


(31-2 ± 4). The number of red cells per 100 leucocytes rose correspondingly. No significant increase in the absolute number of nucleated cells in the aspirate was observed in our experiments. The red cell maturation index did not materially change.

Administration of 59Fe in doses of 1 [ic/kg (first group) from the first months produced a stimulation of erythropoiesis and a tendency for the

00

I 40 o '■£ 20 en

u 1-2 3-4 6-8 10-11 Monlins

FIG. 2. Changes in the number of reticulocytes in the bone marrow. 1 —control; 2—administration of stable iron; 3—administration of 10 μο/kg 59Fe;

4—administration 1 μο/kg 59Fe

relative number of erythroblastic cells (cf. Fig. 1) and reticulocytes (Fig. 2) in the bone marrow to increase was observed.

Whereas in the control animals the mean reticulocyte count in the first 4 months was 32 ± 3·1°/οθ5 in the experimental animals it was 49 ± 4-3°/00. Reticulocytosis was also observed at later periods but not in all animals (10-12 months). The red cell and haemoglobin content of the peripheral blood was also somewhat increased. The red cell indices were within normal limits during the first 8 months, but after 9-10 months macrocytosic leptocytes and anisocytosis appeared.

With administration of the larger 59Fe dose—10 μο/kg (second group)— the relative and absolute number of cells of the erythroblastic series did not increase. Persistent reticulocytosis in the peripheral blood and increase in the red cell count did not appear, but in the first 4 months the number of reticulocytes in the bone marrow rose to 46 + 5-7°/00 (cf. Fig. 2). From the first month of the experiment, qualitative changes in the red cells were noticed— significant anisocytosis and formation of macrocytic leptocytes. The haemoglobin level and the colour index both increased in this group, as in the animals receiving stable iron, from the first month but was more marked.

Thrombopoiesis. No statistically significant changes in the number of platelets in the blood of the experimental animals during prolonged administration of 59Fe were observed.

Leucopoiesis. The experimental animals showed no significant changes in neutrophilic leucopoiesis. Neutrophil maturation in the bone marrow was unchanged.

The absolute neutrophil number in the peripheral blood of animals of all

fa ï

Effect of Prolonged 59Fe on Haemopoiesis 55

four groups varied throughout the experiment within similar limits. However, in animals of the second experimental group (10 μο/kg), by the end of the first year, a significant number of cells with fragmented nuclei had appeared (up to 8-13 per cent). In the control animals, neutrophils with nuclear fragmentation did not exceed 0-5-1 per cent.

The greatest deviations from normal in the experimental rabbits were in the absolute number of lymphocytes.

In the control rabbits, and those receiving stable iron, the absolute lymphocyte number did not change significantly throughout the experiment and the number of lymphocytes did not fall below 4000 per mm3 of blood (Fig. 3). In the control group the mean lymphocyte count before the experiment was 6500 ± 370; after 9-12 months, 6500 ± 400; and after 15-17 months, 5500 ± 330. In the rabbits receiving stable iron solution, the mean lymphocyte count before the experiment was 6200 ± 340; and after 9-12 months; 6300 ± 580. And although after 15-17 months it fell somewhat more than in the control group to 4900 + 610 this difference cannot be considered significant.

In the rabbits of the second experimental group (59Fe 10 ^clkg) disturbed lymphopoiesis was observed from the first months of the experiment. During the first 8 months these appeared mainly as a periodic fall in the absolute lymphocyte number with a subsequent return to the initial level or even slightly above. Nevertheless, the mean number of lymphocytes in the group during the first 4 months fell from 6500 + 240 to 5100 ± 160. The fall in the lymphocyte count began to be especially marked from the 9th month of the

Months

FIG. 3. Changes in the absolute number of lymphocytes in the peripheral blood. 1 —control; 2—administration of stable iron; 3—administration of 10 [Lcl,kg 59Fe;

4—administration 1 μΰ/1<£ 59Fe

experiment (Fig. 3). At this period lymphopenia became more frequent, its duration increased and the return of the absolute lymphocyte count to above its initial level was no longer observed. In peripheral blood smears at this period increased numbers of decaying lymphocytes were seen (from 3-4 to 20 per cent in some animals).

Qualitatively changed lymphocytes appeared, as for example, mature lymphocytes with intensely basophilic cytoplasm (from 0-5 to 6 per cent).


Such cells were found very rarely in the control animals. Moreover, up to 3-5 per cent of lymphocytes in the experimental animals had nuclei extended into the form of a rod, which was never observed in the control rabbits. Nor were such cells seen after administration of other isotopes.

During the second year lymphopenia in animals of the second experimental group progressed, and after 15-17 months the absolute lymphocyte count fell to a low level, on average 3500 ± 280. However, the cytological changes described above are less marked at this period.

In the rabbits of the first experimental group (59Fe 1 μ^1<£) the absolute lymphocyte count fell at about the same rate as in animals of the second group during the first year, but in the second year the decline was less steep so that after 15-17 months the mean count in this group was higher: 4300 + 430, i.e about the lower limit of normal. Qualitative changes in peripheral blood lymphocytes were less pronounced.

Study of contact preparations of the spleen of second group animals revealed no changes in lymphopoiesis. But calculation of the lymphogram disclosed certain peculiarities by comparison with the control animals.

Thus, contact preparations of the spleen of healthy control animals always showed, apart from lymphocytes, a small number of neutrophils (average 7 per cent), of which about 0-5 per cent were young forms—promyelocytes, myelocytes and juveniles. Sometimes a small number (less than 1 per cent) of normoblasts were observed, and also about 0-5 per cent plasma cells.

These results are in agreement with the lymphograms given by I.A.Po-berii (1957). In rabbits of the second experimental group killed in the first year, the overall number of neutrophils in the spleen was higher than in the control animals (11-1 per cent); the percentage of young forms (2*6) was especially high. Also found was an average 2-7 per cent of cells of the red series; apart from haemoglobinized normoblasts, which were also found rarely in the control animals, early erythroblasts and even proerythroblasts were observed. Possibly the normoblasts frequently seen in the peripheral blood at this time are connected with the presence of erythropoiesis in the spleen.

The number of plasma cells (average 1-8 per cent) was somewhat increased by comparison with the control. At later periods the number of neutrophils and cells of the red series in the spleen declined, while the plasma cells doubled (average 3*7 per cent).

In spleen preparations from the first group animals (59Fe 1 μ-c/kg) somewhat more neutrophils (about 10 per cent) and plasma cells (3-5 per cent) were found than in the controls. The number of cells of the red series was the same as in the control animals.

Extramedullary haemopoiesis was also found in other organs. Thus, a significant number of myeloblasts, myelocytes, juvenile neutrophils and also young cells of the red series were found during the first year in liver preparations. A smaller number of young blood cells was found in preparations from the kidneys and lungs.

These observations gave grounds for supposing that one of the earliest


reactions to systematic administration of 59Fe is the formation of foci of extramedullary haemopoiesis not only in the spleen but also in other organs.

Thus, systematic observation on the state of haemopoiesis during prolonged administration of 59Fe revealed the earliest changes in erythropoiesis and lymphopoiesis.

Comparison of changes in erythropoiesis during daily administration of stable iron and two different doses of 59Fe (1 and 10 μΰ/Ι^) shows that, under our experimental conditions, the effect of the active isotope on erythropoiesis is associated with not only the amount of activity administered, but also to a certain degree, with the physiological background which is created by daily administration of elemental iron.

Thus, administration of stable iron (1-4 mg/kg) in our experiments produced primarily an increase of haemoglobin, which is in complete agreement with the numerous results in the literature. However, the impression exists that iron is not only a material used in the construction of haemoglobin but is also a specific stimulus of erythropoiesis (G.K.Lavskii, 1934; S.Ya.Rik-man, 1952; E.Shtarkenshtein, 1935).

This suggestion explains, to some extent, the relative increase in the number of erythroblastic cells of the bone marrow observed in our experiments after 8-10 months of administration of stable iron. In all probability, the more pronounced posthaemorrhagic hyperplasia of the erythroblastic part of the bone marrow observed in animals of this group (cf. our later article in the present collection) is a manifestation of the heightened reactivity of this part of the bone marrow.

A definite stimulatory effect was observed with administration of small amounts of 59Fe (1 μc/kg), i.e. when the effect of iron as an element was combined with small amounts of ionizing radiation (0Ό4 rads per day). In animals of this group, as well as the rise in the haemoglobin level, the capacity of the bone marrow to form cells also increased. Together with a relative enlargement of the erythrocytes in the bone marrow the number of young red cells also increased (reticulocytosis). The number of red cells in the peripheral blood also increased. Signs of impairment of erythropoiesis in the form of qualitative changes in the red cells appeared only after 9-10 months.

With administration of the larger 59Fe dose (10 μc/kg), producing general body irradiation of, on average, 0*3 rads/day, impairment of red cell formation became apparent. Moreover, V.L.Ponomareva detected certain qualitative peculiarities of the red cells formed during erythropoiesis stimulated by bloodloss ; haemoglobin concentration was lower than in the control animals. Apparently, prolonged accumulation of 59Fe in the bone marrow may lead to impairment of the processes of haemoglobin synthesis. In this connection an attempt was made to detect differences in the haemoglobin structure of the experimental animals using spectrophotometric analysis carried out at our request in Prof. G.V.Derviz's laboratory. The haemoglobin of the experimental animals had the same Huffner coefficient as that of the controls (1, 65), and no pathological pigments were found. However,


this does not exclude the possibility of more subtle changes in the haemoglobin molecule during prolonged administration of 59Fe.

It would seem that the neuro-humoral regulation of erythropoiesis was disturbed during our observation period. Since the 59Fe was administered orally it can be assumed that impairment of gastric secretory function had some part in this, and also the spleen, in which a considerable quantity of 59Fe is deposited (cf. G. A. Avrunina's article in the present collection).

It is known that pathological changes in the spleen lead not only to disturbance of lymphopoiesis but can also have an effect on medullary haemopoiesis. The regulatory function of the spleen in this respect is confirmed by many writers (I.M.Azbukina, 1938; M.A.Kalnyn', 1955; E.L.Kan, 1954; R.A.Kukainis, 1950; T.A.Lebedeva, 1940; N.S.Rozanova and E.A.Zhu-kova, 1955; V.B. Färber, 1946; N.A.Fedorov et al, 1956; M.G.Kakhete-lidze, 1952; and others). The important role of the spleen in the restoration of normal haemopoiesis after acute radiation sickness produced by external irradiation has also been demonstrated. Screening of the spleen facilitated more rapid restoration of peripheral blood indices and resulted in a milder type of sickness and lower mortality rate (M. S.Lapteva-Popova and Yu.V. Venitskovskii-Zolotykh, 1960; V.Ya.Lavrik and G.A.Levchuk, 1958; N.I. Shapiro et al., 1955; and others).

An analogous effect was obtained by administration of pulverized spleen to animals whose abdominal cavity had been irradiated (A. G. Karavanov et al, 1959; N.I.Shapiro et al, 1955; V.P.Teodorovich, 1958).

In our experiments impairment of lymphopoiesis during administration of 59Fe in an amount of 10 μc/kg was very marked and consisted in a gradual decline of the absolute number of lymphocytes in the peripheral blood. For elucidation of the character of the, at first transient, and then permanent lymphopenia we compared the condition of the peripheral blood and lym-phoid tissue according to E.S.Gaidova's histological results (cf. the article in the present volume) in healthy animals of the different groups dying in the first and second years. In the control rabbits, both in the first and second years, the number of lymphocytes in the peripheral blood and the condition of the lymphoid tissue were normal. In animals receiving 10 μc/kg 59Fe in the first year a significant number of animals with lymphopenia were observed, not accompanied, however, by any abnormalities of the lymphoid tissue. In animals of this group in the second year the lymphopenia had become more pronounced and a more or less marked reduction of lymphoid tissue in the spleen was observed. Considerable impairment of the haemo-poietic capacity of the lymphoid tissue was also detected in experiments using functional stresses; thus after bloodloss and parturition prolonged absolute lymphopenia developed in the experimental animals.

The sole sign of impairment of neutrophilic myelopoiesis by 59Fe was the appearance of neutrophils with nuclear fragmentosis.


CONCLUSIONS

1. Daily oral administration to rabbits of l-4mg/kg stable iron in the form of FeCl3 produced, in the first months of the experiment, an increase of haemoglobin, and at later periods (after 12-14 months) a relative increase in the erythroblastic part of the bone marrow. No changes in neutrophiHc leucopoiesis and lymphopoiesis were observed in these animals.

2. Administration of 59Fe in doses of 1 μΰ/kg daily produced temporary stimulation of erythropoiesis—a parallel increase in the number of red cells and haemoglobin, reticulocytes, chiefly in the bone marrow, and a relative increase in erythroid series in the bone marrow. Qualitative changes in the red cells appeared after 8-9 months. No qualitative or quantitative changes of the neutrophils were observed. Depression of lymphopoiesis became marked towards the end of the first year. The number of platelets was unchanged.

3. Administration of 10 μc/kg 59Fe daily led to impairment of haemopoiesis as a whole. Functional changes in the erythroblastic tissue were manifested in the form of qualitative changes of the red cells (macrocytosic lepto-cytes, anisocytosis). Increase of the haemoglobin level and colour index occurred in parallel with an increase in red cell size. No changes in neutrophil number in the peripheral blood were observed but by the end of the first year qualitative changes in the nucleus began to appear in the form of nuclear fragmentation.

Depression of lymphopoiesis in the form of lymphopenia and qualitative changes of the lymphocytes began to appear from the second month of administration.

A greater or lesser failure of all the haemopoietic processes was revealed when functional stresses were applied.

R E F E R E N C E S

A Z B U K I N A I . M . , Trudy Tomsk, med. inst., 6, 379-385 (1938). BELOBORODOVAN.L., Toxicology of Radioactive Substances (Materialy po toksikologii

radioaktivnykh veshchestv), vol. 2, pp. 39-52, Moscow (1960). BELOBORODOVAN.L. and BARANOVAE.F. , Toxicology of Radioactive Substances (Mate

rialy po toksikologii radioaktivnykh veshchestv), vol. 1, pp. 171-193, Moscow (1957). FARBER V.B., Klin, med., 24 (4-5), 46-54 (1946). F E D O R O V N . A . , NAMYATYSHEVA A.M. and K A K H E T E L I D Z E N . G . , Trudy Inst, gemat. i

pereliv. krovi, 1 (10), 16 (1956). KAKHETELIDZE M. G., An experimental pathological investigation of the haemopoietic fac

tor of the stomach using a new method (EksperimentaPno-patologicheskoe issledovanie gemopoeticheskogo faktora zheludka s pomoshch'yu novogo metoda). Dissertation, Tbilisi (1952).

KUKAINIS R. A., The role of the spleen in haemopoietic processes (under normal conditions and with certain types of experimental anaemia) (Materialy po voprosu o roli selezenki v protsessakh krovotvoreniya (v norme i pri nekotorykh vidakh eksperimental'noi anemii)). Dissertation, Riga (1950).

TRS 5


KALNYN' M. A., The effect of spleen denervation on the haemopoietic processes (Vliyanie denervatsii selezenki na protsessy krovotvoreniya). Dissertation, Riga (1955).

K A N E.L., Byull. eksper. biol. med., 3, 29-33 (1954). KARAVANOV A.G. et al, Vrach. deh, 1, 45-52 (1959). LAPTEV A-POPOVA M.S. and VENITSKOVSKII-ZOLOTYKH Yu. V., Med. radioL, 5, No. 2, 3-12

(1960). LAVRIK V . Y A . and L E V C H U K G . A . , Vrach. deh, 11, 1169-1174 (1958). LAVSKII G.K., Sov. prob, gematol. i pereliv. krovi, 9-10, 145-156 (1934). LEBEDEVA T. A., The effect of the spleen on blood regeneration after heavy bloodloss (O

vliyanii selezenki na regeneratsiyu krovi posle obil'nogo krovopuskaniya). Transactions of the Perm State Stomatological Institute, vol. 2 (1940).

P O B E R I I I . A . , Voprosy pitaniya, 16, No. 4, 40-47 (1957). RIKMAN S.YA. , Haemopoiesis in children suffering from rickets and anaemia treated with

vitamin D and iron preparations (Krovotvorenie u detei, boPnykh rakhitom i anemiei, pri kompleksnom lechenii vitaminom D i preparatami zheleza). Dissertation, Moscow (1952).

ROZANOVA N. S. and ZHUKOVA E. A., Sov. prob, gematol. ipereliv. krovi, 31 , 85-86 (1955). SHAPIRO N. I . et al., Collection of Works on Radiobiology (Sbornik rabotpo radiobiohgii),

Moscow (1955). SHTARKENSHTEINE., Problems of Biology and Medicine (Problemy biologii i meditsiny),

pp. 339-347, Moscow (1935). TEODOROviCH V.P., Aktual. voprosy pereliv. krovi, Leningrad, 6, 109-111 (1958).

CHANGES IN ERYTHROPOIESIS DURING PROLONGED ADMINISTRATION OF 59Fe

V. L. PONOMARE VA

CHANGES in the red blood during chronic administration of small doses of 59Fe are of particular interest since radioactive iron (like its stable isotope), is incorporated into the haemoglobin molecule, and must participate directly in the process of erythropoiesis.

It is already known that ionizing radiation has a considerable effect on erythropoiesis. Previous work has demonstrated the great sensitivity of the erythroblastic part of the bone marrow to X-radiation (L.G.Berman, 1946; M.S.Lapetva-Popova, 1954; V. V.Sokolov and V.A.Gubin, 1954; I.T.Aba-sov, 1956; M.N.Pobedinskii, 1955; A.A.Bagdasarov, 1959; I.P.Mishchen-ko, 1928; G.V.Voskoboinikov, 1954; I. D. Makulova and I.M.Velikson, 1957 ; M. Bloom and W. Bloom, 1947 ; Brecher, Endicott, Gump and Brawner 1948).

Early impairment of erythropoiesis following internal irradiation has been shown in our laboratory in experiments using 60Co and certain other radio-isotopes in amounts similar to those now being studied (N. L. Beloborodova and E. F. Baranova, 1957; N. L. Beloborodova, 1960).

The present work is part of a collective investigation of the effect of 59Fe on the blood.

In order to detect early changes in erythropoiesis, apart from counting the numbers of red cells, reticulocytes, and estimating the haemoglobin level, the shape, volume, anisocytosis and osmotic properties of the red cells were investigated. The diameter of erythroblasts and normoblasts in the bone marrow was also measured.

The experiments were carried out on rabbits. The rabbits of the first experimental group (12 animals) received daily 1 μc/kg 59Fe in the form of a solution of 59FeCl3; the rabbits of the second experimental group, 10 [xc/kg 59Fe (19 animals). Of the two control groups one served as a biological control (19 animals) and the second (30 rabbits) received stable iron daily in the same quantity and compound (FeCl3) as the animals of the second experimental group.

Measurement of the red cells is a precise and sensitive means of studying the state of erythropoiesis and especially of detecting early haematological reactions. It is known that this qualitative characteristic changes earlier in certain pathological conditions than do the numerical indices of the red blood. Microcytosis developed earlier in pregnant rabbits suffering from

61


iron-deficiency anaemia than the decline in haemoglobin and red cell number. Moreover, a series of writers show a direct correlation between the size of peripheral blood red cells and the size of their precursors in the bone marrow (G.A.Alekseyev, 1958; Z.I.Atakhanova, 1952; L. M. Fridman, 1955; V.V.Progonnaya, 1955; S.A.Troitskii, 1959). Thus, measurement of red cell size detects, to a certain extent, qualitative shifts in the haemopoietic organs. Intensification of the erythropoietic processes results in the entry into the peripheral blood of red cells of large diameter. E. M. Semenskaya (1948) and L. M. Fridman (1955) detected macrocytosis in the regenerative period following bloodloss; V.A.Gubin (1954) and V.V.Sokolov, during restoration of erythropoiesis in animals suffering from acute or chronic radiation sickness.

On the other hand, when the number of ageing elements in the peripheral blood increases, a tendency to microcytosis and spherocytosis is observed. This has been observed by E. M. Semenskaya (1946) after bloodloss, when the number of microcytes increased because of the emergence of red cells from the depot. V.A.Gubin (1954) and V.V.Sokolov (1952) have also observed this phenomenon during depression of the haemopoeitic processes following irradiation. Iron deficiency is manifested by microcytosis, as occurs in hypochromic anaemias (G.A.Alekseyev, 1958; L.M.Fridman, 1955; V.V.Progonnaya, 1955; E.LIvanyuk-Belugina, 1957).

At the same time it is known that in deficiency of the antianaemic factor (vitamin B12) macrocytosis develops, as for example in Addison-Beirmer anaemia and also in diseases of the liver.

It may thus be concluded that red cell measurement is a valuable supplementary method of investigation enabling the early detection of changes in erythropoiesis. It is of especial importance in studying the chronic effect of ionizing radiation. However, only in isolated instances has attention been given to the qualitative characteristics of the red cells.

A. A. Bagdasarov (1959) has observed the development of macrocytosis and a reduced number of reticulocytes in X-ray workers (in some of those investigated) and spherocytosis in others.

I.D.Makulova and I.M.Velikson (1958) demonstrated an increase of red cell volume associated with spherocytosis in patients with early signs of radiation sickness.

N.A.Chulina (1959) has found an increase in red cell volume together with increased erythrocyte diameter but without spherocytosis in persons exposed to ionizing radiations.

M. S. Lapteva-Popova (1954) has observed the development of macrocytic anaemia in animals in the terminal stages of chronic radiation sickness.

The literature concerning changes in red cell osmotic fragility following irradiation deals mainly with an acute single exposure in vivo or in vitro, in doses of the order of several hundred roentgens.

The majority of references are concerned with the haemolytic effect of X-irradiation. Some authors consider the increase in the bilirubin of the blood and urine of people and animals, subjected to radiation, a sign of increased

Changes in Erythropoiesis during Administration of59Fe 63

red cell haemolysis (Kh. Kh. Vlados and E. A. Bondarenko, 1934 ; M. G. Shchi-tikova, 1958), whilst others regard the increased iron deposition in the cells of the reticulo-endothelial system to be of greater significance (P. V. Sipovskii, 1936).

Increased osmotic fragility of the red cells following X-irradiation has been demonstrated by a series of writers using different methods (A. P. Belousov, 1957; I.A.Terskov and LI.Gitel'zon, 1957; K.S.Trincher, 1959). E.E.Do-brovol'skii and L.N.Obrucheva-Dobrovol'skaya (1926), in experiments in vitro, found reduced red cell osmotic fragility after irradiation.

No publications referring to the effect of chronic internal administration of radioactive isotopes on the cytometrical qualities of the red cells and their osmotic properties have been found.

M E T H O D S

An eye-piece micrometer was used for measuring red cell diameter. One hundred cells in the smear were counted : 50 at each end of the smear. Mean red cell volume was calculated according to haematocrit values. In addition, cell thickness was determined and the sphericity index calculated—the ratio of diameter to thickness. An increase denotes a tendency to leptocytosis; a decrease, to spherocytosis.

Since anisocytosis may be directly connected with impairment of normal formation of cells of the erythroblastic series, the degree of anisocytosis was specially determined using the method suggested by A.P.Egorov and M. F. Aleksandrovaya.

In parallel with these investigations the fragility of the red cells was determined in relation to hypotonie solutions of NaCl.

There are many methods of determining red cell osmotic fragility. They can be divided into the macroscopic and the microscopic.

Among the microscopic is M. V.Yanovskii's method (1900). We used this method but modified it somewhat, increasing the number of NaCl solutions to six (1, 0-55, 0-50, 0-48, 0-45 and 0-40 per cent).

Having obtained curves of red cell lysis, which normally have an S-shape, we then determined mean fragility, i.e. the point at which 50 per cent of red cells are haemolysed.

In addition, with more marked shifts in erythropoiesis, taking place after application of functional stress (parturition, bloodloss), changes were looked for in the ratios of groups of red cells with varying fragility. For this purpose all the red cells were divided according to their fragility into three groups : very fragile haemolysing in 0-55 per cent NaCl), moderately fragile (haemo-lysing in 0-50-0-48 per cent NaCl) and resistant (not haemolysed at 0-45 per cent NaCl).


R E S U L T S

In the control group the variation in number of red cells and haemoglobin level was negligible throughout the observation period (red cells about 5,000,000 per mm2 haemoglobin about 13 g per 100 ml), increasing chiefly in the spring-summer months, this increase usually unaccompanied by any substantial change in the colour index.

Mean red cell diameter, obtained from 40 normal rabbits, was 6-40 ± 0Ό51 μ, mean volume 65 ± 0-9 μ3. The figures obtained for mean cell volume agree with those of P.A.Korzhuev (1949), who observed a volume of 66 μ3. Values for mean cell diameter given by B.K.Lemazhikhin and G.M.Frank (1955) (6·5-6·6 μ) are somewhat higher than our figures.

In the group of control rabbits mean red cell diameter and volume values remained close to the normal figure.

To determine red cell distribution by diameter we constructed Price-Jones curves. The apex of such a curve shows the most frequently occurring diameter in a given group while the width of the base indicates the degree of anisocytosis. In the control rabbits the largest group of cells were of diameter 6 μ (49-54 per cent). Subsequently, for a period of 14-15 months, red cell diameter distribution did not change significantly, and nor did the width of the base of the curve, expressing anisocytosis.

Mean red cell osmotic fragility throughout the observation period in the control group fluctuated within the limits 0-45-0-50 per cent NaCl.

In the group receiving stable iron the red cell count throughout the observation period, as in the control group, remained around 5,000,000 per mm3 of blood.

Red cell size showed an increase in mean diameter by the end of the first month from 6-29 + 0-03 to 6-47 ± 0-04 μ.

In analysing the results obtained it appeared that the increase of mean red cell diameter throughout the group as a whole was due chiefly to those rabbits (9 of 20) which showed, prior to the experiment a tendency to micro-cytosis (mean red cell diameter from 5-89 to 6-29 μ).

In this group of animals mean red cell diameter had increased by the end of the first month of the experiment from 6-15 ± 0-04 to 6-53 ± 0-053 μ. By the third month it had fallen to a normal mean—6-46 μ.

In the other animals, having a normal mean red cell diameter before the experiment, diameters were either unchanged or slightly increased.

By the end of the first month of the experiment the haemoglobin had risen sharply (from 12-0 ± 0-21 to 14-0 ± 0-25 g per 100 ml) as had the colour index (from 0-75 ± 0-02 to 0-88 ± 0-02).

In subsequent months, the haemoglobin level and colour index declined but at the end of the experiment were still somewhat higher than in the control.

Thus, the effect of stable iron was chiefly to increase haemoglobin formation.


Absorption of stable iron in a chloride compound (FeCl3) can be assumed from the results of G. A. Avrunina (cf. the article in the present collection), who studied uptake of 59Fe from the same chemical compound. The maximum level of blood activity (i.e. greatest degree of saturation of the blood with 59Fe) was established by this writer on the 45th-60th day from the beginning of the experiment. Thus, by the end of the first month, administered iron has already been fairly well incorporated into the general iron metabolism of the body. The rapid inclusion of administered 59Fe into all the cells of the erythrocyte series has been shown by M. Austoni (1954).

FIG. 1. Changes in the colour index during prolonged administration of 59Fe. 1—administration of l(^c/kg 59Fe; 2—stable iron;

3 — 1 μο/kg 59Fe; 4—control

In the group of rabbits receiving the large 59Fe dose (10 μο/kg) the red cell count did not materially alter throughout the experiment, remaining on average within the limits 5,500,000-4,700,000 per mm3. However, from the first month, as in the rabbits receiving stable iron, the haemoglobin began to rise. By the 6th month the haemoglobin content in this group had risen 1-7 g per 100 ml above the initial level, after which it declined; however, up to the 20th month it continued to fluctuate around a level higher than the original.

Thus, the total haemoglobin content in the group receiving 59Fe increased by almost the same extent as in the group receiving stable iron. But the change in the colour index, expressing the haemoglobin content of each red cell, was different in the two groups. Whereas in the stable iron group a significant increase of the colour index was observed only in the first 3 months of the experiment, in the 59Fe group this increase, which became statistically valid in the 1st month, reached a maximum by the 6th month (from 0-79 ± 0Ό1 to 0-88 ± 001), after which it declined, although remaining, up to the end of the experiment, at a higher level than in the other three groups (Fig. 1).*

It should be pointed out that red cell saturation with haemoglobin at this time was the same as in the control group, i.e. haemoglobin concentration in the red cells was not increased, the higher colour index being connected

* In Figs. 1, 2 and 4 each point represents a group mean calculated for the period marked on the abscissa.


with increased red cell size. And indeed, the increase in haemoglobin content of the red cells ran parallel to the increase of their mean diameter. This increase became statistically valid by the 3rd month and had reached a maximum by the 6th month (from 6-38 ± 0-04 to 6-73 ± 0-04 μ), after which mean diameter declined somewhat. However, it remained above that of the other groups up to the end of the experiment (Fig. 2).

Construction of Price-Jones curves for different months of the experiment showed, that in the experimental group, the shape of the curve begins to change from the 1st month. The apex of the curve is flatter and by the 3rd

6-8

QJ

1 H

1 6-2

I 60

^ssiC'.r*'

1 _

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1-3 4-6 7-10 13-14 Months

FIG. 2. Changes in mean red cell diameter during prolonged administration of 59Fe.

1—administration of l(^c/kg 59Fe; 2—stable iron; 3—1 μο/kg 59Fe; 4—control

month a predominance of red cells of 7 μ diameter becomes apparent. An-isocytosis has increased by this time (because of an increase of both micro-cytes and macrocytes). Later, the percentage of red cells of 7 μ diameter continues to increase (in the 6th month from 36 to 51) (Fig. 3).

A statistically valid increase of anisocytosis in this group can be seen using A.P.Egorov's method from the 3rd to the 13th months of the experiment (Fig. 4).

Red cell volume increased somewhat, beginning from the 4th month, but significantly less than diameter. In this connection, in the group receiving the large 59Fe dose increased numbers of leptocytes appeared from the 3rd month. This change of shape was maintained up to the 12-13th month ; by the 14th month the number of leptocytes had decreased. Reticulocytosis was absent and the number of reticulocytes throughout the observation period fluctuated within the same limits as in the controls.

FIG. 3 (opposite). Red cell diameter distribution curves (Price-Jones curves) at different months of the experiment (abscissa—red cell diameter; ordinate—number

of red cells). a—before the experiment; b—1 month after commencement of experiment;

c— 2 months after; d—3 months after; e—6 months after; f—7 months after; g—10 months after; h—13-14 months after

1—control; 2—administration of l(^c/kg 59Fe


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4 5 6 7 8 9 μ 4

FIG. 3a-h.

5 6 7 8 9μ


In the group of animals receiving the small 59Fe dose (1 μ^1<£) the reaction to administration of the isotope in the first few months took the form of a stimulation of erythropoiesis. This was seen as an increase of the red cell count (on average by 900,000 mm3) and haemoglobin level (on average by 1*6 g per 100 ml) in the first 4 months, slight reticulocytosis, and also hyper-plasia of the erythroid series in the bone marrow. The increase of these indices was maintained in some rabbits from 2 to 8 months, after which the figures declined to their initial level. Colour index and red cell haemoglobin concentration index were unchanged. A statistically valid increase of mean cell

FIG. 4. Degree of anisocytosis during prolonged administration of 59Fe. 1—administration of 10 μο/kg 59Fe; 2—stable iron;

3 — 1 μο/kg 59Fe; 4—control

diameter (from 6-38 ± 0-01 to 6-56 ± 0Ό5 μ) with increased leptocytosis and anisocytosis appeared in these animals later (only by the 9-10th month of the experiment) and was less pronounced than in the group receiving the large 59Fe dose.

Mean osmotic fragility of the red cells did not differ from the control group.

In order to clarify the origin of macrocytic leptocytes in the peripheral blood, the diameters of normoblasts and erythroblasts in the bone marrow were measured. Measurements were made of 100 cells in a bone marrow smear (50 late normoblasts and 50 intermediate normoblasts).

The measurements showed that in rabbits receiving the large 59Fe dose the intermediate and late normoblast diameter distribution curves shift towards cells with a large diameter from the 2nd month of administration. This shift is maintained for 3 months but by the 6th month these curves are almost alike for the experimental and control groups (Fig. 5).

Thus, daily administration of 1-4 mg/kg stable iron produced increased haemoglobin formation without a significant increase of the number of red cells. This was accompanied by some increase of red cell size and change of shape towards thinner cells (leptocytes).

A number of writers have indicated that the primary effect of treatment of anaemia with iron is to raise the haemoglobin level (M. S. Dul'tsin, I.I.Yu-rovskaya and K.A.German, 1934; M.N.Volk, 1938). But for us, the work of A.I.Ivanov (1941) is of particular interest, since he found an increase of haemoglobin not only in anaemic but also in healthy children receiving iron preparations orally.


We have been unable to find in the literature any reports of change of red cell shape and volume after iron administration to a healthy individual.

Administration of 1 μ^1<£ 59Fe(iron weight content 0-1-0-4 mg/kg) gave rise to a parallel increase of the red cell count and haemoglobin level and a temporary increase of the number of reticulocytes in the peripheral blood during the first few months of the experiment.

Analysis of bone marrow changes (cf. the article by N. L. Beloborodova, E.K.Red'kina and V.L.Ponomareva in the present collection) suggests a stimulation of erythropoiesis in this group of animals. This reaction, apparently, is caused by small doses of ionizing radiation. A similar effect has been demonstrated by many workers during repeated exposure to small doses of external irradiation (M. Dmitrov and G. Markov, 1956 ; Ts. D. Sindrer and E.T.Teselkina, 1935; M.S.Lapteva-Popova, 1959; I.S.Glazunov and P.M. Kireyev, 1958; and others). However, the effect of the element iron itself is important since prolonged administration produces hyperplasia of the red cells in the bone marrow (cf. the article by N.L. Beloborodova et al.).

In considering the effect of administration of the large radio-iron dose (10 μc/kg) the effect of the carrier iron, the content of which in these experiments was 1-4 mg/kg, should be taken into account. This carrier iron may explain the increase of haemoglobin during administration of 59Fe. But in this group, as well as increased haemoglobin formation, significant changes in the shape and size of the red cells were observed.

I ^ ^ l

1 8 9 10 11 12μ Diameter

9 10 11 12 13 14 15/1 Diameter

FIG. 5. Bone marrow late and intermediate normoblast diameter distribution curves.

1—before the experiment; 2—after 2 months' administration of l(^c/kg 59Fe; a—late normoblasts; b—intermediate normoblasts

The early increase in size of the red cell precursors in the bone marrow, the appearance of macrocytic leptocytes and increased anisocytosis of peripheral red cells (with a normal reticulocyte number) suggests the presence of a harmful effect of 59Fe on the red cells, possibly with impairment of red cell formation.

This assumption is confirmed by the results obtained when functional stresses in the form of bloodloss and parturition were applied to the haemo-poietic organs (cf. the appropriate article in the present volume). At the


time when the anaemia, produced by bloodloss and parturition, in the experimental group was greatest, qualitatively changed red cells appeared (giant, spherical forms) with increased fragility to hypotonie solutions of NaCl. These findings were not observed in the control group.

Apart from qualitative changes of the red cells in the experimental animals, under certain conditions their formation was retarded, as a result of which anaemia following parturition was more prolonged than in the controls.

All that has been said above leads us to agree with A.A.Bagdsaarov (1959) that the appearance of macrocytosis, with the absence of reticulocytosis, during prolonged administration of small doses of ionizing radiation may be a sign of deficiency in the erythropoietic system of the bone marrow.


1. In the group of control animals the red blood indices throughout the observation period fluctuated insignificantly. A seasonal increase of red cell number and haemoglobin, without increase of the colour index, was observed.

2. Daily administration of stable iron produced a temporary increase in haemoglobin. The other indices were close to those in the control group.

3. Administration of 10 \^ojkg 59Fe produced, apart from raising the haemoglobin level, a series of early qualitative changes of the red cells in the form of increase in the number of red cell precursors in the bone marrow and the appearance of macrocytic leptocytes with increased anisocytosis of the red cells in the peripheral blood.

4. Administration of a smaller quantity of the isotope (1 μο/kg) resulted in a stimulation of erythropoiesis during the first months of the experiment. Macrocytic leptocytes and anisocytosis of the red cells, indicating impairment of erythropoiesis, appeared later and were less pronounced than in the rabbits receiving the large dose.

5. No changes were found in osmotic fragility during administration of stable and radioactive iron (1 and 10 [xc/kg).

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radioaktivnykh veschestv), vol. 2, Moscow (1960). BELOBORODOVAN.L. and B A R A N O V A E . F . , Toxicology of Radioactive Substances (Ma

terialy po toksikologii radioaktivnykh veschestv), vol. 1, Moscow (1957). BELOUSOV A.P., Vest. rent, i rad., 3 (32), 5-14 (1957).

Changes in Erythropoiesis during Administration of59Fe 71 BERMAN L.G., The effect of X-rays on haemopoiesis (Vliyanie rentgenovskikh luchei na

funktsiyu gemopoeza). Dissertation, Moscow (1946). BLOOM M. and BLOOM W., / . Lab. Clin. Med., 32, 654-659 (1947). BRECHER J., ENDICOTT K., G U M P H. and BRAWNER H., Blood, 3, 1259-1274 (1948). CHULINA N. A., Proceedings of a conference of young scientific workers, 12-14th Novem

ber 1959. Inst. gigiena truda i profzabolevanii A M N SSSR (1960). DMITROV M. and MARKOV G., IZV. Inst. biol. Bolg. AN, 7, 231-278 (1956). DOBROVOL'SKIIE.E. and OBRUCHEVA-DOBROVOL'SKAYAL.N., Experimental and Clinical

Radiology (EksperimentaVnaya i klinicheskaya rentgenologiya), vol. 1, pp. 76-99, Kharkov (1926).

D U L ' T S I N M . S . , YUROVSKAYAI.I . and GERMAN K.A., Terap. arkh., 12 (6), 118-134 (1934).

FRIDMAN L.M., Collected Works of the Mukhadze Blood Transfusion Institute, 4, 103-109 (1955).

FRIDMAN L.M., Osmotic and mechanical red cell fragility in anaemic conditions (Osmo-ticheskaya i mekhanicheskaya rezistentnosf eritrotsitov anemichekikh sostoyaniyakh). Dissertation, Tbilisi (1955).

GITEL'ZON 1.1, and TERSKOV I. A., Differential investigation of red cell condition according to osmotic fragility (Differentsial'noe issledovanie sostoyaniya eritrotsitov po ikh osmo-ticheskoi stoikosti). Sectional Proceedings of 5th Scientific Conference of Tomsk University, Tomsk (1956).

GLAZUNOV I.S. and KIREYEV P.M., Sov. med., 4, 49-54 (1958). GUBIN V. A., Dokl. AN SSSR, 20 May (1954). IVANYUK-BELUGINAE.I., New Methods of Diagnosis in Oncology and Radiology (Novye

metody diagnostiki v onkologii i rentgenologii), pp. 182-187, Kiev (1957). I V A N O V A . I . , Trudy Samar. med. inst., 5, 41 (1941). KORZHUEV P.A., Biokhimiya, 14 (4), 338-340 (1949). LAPTEVA-POPOVA M.S. and K R A Y E V S K I I N . A . , Prob, gematol. ipereliv. krovi, 4 (12), 3-14

(1959). LAPTEVA-POPOVAM.S. and VENITSKOVSKII-ZOLOTYKH Yu.V., Med. radiol, 5 (2), 3-12

(1960). L E M A Z H I K I N B . K . and FRANK G.M., Trudy 1st. biofiz. AN SSSR, 1, 276-287 (1955). MAKULOVAI .D . , Proceedings of the 30 th Anniversary Session of the Leningrad Inst. of

Hygiene of Work and Occupational Diseases, pp. 50-56 (1957). MISHCHENKO I. P., Experimentaland Clinical Radiology (EksperimentaVnaya i klinicheskaya

rentgenologiya), vol. 2, pp. 58-66, Khar'kov (1928). POBEDINSKI IM.N. , Vrach. delo, 3, 233-236 (1955). PROGONNAYA V. V., The clinical importance of determining size and shape of the red cells

in certain haematological syndromes (Klinicheskoi znachenie opredeleniya velichiny i formy eritrotsitov pri nekotorykh kliniko-gematologicheskikh sindromakh). Dissertation, Kiev (1955).

SEMENSKAYAE.M., Anisocytosis in anaemic conditions (Anizotsitoz pri anemicheskikh sostoyaniyakh). Dissertation, Tbilisi (1946).

SEMENSKAYA E. M., Transactions of Tbilisi Research Inst. of Blood Transfusion, pp. 118-129 (1948).

SHCHITIKOVA M.G., Patol.,fiziol. i eksper. terap., 1, 22-27 (1958). SINDRER Ts.D. and TESELKTNA E.T., Occupational Hygiene in Radium Production (Gigiena

truda v proizvodstve radiya), pp. 79-92, Moscow-Leningrad (1935). SIPOVSKIIP .V. , Vest, rengenol. i radiol, 16 (5), 373-379 (1936). TESELKTNA E. T., Occupational Hygiene in Radium Production (Gigiena truda v proizvodstve

radiya), pp. 127-142, Moscow-Leningrad (1935). TERSKOV I.A. and GITEL'ZON 1.1., Biofizika, 4, 523-534 (1957). TRINCHER K.S. , Biofizika, 4 (1), 78-83 (1959). T R O I T S K I I S . A . , The relationship between red cell count and diameter (O vzaimocvyazi

izmenenii kolichestva eritrotsitov i ikh diametra). Proceedings of IVth Ail-Union Conference of Medical Laboratory Technicians, Moscow (1959).


TSIBULEVSKAYAL.G., Vrach. deh, 6, 507-512 (1949). V E L I K S O N I . M . , M A K U L O V A I . D . , SHCHEGLOVA A.V. and RODOTOVSKAYAR.D. , Clinical

aspects of the chronic effect of ionizing radiation (K klinike khronicheskogo vozdeistviya ionisiruyushchei radiatsii). Transaction of a scientific session devoted to achievements during 1955, Leningrad (1958).

VLADOS K H . K H . and BONDARENKO E. A., Sov. prob, gematol. ipereliv. krovi, 15, 848-862 (1934).

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VOSKOBOINIKOVG.V., Changes in the activity of blood catalase and the erythropoietic function of the haemopoietic organs following irradiation (Izmenenie aktivnosti katalazy krovi i eritropoeticheskoi funktsii organov krovotvoreniya pod deistviem ionisiruyush-chego izlucheniya). Dissertation, Moscow (1954).

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YANOVKSII M. V., Transactions of the Society of Russian Doctors 1888-1890, p. 27.

HAEMOPOIESIS AFTER BLOODLOSS IN RABBITS DURING PROLONGED

ADMINISTRATION OF 59Fe

N . L . B E L O B O R O D O V A , E. K. R E D ' K I N A and V . L . P O N O M A R E V A

THIS paper is concerned with the investigation of the regenerative capacities of the haemopoietic organs in animals which daily for 6-7 months have received orally a solution of 59FeCl3.

As our previous investigations have shown (N. L. Beloborodova and E. F. Baranova, 1957), one of the physiological stresses which detects deficiencies in the haemopoietic organs is bloodloss, since it reveals, to a considerable extent, the body's reactivity and its capacity to mobilize its defence mechanisms. One of the compensatory reactions is the increased erythro-poiesis to replace the lost red cells.

The reaction of the normal body to haemorrhage has been fairly well studied both experimentally and in blood donors. Even in the last century numerous investigators discovered that venesection results in a fall in the number of red cells which progresses for several days. As was shown by special experiments, dilution of the blood by tissue fluid entering the blood stream plays an important role in this phenomenon (M.Yu. Rappoport, 1948; I.R.Petrov, 1945; G.A.Guseinov, 1954; G.A.Barashkov, 1953; and others). But this is not the only cause. It is established that at a certain time after haemorrhage, the red cell breakdown in the reticulo-endothelial system is increased.

V. M. Rokitskii first drew attention to this aspect in 1899, and the problem was later studied by A.A.Bogomolets' pupil Ya. G. Uzhanskii (1932), who considered erythrodieresis a necessary factor in the regulation of erythro-poiesis. The existence of red cell breakdown after haemorrhage is clearly revealed by histological investigations (Ya.G.Uzhanskii, 1946; O.A.Levin, 1932; E.I.Egorova, 1947) and by special biochemical investigations (V. V. Ko-val'skii et al, 1938).

In addition to the entry into the blood stream of tissue fluid the mobilization of stored blood cells may occur, which would explain the appearance, soon after haemorrhage, of red cells with a reduced diameter (E. M. Semenskaya, 1946).

As a result of the simultaneous dilution of the blood, breakdown of red cells and emergence of stored red cells rich in haemoglobin, the fall of the red cell count and haemoglobin level is not correlated directly with the amount of blood lost (V. M. Rokitskii, 1899; N.Kh.Tolmachev, 1937;

73


G.A.Barashkov, 1953; and others). This would also explain the absence of an agreed theory as to the relationship between the fall in the red cell count and the haemoglobin concentration in the peripheral blood, and also the steps by which these indices are restored (N.M.Polonskii, 1947; M.A.Volin and S.N.Sorochkina, 1935; O.I.Komareva, 1946; and others). Restoration of the red cell count after moderate bloodloss begins on the 3rd-5th day. All the investigators note reticulocytosis at this stage, the curve often having an irregular character (P. D. Gorizontov, 1930; E. M. Semenslaya, 1946; N.G.Tsel'tner, 1939; M.I.Belen'kaya, 1952; and others). A.B.Kheifits and A.A.Poroshin (1937), studying qualitative changes of the reticulocytes after haemorrhage, found that, in the first few days, more mature forms enter the peripheral blood from the bone marrow reserve, while the number of young reticulocytes begins to increase later. The number of reticulocytes in the bone marrow also increases (A.B.Kheifits, 1939). All workers investigating the morphological composition of the bone marrow after haemorrhage (A. D.Ti-mofeyevskii, 1913; G.LAlekseyev, 1953; K.N.Klimova, 1954; I.I.Tarasov, 1953; M.I.Belen'kaya, 1952; and others) have observed hyperplasia of the erythroid tissue on the 2nd-3rd day. A little later (on the 3rd-6th day) definite myelocytic reaction began, accompanied by an increase in the number of young granulocytes.The increase in the number of haemocytoblast mitoses (I. S. Lindenbaum, 1929; E.I.Egorova, 1947) is evidence of an increase in the proliferative processes of the haemopoietic tissue.

The reaction of the spleen and lymph nodes to haemorrhage has been less well studied. Histological analysis has shown that loss of blood results in increased lymphopoiesis, and also in the formation of foci of extramedullary haemopoiesis (E.I.Egorova, 1947; O.A.Levin, 1932; A.A.Makarova et al., 1926). Lymphocytosis in the peripheral blood was noticed by one of the first early investigators (G. L. Antokonenko, 1893) and later confirmed (Yu. M. Ir-ger and R.Yu. Dobruskina, 1934).

The part played by iron and especially its inorganic salts in red blood regeneration has still not been completely explained, despite the considerable amount of work which has been done in this field. Earlier work revealed the beneficial effect of iron salts on blood regeneration after bloodloss against a background of artificially induced anaemia (Woltering, 1895; M.I.Il'yashev, 1901 ; Aporti, 1900; S.Tartakovskii, 1903, and others). In more recent studies with blood donors, administration of iron salts had no effect (D.I.Rafel'son and L.I.Ashkinazi, 1955; G. V. Tumanovskaya, 1943). However, there is considerable evidence demonstrating the importance of iron in the haemopoietic processes generally and in the regenerative processes in particular, including much work on iron metabolism in anaemias, including post-haemorrhagic anaemias.

Thus, bloodloss has a very marked effect on the whole haemopoietic process, changing the course of all its phases : breakdown and proliferation are increased, as are also maturation and release of mature cells. In addition, bloodloss leads to the development of compensatory phenomena in the haemopoietic tissue.

Haemopoiesis after Bloodloss in Rabbits 75

In view of all these changes particular attention is drawn to bloodloss as a means of detecting functional deficiencies of the haemopoietic organs.

In our experiments, venesection was carried out on three groups of animals

(1) control rabbits (9 animals); (2) rabbits which had received daily for 7 months 10 μο/kg 59Fe in the

form of 59FeCl3 (8 animals); (3) rabbits which had received stable iron (FeCl3) in the same quantity by

weight and for the same period as animals of the second group (11 animals). The composition of the peripheral blood before bloodloss in the control

rabbits and those receiving stable iron was approximately similar, the indices fluctuating within normal limits (haemoglobin 13-6 g per 100 ml, mean red cell diameter 6-29 μ, mean red cell volume 65 μ3, anisocytosis 1-3).

In rabbits receiving 59Fe (10 (xc/kg) just before bloodloss a higher haemoglobin level (14-5 g per 100 ml), greater mean diameter (6-7 μ) and volume (70 μ3), leptocytosis and anisocytosis (1-7) were observed. The average neutro-phil count was the same as in the control group. The number of lymphocytes was somewhat lower (5600) than in the control animals (6200).

The animals were bled 1 per cent of body weight from the marginal vein of the ear. The peripheral blood was examined for 2 successive days directly after bloodloss, and thereafter on the following 3, 4, 5, 10, 17 and 25 days. A bone marrow puncture from the ilium was carried out before bloodloss and also 3, 5 and 10 days after. A.P.Egorov's method was used to stain bone marrow reticulocytes.

R E D C E L L C H A N G E S

The first day after bloodloss the red cell count in the control group fell on average from 5-3 + 0-11 to 4-0 ± 0-16 millions per mm3 (by 25 per cent); haemoglobin, from 13-22 ± 0-29 to 10-62 ± 019 g per 100 ml (by 20 per cent) (Fig. 1) and the haematocrit from 37 per cent ± 1-24 per cent to 25 per cent ± 1-01 per cent (by 32 per cent). In addition, some increase occurred in the colour index from 0-74 + 0-02 to 0-81 + 0-02 and especially in the red cell haemoglobin saturation index—from0-70 ± 0-02 to 0-85 + 004(Fig.2). In view of the fall in mean red cell volume (in 7 of 8 rabbits) it can be supposed that on the first day following bloodloss smaller red cells more saturated with haemoglobin are still emerging from the storage depots.

On the second day the fall in the haemoglobin level was maximal, but at the same time the process of red blood regeneration began, as shown by the 100 per cent increase the reticulocyte count (from 26 to 52°/00) and the appearance of normoblasts in the peripheral blood) on average up to 100 cells per mm3 blood). Mean red cell diameter also began to increase, chiefly because of an increase in the percentage of polychromatophilic macrocytes (red cells of 8 and 9 μ diameter). The colour index and saturation index, although falling slightly, nevertheless remained above the initial level. By the 3rd-5th day the red blood regeneration was at its maximum: the number of all types TRS 6


of red cells in the bone marrow had increased (from 78,000 ± 10,000 to 147,000 ± 28,000 per mm3 aspirate on the 5th day), the percentage of red cell mitoses had risen from 0-8 + 0-1 to 13 ± 0-2 (or from 1-79 to 2-24 per 100 erythroblasts), and the number of reticulocytes of the bone marrow had increased (from 45 + 6 to 104 ± 12°/00) and of the peripheral blood (from 26 to 78°/oo, or from 140,000 to 350,000 per mm3, i.e. almost three times the original level) (Fig. 3). The haemoglobin level in the peripheral blood began to rise from the 3rd day; and the red cell count, from the 4th day after blood-loss. With the increase in the number of reticulocytes mean red cell diameter increased (from 6-35 ± 0Ό8 to 6*73 ± 0Ό7 μ), chiefly because of a rise in the percentage of macrocytes (from 4Ό + 1-2 to 17 ± 2) (Fig. 4). Probably connected with this was the increase in the group of red cells showing less osmotic fragility (at maximum on the 5th-7th day from 12 ± 2-5 to 28 ± 3-27 per

FIG. 1. Changes in haemoglobin level in the peripheral blood in rabbits after bloodloss (as a percentage of the initial level). 1—control; 2—administration of stable iron;

3—administration of radioactive iron (59FeCl3 Ì0μc|kg)

10 15-17 Days

25

FIG. 2. Changes in red cell haemoglobin saturation after bloodloss. 1—control; 2—administration of radioactive iron (59FeCl3 l(^c/kg)

cent). By the 10th day the total number of cells of the red series in the bone marrow and their number in mitosis had reached the original level. But despite the almost complete restoration of the red cell count and haemoglobin by this time (95 per cent of the initial level), the number of reticulocytes in the bone marrow (81 ± 10°/00) and in the peripheral blood was still increased. Clearly this is the reason for the mean red cell diameter, although considerably redu-


ced, still remaining above the initial figure. By the 15th-17th day the red cell count and the haemoglobin level had reached their original values. Mean red cell diameter, reticulocyte count and the number of cells having greater osmotic resistance were still somewhat above the initial values.

^350h £ Ì300 ^250

£ g 150 £§ioo

1

I I I I

10 Days

15 25

FIG. 3. Changes in the reticulocyte count after bloodloss. 1—control; 2—administration of stable iron; 3—administration of radioactive iron

(59FeCl3l(^c/kg)

In the group of rabbits receiving stable iron, the response to bloodloss was similar to that of the control group and occurred over the same time intervals. By the 10th day, 92 per cent of red cells and haemoglobin had been restored; by the 17th day 100 per cent. But in this group the appearance of smaller and more saturated red cells on the first day was not as clearly marked as in the control group. The index of red cell haemoglobin saturation was almost unchanged in this group. From the 1st to the 7th day after bloodloss the number of normoblasts observed in the peripheral blood of these animals

Days

FIG. 4. Incidence of macrocytosis after bloodloss. -control; 2—administration of stable iron ; 3—administration of radioactive iron

(59FeCl3 ΙΟμο/kg)

was considerably greater than in the controls (on average up to 700 cells per mm3). Hyperplasia of the red cell series of the bone marrow on the 5th day was somewhat more pronounced than in the control group (cf. the Table 1).

In the group of rabbits receiving 59Fe the initial fall in the red cell count, haemoglobin and haemotocrit was not substantially different from that observed in the control groups (cf. Fig. 1). As in the controls the colour index


TABLE 1. Changes in

Time of examination

Before bloodloss

5 th day after bloodloss

10th day after bloodloss

Group

Control Receiving stable iron Receiving 5 9Fe (ÌOμc|kg)

Control Receiving stable iron Receiving 5 9Fe (10 μο^ξ)

Control Receiving stable iron Receiving 5 9 Fe (10 μΰ/1<£)

No. of nucleated

cells per mm3

bone marrow aspirate

276 176 230

317 321 182

261 278 206

Total no. of erythroblasts

per 100 nucleated

cells

38-4 38-5 34-5

56-5 56-8 47-3

36-4 50 48-3

per mm3

aspirate (thousand)

78-0 100-0 68-0

147-0 181-0 89-0

108-0 138-0 95-5

and haemoglobin saturation index (cf. Fig. 2) increased during the first 2 days after bloodloss. Some reduction of mean red cell diameter without significant volume changes, i.e. similar to the controls, was observed, and more saturated red cells appeared, clearly having been stored earlier. But by comparison with the controls a more pronounced tendency to spherocytosis was noticeable. Thus, in the experimental animals the sphericity index was 2-3, whereas in the controls it was 3-8.

The chief differences in reaction to bloodloss in the experimental animals appeared later. Thus, the compensatory hyperplasia of the erythroid portion of the bone marrow, which began on the 3rd day in both control groups, was absent in the experimental group. The slight increase of the absolute number of erythroid bone marrow cells observed in these animals was not statistically significant.

The retarded and slow reaction of the erythroblasts of the bone marrow in animals receiving 59Fe was also manifested by a lower (by comparison with the controls) reticulocytic response both in the bone marrow and in the peripheral blood. The reticulocyte count rose more slowly and its maximum was lower (in the experimental animals—80 ± 12°/00 on the 10th day; in the controls—104 + 12°/00 on the 5th day). This is clearly shown when the reticulocyte count is expressed not per thousand but in absolute figures, i.e. per mm3 of blood (cf. Fig. 3).

Furthermore, in the animals of the experimental group, as in those receiving stable iron, a large number of normoblasts appeared in the peripheral blood on the 2nd day after bloodloss (on average up to 550 normoblasts per mm3).

By the 3rd day some increase in erythropoiesis was noticed. By this time the number of red cell mitoses in the bone marrow had increased from 0-7 ±0-2 to 1-7 ± 0-4 per cent (or from 1-98 to 3-54 per 100 erythroblasts), the reticulocyte count in the peripheral blood had increased (from 25 to 59°/0o),


Erythropoiesis after Bloodloss

Different erythroblast generations as percentages of total no. of erythroblast s

proery-throblasts

1-8 2-3 1-7

2 1 2 1 2-7

1-9 1-2 2-3

early normo-blasts

4-2 3-6 5-4

3-9 3-5 3-8

4-6 3-2 3-5

intermediate normoblasts

44-3 46-3 40-6

42-1 48-4 44-3

43-9 45-8 45-2

late normoblasts

49-7 47-8 52-3

51-9 46-0 49-2

49-6 49-8 49-0

Maturation index of erythro

blast protoplasm

0-063 0-063 0-08

0-064 0-059 0-067

0-071 0-046 0-061

No. of ery-throblasts in mitosis (per 100 erythro-blasts)

1-79 1-78 1-98

2-24 2-23 1-01

2-15 1-76 1-22

No. of reticulocytes (°/oo)

marrow aspirate

45 53 27

110 100 54

87 87 72

peripheral blood

26 24 25

78 68 39

70 58 47

No. of red cell s in peripheral

blood per mm3

5-3 5-6 5-4

4-2 4-7 4-0

5-0 5-1 4-7

and a tendency for the haemoglobin to increase had appeared (cf. Fig.l). But then, by the 5th day, the number of mitoses fell to the original level and the reticulocyte count in the peripheral blood and fallen by 20 per cent. The haemoglobin now reached its lowest level (10-7 ± 0-18 g per cent, 73 per cent of the original level). Complete restoration by the 15th—17th day, as in the controls, did not occur. The red cell haemoglobin saturation index on the 25th day remained lower (0-68 ± 0-009) than in the controls (0-75 + 0-031) (cf. Fig. 2). Mean red cell diameter increased in parallel with the retoculocyte count in the experimental animals as in the controls. The number of macro-cytes reached 21 ± 2 per cent (cf. Fig. 4). It was characteristic for this increase to be due not only to cells of 8 and 9 μ diameter but also to cells of 10 μ, which were absent in the control group.

The group of red cells more resistant to hypotonie NaCl solutions was increased from the 2nd to the 25th day after bloodloss, as in the controls.

In analysing post-haemorrhagic changes in erythropoiesis observed in our experiments we can detect two phases, taking as a basis the data referred to above. The first phase comprises a fall in the red cell count and haemoglobin level, connected with the loss of red cells, dilution of the blood by interstitial fluid and accelerated red cell destruction. The regeneration of the red cells has not yet reached maximum. The second phase begins with the increased regeneration of the red cells and haemoglobin and is directly dependent upon the activity of the bone marrow.

Analysing our data from this point of view we find that the primary changes after bloodloss, linked with dilution of the blood and red cell destruction are almost alike in the control and experimental groups.

Differences in the reaction to bloodloss of the animals receiving 59Fe appeared later, in the second phase, the development of which, as we have already indicated, is directly connected with the regenerative capacity of the bone marrow.


In the control animals bloodloss produces a marked increase in erythro-poiesis, a compensatory increase in the red cell series of the bone marrow, an increase of mitotic activity of erythroblastic cells and reticulocytosis in the bone marrow and in the peripheral blood.

In the animals receiving 59Fe the regenerative processes in the bone marrow were retarded and less vigorous than in the controls; this is shown by the absence of hyperplasia of the erythroid series of the bone marrow and less reticulocytosis. But despite this the restoration of the red cell count was achieved in a period similar to that of the controls. The haemoglobin level was restored more slowly.

Measurement of diameter, size and thickness of the red cells after bloodloss revealed a series of qualitative changes which might be a consequence of the impairment of normal red cell genesis by 59Fe. Thus, bloodloss led to the formation of very large red cells along with less reticulocytosis than in the controls. In addition, red cell saturation with haemoglobin throughout the whole observation period was lower after bloodloss than in the controls. This latter circumstance gives some grounds for believing that administration of 59Fe leads to some impairment of haemoglobin synthesis.

The administration of stable iron in the form of FeCl3 did not significantly alter the degree of red cell regeneration.

W H I T E C E L L C H A N G E S

During the first 4 days after bloodloss in the control animals an increase of the number of leucocytes in the peripheral blood was observed, on average from 10,000 ± 800 to 14,000 ± 1200 per mm3. This was chiefly due to an increase in the lymphocyte count (Fig. 5). Thus, during the first 2 days the lymphocyte count increased from 7500 ± 300 to 8300 ± 700 per mm3. In the following days it fell a little (on average to 5600 ± 810), while in some individuals temporary lymphopenia was observed on the 7th-10th day, when the lymphocyte number was less than 4000 per mm3.

In the rabbits receiving stable iron the lymphocyte count after bloodloss was not significantly changed, increasing only slightly on the 2nd-5th day.

In animals receiving 59Fe the number of lymphocytes increased only on the first day after bloodloss (from 6200 ± 500 to 7300 + 600 per mm3), and then fell considerably in most animals (to 3200-3400 per mm3). The greatest fall was observed on the 4th and 7th-10th days (cf. Fig. 5). After 15-25 days the lymphocyte count in these animals increased but still remained less than the initial level.

In the first few days after bloodloss atypical lymphocyte forms (up to 5-6 per cent) appeared in the peripheral blood of the experimental animals (with a nucleus drawn out into the shape of a rod or divided into several segments, with vacuolized nucleus or protoplasm; sometimes binuclear lymphocytes were found, and also lymphocytes with a markedly basophilic protoplasm). Such atypical cells were found extremely rarely, and in very


small numbers, in the control animals. It should also be noted that in the first 3 days an increase of the number of decaying lymphocytes (up to 15 per cent) was observed in the animals of all the groups.

Lymphocyte numbers in the bone marrow also varied. Thus, before blood-loss in rabbits receiving 59Fe the relative and absolute count of lymphocytes in the bone marrow was lower—19*3 per cent, or 21,000 per mm3 aspirate— than in the controls—29 per cent, or 70,000 per mm3 aspirate. After bloodloss in the control rabbits the relative and absolute count of lymphocytes fell somewhat on the 3rd and 5th days and increased on the 10th day almost to its initial level (23 per cent or 60,000 per mm3). In the animals receiving stable

E

I so I 7-0 § 6Ό o § 5-0 QJ

& 4-0 o

§ 3-0 ^ 1 2 3 4 5 6 7 8 9 10

Days FIG. 5. The absolute lymphocyte count after bloodloss.

1—control; 2—administration of radioactivity iron (59FeCl3 l(^c/kg) iron the absolute lymphocyte count increased from 26,000 to 45,000 per mm3

blood on the 5th day and remained at this level up to the 10th day. In rabbits receiving 59Fe the lymphocyte count fell somewhat on the 3rd-5th day and dropped sharply on the 10th day (10-2 per cent, or 11,000).

Changes in the number of neutrophils after bloodloss were also different in the different groups. Thus, in the controls only on the first day was some increase of the neutrophil count observed (on average from 3400 to 5000 per mm3 blood) without a leftwards shift of the neutrophil nuclear lobe count. In the following days the number of neutrophils was close to the original level. Apparently, the neutrophil leucocytosis of the first day is redistributive in character.

In rabbits receiving stable iron a slight, more or less even and relatively prolonged neutrophil leucocytosis was observed : the absolute neutrophil count increased from 3700 ± 460 to 5000 ± 620 per mm3 and was maintained at this level until the 10-15th day. The absolute number of cells of the neutro-philic series in the bone marrow was not significantly changed, but on the 3rd and 5th days their percentage fell because of the increase of the red cell series. The white cell maturation rate was also almost unchanged. In animals which received 59Fe before bloodloss, the curve of the absolute neutrophil count had two summits—on the lst-3rd day (from 3900 ± 660 to 6200 ± 700 per mm3) and slightly lower (up to 5200 per mm3) on the 7th-10th day. Posthaemorrhagic leucocytosis in these animals, beginning from the 2nd day,

- ^ \ A _ v.

Z I L_

V I 1

Λ ν

I

\ v^^

J 1

^

1

_ ! L_


was caused not by lymphocytosis as in the controls but by neutrophil leuco-cytosis. However, no signs of increased granulopoiesis were observed. We did not observe hyperplasia of the granulocytic series in the bone marrow in any of the groups, clearly because of the comparatively small (1 per cent by weight) amount of blood removed.

Thus, bloodloss also revealed in rabbits subjected to prolonged 59Fe administration certain changes in leucopoiesis, especially in lymphopoiesis. The profound and fairly prolonged lymphopenia which developed after bloodloss in these animals is evidence of impaired lymphopoiesis. In this way bloodloss confirmed our assumption of lymphopoietic deficiency, deduced from the short-lived lymphopenia observed in the majority of animals of the experimental group. Since no morphological changes in the lymphoid tissue were as yet apparent the impairments were obviously functional in character. The lymphoid tissue still retained its compensatory capacities, since the lymphopenia observed in the chronic experiment was transitory, while 15-20 days after bloodloss a slow increase of the lymphocyte count began.

Apparently, prolonged administration of 59Fe also damaged the mega-karyocytic system of the bone marrow. Thus, in the control animals on the 7th-8th day after bloodlos, an increase in the number of platelets was observed, on average from 200,000 to 450,000 per 1 mm3. In animals of the experimental group the number of platelets on the 10th day after bloodloss was lower than the original figure.


1. The decline of the red cell count and haemoglobin level in control and experimental animals receiving prolonged 59Fe administration was substantially the same.

2. In the experimental animals a retarded and less vigorous development of compensatory posthaemorrhagic reactions in the bone marrow was observed. However, the restoration of the red cell count in the peripheral blood was achieved in approximately the same periods as in the controls. The haemoglobin level was restored more slowly.

3. After bloodloss, impairment of the regenerative capacity of the erythro-blastic part of the bone marrow resulted in the formation of qualitatively changed red cells containing less haemoglobin in the experimental animals.

4. In animals receiving 59Fe impaired lymphopoiesis was observed after bloodloss as shown by the absence of pronounced posthaemorrhagic lymphocytosis and the development of absolute lymphopenia.

5. In the experimental animals the absence of thrombocytosis after bloodloss indicated changes in thrombocytopoiesis.

6. Administration of stable iron had no significant effect on the recovery of the peripheral blood after bloodloss.

Haemopoiesis after Bìoodìoss in Rabbits 83

REFERENCES

ALEKSEYEV G.I., Ter. arkh., 25 (5), 76-84 (1953). ANTOKONENKO G.L., Changes in the morphological composition of the blood and certain

changes of the tubular bone marrow following large-scale haemorrhage (Ob izmeneniyakh morfologicheskogo sostava krovi i nekotorykh izmeneniyakh kostnogo mozga trub-chatykh kostei pod vliyaniem bol'shikh krovopuskanii (eksperimentaPnye issledo-vaniya)). Dissertation, SPB (1893).

APORTI F., Über die Entstehung des Hämoglobins und der roten Blutkörperchen, Centralblatt für innere Medizin, vol. 2, p. 41 (1900).

B A R A S H K O V G . A . , The body's reaction to bloodloss and the diagnosis of internal haemorrhages (Reaktsiya organizma na krovopoteryu i diagnostika vnutrennikh krovo-techenii). Dissertation, Leningrad (1953).

BELEN'KAYA M.I. , Trudy Kievskogo inst. pereliv. krovi, 1, 122-124 (1952). BELOBORODOVAN.L. and B A R A N O V A E . F . , Toxicology of Radioactive Substances (Mate-

rialy po toksikologii radioaktivnykh veshchestv), vol. I, Moscow (1957). EGOROVA E.I. , Histomorphological reaction of the hypophysis and the thyroid gland to

bloodloss and regeneration in rabbits (Gistomorfologicheskaya reaktsiya gipofiza i shchitovidnoi zhelezy na krovopuskanie i regeneratsiyu krovi u krolikov). Dissertation, Ashkhabad (1947).

G O R I Z O N T O V P . D . , Vest, khir., 62-63, 9-15 (1930). GUSSEINOV G. A., The influence of the central nervous system on speed of blood restora

tion after massive bloodloss (Materialy k voprosu o vliyanii tsentral'noi nervnoi sistemy na skorost' vosstanovleniya krovi posle massivnogo krovopuskaniya). Dissertation, Moscow (1954).

IL'YASHEV M.I. , The effect of salts of various heavy metals on the morphological composition of the blood and haemoglobin formation (O vliyanii solei razlichnykh tyazhelykh metallov na morfologicheskii sostav krovi i obrazovanie gemoglobina). SPB (1901).

IRGER Y U . M . and DOBRUSKINA R . Y U . , SOV. vrach. gaz., 18, 1330-1335 (1934). KHEIFITS A.B., Collected Works of the Rostov District Blood Transfusion Research Institute,

1, 21-32 (1939). KHEIFITS A.B. and POROSHIN A.A., Klin, med., 15 (3), 399-405 (1937). KLIMOVA K. N. , Medullary haemopoiesis in donors after loss of different amounts of blood

(Kostnomozgovoe krovotvorenie u donorov posle vzyatiya razlichnykh doz krovi). Dissertation, Leningrad (1954).

KOVAL'SKII V. V. et al, Klin, med., 16 (8), 976-980 (1938). KOMAROVA O.I., The effect of X-rays on the morphological composition of the blood after

bloodloss (Vliyanie luchei Rentgena na morfologicheskii sostav krovi posle krovopoteri). Dissertation, Sverdlovsk (1946).

LEVIN O.A., Vest, khir., 26, 76-77, 96-104 (1932). LINDENBAUMI.S . , Vest, khir., 16-17 (48^9) , 323-330 (1929). MAKAROVA A.A. et al., Experimental anaemia from bloodloss (Eksperimental'naya ane-

miya ot krovopuskaniya). Transactions oflXth Congress of Therapeutists U.S.S.R., Moscow-Leningrad, pp. 304-323 (1926).

PETROV I.R., Acute Bloodloss and its Treatment with Blood Substitutes (Ostrava krovopo-terya i ee lechenie krovezameshchayushchimi sredstvami), Leningrad (1945).

POLONSKII N .M. , Byull. eksper. biol. i med., 24, 1, No.7, 63-67 (1947). RAPPOPORT M . Y U . , Bloodletting (Krovopuskanie), Moscow (1948). RAFAL'SON D. I . and ASHIKNAZI 1.1., Current problems of blood transfusion (AktuaVnye vo-

prosy perelivaniya krovi), 4, 9 (1955). R O K I T S K I I V . M . , Blood changes after profuse bloodloss (K voprosu ob izmenenii krovi

posle obiPnogo krovopuskaniya). Dissertation, SPB (1899). SEMENSKAYAE.M., Anisocytosis in anaemic conditions (Anizotsitoz pri anemicheskihk

sostoyaniyakh). Dissertation, Moscow (1946).


TARASOVI. I . , Effect of bloodloss and blood substitute fluid on the activity of the bone marrow in dogs (Vliyanie krovopuskaniya i krovezameshchayushchei zhidkosti na funtional'nuyu deyatel'nost' kostnogo mozga u sobak). Author's abstract of Dissertation, Saratov (1953).

TARTAKOVSKII S.O., Absorption and Assimilation of Iron (O vsasyvanii i usvoenii zheleza), Kiev (1903).

TIMOFEEVSKII A.D. , The Morphology of the Bone Marrow in Anaemias (Morfologiya kostnogo mozga pri anemiyakh), Tomsk (1913).

TOLMACHEV N . K H . , Prob, endokrin., 2 (1), 35-46 (1937). T S E L ' T N E R N . G . , Vrach. deh, 6, 399 (1939). TUMANOVSKAYA G. V., Blood regeneration in donors after intake of iron preparations

(Regeneratsiya krovi u donorov posle priema preparatov zheleza), Transactions oflzhev State Medical Institute, 6, 129 (1943).

UZHANSKII Y A . G . , Vrach. deh, 23-24, 1049-1056 (1932). UZHANSKII Y A . G . , Uspekh. sov. biol, 22 (1), 61-73 (1946). VOLIN M. A. and SOROCHKINA S.N., Ter. arkh., 13 (5), 77-84 (1935). WOLTERINGH. , Zschr. fürphysiol, Chemie, 21 (2-3), 186 (1895).

THE EFFECT OF PREGNANCY AND PARTURITION ON HAEMOPOIESIS

IN RABBITS DURING PROLONGED 59Fe ADMINISTRATION

N. L. B E L O B O R O D O V A , V. L. P O N O M A R E V A and E. K. R E D ' K I N A

THE STUDY of haemopoiesis during pregnancy and parturition in animals subjected to prolonged exposure to radioactive isotopes is of particular interest since in this physiological state haemopoiesis is achieved under difficult conditions of increasing iron deficiency and impairment of the endocrine equilibrium. It has been shown more than once in the work of our laboratory that pregnancy and parturition may reveal a concealed deficiency of the haemopoietic organs in experimental animals subjected, over prolonged periods, to the action of small amounts of radioactive isotopes (N. L. Beloborodova and E. F. Baranova, 1957; N. L. Beloborodova and E. K. Red'kina, 1960). The existing literature on haemopoietic changes during normal pregnancy and in the post-partum period consists mainly of the results of clinical observations. There are very few reports concerning laboratory animals.

Changes in leucopoiesis connected with pregnancy are not well documented and the data of different writers are contradictory (A. K.Yanichenko, 1927; T.N.Ankudinova, 1958; R.P.Milyashkevich, 1931; O.D.Boldyreva, 1948; M.A.Daniakhii, 1937; E.M. Liozina, 1952; and others). More clear-cut deviations from normal during pregnancy are observed in erythropoiesis. Thus, M.A.Daniakhii (1937, 1938), N.N.Myasnikov (1937), E.M.Liozina (1925), L.E.Gurtovoi (1956), T.M.Gurovskaya (1955) and others report a marked reduction of haemoglobin and red cells towards the end of pregnancy, accompanied by proliferation of the erythroblastic part of the bone marrow. With increasing duration of pregnancy an increase in the number of early normoblasts is observed (E.M.Liozina, 1952; O.D.Boldyreva, 1948; M.A.Daniakhii, 1938). The hyperplasia of the red cell series of the bone marrow is considered to be ineffective by the majority of authors since, by the end of pregnancy, the number of reticulocytes in the peripheral blood does not exceed normal or is even a little below, and less mature forms and even normoblasts appear. The number of mitoses in the marrow is increased significantly. This ineffective hyperplasia is a result of impairment of the maturation of cellular elements, produced by iron deficiency resulting from the increased utilization of the latter during pregnancy. Therefore young baso-philic red cells, unsaturated with haemoglobin, accumulate in the bone mar-

85


row and delay the final process—the release of mature red cells. E. M.Liozina (1952), N. N. Myasnikov (1957) and others report on iron-deficiency anaemia during pregnancy. It is well known that the administration of iron preparations improves this type of anaemia.

We have studied haemopoiesis during pregnancy in rabbits following prolonged oral administration of relatively small amounts of 59Fe. Two groups of female rabbits of the same age were selected for the experiment : 8 rabbits had received daily 10 μΰ/kg 59FeCl3 for 10 months prior to onset of pregnancy and 5 rabbits were used as controls. Both the control and experimental rabbits were inseminated by control males. All the control and 7 of the experimental females gave birth at term (30-31-day pregnancy). The number of offspring and their weight was substantially the same in both groups (average 5 offspring weighing 34-36 g). In one experimental female, pregnancy terminated prematurely and was followed by chronic anaemia of the same type as that which occurred post-partum in animals of this group.

The condition of the peripheral blood was studied at intervals throughout pregnancy; afterwards daily for 5 days and thereafter on the 7th, 10th, 15th, 20th and 25th days. In order to examine the morphological composition of the bone marrow a narrow puncture of the ilium was carried out before pregnancy, and on the 10-14th and 20-24th day of pregnancy and 1, 3, 5, 10 days after parturition. The morphological composition of the aspirate was studied in smears stained May-Grunwald-Romanovskii ; the number of marrow reticulocytes was counted.

T H E P E R I P H E R A L B L O O D AND BONE M A R R O W P I C T U R E D U R I N G PREGNANCY

At onset of pregnancy, i.e. 10 months after commencement of 59Fe administration, qualitative changes of the red cells in rabbits of the experimental group were less marked than during the first months of 59Fe administration. Mean red cell volume and diameter, increased at the beginning of the prolonged experiment, had again declined, although the latter along with anisocytosis and leptocytosis remained greater than in the control animals. With the reduction of mean red cell size the total haemoglobin level was also somewhat reduced, although the haemoglobin saturation coefficient of each red cell remained the same.

As far as the white cells are concerned, in the first months of 59Fe administration, absolute lymphopenia arose periodically in the experimental animals and in addition the average absolute lymphocyte count fell gradually in the whole group. Prior to pregnancy, i.e. after 10 months of daily 59Fe administration, it was 4700 ± 500 per mm3 blood. In the control animals at this time there were 5500 + 600 lymphocytes per mm3. The absolute neutro-phil number in the peripheral blood was similar in both groups.

The morphological composition of the bone marrow and the number of reticulocytes in marrow aspirates were substantially similar in the two groups.

Effect of Pregnancy and Parturition on Haemopoiesis in Rabbits 87

HAEMOPOIESIS D U R I N G P R E G N A N C Y AND AFTER P A R T U R I T I O N

Erythropoiesis. All changes in erythropoiesis observed in the control rabbits during pregnancy may be explained, in agreement with the literature, by the gradual onset of iron deficiency. The first sign of erythropoietic impairment was an increase of the number of microcytes (diameter less than 6 μ), beginning from the 10—12th day of pregnancy. An increase of the number of basophilic erythroblasts at this time and of the erythroblast maturation index

%

100

90

1 so 1 70

60

1

I I I I 1 U J 1 1 1 1 1 1 1 1 L 9-11 14-16 19-21 24-26

Ooys of pregnancy 30l2 4 6 8 10 12141618 20

Days post-partum

FIG. 1. Changes in the red cell count (as percentages of the initial count) in rabbits during and after pregnancy.

1—control rabbits; 2—rabbits after administration of 59FeCl3 (l(^c/kg)

were not statistically significant; no hyperplasia of the red cell series was observed. The number of reticulocytes in the peripheral blood increased up to the 19-20th day of pregnancy (up to 56°/00 on average).

In the second half of pregnancy signs of iron deficiency began to appear and the number of basophilic erythroblasts increased somewhat. The total number of cells of the erythroblastic series reached 52-9 ±2-1 per cent of all nucleated cells of the bone marrow. The number of mitoses of erythroblastic cells was materially unchanged during pregnancy. In the last days of pregnancy the number of reticulocytes in the peripheral blood began to decline (to 1 l°/oo on the 29th day) and the number of microcytes was maximal (from 8Ό ± 2-7 to 19 ± 3-4 per cent). A slight fall in the number of red cells (not more than by 700,000 per mm3 blood) was observed in some control rabbits only during the last days of pregnancy (Fig. 1).

The haemoglobin level during pregnancy changed in parallel with the red cell count and so the colour index was almost unchanged (Fig. 2).

Certain qualitative changes of the red cells could also be observed, namely a tendency to spherocytosis beginning from the 10-12th day of pregnancy. By the 19-22nd day the sphericity index had fallen from 2-94 ± 0-14 to 2-66


± 0Ό6 (Fig. 3). Clearly in parallel with this, at the same period the group of red cells least resistant to hypotonie solutions of sodium chloride increased somewhat. This is in agreement with results in the literature that red cell fragility increases during pregnancy (N. M. Nikolayev, 1936). On the first day after parturition, inhibition of erythropoiesis was observed in the control rabbits. Acute reticulocytopenia arose—the number of reticulocytes in the

0-90 X

*§ 0-80

| 070 o

U 0-60

1

- Γ < Ξ Γ Ξ ^

s- " ■ " " "

I

v—

1 1 1 1 1 1 1

2

1

I I l

9-11 14-16 19-21 24-26 Days of pregnancy

30 2 4 6 8 10 12 141618 20 i c Days post-partum

FIG. 2. Changes in the colour index in rabbits during and after pregnancy. 1—control rabbits; 2—rabbits after administration of 59FeCl3 (l(^c/kg)

9-11 14-16 19-21 24-26 Days of pregnancy

30l2 4 6 8 10 1214 1618 20 Days post parfum

FIG. 3. Changes in the red cell sphericity index in rabbits during and after pregnancy.

1—control rabbits; 2—rabbits after administration of 59FeCl3 (l(^c/kg)

bone marrow fell to 10-3 + 2°/00 · On the 5th day the reticulocyte count had returned to normal. The number of red cells remained depressed only for 1 day after parturition (to 4,300,000 on average).

A slow increase in the red cell count had begun on the 2nd day and the return to the initial level was completed between the 10th and 15th post-partum days. The haemoglobin level lagged behind and restoration was not completed until the 20th day. A slight decline of the colour index was observed (Fig. 2) and of the red cell haemoglobin saturation index connected, apparently, with the slow recovery of the body's normal iron balance. The increased numbers of microcytes were present up to the 20th day after par-


turition. In the post-partum period a tendency to spherocytosis was noted and an increased number of red cells with increased osmotic fragility remained. In addition and parallel with the increased numbers of reticulocytes (from 26 ± 2-7 to 62 ± 7-8°/00? reaching maximum on the 10th day after parturition), the number of more resistant red cells also increased (on average from 14 to 29 per cent). The number of highly resistant red cells remained increased up to the 20th day after parturition.

Changes in erythropoiesis during pregnancy in rabbits of the experimental group were as follows.

In the first half of pregnancy, as in the control females, retarded maturation of the erythroblast protoplasm was absent. But already pronounced relative hyperplasia of the red cell series of the bone marrow was observed (the number of erythroblastic cells increased from 34-8 + 1-3 to 43*5 ± 3*9per cent).

In the second half of pregnancy retarded maturation of erythroblast protoplasm was more pronounced than in the control females (the maturation index increased from 0045 ± 0005 to 0065 ± 0005). The number of erythroblastic cells in the bone marrow rose to 54 + 3-9 per cent.

Changes in the red cell count in the experimental rabbits during pregnancy differed substantially from what was observed in the controls (Fig. 1). Thus, already by the 19-20th day of pregnancy the red cell count had fallen from 5,400,000 ± 870,000 to 4,700,000 ± 150,000 per mm3, i.e. on average by 800,000 per mm3. In the last few days of pregnancy the mean fall was 1,000,000 per mm3. At the same time the colour index increased markedly.

Taking into account the greater increase of red cell sphericity in comparison with the controls (the sphericity index fell on the 24-26th day of pregnancy from 3-3 ± 0-44 to 2-5 + 0-16) it may be suggested that accelerated red cell breakdown played some part in the development of anaemia during pregnancy in the experimental animals (Fig. 3).

On the first day following parturition, haemopoiesis in the experimental animals was, according to the myelogram, similar to that of the control animals. However, the red cell count was restored more slowly. Thus, having fallen on the first day after parturition by 1,400,000 per mm3 it continued to fall on the 2nd and 3rd days. The maximal fall of the red cell count was 1,800,000 per mm3 i.e. it was 2\ times greater than in the control animals (in which the maximal fall was 700,000mm3 on the first day following parturition). Characteristic of the anaemia which developed in the experimental animals was the appearance of red cells of large volume (mean volume increased from 64 ± 1-3 to 83 + 4-4 μ3) and greater sphericity (spherical index on average 2-5). Correspondingly, there was a considerable increase of the colour index (from 0-76 ± 0-026 to 1-0 ± 0072) (Fig.2). Clearly the increased red cell sphericity explains the increased number of less resistant red cells in relation to hypotonie sodium chloride solutions (from 7-0 + 2-3 to 26 + 7-4 per cent). It is possible that the restoration of the red cell count was also impeded by the accelerated breakdown of the pathological cells described above. The


return to normal of the peripheral blood count had not been completed in the experimental rabbits even by the 20th day after parturition.

Thus, in the experimental rabbits the impaired erythropoiesis during pregnancy and after parturition was twofold. Firstly, it is due to iron deficiency and manifested microcytosis, reticulocytopenia, retarded red cell maturation in the bone marrow and so on. These changes were observed in the control animals. The relative hyperplasia of the red cell series of the bone marrow which developed in the experimental animals in the first half of pregnancy, i.e. before the appearance of serious signs of iron deficiency, was clearly compensatory in character.

Secondly, the impaired erythropoiesis is due to the prolonged effect of 59Fe on the marrow cells and manifested by prolonged anaemia in the rabbits of the experimental group during pregnancy and after parturition the anaemia was associated with increased colour index due to the presence of macrocytes and spherocytes, the latter showing increased haemolysis, in the osmotic fragility tests.

Neutrophil leucopoiesis. In the control rabbits no significant changes in neutrophil maturation in the bone marrow or changes in the absolute and relative numbers of neutrophils in the peripheral blood were observed. However, the neutrophil nuclear lobe count shifted gradually to the right during the second half of pregnancy. On the first day following parturition the absolute neutrophil count increased and a variable neutrophilic leucocytosis followed. This leucocytosis evidently was redistributive in character since a pronounced left shift of the neutrophil nuclear lobe count was not observed. The right shift of the neutrophil lobe count, developing during pregnancy, gradually declined after parturition. Qualitative changes of neutrophils in the form of increased nuclear fragmentation were observed only on the first days after parturition (on average up to 2-3 per cent).

In the experimental animals neutrophil leucocytosis with a shift to the right in the lobe count occurred between the 12th and 20th days of pregnancy (Fig. 4). Nuclear fragmentation of peripheral blood neutrophils was observed more frequently than in the controls, and began during the first half of pregnancy. By the 20-25th day of pregnancy the mean number of neutrophils with changed nuclei reached 11-5 per cent (range 6 to 19 per cent) and remained at a high level (average 6-5-5 per cent) for 10 days after parturition. A small number of neutrophils with nuclear chromatin breakdown was observed.

Maximum neutrophil leucocytosis (average 6700 mm3), accompanied by a right shift in the lobe count was found on the first day after parturition. On subsequent days the number of neutrophils in the blood declined but the number of cells with a low lobe count increased (Fig. 4).

Thus, changes in neutropoiesis in the experimental animals differed somewhat from those of the control group. The prolonged left shift of the neutrophil lobe count after parturition suggests an impairment of the processes of release of neutrophils from the bone marrow, since no significant changes of maturation processes in the bone marrow could be detected.


Lymphopoiesis. A gradual decline of the absolute lymphocyte number was observed throughout pregnancy in the control rabbits (from 5600 + 600 before pregnancy to 4100 + 200 per mm3 on the 26-30th day of pregnancy). After parturition, their number again increased (Fig. 5).

024

0·20

0-16

0-12

0-08

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-STO

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/ 0 \ 1b />-* N^

1 1_ 9-11 14-16 19-21 24-26

Days of pregnancy 30 3 5 7 10 15 20 ^ c Oays post-parfum

F re 4. Changes in the absolute count and maturation index of neutrophils in rabbits during and after pregnancy.

Control: la—absolute neutrophil number; lb—coefficient of neutrophil nuclear shift. Administration of l(^c/kg 59Fe: 2a—absolute neutrophil number; 2b—co

efficient of neutrophil nuclear shift

Ì6-0 l _

21 B 5 - 0 a 3 4-0 £ S 3-0 o

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1 L_i_] 1 1 1 L

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_J 1 U-J L 9-11 14-16 19-21 24-26 29-30 2 4 6 8 10 12 1416 10 20

Days of pregnancy . Days post-partum

FIG. 5. Changes in the absolute lymphocyte count in rabbits during and after pregnancy.

1—control; 2—after administration of 59Fe (lC^c/kg)

In all the animals of the experimental group persistent absolute lympho-penia developed during the second half of pregnancy (2500 ± 200 per mm3). After parturition lymphopenia persisted in these animals for 10 days and only after 15 days did the lymphocyte count return to its initial level. In the control females, qualitative changes of lymphocytes were very slight and were only observed on the first days following parturition (appearance of TRS 7


mature lymphocytes with intensely basophilic cytoplasm—up to 1 per cent). In the experimental rabbits these changes were much more marked. During pregnancy 3-4 per cent of lymphocytes with basophilic cytoplasm were observed and these were also found, but in smaller numbers, during the recovery period. Lymphocytes with nuclear fragmentation were found in almost all the experimental rabbits after parturition.


1. In the control rabbits pregnancy produced marked changes in haemopoiesis, which returned to normal relatively quickly after parturition. The most significant changes were in erythropoiesis, and were associated with iron deficiency (anaemia, microcytosis, reticulocytopenia etc.). The fall in the absolute lymphocyte count to the lower limits of normal which was observed at this time was replaced after parturition by absolute lymphocytosis.

2. Functional deficiencies of the haemopoietic organs of the experimental animals were manifested during pregnancy and after parturition in more profound, and sometimes qualitatively different changes from those of the control. Thus, anaemia during pregnancy occurred earlier, and became more severe for several days after parturition, and was accompanied by pathological changes in the red cells. The absolute lymphocyte count during pregnancy declined and a pronounced lymphopenia developed. Post-partum lymphocytosis was not observed.

REFERENCES

ANKUDINOVA T.N., Akush. i ginek., 1, 42-44 (1958). BELOBORODOVA N. L. and BARANO VA E. F., The Toxicology of Radioactive Substances (Ma

terial)? po toksikologii radioaktivnykh veshchestv), vol. 1, Moscow (1957). BELOBORODOVA N.L. and RED'KINA E.K., The Effect of Ionizing Radiation on Pregnancy,

Foetal Condition and Newborn ( Vliyanie ioniziruyushchei radiatsii na techenie beremen-nosti, sostoyanie ploda i novorozhdennogo), Leningrad (1960).

BOLDYREVA O.D., The myelogram of iliac bone marrow puncture in women during pregnancy and haemorrhage (Mielogramma podvzdoshnogo punktata kostnogo mozga u zhenshchin pri fiziologicheskom techenii beremennosti i krovopoteryakh). Dissertation, Stavropol' (1948).

BOLDYREVA O.D., Akush. i ginek., 2, 20-24 (1949). DANIAKHII M.A., Akush. i ginek., 1, 55-63 (1937). DANIAKHII M. A., Pregnancy and Haemopoiesis (Beremennosf i krovotvorenie), pp. 73-87,

Tashkent (1938). GUROVSKAYA T.M., Akush. i ginek., 4, 26-30 (1955). G U R T O V O I L . E . , Akush. iginek., 5, 23-25 (1956). LIOZINA E. M., Normal and Pathological Haemopoiesis during Pregnancy (Krovotvorenie pri

beremennosti v norme i patologa), Kiev (1952). MILYASHKEVICHR.P. , Akush. iginek., 3, 236-242 (1931). M Y A S N I K O V N . N . , Akush. i ginek., 1, 44-48 (1957). N I K O L A E V N . M . , Akush. iginek., 11, 1275-1281 (1936). YANICHENKO A.K., Changes in the leucocytic formula connected with pregnancy (Izme-

nenie leikotsitarnoi formuly v svyazi s beremennost' yu). Proceedings of VHth All-Union Congress of Obstetricians and Gynaecologists, pp. 705-727, Leningrad (1927).

PERIPHERAL BLOOD CHANGES IN WHITE RATS FOLLOWING INTRATRACHEAL INJECTION

OF VARIOUS 59Fe COMPOUNDS


IN EXPERIMENTS on distribution of different 59Fe compounds in animals after intratracheal injection, results have been obtained indicating prolonged retention of the substance in the lungs and a peculiar distribution in other organs and tissues. Consequently, certain changes in the lungs and throughout the body might be expected. Because of the selective accumulation of 5 9Fein the bone marrow, which is one of the most radio-sensitive organs, it was felt that, in order to characterize the radiotoxicological effects of 59Fe, a study of haematological changes should be undertaken.

The literature dealing with haemopoietic reactions to internal radioactive substances is scanty. The most fully studied aspect of this problem has been the reaction of the haemopoietic system to internal administration of radioactive substances in amounts producing acute and subacute radiation injury. The work of A. P. Egorov, I. K. Petrovich, G. N. Teplinskaya, L. N. Burykina, M. I. Nemenov and others have shown that this reaction is basically similar to the reaction as a result of external radiation. The changes in the haemopoietic system may vary because of the different physico-chemical properties of the various compounds and the peculiarities of their behaviour in the body. These differences are more pronounced in chronic forms of radiation sickness, produced by prolonged internal administration of different radioactive substances in doses close to the maximum permissible (N. L. Beloborodova, E. F. Baranova, G. N. Teplinskaya and others). A connection between the haemotological changes, the mode of entry into the body and character of administration can be detected.

In the work of R. E. Kavetskii, L.B.Stolyarova and R.D.Nikitenko there are indications that following a single intravenous injection of trace amounts of 59Fe in rabbits (7-5-3-75 μc per animal) marked changes in the leucocytes are observed even in the first few hours. After 4 hr the authors detected a reduction in the leucocyte count caused by a fall in the numbers of lymphocytes. During the next 2 days the development of a neutrophil leucocytosis was observed. By the 8th day a gradual recovery occurred although even after 10 days a certain increase over the original level could be detected. We have found no references in the literature which has reached us dealing with

93


the prolonged effect of 59Fe on the blood system. Furthermore, there are no reports on the effects on haemopoiesis of radioactive substances entering through the respiratory tract and retained for long periods in the lungs.

A study was made of changes in peripheral blood of 54 white rats after intratracheal injection of soluble and insoluble 59Fe compounds. A further 16 animals were used as controls.

Every month for 15 months the haemoglobin level, red cell and reticulocyte counts and total and differential counts were determined. The cytological changes which occurred were noted.

TABLE 1. Morphological Composition of Rat Blood before 59Fe Injection

Content of formed elements

in the blood

Mean Maximum Minimum

fi

<U o

Ό Ξ tf 5

7 8-8 5-86

e ο ο ω

"s ε O M

87 98 81

''ο ο t/5 <υ >. υ

υ

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e« α

§i 3 2

H4 £ ,

12-5 16-8 6-8

6 S

5 g,

6 2

9-5 13-7 5-6

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0-3 0-5 0-2

S ε α

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8 | 00 ϋ

0-1 0-2 0

ε ε α>

1 s O A

0-4 0-9 0-1

Neutrophils (thousand per mm3)

unseg-mental

0-15 0-4 0-1

segmented

2-5 4-6 1-1

total

2-65 5 1-2

TABLE 2. Experimental Scheme

No. of group

1 2 3 4 5

Number of animals in group

9 10 10 10 15

Substance injected

5 9 Fe citrate 5 9 Fe oxide 5 9 Fe oxide 5 9 Fe oxide 5 9Fe oxide

Activity

20 27-5

3-36 1-06 0-03

three times

The data in the literature on the morphological composition of the blood of healthy rats shows great variation, possibly due to differences in the age of the animal and to seasonal variations of particular indices, and also to different methods of analysis. With this in mind, we tried to determine the normal morphological composition of the blood in the animals used in the experiment. For this purpose the blood of all animals was examined two or three times prior to administration of 59Fe. The results we obtained, presented in Table 1, differ somewhat from those found in the literature, but we felt justified in taking our figures as the normal for this group of rats.

The lymphocytes comprised 75 per cent and the neutrophils 21 per cent of the total leucocytes. The red cell count varied between 5,380,000 and 8,040,000 per mm3 (average 6,800,000), and the haemoglobin from 77 to 91 per cent (average 85 per cent).

Peripheral Blood Changes in White Rats 95

Prolonged observation of the control animals (which also had monthly blood counts) showed that, with increase in age, some increase in the neutro-phil count occurs, on average up to 3500-4000 per mm3, with a relatively unchanged lymphocyte count. During the 15-month experimental period the total number of leucocytes varied between 6700 and 18,200 per mm3 neutro-phils 1100 and 6100 per mm3 and lymphocytes—5300 and 13,700 per mm3.

For intratracheal injection, 59Fe citrate was used as a soluble compound and a finely dispersed powder of 59Fe oxide as an insoluble compound. The experimental scheme is presented in Table 2.

C H A N G E S IN THE P E R I P H E R A L B L O O D OF R A T S AFTER I N T R A T R A C H E A L I N J E C T I O N

OF 59Fe C I T R A T E

The red cell count in the experimental animals varied from 6,600,000 to 8,900,000 per mm3, i.e. it remained within normal limits. The haemoglobin varied from 84 to 99 per cent (14—16-6 g per cent), which is also within normal limits, observed in the control group. However, it should be noted that in the animals receiving 59Fe, beginning from the 4th-6th months, the red cell count and haemoglobin level increased somewhat relative to the values

S 0 1 2 3 4 5 6 7 8 9 10 11 1213 % 15 Months

FIG. 1. Fluctuations in the leucocyte count in animals which had received 59Fe citrate and control animals.

1—control animals; 2 and 3—experimental animals

observed in the controls, although remaining within the upper limit of normal. At the same time a small decline of the colour index to 0*56-0-54 (control 0-6) was observed.

The results obtained are concommitant with a slight stimulation of erythropoiesis, an assumption supported by a rise in the reticulocyte count to35-37°/0 0.

The earliest leucocyte changes, in animals receiving 59Fe citrate, consisted of sharp oscillations of the total count, primarily upwards. The range of fluctuation was considerably wider than in the control group, and in certain


TABLE 3. Number of Leucocytes in the Peripheral Blood (thousand per mm3)

No. of

animal

90 91 92 93 94 95 96 97 98

Number of leuco

cytes before injection of 5 9Fe citrate

12-9 14-9 13-6 15-2 10-1 10-6 14 11-2 9-8

Number of leucocytes

months

1

21-2 15-9 9-7 9-2

17-7 13-1 12-1 16-3 15-7

2

22-4 13-9 12-7 19-9 8-6

22-1 19 8-5

14-5

3

19-9 20-1 18-5 16-1 23-7 10-9 20-7 13-1 8-4

4

18-9 11-7 13-5 15-3 18-2 16-5 12-3 12-4 19-9

5

11-6 18-1 11-4 28-2 16-3 8-9

17 6-1

17-4

6

15 12-4 6-5

16-8 12-8 18-3 18-7 11-2 16-4

7

13-6 12-1 14-2 20-1 25-1 11-6 13-5 14-1 13-8

cases a leucocytosis of up to 25,000-28,000 per mm3 was observed (Fig. 1) The times of onset of leucocytosis varied between animals, which once again underlines the dissimilar sensitivity of animals to ionizing radiation. Leucocyte changes in animals of this group are shown in Table 3.

As can be seen from our results, the leucopenia which is so characteristic of pronounced radiation injury, was not observed. Only in a few animals was

E E

26 24 22 20 18 16 14 12 10 8 6 4 (

/ \ / \ 1 \ / \ / x / \ Λ

-Λ / %/ \ Λ / \ h\ \ f\ V \J ^

I 1 I . 1 1 1 1 I 1 1 1 1 1 1

3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Months

FIG. 2. Fluctuations in the total leucocyte and lymphocyte counts after injection of 59Fe citrate.

1—total leucocytes; 2—lymphocytes

a temporary fall in the leucocyte count to 6000 per mm3 observed. On the other hand leucocytosis above 20,000 was a comparatively frequent occurrence and was observed in 6 of 9 animals.

Comparing the total leucocyte and lymphocyte counts shown in Fig. 2, it can be seen that in the majority of animals, especially in the first 6-8 months after injection, the increase in the total leucocyte count was due to an absolute and relative lymphocytosis.


of Rats after Intratracheal Injection of59Fe Citrate

after 59Fe citrate injection

8

17-4 21-3 17-8 17-6 17-3 15-3 14-2 9-3

18-8

9

17 16-4 15-9 13-8 11-2 6-2

15-7 16*2 12-8

10

20-3 12-6

16-8 14-3 18-3 15-1 21-4

11

12-3 9-5

9-2 14-1 10-1 16-6 15-3

12

17-8 10-5

12-5 16-2 12-6 12-5 17-4

13

16-2 16-1

18-2

17-3 16-3

14

14-5 15-3

13-3

17-6 12-1

15

17-3 12-3

12-8

14-3 11-8

The fluctuation of the lymphocyte count in the experimental group was greater than in the control. Whereas in the control group the number of lymphocytes varied from 5300 to 13,700 per mm3 in the experimental group the variations were from 4500 to 15,200 per mm3 blood.

The neutrophil count after administration of 59Fe citrate tended to rise. A not infrequent occurrence at all stages of the experiment was an absolute neutrophil leucocytosis up to 7000-11,000 per mm3. Neutrophil leucocytosis above 6000 per mm3 was observed in 7 of 9 animals and in 6 of these more than once. Pronounced neutropenia was not observed.

The number of eosinophils varied from 200 to 1400 per mm3, which exceeded the upper limit of variation in the control group.

In 7 animals, beginning from the 4th-6th months, an increased number of cytological changes in the formed blood elements was observed, expressed by fragmentation, hypersegmentation and pyknosis of neutrophil nuclei and lymphocyte cytolysis. It should be observed that even when a neutrophil leucocytosis was present, no increase was detected in the number of un-segmental and juvenile forms. The presence of hypersegmented forms indicates accelerated neutrophil maturation, one of the early manifestations of radiation injury of the blood.

C H A N G E S IN THE P E R I P H E R A L B L O O D AFTER A S INGLE I N J E C T I O N OF 59Fe O X I D E

In animals which had received a single injection of 59Fe oxide of either 27-5, 3-36 or 1-06 μο, changes in the blood picture were similar in type, revealing no qualitative differences associated with the magnitude of the dose received, thus enabling presentation of the results obtained in this part of the experiment without reference to the amount of radioactivity administered.


In the experimental animals during the observation period, no cases of anaemia, characteristic of severe forms of radiation sickness, were detected. On the other hand, in a number of animals beginning from the 4th-6th months, an increase of the red cell count to 9,000,000-9,800,000 per mm3 was observed, which exceeded the maximum number found in the controls (8,800,000). The haemoglobin level fluctuated from 78 to 106 per cent, also exceeding the range of variation in the control group. At the same time a reduction in the colour index to 0-52-0-55 was observed, but this is still within the normal limits.

No constant changes were found in the number of reticulocytes. These varied between 14 and 440/00, which is slightly above the normal range.

The deviations from normal of the red blood indices mentioned above were inconstant and were not observed in all animals of the group. Thus, of the 30 animals, an increase of the red cell count above 8,800,000 per mm3 was observed in 16, haemoglobin level above 98 per cent in 11, and reticulocytosis above 35°/00 in 7 animals.

The most frequent change was an increase of the red cell count, which was observed in 53 per cent of the animals.

Animals which received 59Fe oxide displayed sharp fluctuations of the number of leucocytes, chiefly upwards. The range was from 6200 to 29,000 per mm3.

Leucocytosis in the experimental animals was one of the most frequent peripheral blood changes, as can be seen in Table 4.

TABLE 4. Frequency of Raised Leucocyte and Red Cell Counts in the Experimental Animals

Number of animals in group

30

Number of animals in which leucocytosis above 20,000 per mm3

was detected

on one occasion

8

two occasions

11

three or more occasions

5

total

24

Number of animals in which red cells above 8,800,000 per mm3

were detected

on one occasion

6

two occasions

8

three or more occasions

2

total

16

As Table 4 shows, leucocytosis was observed in 80 per cent of animals. The leucocytosis occurred at variable intervals after administration of the radioactive material, and was usually of short duration. Figure 3 shows the total leucocyte count in three animals over a period of 15 months.

It can be seen that the recurrence of leucocytosis is irregular. At the time when leucocytosis was observed in some animals, in others the leucocyte number remained normal. By the end of the observation period there was some stabilization of the leucocyte count at the upper limit of normal. However, in some animals, leucocytosis occurred even in the later stages of the experiment.


Pronounced leucopenia was not observed. A slight, temporary fall of the leucocyte count to 6200 per mm3 was observed in 3 animals only.

Analysis of the differential counts showed that the fluctuations of leucocyte numbers referred to above, especially in the first 8-10 months, were due

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Months

FIG. 3. Fluctuations of the total leucocyte count in 3 rats which had received 59Fe oxide.

1—rat no. 1; 2—rat no. 2; 3—rat no. 3

E 24 E 22 ÏL20

I« 3 16 E 14 ~ 1 2 5 10 ° 8 QJ c

f L

^ 2

1

- \ ' \ - \ - /A \ ΓΑΜ // \V - A - / \

1 1 L

A V \ /\ j / \ \ / ^ \ A A V

2

- ' V V 1 1 1 1 i 1 1 1 1 1 L _

1 2 3 4 5 6 7 <3 9 10 11 12 13 14 Months

FIG. 4. Changes in numbers of leucocytes, lymphocytes and neutrophils in animals which had received a single injection of 59Fe oxide. 1—total leucocytes; 2—neutrophils; 3—lymphocytes

mainly to an increase of the number of lymphocytes in the later stages, an absolute increase of the number of neutrophils (Fig. 4) contributed significantly to the leucocytosis.

The fluctuation on the number of lymphocytes was considerably greater in the experimental animals than in the controls. Whereas in the control animals the lymphocyte count varied from 5000 to 13,700 per mm3 in the experimental animals, the range was from 3800 to 19,000 per mm3.


In addition to sharp fluctuations in the lymphocyte count, an increased number of cytological changes was observed, indicating a disturbance of the lymphopoietic processes. Such changes as cytolysis, fragmentation, and nuclear vacuolation were found in the majority of animals after the 3rd-4th months.

In the experimental animals, especially during the later months, absolute neutrophil leucocytosis was a frequent occurrence. Neutrophil leucocytosis (above 6000 per mm3) was observed in 19 of 30 animals which indicates the relative frequency of this manifestation. In most cases the increase of neutrophils was short-lived. The number of neutrophils fluctuated between 1500 and 11,800 per mm3.

It must be pointed out that even with pronounced neutrophil leucocytosis a left shift of the neutrophil lobe count was not observed, although this is the index of any alteration of neutrophil maturation. The increase in the number of neutrophils with nuclear fragmentation and hypersegmentation confirms this situation.

During the first 6-8 months some animals displayed an eosinophilia up to 1400-1900 per mm3 (8-10 per cent). Such eosinophilia was not observed in the control animals. In the later stages, the number of eosinophils remained within normal limits.

B L O O D INDICES OF R A T S AFTER A T R I P L E I N J E C T I O N OF 003 μc 59Fe O X I D E

Animals receiving 59Fe oxide in small doses showed some increase in the red cell count and haemoglobin level. The increase was more pronounced than in the previous groups. In a number of animals the increases of red cells and haemoglobin occurred in the 3rd-4th month and persisted. Changes of red blood indices in individual animals are shown in Table 5.

TABLE 5. Number of Red Cells (million per mm3) and Haemoglobin Level (Percentage)

No. of animal

42

46

57

44

52

Before injection

6-93 86

7-77 91

6-58 82

6-98 86

7-4 85

1

7-54 92

7-42 92

7-03 88

7-93 97

7-51 92

2

7-92 96

7-96 93

7-63 91

8-26 99

8-73 97

3

8-95 103

9-06 100

8-12 93

8-62 96

9-31 96

4

8-83 100

7-98 98

8-92 96

8-42 100

9-93 99

Months after

5

9-24 98

7-93 96

10-2 102

8-98 102

8-59 97

7

9-1 99

8-94 102

8-36 99

9-53 104

8-31 95

The numerator of each fraction signifies the number


Of the 15 animals of this group an increase in the red cell count above 8,800,000 per mm3 was seen in 10, and haemoglobin above 98 per cent in 7. The colour index remained within normal limits.

The reticulocyte count fluctuated between 15 and 30 per thousand, not differing in this way from the control group. The findings outlined above indicate some stimulation of erythropoiesis without any evidence of impaired production.

The fluctuation in the leucocyte count of rats which received 59Fe oxide in small amounts was narrower than in animals receiving larger amounts. The leucocyte count varied from 7200 to 21,500 per mm3, which only slightly exceeds the range in the control group. Leucocytosis above 21,500 (24,000 and 26,000 per mm3) was only on one occasion in only 2 animals. It should be noted that although the indices did not often go beyond physiologically normal limits, the month to month fluctuation of the leucocyte count in individual animals was fairly marked (Fig. 5), indicating a definite instability of the white blood system.

In the overwhelming majority of animals leucocytosis was caused by an increase in the number of lymphocytes. The lymphocyte count varied between 5300 to 16,000 per mm3 except, in the two animals with leucocytosis mentioned above, in which the lymphocyte count reached 18,000 per mm3. Absolute lymphocytosis above 13,700 per mm3 was observed in 7 animals and in 4 of these on more than one occasion.

The number of neutrophils varied between 1500 to 10,000 per mm3, which is somewhat above the normal range. However, the frequency of neutrophil leucocytosis in animals of this group was less than in the previous groups. A neutrophil leucocytosis above 6000 per mm3 was observed in 4 of 15 animals. At different periods an eosinophilia of up to 1600 per mm3 was noticed in certain animals.

Qualitative changes of the formed elements of the blood in the form of

of Individual Animais which Received three Doses ofO-03 ßc 59Fe203

injection

8 8-69 101 7-95 96 7-55 96 8-87 98 8-76 98

9 8-55 98 8-56 99 9-84 99 8-5 95 8*91 97

10 8-31 96 8-64 99 8-8 98 9-1 97 9-05 97

11 8-92 100 8-93 98

9-23 101 8-75 96

12 8-93 97 9-03 100

9-18 99 8-84 98

13

9-31 99 8-64 97

8-9 100

14

8-92 95 8-49 98

8-56 96

15

9-22 99 8-6 96

8-42 97

of red cells, the denominator-haemoglobin.


cytolysis, and nuclear fragmentation and pyknosis, were found in these animals beginning from the 5th-6th months, but not as frequently as in the animals which received preparations of greater activity.

Thus, intratracheal injection of rats with different 59Fe compounds produced detectable changes in the peripheral blood, manifested by sharp fluctuations in the number of lymphocytes and neutrophils, usually an increase,

6 7 8 9 10 11 1? 13 14 15 Months

FIG. 5. Changes in the total leucocyte, lymphocyte and neutrophil counts in rats which received three doses of 0-03 μc 59Fe oxide.

1 —total leucocytes; 2—lymphocytes; 3—neutrophils

F 3

59Fe citrate 2ΰμζ 59Fe oxide ?7-5-i-06pc 59Fe oxide 0·03μο3 VZ\ Once ES3 More than once

FIG. 6. Frequency of occurrence of increased numbers of white blood cells in animals of different groups (percentages).

some rise in the red cell count and haemoglobin level, and qualitative changes in the leucocytes.

The changes mentioned were observed in all the groups of experimental animals, but the frequency and degree of the alterations varied (Fig. 6).

Sharp fluctuations in the numbers of leucocytes were observed from the first months of the experiment, although the normal range of variation was not exceeded by all animals.


The most characteristic change which followed injection of 59Fe soluble citrate was an increase in the neutrophil count, which was found in 77 per cent of the animals of this group.

Leucocytosis, caused by an increase of the number of lymphocytes and neutrophils, was most frequently observed in animals which received 59Fe oxide in an amount of 27-5-1Ό6 μο. Leucocytosis was observed in 80 per cent of animals of this group, whereas in the group receiving 3 injections of 0Ό3 [LC it was found only in 53 per cent.

Qualitative changes appeared from the 3rd-6th months and were, more persistent. Such qualitative changes in peripheral blood cells indicate that in addition to a stimulation of haemopoiesis, manifested by an increase in the number of formed blood elements in the peripheral blood, some alterations in blood formation occur, leading to the appearance of pathological forms.

In this later stage, beginning from the 9th-10th month, these changes begin to regress due probably to a gradual reduction of the radioactivity of the injected substance, and the action of compensatory mechanisms in the body.


1. Intratracheal injection of white rats with various 59Fe compounds of activity from 27-5 to 1-06 μc produced quantitative and qualitative changes in the leucocytes, which may be the result of early haemopoietic damage.

2. The blood changes consist of sharp fluctuations in the lymphocyte and neutrophil counts, mainly upwards, and structural changes in the lymphocytes.

3. An increase in the red cell count and haemoglobin level was z constant finding following the administration of 59Fe to rats.

4. At the dose levels of radioactivity administered, no pronounced differences were observed between the soluble citrate and the insoluble oxide.

5. Three injections of 0Ό3 μο of 59Fe oxide gave rise to peripheral blood changes but these were less pronounced and appeared less regularly than in the animals which received preparations of greater activity.

R E F E R E N C E S

BELOBORODOVA N.L. , Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, pp. 39-53, Medgiz (1960).

BELOBORODOVA N.L . and BARANOVA E.F. , Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 1, pp. 162-166, Medgiz (1957).

B U R Y K I N A L . N . , Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 1, pp. 61-76, Medgiz (1957).

BURYKINA L. N. et al, The long-term effects of contamination by small doses of radioactive substances in the chronic experiment (Otdalennye posledstviya porazheniya malymi dozami radioaktivnykh veshchestv v khronicheskom eksperimente). Report on 2nd UNO International Conference on the uses of atomic energy for peaceful purposes, Geneva (1958).


EGOROV A. P. and BOCHKAREV V. V., Haemopoiesis and Ionizing Radiation (Krovotvorenie i ioniziruyushchaya radiatsiya), Medgiz (1954).

KAVETSKII R.E. et al., Effect of small (trace) doses of radioactive substances on morphology and biochemistry of the blood (Vliyanie malykh (indikatornykh) doz radioaktivnykh veshchestv na morfologicheskii i biokhimicheskii sostav krovi). Proceedings of All-Union Conference on the application of radioactive and stable isotopes and radiations in the national economy, Moscow (1957).

NEMENOV M.I . and GUREVICH R.G. , Vest, rentgen. i radiol., 9 (1), 7-17 (1931). PETROVICH I.K., Changes in the blood picture in animals at long intervals after adminis

tration of radioactive substances (Izmenenie kartiny krovi z zhivotnykh v otdalennye sroki posle vvedeniya v organizm radioaktivnykh veshchestv). Abstracts from a conference on long-term effects of radiation injury, Medgiz (1956).

STOLYAROV L.B. and NIKITENKO R.D. , Experimental Application of Radioisotopes in Medicine (Opytprimeneniya radioaktivnykh izotopov v meditsine),pp. 200-204, Kiev (1955).

TEPLINSKAYA G.N., Changes in the peripheral blood picture of rats after single and prolonged administration of different doses of 9 0Sr (Izmenenie kartiny perifericheskoi krovi krys pri odnokratnom i dlitePnom vvedenii razlichnykh doz strontsiya-90). Abstracts of work devoted to radiostrontium. Moscow (1959).

CHANGES IN CARBOHYDRATE METABOLISM AND THE SERUM PROTEIN FRACTIONS

DURING PROLONGED ADMINISTRATION OF 59Fe

R. L. O R L Y A N S K A Y A

AT THE present time there are many reports in the literature on the effect of acute radiation injury on metabolism of the carbohydrates and proteins (I.I.Ivanov et ai, 1958; L. S. Cherkasova et al, 1959).

However, there is little information concerning biochemical changes in the body during prolonged exposure to ionizing radiation, especially, following internal administration of radioisotopes.

Moreover, prolonged chronic poisoning by radioactive isotopes may give rise to changes in the functional condition of the liver, pancreas and other organs, which would result in changes in carbohydrate and protein metabolism.

In acute radiation sickness the total protein content of the serum is unaltered but the literature contains contradictory reports about the quantity of the different proteins which make up this total, and little information is available concerning the serum proteins following prolonged administration of radioactive isotopes. Reports concerning changes in carbohydrate metabolism are scanty. Therefore, we thought it desirable to make a study of changes in the total and differential protein content, and also the blood sugar level in rabbits during prolonged 59Fe administration. These points have been investigated in rabbits which received 59Fe for a prolonged period and the results reported below.

M A T E R I A L S AND M E T H O D S

Four groups, each consisting of at least 10 young rabbits of both sexes, which had previously been under haematological examination were selected for the experiment. Animals of all 4 groups were kept under similar conditions and diet (a description of the groups is given in the introductory article by E. B. Kurlyandskayain the presenti volume).

The sugar content of the blood was determined by Hagedorn-Jensen's method (S.D.Balakhovskii, 1955). The blood sugar was determined in animals of all groups after loading with glucose at intervals of 30, 60, 120 and 180 min. Glucose was administered orally as a 50 per cent solution at the

105


rate of 2 g per kg of body weight (N.I.Vinogradova and E. D. Grishchenko, 1960). The total serum protein was determined by refraction. The different serum protein fractions were determined by paper electrophoresis (A.E. Gurvich, 1955). Separation of the fractions was carried out in a veronal-medinal buffer at pH 8-6. Time of run was 18 hr. The electrophoretogram was stained with a 0-04 per cent solution of acidic blue-black stain in methyl alcohol. The unadsorbed stain was washed off with 10 per cent acetic acid containing 4 per cent phenol. For quantitative determination of the protein fractions the stain absorbed by the protein was eluted from the paper bands with 0*1 NNaOH and the amount of stain measured with a photoelectric colourimeter model PEC-M.

R E S U L T S

The figures given below represent mean values of measurements for any given group and are statistically valid.

The blood sugar level in rabbits of the first group (1 μc per kg) from the 1st to the 9th months was almost unchanged at 113 ± 2-5 mg per 100 ml. At the 9th month some decrease was noted (102 ± 4 mg per 100 ml), but by the 16th month the blood sugar had returned to its original level.

In second group rabbits (10 μc/kg) the blood sugar level from the first to the 9th month was almost unchanged at 125 ± 0-86 mg per 100 ml. At the 12th month a fall to 91 mg per 100 ml was observed, and by the 16th month, despite some increase (109 mg per 100 ml), the blood sugar level was still below its original value.

In rabbits of the third group (control animals which received a solution of FeCl3 containing non-radioactive iron) the blood sugar level was unchanged at 112 ± 1-4 mg per 100 ml for the first 9 months of the experiment. By the 12th month it had risen to 126 mg per 100 ml.

In fourth group rabbits (physiological control) from the first to the 12th month of the experiment the blood sugar level was almost unchanged. At the 9th month it was 124 ± 1 mg per 100 ml; and at the 12th month, 118 mg per 100 ml.

Thus, after the first 9 months of the experiment the blood sugar level of rabbits of the first group had fallen by 9-8 per cent from the initial value, of the second group 27-2 per cent; and in rabbits of the fourth group 4-9 per cent.

These results show that the greatest fall in the blood sugar level occurred in rabbits of the second group. However, even in this group can only say that there is a tendency for the blood sugar level to fall, since the values obtained fall within the lower limits of the normal range.

In the first group (1 μ ΰ ^ ) the blood sugar is at its maximum (except at 6 months) after 30 min (Fig. 1). After 60 min some decline can already be observed, and later the level descends more steeply, reaching the initial value after 3 hr. The greatest increase of the curve by comparison with the resting level occurs after 9 months (by 80 mg per 100 ml).

Changes in Carbohydrate Metabolism and Serum Protein Fractions 107

In the second group (10 μο/kg) the curve reaches its maximum height after 60 min (Fig. 2), but the curves of this group differ in type from the others— they are flatter. The greatest rise of the curve occurs at the 6th month (71 mg per 100 ml). The curves have not returned to the initial level 3 hr after glucose administration.

200 r

I I I 1 I ! L 30 60 120 180

Time (min)

FIG. 1. Blood sugar curves of rabbits receiving 1 μο/kg 59Fe at different intervals after commencement of administration of 59Fe.

1—initial; 2—after 3 months; 3—after 6 months; 4—after 9 months; 5—after 12 months; 6—after 16 months

As has already been pointed out above, the resting blood sugar level of second group rabbits after 12 months is lower than in animals of other groups. Consequently, the sugar curve lies below the others, although it is of similar type.

The sugar curves for groups 3 and 4 (stable iron and physiological control) are very similar in character and values at all times at which they were investigated. Therefore, we have only included curves for the third group in this report (Fig. 3).

For this group the curve reaches its highest point after 60 min (apart from the 12th month) and returns to its initial level after 3 hr. The greatest rise relative to the original level occurs at the 12th month (by 49 mg per 100 ml).

The total serum protein in rabbits of the first group (1 μc/kg) over a period of 12 months varied from 6-37 g per 100 ml (initial) to 6-39 g per 100 ml (12th month). A slight increase was observed only at the 6th month (6-75 g per 100 ml). During these months, in animals of the second group the total protein content slowly increased from 6-55 g per 100 ml (initial) to 7-12 per 100 ml by the 6th month, and then fell again to 6-49 per 100 ml by the 12th month. In the 3rd and 4th groups during these same months the TRS 8


FIG. 2. Blood sugar curves of rabbits receiving 10 μο^ per day at different intervals from commencement of administration of 59Fe.

1—initial; 2—after 3 months; 3—after 6 months; 4—after 9 months; 5—after 12 months; 6—after 16 months

200 V

I I i I I I L

30 60 90 120 150 18. Time (min)

FIG. 3. Blood sugar curves of rabbits receiving a solution of stable iron (FeCl3). 1—initial; 2—after 3 months; 3—after 6 months; 4—after 9 months;

5—after 12 months


serum protein level was unchanged (respectively 6-39 g and 6-40 g per 100 ml). Thus, only in rabbits of the second group was some increase of total protein content observed at the 6th month, equal to 9 per cent of the initial value.

Analysis of data obtained by electrophoresis in the first and second groups disclosed a decline of the serum albumin and some increase of the serum globulins. The albumin globulin ratio calculated from these data also shows significant changes in the ratios of the serum protein fractions in rabbits of the first and second groups, which were not detected in the third and fourth

£ 60 |-

I I I I L 0 . 3 6 S 12

Months

FfG. 4. Changes in the albumin/globulin ratio calculated as a percentage of the initial value.

groups (Fig. 4). Thus, the A/G ratio at 6 months falls in the first group by 14-1 per cent of its initial value; and in the second group (10 μο per kg), by 22-5 per cent. In this latter group the y-globulin content is raised at 6 months (cf. Table), but at the following determination, the A/G ratio has increased and the content of y-globulin declined (9 months).

In earlier articles (cf. Toxicology of Radioactive Substances, vol. 2, Moscow, 1960) information is given concerning disturbances of carbohydrate (N.I. Vi-nogradova and E. D. Grishchenko, 1960) and protein (E. D. Grishchenko, 1960) metabolism during chronic radiation injury. It is considered necessary therefore to present only recent information which has appeared in the literature.

Changes in carbohydrate metabolism have been investigated by many workers using various doses of external radiation, and at different intervals after irradiation (S. B. Balmukhanov, 1958; D.A.Golubentsev, 1958; R.Ya. Keilina, 1959 ; K. S. Klimenko, 1958 ; and F. M. Tsukrova, 1959). Much less work has been done on changes in carbohydrate metabolism during isotope intoxication (A.V.Fedorova, 1960; A.Ya.Shulyatikova, 1956; N.I.Vi-nogradova, 1960; and E. D. Grishchenko, 1960). These authors indicate the periodicity of changes in metabolism, according to both the dose administered and the interval from beginning of administration of the isotope. Thus, A.V.Fedorova has shown in white rats that, when different amounts of


TABLE 1. Changes in the Serum Protein Fractions

Group of rabbits

First

Second

Third

Fourth

Before the experiment

protein fractions

albumins

52-7 57-1 61-6

59-0 58-3 58-0

57-1 57-8 60-0

58-6 60· 1 58-3

globulins

(X

15-1 14-3 10-6

12-0 11-8 12-2

11-7 15-6 130

13-5 9-9

11-3

P 12-3 14-3 11-4

13-6 14-3 15-7

15-6 14-5 15-0

12-5 13-8 13-7

y

20-1 14-3 16-8

15-4 15-5 14-1

15-6 12-1 12-0

15-7 14-4 16-6

A/G ratio

1-11 1-33 1-57

1-43 1-40 1-38

1-33 1-37 1-50

1-42 1-54 1-64

After 3 months

protein fractions

albumins

51-5 56-3 57-8

58-8 55-8 57-2

55-8 59-6 58-8

59-4 56-8 58-7

globulins

a

160 12-7 9-7

11-1 12-0 11-9

16-3 11-7 12-2

10-0 12-7 12-2

ß 15-6 16-4 12-6

12-4 14-5 14-7

16-3 13-8 13-0

12-7 13-2 13-4

y

16-3 14-9 19-9

17-7 17-6 16-1

11-6 14-9 15-9

17-6 17-3 15-7

A/G ratio

1-27 1-32 1-39

1-43 1-28 1-36

1-24 1-47 1-43

1-46 1-31 1-42

After

protein

albumins

51-7 52-3 55-3

45-9 45-3 48-8

56-5 53-8 59-8

64-5 54-5 65-5

glo

at

17-0 17-6 16-0

12-9 14-1 16-2

13-9 15-9 11-6

14-4 14-3 13-8

radiostrontium are administered, the changes which occur are of similar type but the degree of injury is dependent on dose. The writer observed a temporary fall of the blood sugar level followed by a rise. In acute polonium poisoning A.Ya. Shulyatikova observed a rise of the blood sugar level on the 3rd day, followed by a return to normal by the 8th day. Before death, the blood sugar level again falls. During prolonged 60Co administration to rabbits N. I. Vinogradova and E. D. Grishchenko found that the blood sugar level falls by the 15th month, and then rises (hyperglycaemic reaction).

Our own results are in agreement with those described above. Thus, the decline of the sugar level observed in rabbits of the second group (10 (xc/kg) by the 12th month begins to recover later.

Thus, the direction of changes observed in the blood sugar level of rabbits is similar to that described in the literature, and so can be associated with the effect of radiation from the internally administered 59Fe.

In radiation injury significant disturbances occur in protein metabolism. Thus, N.L.Lipkin (1958) indicates the possibility of changes in the proteins of the tissues. Lethal X-ray doses produce a fall of the nitrogen of the tissue proteins very quickly after irradiation. O. V. Fastyuchenko, L.Ya. Popov and M. G. Nikolaeva (1958) report that even 1 hr after irradiation, a decline of total serum protein is observed, and that the rate of recovery is directly correlated with dose. O. V. Fastyuchenko and B. M. Varshavskii (1957) report an increase in total serum protein and a reduction in the albumin fraction and the A/G ratio.

The literature contains contradictory reports concerning the total serum proteins under radiation conditions. They are difficult to compare because of


Selective Data in Percentages

6 months

fractions

bulins

ß 15-4 14-4 15-3

14-9 14-6 15-5

10-4 14-5 12-8

11-6 13-0 12-7

y

19-4 15-7 13-4

26-4 26-0 21-2

19-1 13-9 15-8

9-5 18-2 8-0

A/G ratio

1-08 1-09 1-24

0-85 0-83 0-95

1-30 1-16 1-25

1-82 1-20 1-89

After 9 months

protein fractions

albumins

56-8 58-8 60-2

60-5 56-7 59-8

56-8 60-5 57-4

61-9 53-9 64-7

globulins

a

11-9 13-2 13-0

14-2 13-7 13-1

15-9 14-4 16-7

13-4 16-9 10-9

ß 11-6 13-2 9-6

12-9 14-2 11-6

11-6 10-8 13-1

12-0 13-4 101

y

19-6 14-8 17-2

11-1 15-4 14-9

15-7 14-8 11-5

12-8 15-8 14-2

A/G ratio

1-31 1-42 1-5

1-54 1-30 1-49

1-31 1-09 1-41

1-67 1-15 1-80

After 12 months

protein fractions

albumins

51-5 53-4 56-3

57-0 54-0 52-8

globulins

a

17-1 111 14-0

14-7 11-4 11-6

ß 11-4 13-3 10-9

9-5 15-4 121

y

20-0 22-2 18-8

19-2 19-2 23-6

A/G ratio

1-06 1-15 1-29

1-33 1-17 111

differing radiation conditions, different sources and various investigation periods. Thus, L. G. Sherman (1960) reported that with doses of 50, 100, 200, and 400 r the total serum protein is almost unchanged, but that there are significant changes in the A/G ratio: the albumin fraction declines considerably and there is a corresponding rise in the globulin fractions.

E. A. Abaturova et al. (1958) have observed hyperproteinuria at the height of radiation sickness and a predominance of the albumin fraction of the serum. On the other hand, N.A.Zolotukhin, determining protein fractions by two methods (salting-out and electrophoresis in a Tiselius apparatus) finds a reduction of the albumin fraction and a corresponding increase of globulins, the degree of change being proportional to dose.

V.D.Blokhina (1960), determining protein fractions with a Tiselius apparatus in animals irradiated with different X-ray doses, found a reduction of the serum albumin and increased <%- and ^-globulins. V.P.Moiseeva (1958) also gives data showing a fall in the albumin fraction and increase of globulins, especially marked in the period preceding death of the animal. E. D. Grishchenko (1960), in an article on the effect of prolonged oral administration to rabbits of 60Co, draws attention to the pattern of development of changes in protein metabolism.

We were unable to detect significant changes in the total serum protein during the 16 months of observation. Some increase was seen in second group animals at the 6th month but this was small, not exceeding 9 per cent of the initial level, and of short duration.

However, the albumin/globulin ratio underwent considerable changes which were especially marked in second group of animals.

The A/G ratio was reduced for many months because of a fall of albumin


in the serum. Our results are in complete conformity with those obtained by other writers.

It might be thought that changes in the amount of albumin are connected with disturbances of liver function, where most serum proteins, especially the albumins, are synthesized.

Confirmation of this proposition is provided by the results of E.D.Grish-chenko, who found that changes in the fractional composition of the serum proteins during chronic administration of 60Co, are linked with an impairment of albumin synthesis in the liver. Firm proof is given by N.V.Syro-myatnikova's investigations (1958), which demonstrated a disturbance of the protein-forming capacity of the liver when amino acids were used as a functional stress in acute radiation sickness. The relative increase of y-globulins detected is also in agreement with data in the literature. We were unable to detect any changes in other protein fractions, especially the ß-globulins, with which, according to Paoletti (1957) and T. M. Ostroukhova (1958), 59Fe binds.


1. During prolonged administration of 1 and 10 μο/kg 59Fe to rabbits for 16 months, the blood sugar level changes little. Only at the 12th month is some decline observed in animals receiving 10 μο/1<£ and in these animals the sugar curve is of hypoglycaemic type.

2. During prolonged administration of 59Fe to rabbits, no change was observed in total serum protein. In rabbits receiving 10 μο/1<£ 59Fe a decline of the serum albumin and a corresponding increase of serum globulins was observed in the 6th month. The A/G ratio first falls and then, after a few months, returns to normal.

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ABATUROVA E.A. et al., The Effect of Ionizing Radiation on the Animal Body (Deistvie ioniziruyushchego izlucheniya na zhivotnyi organizm), Kiev (1958).

BALAKHOVSKIIS.D. and BALAKHOVSKIII .S. , Methods of Chemical Analysis of the Blood (Metody khimicheskogo analiza krovi), Moscow (1955).

BALMUKHANOV S.B., The effect of adrenalin on the blood sugar level in radiation sickness (O vliyanii adrenalina na uroven' sakhara v krovi pri luchevoi bolezni). Works of the Department of Radiology of the Kazakh Medical Institute, vol. 1, p. 99, Alma-Ata (1958).

BLOKHINA V.D., Early Reactions of the Body to Radiation (Issledovanie rannikh reaktsii organizma na radiatsionnoe vozdeistvie), Moscow (1960).

CHERKASOVA L.S. et ai, Expenditure and replacement of carbohydrates in the body and certain features of their metabolism in radiation injury (O raskhodobanii i vospolnenii uglevodov v organizme i nekotorykh osobennostyakh ikh obmena pri luchevykh po-razheniyakh). Transactions of XIII Scientific Session of the Institute of Nutrition AMN SSSR, p. 74, Moscow (1959).

FASTYUCHENKOO.V. and VARSHAVSKIIB.M. , Changes in some indices of protein metabolism during chronic and acute exposure to ionising radiation (Izmeneniya nekotorykh pokazatelei belkovogo obmena pri khronicheskom i ostrom vozdeistvii ioniziruyush-


chego izlucheniya). Transactions of an All-Union Conference on Medical Radiology, Moscow (1957).

FASTYUCHENKO O. V. et al., Early changes in the protein composition of the serum in acute radiation sickness (Rannie izmeneniya v belkovom sostave syvorotki krovi pri ostroi luchevoi bolezni). Transactions of a conference on "Early mechanisms of radiation injury", p. 20, Kharkov (1958).

FEDOROVA A. V., Some biochemical reactions of the body to chronic and single administration of 9 0Sr (Nekotorye biokhimicheskie reaktsii organizma pri khronicheskom i odnorazovom postuplenii 9 0Sr). Report of a conference of the Institute of Radiation Hygiene on achievements in 1959, Leningrad (1960).

GOLUBENTSEVD.A. , Early changes of carbohydrate-phosphorus metabolism in acute radiation injury and their pathogenic significance (Rannie izmeneniya uglevodno-fos-fornogo obmena pri ostrykh radiatsionnykh porazheniyakh i ikh patogeneticheskoe znachenie). Transactions of a conference on "Early mechanisms of radiation injury", p. 27, Kharkov (1958).

GRISHCHENKO E.D. , Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, p. 65, Moscow (1960).

GURVICH A.E., Lab. deh, 3, 3 (1955). IVANOV I.I. et al, Metabolism in Radiation Sickness (Obmen veshchestvpri luchevoi bolez

ni), Moscow (1956). K E I L I N A R . Y A . , Early changes in carbohydrate metabolism (O rannikh izmeneniyakh v

uglevodnom obmene). Transactions of a conference on "Early mechanisms of radiation injury", p. 29, Kharkov (1958).

KEILINA R . Y A . , Biokhimiya, 24 (6), 266 (1959). KLIMENKO K.S., The Effect of Ionizing Radiation on the Animal Body (Deistvie ionizi-

ruyushchego izlucheniya na zhivotnyi organizm), p. 63, Kiev (1958). LIPKIN N . F . , Idem, p. 89. MOISEEVA V.P., Aktual. vopr. pereliv. krovi. 66, 63 (1958). OSTROUKHOVA T. M., Aktual. vopr. pereliv. krovi, 6, 63 (1958). P A O L E T T I C , C. R. Acad. Sci., 245 (3), 377 (1957). SHERMAN L. G., Comparative characteristics of some biochemical ingredients of the serum

of white rats after a single X-irradiation (Stravnitel'naya kharakteristika nekotorykh biokhimicheskikh ingredientov syvorotki krovi belykh krys posle odnorazovogo rent-genovskogo oblucheniya). Reports of a Conference of the Institute of Radiation Hygiene on achievements in 1959, p. 26, Leningrad (1960).

SHULYATIKOVA A . Y A . , Comparative study of changes in carbohydrate metabolism of animals following acute and prolonged polonium intoxication (Sravnitel'naya otsenka iz-menenii uglevodnogo obmena u zhivotnykh pri ostrykh i otdalennykh srokakh gibeli ot polonievoi ontoksikatsii). Transactions of an Ail-Union Conference on Medical Radiology, p. 54, Moscow (1956).

SYROMYATNIKOVA N. V., Aktual. vopr. pereliv. krovi, 6, 89 (1958). TSUKROVA F.M., Med. radiol, 10, 80 (1959). VINOGRADOVA N . I . and GRISHCHENKO E.D. , Toxicology of Radioactive Substances

(Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, p. 65, Moscow (1960).

THE EFFECT OF PROLONGED INTERNAL ADMINISTRATION OF 59FeCl3

ON THE RABBIT ELECTROCARDIOGRAM O. A. S A I T A N O V

MANY experimental and clinical reports have recently been published concerning changes in the cardiovascular system following acute and chronic exposure of the body (external and more rarely internal) to ionizing radiation (V. A. Fanardzhyan, K. A. Kandaryan et al, 1960; V. K. Sel'tser and N. V. Lo-seva, 1960; B.B.Moroz and S.P.Grozdov, 1960; S.P.Grozdov, 1961; A.O. Saitanov, 1958-1960; S.I.Teplov, V.S.Sverdlov and B.F.Korovkin, 1959; V.I.Korchemkin, 1959; T. S. Seletskaya, 1959; I.N.Bukhalovskii, 1959; I.S.Glazunov and P. M. Kireev, 1958; E. D. Semiglazova, 1958; LM.Velik-son, 1957; N.S.Molchanov, 1957; and others).

These authors report that electrocardiographic changes often occur before a clinically pronounced picture has developed and, in a number of cases, are an early sign of exposure to ionizing radiation. Whitfeld and Kunkler (1957), Saphir (1947), Utsumi, Kakefueda and Saito (1958), T. S. Seletskaya (1959) and others have noted the considerable sensitivity of the heart even to therapeutic doses of X-irradiation (from clinical and electrocardiographic data), thus confirming the conclusions of earlier investigators on the susceptibility of the heart to ionizing radiation (Yu.I.Arkusskii et al, 1937; Prosser, 1947; L.Gempel'man, G.Lisko and D.Gofman, 1954; A.V.Lebe-dinskii, 1955-1956; A.V.Kozlova et al, 1957; E.I.Vasil'eva, 1957; K.A. Kandaryan, 1958; and others).*

Some writers (S. P. Grozdov, 1958 ; N. D. Stetsenko, 1959 ; and others) have shown that a more stable electrocardiogram accompanies a favourable course of the sickness.

We have been unable to find, in the literature which has reached us, any work devoted to the electrocardiographic investigation of the effect of prolonged internal administration of 59Fe on the cardiovascular system. We therefore set out to examine heart activity as shown by electrocardiograms in rabbits subjected to prolonged (for 2 years) internal administration of 59FeCl3.

Data on 59Fe,the experimental groups of animals and methods of administration are given in the introductory article by E.B.Kurlyandskaya.

* Cf. the literature review in the article by A.O. Saitanov in Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, p. 102, Moscow (1960).

114

Effect of Prolonged Internal Administration of 59FeCl3 115

R E S U L T S FOR THE C O N T R O L G R O U P

As a control group we took the 10 animals which received no iron preparations (fourth group) but which lived under the same conditions and over the same periods as the experimental animals, and also the 10 rabbits which received stable iron (third group) in amounts equivalent to the 59Fe dose in the second group.

Analysis of electrocardiographic results in both subgroups always showed sinus rhythm. In the physiological control group the heart rate varied between 220 to 330 per min (average 267 per min), while the corresponding figures for the stable iron group were 180-320 per min (average 280 per min).

The mean (statistical) heart rate in the control group was 274 ± 6 per min. In 2 animals, receiving stable iron, pronounced arrhythmia appeared at the 8th-9th month—180-270 and 180-300 contractions per min. In one rabbit of this group at the 8th month transitory ventricular electrical alternation was observed, which is sometimes seen in healthy animals. In 8 rabbits, the electrical axis was longitudinal and in 5 inclined to the right, but in 2 animals a slight tendency to a left inclination was observed. The P wave was positive. In 6 rabbits (2 from the physiological control and the other 4 receiving stable iron) intra-atrial conductivity periodically reached the upper limit of normal. In these animals, at the 12th month, a slight voltage increase of the P wave was observed. Atrio-ventricular conductivity slowed (to 0Ό9 sec) at the 2nd month only in one rabbit receiving stable iron. On average, atrioventricular conductivity was 0Ό6 ± 0015 sec.

In 2 control rabbits pronounced Q waves appeared in the standard I and left thoracic leads at the 7th month.

TABLE 1. Chief Characteristics of the Electrocardiograms of the Control and Experimental Rabbits

mean statistical results

Group

Control

Receiving ^ c / k g 5 9 F e C l 3

Receiving 1 μο/kg 5 9FeCl3

Number of ex

periments

104

118

103

Interval duration (sec)

P-Q

0 0 6 ± 0-015

0-0765 ± 0026

0076 ±0-002

QRS

0-035 ± 0-0015

0-045 ± 0-00132

0-037 ± 0 0 0 1

QRST

0-136 ± 0-0026

0-142 ± 0-0054

0-140 ± 0-006

Height

(mV)

0-26 ± 0-01

0-16 ± 0-013

0-21 ± 0015

Slight voltage fluctuation of the QRST complex appeared at the 5th-15th months in the 10 rabbits receiving stable iron. A slight decline of the complex at the 7th month was observed in 1 rabbit receiving stable iron, and transitory crenulation of the S wave in 2 rabbits of the control group. In 1 rabbit


of this group at the 9th month a transitory change in the configuration of the QRS complex Sforn i was seen. The mean value of intraventricular conductivity was 0035 ± 0-0015 sec.

The S-T interval was isoelectric. The Twave in most animals was positive in all leads. The T wave was on average 0-26 ± 0-01 mV. In 3 animals the S-T interval was inclined obliquely upwards. The electrical systole was temporarily prolonged (0-18 sec) only in 1 rabbit. In all the others it was normal and depended on the number of contractions. No disturbances in excitation could be detected in the control group. In a few rabbits slight changes in intraventricular conductivity could be observed (crenulation of the S wave, appearance of the Q wave), but these bore the signs of physiological fluctuations. In the great majority of animals the time values of the waves, and intervals, were fairly stable, sometimes fluctuating within 0-01-0-02 sec according to heart rate. Thus, the electrocardiographical data obtained for the control group were in agreement with the normal electrocardiogram described by various writers (Slapok and Germanek, 1957; E.Lepeshkin, 1957; L.P.Peresad'ko, 1953; Massman and Optits, 1954; A.O.Saitanov, 1960; and others).

R E S U L T S FOR THE F I R S T EXPERIMENTAL G R O U P

The first group of rabbits received 1 μc/kg 59FeCl3. The heart rate was raised between 120 to 310 per min (average 272 + 2-8 per min). In one rabbit at the 7th month transitory ventricular alternation was observed and in another at the 2nd month ventricular extrasystoles occurred for a period.

The electric axis was inclined to the left in 3 rabbits, in 2 of which considerable movement of inclination was observed. Electrocardiograms with right inclination were found half as often as in the controls. This indicates a transition of longitudinal and right type (through normal) to a left inclination.

P wave. In 12 rabbits of 20 in this group a well-pronounced acuminate P wave was observed (Figs. 1 and 2).

In 9 rabbits intra-atrial conductivity somewhat exceeded the upper limit of normal (0-05 sec). In one of these animals at the 6th month a very high (more than 0-6 mV) and extended (0-09-0-10 sec) wave was seen, indicating intra-artial block, but this rapidly disappeared (Fig. 3).

In 7 rabbits the upper limit of conductivity was recorded at the 7th-12th months.

P-Q interval. In 4 rabbits atrioventricular conductivity fluctuated between 005 and 0-10 sec, in 3 it exceeded the upper limit of normal (008 sec), and in 2 it was clearly retarded (0-09-0-10 sec). On average atrioventricular conductivity was 0-076 ± 0-002 sec. In the majority of animals the retardation was observed at the 6th-14th months.

Q wave. In 2 animals at the 2nd-6th months a pronounced Q wave appeared (more than 1-5 mV). In one rabbit the Q wave reached 0-6-0-5 mV in

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E # k t o j Prolonged Administrotion of Radioactive Iron 47

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Effect of Prolonged Internal Administration of59FeCl3 119

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F I G . 3. Electrocardiograms in standard and thoracic leads. Rabbit receiving ^ c / k g 5 9 F e C l 3 .

a—before administration; b—after 6 months: pronounced Pi_m wave, pronounced and extended P C R , C R 5 wave, high and wide P C R 4 and giant P C R 5 (0-4 mV); very low Γι_π wave and reduced 7 C R , C R 1 _ 4 _ 5 ; c—at 9th month: P wave has become considerably lower, P C R 4 wave acute, T wave approximately normal size, but

remains lower than initially (Tm wave flattened)


the CR lead. At the same time a retardation of intra-atrial conductivity and decline of the T wave were observed in this animal (cf. Fig. 3). After a few months the electrocardiogram became normal. This indicates that in this group, as in the group receiving the larger 59Fe dose, transitory functional changes occurred in the electrocardiogram.

QRS complex. In 11 of the 20 rabbits of this group, at the 8th-9th months, a temporary, moderate increase in height of the R and S waves was observed. In 1 rabbit the increase was considerable in the thoracic leads. In 2 animals at the 9th-13th months a slight decline of the QRS complex was seen. In 9 rabbits intraventricular conductivity reached the upper limit of normal (0Ό5 sec) twice as often as in the control group. It averaged 0-037 ± 0Ό01 sec. No clearly pronounced retardation of intraventricular conductivity was observed in this group (cf. Table 1).

S-T interval. In 3 rabbits, at the 8th-9th months, the S-T was slightly obliquely inclined downwards. In 2 animals this lowering was accompanied by a pronounced reduction of, or biphasic ( + ) Twave (cf. Fig. 3), indicating a change in the bioelectrical processes of the ventricular myocardium. In 1 rabbit the Twave was high with a sharp apex, which might also indicate a similar change (B. B. Moroz, S. P. Grozdov et al.).

T wave. In 10 rabbits, at the 8th-9th months, a transitory, moderate enlargement of the Twave was observed. In the other 10 animals a tendency to decline of the T wave was seen, primarily in the I—II standard and left (CR4 _ 5) thoracic leads (cf. Fig. 3). In 3 of these rabbits the decline and smoothing out of the Twave was more pronounced at the 14th and 19th months. On

TABLE 2. Comparative Data on Changes in the Size of Waves, Length, Shape and

Changes in wave size, conductivity and position

of intervals

Enlargement of P wave Enlargement of Q wave Enlargement of QRS complex Enlargement of T wave Reduction of T wave Reduction of S-T interval (or change of

shape), accompanied by reduced, flattened or biphasic ^ T waves

Retardation of intra-atrial conductivity (0-08-0· 10 sec)

Retardation of intraventricular conductivity (0-05-0-08 sec)

Group

control

months of observation

1-5

2 1 2 2 -

1

—

6-11

— 1 6 4 -

—

12-17

4

2 4 1

—

18-20

— ----

—

total cases

6 2

10 10 1

1

—

months of most

pronounced changes

12-14 2-6 5-15 5-15 11

2

—


average, the height of the Twave was 0-21 + 0Ό15 mV. In some cases the T wave became high and sharp-peaked. Changes in the T wave became apparent especially in rabbits with signs of chronic radiation exposure and showing haematological changes (N. L. Beloborodova, V. L. Ponomareva and E.K.Red'kina).

The enlargement and acuity of the Twave initially, and its flattening at later stages is possibly connected with disturbances in the electrolyte balance. Ellenwood and co-workers (1957) have observed an increase of potassium and decline of calcium in the early period and a fall in potassium content at later stages of acute radiation injury.

Ventricular systole. The ventricular electrical systole fluctuated from 0T3 to 016 sec according to frequency of contractions. On average, g - J w a s 0-14 ± 0-006 sec. However, it revealed normal limits more often (7 animals) than in the controls (4 animals).

This surpassing of upper normal limits was observed in 4 animals at the 8th-18th months; and in 3, at the 16th—18th months of administration. In these rabbits a slight decline of the terminal part of the ventricular complex was observed (primarily of the Twave). This decline (flattening) was particularly marked in rabbits with signs of chronic radiation injury.

Continuing study of more than 214 electrocardiograms of the experimental group did not reveal any significant changes in rhythm and number of contractions by comparison with the control rabbits.

The transitory enlargement of the P, QRS and T waves observed by us have been described by a number of authors in acute exposure to 32P, 89Sr

Position of Intervals in Electrocardiograms of Control and Experimental Groups

Group

receiving K^c/kg5 9FeCI2 receiving 1 [LC/k£ 59FeCl3

months of observation

1-5

4 1 3 2 1

1

3

1

6-11

3 2 4 3 5

1

6

2

12-17

2 -4 3 6

2

3

2

18-20

1 1 --—

-

-

-

total cases

10 4

11 8

12

4

12

5

months of most

pronounced changes

5-7 7-10 4-12 6-14 7-15

7-17

7-10

4-14

1-5

2 -1 1 1

-

1

-

6-11

7 2 8 8 5

2

3

-

12-17

3 -1 1 2

1

1

-

18-20

— -1 -2

-

-

-

total cases

12 2

11 10 10

3

5

-

months of most

pronounced changes

7-12 7-10 8-9 8-9

14-19

8-14

6-14

-


and X-rays (I. N. Bukhalovskii, 1959, and others), chronic administration of 60Co (A. O. Saitanov, 1960), intravenous or subcutaneous injection of rabbits with 0·1 μΰ/kg polonium (B.B.Moroz and S.P.Groz-dov, 1960) and X-irradiation of rats (Caster, Armstrong and Simonson, 1957).

The appearance, during the period from 2 months to 12 months of administration, of certain signs indicating changes in nervous regulation of the heart (extrasystoles, high and long P and Q waves, transitory retardation of intra-atrial and intraventricular conductivity, etc.) is apparently connected with changes in autonomie ennervation and with a change in the functional condition of the nerve centres which regulate cardiac activity, and also, possibly, with hormonal endocrine effects (M. G. Durmish'yan et al.). Changes in nervous control of blood circulation after irradiation have been observed by N.A.Kurshakov (1954), A.V.Lebedinskii (1956), M.Dmitrov and T.Markov (1956), I. M. Kireev and I. S. Glazunov (1958), V. K. Sel'tser and N. V. Lo-seva (1960), and others.

In the majority of animals a gradually increasing left axis deviation was observed. This position of the axis was seen in the experimental group twice as often as in the controls. The appearance of sinistralgrams in irradiated rabbits has been reported by V.A.Fanardzhyan et al. (1960), B.B.Moroz and S.P.Grozdov (1960), A.O.Saitanov (1960), and others. This is linked, apparently, with greater damage to the myocardium of the left ventricle because of its greater functional load, as is shown by the results of a number of writers (L. Gempel'man, G.Lisko and D.Gofman, 1954; C.N.Sergeev and N.I.Bukhalovskii, 1959; V.A.Fanardzhyan et al, 1959; A.O.Saitanov, 1958, 1960, 1961).

The decline of the terminal part of the QRST complex (primarily the T wave) was marked from the 15th month in rabbits receiving 10 μc/kg 59Fe and somewhat later (from the 18th month) in the animals receiving 1 \xcjkg. Changes in the terminal part of the ventricular complex were more often observed in the standard I and in the 4th-5th thoracic leads, resembling the picture of dystrophic lesions, and, sometimes, of acute circulatory impairment or focal damage of the myocardium. Such changes are described in the literature following exposure to X- and y-rays (especially to the heart and head regions) of experimental animals, and in man after therapeutic X-irradiation (Yu.I.Arkusskii and M.I.Mints, 1937; Whitfeld and Kunkler, 1957; T. S. Seletskaya, 1959; Utsumi, Kakefueta and Saito, 1958 ; and others). Changes in the bioelectric processes of the cardiac muscle caused by irradiation are apparently primarily connected with a change in oxidative-restorative processes (Caster, Armstrong and Simonson, 1957; S.I.Teplov, V. S. Sverdlov and P. F. Korovkin, 1959 ; V. A. Fanardzhyan, 1960 ; and others). There is a basis for believing that these changes are produced by a disturbance in neuro-endocrine control and a change in the function of the supra-renals (M. G. Durmish'yan et al, 1961; Ya.I.Azhipa and T. A. Filyashina, 1961) with an impairment of the electrolyte balance (Ellenwood, Wilson and Coon, 1954; Raab, W., 1959; T. I. Ivanchenko, 1959), leading to alterations


of the ionic gradient of the electrolytes of the cardiac muscle and of the bioelectric processes.

In a number of cases oedema of the myocardium has further impaired the functional capacity of the heart (cf. the article by E. S. Gaidova in this volume). It is possible that in rare cases vascular impairment may be of significance (S.S.Vail', T.A.Zedgenidze and D. S. Sarkisov, 1956).

In most rabbits with low T waves a tendency to increase was observed, and, in some, an enlargement of the ventricular electric systole. The clinical and haematological examinations of these animals carried out by N. L. Beloboro-dova, V. L. Ponomareva and E.K.Red'kina (cf. the article in this volume) reveal the marked chronic effect of ionizing radiation. Thus, associations were established between size of dose, time of irradiation, degree of injury and character of changes in the electrocardiogram.

R E S U L T S FOR THE SECOND E X P E R I M E N T A L G R O U P

The rabbits of the second group received 10 μc/kg 59FeCl3. In 18 of the 20 animals there was sinus rhythm and the heart rate varied between 190 to 320 per min (statistical mean 258 ± 4 per min). Thus, by comparison with the controls (274 contractions per min) a slight difference was observed, but this was not statistically significant. In one rabbit, at 2nd-5th months for a temporary period atrial extrasystoles occurred ; and in 3 animals in the period 5-11 months, transitory ventricular electrical alternation occurred.

Electric axis. In 5 rabbits it was deviated to the left.* In 3 of these the left deviation was marked and was accompanied by discordant dislocation of the S-T interval and divergence of the axes of the R, S and T waves. A left deviation of the axis occurred twice as often in this group as in the controls.

P wave. In 10 rabbits generally at the 5th-8th months a transitory enlargement and acuity of the P wave was observed (reaching, and sometimes exceeding the T wave). In 7 animals, intra-atrial conductivity somewhat exceeded the upper limit of normal (0Ό5 sec). In 1 rabbit conductivity was considerably longer than normal (0-06 sec), indicating pronounced retardation of intra-atrial conductivity.

Interval P-Q. In 12 rabbits (more than half the animals of this group) atrio-ventricular conductivity fluctuated between 0-05-0-09 sec. A tendency to retardation of atrioventricular conductivity (P-Q 0-08 sec) was observed in 9 rabbits. In half this number of animals, the retardation appeared at the 6th-7th month. In 3 rabbits, the interval reached a maximum 0-09 sec, indicating significant retardation of atrioventricular conductivity. On average, conductivity was 0-0765 ± 0-0026 sec (cf. Table 1).

* In view of the fact that in the low-voltage rabbit electrocardiogram it is not possible to determine accurately the inclination of the cardiac electric axis according to the angle a, the thoracic leads were used in addition. The ratio of the initial and terminal inclination of the RI S and RI I waves in the right and left thoracic leads was calculated to determine axis deviation. TRS 9


Q wave. A pronounced (more than 0-15 mV) Q wave appeared in 4 rabbits, in 2 of these at the 7th month and in 1 at the 2nd month. In this last animal very large waves appeared in the thoracic leads: in CR, 0-6 mV; and in CRX, 0-5 mV; at the 5th month, these waves disappeared (Figs.4 and 5). This indicates that the appearance of high Q waves is connected with functional changes in the atrial myocardium.

QRS complex. In 11 rabbits, i. e. more than thalf the total of this group, at the 4th-12th months, and in one at the 2nd month, a transitory fluctuation in height of the complex was seen in the form of moderately increased amplitude of the R and S waves. In 3 animals, at the 7th-12th months, there was a slight decline in voltage and in one case a considerable decline.

Division of the QRS complex was observed in 1 rabbit. In 5 animals conductivity was somewhat above normal (0*05 sec), and in 5 it was considerably retarded (0-08 sec). Mean conductivity was 0Ό45 ± 000132sec. In most animals, retardation of conductivity was observed at the 6th—11th months. This retardation was often accompanied by a slight decline in rhythm frequency. In many animals conductivity became normal at times. In 1 rabbit with pronounced sings of chronic radiation injury at the 14th month a partial left bundle block appeared against a background picture of deficient blood supply and, possibly, local lesions in the myocardium of the left ventricle (cf. Figs. 4 and 5).

S-T interval. In 5 rabbits at the 5th-17th months a slight (by 1-1-5 mm) fall of the S-T in the form of a straight or convex interval was observed. This decline was accompanied in 4 animals by a flattened or biphasic T wave, and in 1 animal by an inverted Twave (cf. Figs. 4 and 5).

T wave. In 8 animals, chiefly at the 6th-14th months, a transitory, moderate enlargement of the T wave was observed. In a considerable number of animals of both groups (1 and 10 μΰ/kg 59Fe) the Twave had the characteristic shape of an equilateral triangle with a sharp apex (Figs. 2 and 3). A slight decline of this wave was seen in 12 rabbits (in 6 of these at the 9th-15th months). This fall of the T wave was often accompanied in both groups by sharp P waves (cf. Figs. 2 and 3). Flattened or weakly biphasic T waves were observed in 4 rabbits, in 2 at the 5th-7th, and in the other 2 at the 14th month. In one of these the T waves were inverted in the standard I and left thoracic (CJR 4 _ 5 ) leads, while the S-T interval was obliquely and discordantly displaced below, resembling the electrocardiogram in deficient blood supply or focal lesions of the myocardium of the left ventricle (with partial bundle blockage) (cf. Figs. 4 and 5). On average, the height of the T wave (in limb leads) was 0*16 ± 0013 mV, which indicates a significant voltage decline by comparison with the control group. Changes of the T wave were accompanied by a retardation of atrio ventricular conductivity (in 2) and intra-ventricular conductivity (in 1). In 2 of the rabbits mentioned above clinically pronounced changes in the blood system, protein metabolism, etc. were observed.

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Effect of Prolonged Internal Administration of 59EeCI3 127

016 sec slightly exceeding normal (by 0Ό1-0Ό2 sec) and in 3 rabbits it reached 017 sec, significantly above normal (by 0Ό2-0Ό3 sec). On average, g-Twas 0*142 ± 0*0054 sec. An electrical systole of 016 sec was found twice as often, and of 0*17 sec three times as often as in the controls. In 7 animals an extension of the electrical systole occurred during the period from 9 to 14 months. Of the 9 animals with a prolonged systole, the electrocardiogram of 6 displaced a reduced T wave. In the 2 animals with a systole of 0*17 sec clinical and haematological signs of chronic radiation injury were observed.


1. During the first year of the experiment in rabbits receiving 1 and 10 \&\)L% 59FeCl3 signs of changes in the nervous regulation of the heart, transitory changes in intra-atrial and intraventricular conductivity (high and wide P and Q waves) and impairment of excitability (atrial and ventricular extrasystoles) were observed. Electrical alternation of the ventricles was often seen.

2. Intra-atrial, atrioventricular and intraventricular conductivity was at the upper limit of normal or somewhat retarded, while the ventricular electrical systole was somewhat prolonged in almost a third of the experimental animals.

3. Transitory (phasic) changes in height of electrocardiographic waves were observed in the experimental animals. In some rabbits the electrical axis of the heart gradually deviated to the left.

4. Continued observation of electrocardiograms showed the marked effect of prolonged internal administration of 59FeCl3 on bioelectric processes (chiefly on the T wave). This effect begins to be manifested from the 7th month (in certain cases even earlier) and becomes pronounced at the 15th-18th months of administration.

5. The electrocardiographic changes described occurred earlier and in a greater number of animals and were more pronounced in the group receiving ^ c / k g 5 9 F e C l 3 .

6. Changes in the electrocardiogram are not specific for 59Fe administered internally, since they have been observed by us during prolonged administration of similar amounts of 60Co, and by other writers following external and internal (acute and chronic) irradiation from various sources of ionizing radiation.

R E F E R E N C E S

ALEKSENTSEVA E. S. and PERLIN M. S., Changes of cardiac activity and kidneys in radiation sickness (Izmenenie serdechnoi deyatel'nosti i pochek pri luchevoi bolezni). Transactions of the Vitebsk State Medical Institute, Vitebsk (1959).

AZHIPA YA.I . and FILYASHINAT.A., The Effect of Small Doses of Ionizing Radiation on Physiological Activity (Voprosy deistviya malykh doz ioniziruyushchego radiât sii na fiziologicheskie funkt sii), Izd. AN SSSR, p. 8, Moscow (1961).

BuKHALOVSKiiLN., Med. radio!., 4 (9), 24-29 (1959).


CASTER W.O. et al, Amer. J. Physiol, 188 (1), 167-177 (1957). DMITROV M. and MARKOV T., Izv. Inst. biol Volgar. akad. nauk, 7, 231-278 (1956). DURMISH'YAN M.G. et al, The Effect of Small Doses of Ionizing Radiation on Physiological

Activity (Voprosy deistviya malykh doz ioniziruyushchei radiatsii na fiziologicheskie funktsii), Izd. A N SSSR, p. 46, Moscow (1961).

ELLENWOOD L.E. et al, Proc. Soc. Exper. Biol. Med., 94 (1), 129 (1957). FANARDZHYAN V. A. et al., The Effect of Ionizing Radiation on Animals (Deistvie ionizi-

ruyushchikh izluchenik na zhivotnyi organizm), pp. 234-238, Kiev (1960). GEMPEL'MAN L., LISKOG. and GOFMAND. , Acute Radiation Syndrome (Ostryi luchevoi

sindrom) (1954). G L A Z U N O V I . S . and K I R E E V P . M . , SOV. med., 4, 49-54 (1958). GROZDOV S.P., Changes in ECG of rabbits in acute radiation sickness after X-irradiation

or polonium injury (Izmenenie EKG u krolikov pri ostroi luchevoi bolezni posle rent-genovskogo oblucheniya ili porazheniya poloniem). Collected abstracts on radiation medicine in 1958 (1959).

GROZDOV S. P., The Effect of Ionizing Radiation on Animals (Deistvie ioniziruyushchikh isluchenii na zhivotnyi organism), edited by A .A .GORETSKI I , pp. 251-254, Kiev (1960).

GROZDOV S.P., Med. radiol., No. 6, 48-52 (1961). IVANCHENKO T.I., Some features of mineral metabolism in exposure to ionising radiation

(Nekotorye pokazateli mineraPnogo obmena pri deistvii ioniziruyushchei radiatsii). Author's abstract of Dissertation, Moscow (1959).

KANDARYAN K. A., The harmful effect of ionizing radiation (Povrezhdyaushchee deistvie ioniziruyushchego izlucheniya). Section 1 of Proceedings of 7th All-Union Congress of Roentgenologists and Radiologists, Saratov (1958).

KISLOVSKAYAK.L., Collected Abstracts on Radiation Medicine (Sbornik referatov po radiatsionnoi meditsine) Medgiz (1959).

KORCHEMKIN V.l. , Ibid. K R A E V S K I I N . A . , Med. radiol., 5, 75-79 (1960). LAZOVSKAYA A.B., Radiation Sickness and Combined Injury of the Body (Luchevaya bolezrf

i kombinirovannye porazheniya organizma), pp. 89-96, Leningrad (1958). LEBEDINSKII A. V. and YAKOVLEV V. V., Acute Radiation Sickness and its Long ter m Effects

(Ostraya luchevaya bolezri* i ee otdalennye posledstviya), Sukhumi, pp. 8-9 (1959). MOLCHANOV N.S. , The effects of some types of ionising radiation on the cardio-vascular

system (Vliyanie nekotorykh vidov ioniziruyushchei radiatsii na serdechno-sosudistuyu sistemu). Proceedings of VHth Congress of Therapists of the Ukrainian SSR, Kiev (1957).

M O R O Z B . B . and GROZDOV S.P., Collected abstracts on radiation medicine (Sbornik referatov po radiatsionnoi meditsine za 1958), Moscow (1959).

PROSSER S.L. et al, Radiology, 49 (3), 299 (1947). RAAB V., Achievements of Cardiology (Dostizheniya kardiologii), pp. 67-140, Medgiz

(1959). SAITANOV A.O., Clinical and experimental cardiac changes shown by electrocardiograms

in chronic radiation sickness (K voprosu ob izmenenii serdsta po dannym elektrokardio-grafii v klinike i eksperimente pri khronicheskoi luchevoi bolezni). Proceedings of an Anniversary Session of the Institute of Occupational Hygiene and Diseases, Moscow (1957).

SAITANOV A. O., Toxicology of Radioactive Substances (Materialy po toksikologii radio-aktivnykh veshchestv), vol. 2, pp. 102-120, Medgiz (1960).

SAITANOV A. O., Byull eksper. biol. i med., 6, 102-108 (1960). SAPHIR O., A Text on System Pathology, vol. 1, 417-418, New York (1959). SELETSKAYA T.S., Voprosy rentgen. i radiol, 10, 322-330, Moscow (1959). SEL'TSER V. K. and LOSEVA N. V., Cardiac rhythm of rabbits subjected to prolonged effect

of small doses of ionizing radiation (Serdechnyi ritm krolikov, podvergavshikhsya dliteFnomu deistviyu malykh doz ioniziruyushchei radiatsii). Proceedings of 3rd All-Union Conference of Pathophysiologists, Moscow (1960).


STETSENKO N .D . , Functional condition of the heart after internal and external irradiation (Izmeneniya funktional'nogo sostoyaniya serdsta pri vnutrennem i vneshnem obluche-nii). Proceedings of 2nd Congress of Oncologists and 3rd Congress ofRoentgenologists and Radiologists of U.S.S.R., Kiev (1959).

TEPLOV S.I. et al, Med. radiol, 3, 27-33 (1959). UTSUMI K. et al, Acta Path. Jap., 8 (4), 419 (1958). VAIL' S.S. et al, Vopr. patol. serd. i legkikh, Leningrad, 6, 24-44 (1956). VASIL 'EVAE.L , Vest, rentgen. iradiol, 6, 8-13 (1957). VELIKSON I. M., Clinical and physiological investigations of people chronically exposed to

small doses of ionizing radiation (Materialy kliniko-fiziologicheskikh issledovanii u lits, podvergayushchikhcya khronicheskomu vozdeistviyu malykh doz ionisiruyushchei radiatsii). Transactions of a conference of the Central Radiology Institute, Leningrad (1957).

WHITFELD A. and KUNKLER P., Brit. Heart J., 19 (1), 53-54 (1957).

THE EFFECT ON THE HEARTS OF RABBITS OF PROLONGED INTERNAL IRRADIATION

WITH SMALL DOSES OF 59FeCl3

A. O. S A I T A N O V

N OUR previous paper it was shown that even in the first few months after he commencement of administration of 59FeCl3 to rabbits signs of impairment of excitability, conductivity and height of electrocardiographic waves became apparent, and were associated with changes in the nervous regulation of the heart.

By the end of the first year these changes had almost disappeared ; later, signs that repolarization of the myocardium was disturbed were observed— primary and secondary changes in the terminal part of the ventricular complex (mainly of the T wave) of the electrocardiogram.

Similar phenomena have previously been observed by us (1960) during prolonged administration of radiocobalt to rabbits.

At the present time (1961) a number of writers (M.G.Durmish'yan, Ya.I. Azhipa, V. P. Godin and others) believe that considerable radiosensitivity is manifested by the most reactive of the nervous system to the internal administration of small doses of radioactive sodium.

An increase of the radiation dose above the threshold often leads to changes in the character of the effects, to a biphasic development or to a change of the latent period of the response.

These authors emphasize that changes are first manifested in the suprarenal medulla, and later in the cortex. Such alterations in the neuro-endocrine system precede disturbances in haemodynamics (reduction of arterial pressure and other changes) and occur before the haemopoietic system or other systems, are involved.

In this paper we have attempted to determine the condition of the nervous mechanisms engaged in the regulation of cardiac activity during prolonged exposure to small doses of radiation, before any definite disturbances are shown on the electrocardiogram. In a joint work with I. N. Golovshchikova (1960) we demonstrated changes in the condition of the vagus nerve in rabbits after prolonged internal administration of radiocobalt, as a result of which some increase of the depressor reflex to ammonia and reduced sensitivity to adrenalin was observed.

In this connection it was of interest to investigate how these mechanisms are affected by prolonged oral administration of 59FeCl3, the biological effects of which differ from other isotopes, for example 60Co and so on.

130

Effect on Hearts of Rabbits of Prolonged Internal Irradiation 131

We have found no reports in the literature concerned with the effect of 59Fe on heart condition. In order to detect early changes in the nervous regulation of the heart functional tests (ammonia and adrenalin) were used.

The tests were made on rabbits aged 2\ months—before 59Fe administration was begun and then throughout the experiment (18-20 months), i.e. at all phases of the development of chronic radiation injury.

During the experiment cardiac activity was recorded on film with an electrocardiograph (for a detailed description of the animals and experimental methods cf. the articles by E. B. Kurlyandskaya and A.O.Saitanov in the present volume).

The experiments were carried out on 50 animals (24 control and 26 experimental). In all, 285 tests were made, 135 with ammonia and 150 with adrenalin.

Ammonia tests. Using ammonia, studies were made of the olfactory-pharyngeal-cardiac reflex (vagus bradycardia), which is pronounced in rabbits when the mucosa of the respiratory tracts is stimulated. The cardiac component of this reflex, which was the object of study, was manifested by a slowing of the heart rate immediately after application of the stimulus from 250-300 per min to 60-90 per min, followed by a gradual return to the normal rate. It is known that the path of this reflex passes through the receptors and afferent fibres of the trigeminal nerve. Sectioning of both vagus nerves entirely eliminates the cardiac component of the reflex. Electrocardiograms from such rabbits are unchanged when the upper respiratory tracts are stimulated by ammonia fumes. Elimination of this reflex by sectioning indicates that inhibition of cardiac activity by ammonia stimulation depends on excitation in vagus nerve centres and transmission of impulses to the heart. By recording this reflex in healthy animals, and also before and during administration of radioactive substances, it was possible to demonstrate (by inhibition of cardiac activity) how the functional condition of the vagus nerve is changed.

Method. 0-1 ml ammonia water was placed in an olfactometer of 1 litre capacity. Using a rubber squeezer of 50 ml capacity 150 ml ammonia fumes was conveyed to the rabbit's nose in 5 sec. Special attachments were used to maintain a constant distance of 0-5 cm between the animal's nasal apertures and the funnel from which the ammonia vapour issued.

At 5-7 min before the experiment the rabbit's electrocardiogram was taken while the animal was in position on its back. Simultaneously with the administration of ammonia the electrocardiograph was switched on and recording continued until the reaction had ceased and normal heart rate restored.

Results. In total, 135 tests on 50 rabbits were made : 45 tests on the 24 control animals, of which 14 constituted a physiological control (fourth group) and 10 received stable iron (third group), and 90 tests on the 26 experimental rabbits; 35 tests were carried out on 11 rabbits receiving 1 [xc/kg 59Fe and 35 on 15 animals receiving 10 \Lclkg. The material was subjected to statistical analysis.

Inhalation of ammonia vapour in the doses used produced a significant


reduction of the heart rate in the control and experimental groups. The slowing of the heart rate occurred immediately in the animals of the control groups.

In the experimental animals there was a lag in the response to ammonia. In first group of rabbits (1 μ^^£) a lag period was observed in 7 of the 35 tests. These results did not prove to be statistically significant (20 per cent > p > 10 per cent).

In rabbits of the second group (10 μ-c/kg) during the first 6 months a lag period was seen in 3 tests (of 19) in 23 tests out of 30, from the 7th to the 12th months, and from the 13th to the 18th months—in all 6 tests. It was on average 1-8 sec, which was statistically significant (p < 1 per cent). Thus, with increase of the 59Fe dose and prolongation of the duration of the experiment a larger number of animals showed a lag period in the response reaction to ammonia inhalation.

In the first 6 months the heart rate of the first group of rabbits increased (285 + 4-9 per min, p < 1 per cent), and of second group of animals decreased (209 + 10, p < 1 per cent) by comparison with the control group (246 + 2-9 per min). From the 7th to the 18th months the heart rate in the first group of animals did not differ from that of the controls. In the second group of animals, from the 7th to the 12th months, a slight fall in the heart rate was observed (267 ± 4-4), but by comparison with the controls (276 ± 4-8) this was not statistically significant (20 per cent > p > 10 per cent). However, from the 13th to the 18th months, a slowing of the heart rate was observed (270 + 6) which was statistically significant (5 per cent > p > 2 per cent). When ammonia was applied maximal slowing of the heart rate in the first 6 months was greater in the experimental animals : in the first group, by 37*8 per cent (53 ± 3-8 per min, 5 per cent > p > 2 per cent), and in the second group, by 52-3 per cent (42 + 3-9 per min, p < 1 per cent) by comparison with the controls (88 ±1 -3 per min).

In the period from 7 to 12 months the slowing of the heart rate remained, as in the first 6 months, more marked than in the controls: in first group animals, by 35-1 per cent (61 ± 7-8 contractions per min, p < 1 per cent); and in second group animals, by 35-1 per cent (63 ± 5-4 per min, p < 1 per cent), i.e. the difference was less than at the beginning of the experiment.

From the 13th to the 18th months the ammonia reaction changed in both groups. Compared with the controls (63 ± 6-6 contractions per min) in first group rabbits the heart rate increased by 44-4 per cent (91 + 6-9 per cent, p < 1 per cent); and in second group animals, by 98*4 per cent (125 ± 2-9 per min, 5 per cent > p > 2 per cent) (Fig. 1).

The duration of the ammonia reaction during the first 6 months in first group rabbits was greater by 97-8 per cent (93 ±1*3 sec, p < 1 percent); and in second group animals, less by 29 per cent (33 ± 4 sec, 5 per cent > p > 2 per cent) than in the controls (47 ± 5-6 sec).

In the period from the 7th to the 12th months the reaction was considerably extended in both experimental groups; in the first group by 218*5 per

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cent (86 ± 1-6 sec, p < 1 percent) and in the second by 300*6 per cent (109 + 2 sec, p < 1 per cent).

From the 13th to the 18th months the duration of the reaction in the experimental groups did not differ from that in the controls (25 ± 3-8 sec). In first group rabbits it was 8 per cent less (23 ± 2-3 sec), and in the second group, 4 per cent more (27 ± 5*8 sec). These differences were not statistically significant (50 per cent > p > 40 per cent).

Investigation of animals during prolonged administration of 59Fe revealed changes in the heart rate. In rabbits of the first group an increased heart rate was observed ; and in the second group, a slower heart rate during the first 6 months of the experiment with a subsequent return to the original level (from the 7th to the 12th months) and a slight decline towards the end of the experiment (from the 13th to the 18th months). The reflex reaction to ammonia inhalation was also different in the experimental animals. During the first 6 months slowing of the heart rate occurred immediately after inhalation in the experimental animals (as in the controls). In first group rabbits from the 7th to the 12th months and especially towards the end of the experiment a small delay (1-2 sec) appeared in the bradycardia response (lengthening of the lag period). Maximal slowing of the heart rate was observed in the animals of both experimental groups to a significantly greater degree than in the controls throughout the first year of the experiment. From the 13th to the 18th months maximal slowing was less pronounced, and in a certain number of cases reflex bradycardia was abbreviated, or absent altogether.

The duration of the ammonia reaction changed during the experiment. In the first 6 months it was somewhat longer in first group rabbits and reduced in those of the second group. From the 7th to the 12th months the reaction was more prolonged in both groups, but from the 13th to the 18th months it became the same as in the controls.

The control rabbits reacted to ammonia not only by reduced frequency of cardiac contractions but also with a single or grouped extrasystoles (mainly ventricular). In the experimental animals, especially of the second group, extrasystoles have been rarely observed. Thus, it should be noted that reflex bradycardia in the experimental animals during the first year was more pronounced, declined considerably towards the end of the experiment and in certain animals disappeared. Modifications of the reflex consisted in the appearance of a lag period, a reduction in the intensity and duration of the bradycardia and in some reduction in the number of extrasystoles. Consequently, suppression of the vagus reflex did not develop immediately, but gradually, after a brief initial intensification.

Adrenalin tests. Adrenalin is one of the important hormones taking part in the control of the cardiovascular system and especially in the metabolism of the myocardium (Raab, 1959)), increasing its oxygen requirement (Gol-witzer-Meyer and Kroetz, 1940). When adrenalin enters the blood stream the vessels are constricted and arterial pressure rises. The increased arterial pressure acts as a stimulus to the receptors of the aortic and carotid bodies. Im-


pulses from these bodies, and also from the nervous, endocrine and other tissues, give rise to excitation of the vagus nerve centres and vagus inhibition of cardiac activity in the form of bradycardia.

A number of authors (T. V. Grigorovich, 1937; A. V. Lazovskaya, 1958; V.K.Sel'tser and N.V.Loseva, 1960; B.B.Moroz and S.P.Grozdov, 1960; A.O.Saitanov and I.N.Golovshchikova, 1960; D.I.Zakutinskii et al, 1960; and others) have used adrenalin to demonstrate the functional condition of the nervous mechanisms which regulate cardiac activity after exposure to ionizing radiation. Some writers report that preliminary exposure to radiation lowers the threshold of sensitivity to adrenalin and associate this with changes in the adrenal medulla, which can be observed even with small doses of internal radiation (M.G.Durmish'yan, Ya.I.Adzhipa, V.P.Godin et al, 1961).

Methods. A 0-01 per cent solution of adrenalin (dose 4 y/kg weight) prepared as required was injected into the marginal ear vein of rabbits which were placed on a bench. The injection time was a constant 5 sec.

The experiment was performed on 24 control rabbits, of which 14 constituted a physiological control (fourth group) and 10 received stable iron (third group), and 26 experimental animals: 11 receiving 1 μc/kg 59Fe (first group) and 15-10 μο/kg 59Fe (second group). In all, 150 tests were made.

Electrocardiograms were taken before injection of adrenalin. Simultaneously with the injection, the electrocardiograph was switched on and tracing continued until the heart rate had returned to normal.

Intravenous injection of adrenalin produced, after a few seconds, a significant slowing of heart rate in the control and experimental animals. In the animals of the first experimental group (1 μο^) in the period from 7 to 12 months a slight decline in sensitivity to adrenalin was observed together with the appearance of an increased lag period (15-5 ± 1 - 2 sec), statistically significant by comparison with the controls (11-4 sec) (p > 1 per cent).

At the 13th to 18th months in rabbits of this group the retardation reaction was 19-5 per cent more pronounced (157 + 9 per min, p < 1 per cent) than in the controls (195 ±6 -1 contractions per min).

The duration and intensity of the reaction were more marked in animals of the experimental group (period 7-12 months). Greatest duration and intensity were observed in second group rabbits (10 (Jic/kg 59Fe). Towards the end of the experiment (13-18 months) the reflex reaction to adrenalin fell significantly in the experimental animals, the lag period of the bradycardia response was increased, and in some animals more frequently of the second group, no bradycardia was observed at all (Fig. 2). At this period, in the experimental animals, atrial, atrioventricular and ventricular extrasystoles, ventricular rhythm and attacks of paroxysmal ventricular tachycardia were observed. In the control animals only isolated, and more rarely grouped, ventricular extrasystoles were seen.

In the experimental rabbits (especially those receiving 10 [xc/kg 59Fe) injection of adrenalin was sometimes followed by a more pronounced shortening of the S-T interval, and a decline or inversion of the T wave (a change of

ww

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444

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. 2.

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69 136 Toxicology of Radioactive Substances


myocardium repolarization) by comparison with the controls, possibly associated with the dominant effect of sympathomimetic catecholamines (Raab). Thus, the reflex bradycardia following the injection of adrenalin took a course in the experimental group (especially the first) different from that followed in the controls. At the beginning of the experiment, some intensification of reflex bradycardia was observed, later, from the 7th to the 12th months, a lengthened lag period, reduced intensity and duration of bradycardia; and sometimes at the end of the experiment (at the 13th—18th months), absence of bradycardia (suppression of the reflex). Sometimes other changes were also observed, indicating changes not only in the neuroreflex mechanisms but also in the myocardium (the effect on the adreno- and cholinoreactive systems). Apart from ventricular extrasystoles (which were also observed in the control group) atrial and atrioventricular extrasystoles were sometimes seen, and also attacks of ventricular paroxysmal tachycardia. In a number of animals some change in repolarization of the myocardium was observed (decline of the T wave of the ventricular complex). This may indicate biochemical changes in the cardiac muscle (Raab, M. E. Raiskina et al). In comparing the course of changes in reflex control produced by ammonia and adrenalin, the basic similarity of the results obtained should be noted. In both instances, reflex bradycardia was initially intensified and declined towards the end of the experiment. The data concerning cardiac activity in animals during prolonged administration of 59FeCl3 agree with those concerning the development of radiation sickness following X-irradiation (V. P. Cherkasov, 1956), prolonged exposure to radioactive polonium (B.B.Moroz, 1958), 90Sr (V. I. Korchemkin, 1959), 60Co (V. K. Sel'tser and N.V.Loseva, 1960-1961; I. N. Golovshchikova and A. O. Saitanov, 1960) and other radioisotopes (D.I.Zakutinskii et al, 1961).

The chief feature in changes in cardiac reflex activity observed in the experimental animals was the increase in the cardiac vagus reflex at the beginning of the experiment (first 6-12 months) and its decline towards the end of the experiment (13th—18th months), which is possibly associated with changes in the sensitivity of the adrenoreactive or cholinoreactive systems of the myocardium.

A reduction of the inhibitory effect must have great significance in the development of compensatory mechanisms, since it brings in its train the increased power of cardiac contractions, increase of coronary blood flow and oxygen requirement (Golwitzer-Meyer, 1940), and also increased adeno-sine triphosphate metabolism (M. E. Raiskina et al.).

Moreover, the reduction of vagus reflexes indicates that, in different situations of the internal or external environment, the heart can become less sensitive to stimuli which, in ordinary circumstances, lead to a pronounced inhibition of cardiac activity.


CONCLUSIONS

1. During prolonged internal irradiation of rabbits with small doses of 59FeCl3 changes were observed in the functional condition of cardiac activity in response to ammonia inhalation and intravenous injection of adrenalin.

2. These changes were manifested by alterations in the intensity and duration of the cardiac reflex of the vagus nerve and were of a fluctuating character. During the first 6-12 months of the experiment, the reaction occurred rapidly and was accompanied by more marked and prolonged bradycardia. Later, at the 13th—18th months, the lag period increased and the intensity and duration of the reflex declined and certain animals disappeared).

3. At the 6th-12th months of the experiment, injection of adrenalin often gave rise to impairment of the excitation process—appearance of atrial and atrioventricular extrasystoles, and also attacks of ventricular paroxysmal tachycardia.

4. A tendency to change of myocardium repolarization was detected (decline of the T wave of the ventricular complex in response to adrenalin), which may, of course, indicate biochemical shifts in the cardiac muscle.

5. Changes in cardiac reaction to ammonia or adrenalin occurred earlier and were somewhat more pronounced in rabbits receiving 10 μο/kg 59FeCl3 than in the animals receiving 1 μc/kg.

6. The character and direction of changes arising in cardiac reflex reactions of animals undergoing the prolonged administration of small quantities of 59FeCl3 are not specific and have occurred with other radioactive isotopes.

REFERENCES

CHERKASOV V.F., Medical Radiology (Meditsinkaya radiologiya), Moscow, 1 (2), 57-64 (1956).

DURMISH'YAN M. G. et al, The Effect of Small Doses of Ionizing Radiation on Physiological Function ( Voprosy deistviya malykh doz ioniziriuyushchei radiatsii na fiziologicheskie funktsii), pp. 46-50, Izd. AN SSSR, Moscow (1961).

GOLOVSHCHIKOVAI.N., Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, pp. 121-129, Medgiz (1960).

GOLWITZER-MEYER H. and KROETZ C , Pflug. Arch. Ges. Physiol., 241, 248-262 (1940). GRIGOROVICH T. V., Vlth Caucasion Congress of Physiologists, Chemists and Pharmaco

logists (VI Kavkazskii s'ezd fiziologov, khimikov i farmakologov). Authors' abstracts and theses, pp. 17-19, Rostov-on-Don (1937).

KORCHEMKIN V.l., Collected abstracts on radiation medicine for 1957 (Sbornik referatov po radiatsionnoi meditsine za 1957 g.), p. 144, Moscow (1959).

LAZOVSKAYA A. V., Radiation Sickness and Combined Infection (Luchevaya bolezrì i kombinirovannoe porazhenie organisma), pp. 89-96, Leningrad (1958).

MIRZOEV B. M., The Effect of Small Doses of Ionizing Radiation on Physiological Function ( Voprosy deistviya malykh doz ioniziruyushchei radiatsii na fiziologicheskie funktsii), pp. 95-96, Izd. AN SSSR, Moscow (1961).

MOROZ B.B. and GROZDOV S. P. Collected abstracts on radiation medicine for 1958 (Sbornik referatov po radiatsionnoi meditsine za 1958), Moscow (1959).


RAAB V., Achievements of Cardiology (Dostizheniya kardiologii), pp. 67-140, Medgiz (1959).

RAISKINA M.E., Farmakol i toksikol., 1 (31-33), 14 (1951). SAITANOV A. O., Toxicology of Radioactive Substances (Materialy po toksikologii radio-

aktivnykh veshchestv), vol. 2, pp. 102-120, Medgiz (1960). SAITANOV A.O. , Occupational hygiene in work with radioactive substances and sources of

ionizing radiation (Gigiena truda pri rabote s radioaktivnymi veshchestvami i istoch-nikami ioniziruyushchikh izluchenii). Proceedings of an extended symposium, p. 46, Moscow (1961).

SAITANOV A.O. , Physical Factors of the Environment (Fizicheskie faktory vneshnei sredy), pp. 57-71, Moscow (1960).

SAITANOV A. O., The Effect of Small Doses of Ionizing Radiation on Physiological Function (Voprosy deistviya malykh doz ioniziruyushchei radiât sii na fiziologicheskie funktsii), p. 110, Izd. AN SSSR, MOSCOW (1961).

SEL'TSER V.K. and LOSEVA N. V., Ibid., p. 114. ZAKUTINSKIID. I . et ah, Occupational hygiene in work with radioactive substances and

sources of ionising radiation (Gigiena truda pri rabote s radioaktivnymi veshchestvami i istochnikami ioniziruyushchikh izluchenii). Proceedings of an extended symposium, p. 55, Moscow (1961).

TRS 10

MORPHOLOGICAL CHANGES IN RATS AFTER INTRATRACHEAL INJECTION

OF VARIOUS 59Fe COMPOUNDS


IN RECENT years a series of reports have appeared on the behaviour of radioactive substances entering the lungs and the effects they produce on the pulmonary tissue. Interest in these problems is connected with the widespread use of radioactive isotopes in different branches of cience and technology, and the possibility of radioactive aerosols which might enter the lungs through the respiratory tracts.

L.N.Burykina (1957), Cember et al. (1958, 1959), Lisco and Finkel (1949), V.N.Strel'tsova (1956, 1959), T. A. Kochetkova and G.A.Avrunina (1960) have established that injection into the respiratory tracts, mainly of white rats, of 1 4 4CeF3,1 0 3RuO2, Cr 3 2 P0 4 , Ba3 5S04 and other radioactive isotopes produce metaplasia of the bronchial epithelium with subsequent development, in a series of cases, of bronchial cancer of the lungs. It should be noted that, according to M.Shimkin (1957), Horn and Stewart (1952), and Woolley and Wherry (1911), spontaneous lung tumours are rare in white rats. As Cember points out, when radioactive substances enter the respiratory tracts the size of the tissue dose to the lungs, and its distribution in time, constitute one of the determining factors in the development of bronchial tumours. These values in their turn are determined by the chemico-physical properties of the substance concerned (its solubility in the biological media of the body, dispersion of particles, radiation energy and so on).

According to T.A.Kochetkova and G.A.Avrunina (1960), intratracheal injection of rats with chromium phosphate (Cr32P04) in amounts of 0-27,0*1 and 0-07 mc per rat was followed in 11 of 76 animals by the development of keratinizing squamous cell carcinoma of the lungs at periods from 210 to 395 days after injection. A significant number of the animals died early with symptoms of acute or subacute radiation injury. Similar results were obtained by these writers with injection of colloidal gold-198 (198Au).

In the experiments of Cember and co-workers using intratracheal injection of rats with 144CeF3 in amounts of 50, 25, 15 and 5 JAC, development of bronchial cancer was detected in 13 of 93 animals surviving to discovery of the first tumour. In these experiments, the author also observes the death of a significant number of the animals from acute radiation injury during the first weeks after injection. The relatively brief incubation period for tumour

140

Morphological Changes in Rats after Intratracheal Injection 141

development is a striking feature of these results. In 4 cases tumours were detected in rats which died in less than 100 days from injection. The earliest occurrence of a tumour was found in a rat which died after 48 days.

Lisko and Finkel report metaplastic lesions of the epithelium of the bronchial mucosa, developing into bronchial cancer in rats 9 months after inhalation of radioactive cerium oxide (1 4 4Ce203).

From the results of different authors the total radiation doses to the lungs at which development of malignant tumours was observed are extremely variable (Table 1).

TABLE 1. Total Radiation Dose in the Lungs at which Tumours Occurred from the Literature

Substance injected

Cr3 2P04 198Au (colloid) 144CeF3 144Ce

Activity (μο)

100-70 320-150

50-5

Time to first tumour

58 days 36-70 days

48 days 9 months

Radiation dose received by the lungs

(rads)

1300-18,000 4000-13,000 2400-30,000

20;160

Author

ί T.A.Kochetkova \ and G.A.Avrunina

Cember Lisco and Finkel

Yu. I. Moskalev (1959) reports that the chief factors in the carcinogenic effect of radioactive isotopes are the energy and size of the tissue dose, and also the regularity of exposure. Nevertheless, the data so far available do not permit a precise identification of the factors which lead to the development of bronchial tumours, while the problem of whether a threshold exists for the carcinogenic effect of ionizing radiation has not yet been resolved (O. Bryus (1956), M.N.Pobedinskii (1956), and others).

The absence in the literature of information concerning the effect of 59Fe on the lungs, and the importance of this problem for prevention injury to the respiratory organs persuaded us to study, experimentally, the character of the pathological lesions occurring long after injection into the lungs of different 59Fe compounds, and to determine the order of dose of internal radiation of the pulmonary tissue which would lead to the development of malignant tumours.

The 59Fe preparations used in this work contained an admixture of the isotope 60Co of up to 12 per cent in activity, which was taken into account when calculating tissue doses. The amount by weight of the admixture was extremely small.

Preliminary investigation of the distribution of 59Fe in the body after intratracheal injection showed that the injected compounds are retained in the lungs for a long time. This enabled us to follow their radiotoxic effect for 15 months.

The experiments were performed on 80 adult white rats. The animals were divided into 7 groups. The first group of animals received 20 \LC per rat 59Fe


citrate solution once through the trachea. The animals of the second, third and fourth groups were injected with a suspension of finely dispersed 59Fe oxide powder in physiological saline. Each rat received 1 ml suspension with an activity of 27-5, 3-36 and 1*06 μο respectively. The animals of the fifth group received three intratracheal injections of 59Fe oxide suspension of 0Ό3 μα The sixth and seventh groups were controls.

To evaluate tissue doses the behaviour of different 59Fe compounds in the lungs at different periods after intratracheal injection was examined. For this purpose the radioactivity of the organs and tissues of all animals dying or killed at different intervals was investigated. The results obtained are shown in Table 2.

TABLE 2. 59Fe Content of the Lungs at Varying Intervals after Injection Mean Arithmetical Values from 2-4 Animals

No. of group

of animals

1 2 3 4 5

Preparation injected

5 9Fe citrate 5 9Fe oxide 5 9Fe oxide 5 9Fe oxide 5 9 Fe oxide

Activity (μθ

20-0 27-5

3-36 1-06 0-03 Λ

three V injections]

5 9Fe content (percentage of quantity injected)

months 3

5-7 52-8 49-4 56-2

58-3

5

48-7 —

46-2

—

6

3-4 42-5 38-4 49-8

50-4

8

2-3 31-2 41-4 46-4

—

9

36 —

42-5

43-8

10

2-4 — — —

38-3

11

— 27-1 —

—

12

2-3 27-3 29-4 33-5

31-7

15

2-2 28-5 26-8 29-7

—

The data in Table 2 show that 59Fe content of the lungs in all animals usually declined with increasing time after the injection. Removal of iron from the lungs took place slowly, and had not ceased at the end of the observation period. After 15 months, after making allowances for radioactive decay 2-2 per cent of 59Fe citrate and 28-3 per cent of 59Fe oxide remained in the lungs.

It should be noted that at all times the 59Fe citrate was removed from the lungs more rapidly than the oxide. Of the 9*2 per cent iron citrate remaining in the lungs at the end of the first month 7 per cent was removed, while of the 61 per cent oxide, only 32-7 per cent was removed, i.e. little more than half.

The regional lymph nodes contained a significant amount of 59Fe after injection both of the citrate and the oxide. This suggests that at all periods after injection the lymphatics constitute one of the paths of removal of 59Fe from the lungs. The presence of a large quantity of iron in the lymph nodes was confirmed histologically (Fig. 1).

The results of distribution in the lungs of different 59Fe compounds at lengthy intervals after intratracheal injection enabled tissue radiation doses to be calculated.

The dose of ß-radiation received by the lungs was calculated by Marinelli's


method with additions by G.A.Avrunina (1960). The y-radiation dose received by the lungs was not calculated because only insignificant amounts of y-radiation, with high penetrative capacity, are absorbed in the lungs.

No cases of acute radiation injury were detected among the animals during the observation period. However, in the animals which received 59Fe oxide

FIG. 1. Cluster of 59Fe particles in the sinuses of a regional lymph node 12 months after injection of 59Fe oxide.

TABLE 3. Results in the Various Groups of Animals and Radiation Dose in the Lungs

3 o Ö) o 6

1 2 3 4 5

6 7

Vi

«* "S J8.§

9 12 15 10 15

8 11

Preparation injected and activity in μο

5 9Fe citrate (20) 5 9 Fe oxide (27-5) 5 9 Fe oxide (3-36) 5 9 Fe oxide (1-06) 5 9 Fe oxide (0-03)

(three injections) Stable iron oxide Physiological

control

Total 80

Relationship to maximum permissible dose

for single inhalation (Morgan)

More than 100 times More than 250 times More than 30 times More than 10 times At the same level

Rad

iatio

n do

se in

th

e lu

ngs

duri

ng t

he

first

day

(rad

s)

70 206

26 8-5 0-20

Rad

iatio

n do

se in

th

e lu

ngs

duri

ng

15 m

onth

s

587 9790 1296 484 138

Num

ber o

f ani

mal

s dy

ing

duri

ng

15 m

onth

s

1 4 3 3 2

1

13

4>

O KJ

| | 2 o

2 3 3

8


in amounts of 27-5, 3-36 and 1*06 μc a higher mortality rate was observed, caused mainly by inflammatory injuries of the lungs, formation of abscesses and bronchiectases and development of bronchial tumours (Table 3).

P A T H O L O G I C A L LESIONS IN THE O R G A N S OF THE EXPERIMENTAL A N I M A L S *

The organs of animals dying and killed at 3, 6, 8, 9, 12 and 15 months after injection were submitted to morphological examination.

Of the 9 animals which received soluble 59Fe citrate one died. Cause of death on the 390th day after injection was multiple bronchiectases and abscesses of both lungs.

In the animals killed 3-6 months after injection the injected material was found in the lungs in the form of friable, brown accumulations giving a positive iron reaction and situated mainly in the interalveolar septa. Most characteristic of this period were early fibrotic lesions of the pulmonary

FIG. 2. Distribution of 59Fe citrate (20 μο) in rat lung 9 months after injection. Local thickening of interalveolar septa, emphysema.

tissue in the form of local enlargement of interalveolar septa and the appearance of small emphysematous areas (Fig. 2).

In the regional lymph nodes, especially in the sinuses, large clusters of cells with a brown substance giving a positive reaction for iron were found.

* Histological examinations were carried out in the patho-anatomical Laboratory of the Institute of Occupational Hygiene and Disease AMN SSSR under the direction of Prof. P.P.Dvizhkov and with the participation of T.A.Kochetkova.


At the 9th month after 59Fe injection, apart from pronounced fibrotic lesions, there was a marked thickening of blood vessel walls and in a number of animals development of chronic catarrhal-desquamatous bronchitis with a change of the epithelium of the bronchial mucosa. The epithelial cells hypertrophied and the cytoplasm was vacuolated. In other places, where lymphoid follicles protruded into the bronchial lumen, the mucous membrane was thinned and the epithelial cells flattened.

FIG. 3. Suppurative bronchitis. Peribronchial and perivascular fibrosis. Compression of the lumen of the large bronchus. Metaplasia of bronchial epithelium into

stratified squamous. At 12 months after injection of 20 μο 59Fe citrate.

In animals killed 12-15 months after injection the most significant changes were seen in the bronchi. In most animals suppurative bronchitis with formation of multiple bronchiectases and focal pneumonia was observed. The epithelium of the bronchial mucosa was in places flattened and in others, polypose growths were observed. In 2 animals metaplasia of columnar epithelium of the bronchi into stratified squamous was observed (Fig. 3).

In the lungs the diffuse fibrotic process progressed with formation of collagen fibres around vessels and bronchi and causing obliteration of the lumen of some blood vessels. In 2 animals, the ectopie formation of bony tissue in the walls of the large vessels of the lungs and also in the pulmonary tissue itself, was observed (Fig. 4).

All animals killed at later periods displayed trachéal inflammation with infiltration inflammatory cells in the submucosa and degenerative lesions in the cartillage (Fig. 5).

Histological examination of the animals which received insoluble 59Fe oxide in doses of 106, 3-36 and 27-5 μc revealed no marked differences as-


FIG. 4. Pneumosclerosis, Formation of bone in lung tissue 12 months after injection of 59Fe citrate (20 μ^.

FIG. 5. Hyperplasia of submucosal glands and degenerative lesions of trachéal cartilage 12 months after injection of 59Fe citrate (20 μο).


sociated with the amount of radioactivity administered and consequently the results will be presented without consideration of dose.

Of the 37 animals of this group, 10 died during the 15-month observation period. Cause of death of 6 of the animals, which died between the 185th and 246th days after injection, was acute pneumonia with haemorrhage. His-tological examination revealed acute vascular engorgement and bleeding into the alveolar cavities (Fig. 6). The inflammatory cell reaction was weak, suggesting a state of reduced body reactivity.

cavities 7 months after injection of 59Fe oxide (1-06 μο).

Signs of acute congestion and oedema were observed also in the liver, kidneys, brain and spleen, thus indicating the general prevalence of the process.

The cause of death of one animal which died 84 days after injection was acute pneumonia with pyogenic abscesses and septic foci in various organs. No haemorrhagic manifestations were observed in this case.

One rat died 432 days after injection of 59Fe oxide. The cause of death was multiple lung abscesses and bronchiectases with localized foci of suppurative -necrotic pneumonia.

In 2 animals which died 346 and 410 days after injection of 59Fe oxide, cancer of the lungs was detected with métastases in regional lymph nodes and mesentery (these cases will be described below).

Thus, in all animals, the direct cause of death was pulmonary disease, which was the result of the conditions of the experiment, the action of ionizing radiation and the species characteristics of the animals.

In the animals of these groups which were killed 3 months after intratracheal 59Fe oxide injection, the material, as in the experiments with soluble


compounds, was found mainly in the interalveolar septa and in the peri-bronchial tissue, but was deposited in more compact lumps with little cell reaction around these masses.

In different parts of the lungs, often in groups, macrophages were found with inclusions in their protoplasm of fine, dark-brown, or black, particles staining positively by Perles's method.

FIG. 7. Suppurative bronchitis. Metaplasia of bronchial epithelium into stratified squamous. Peribronchial fibrosis. At 6 months after injection of 59Fe oxide

(27-5 μο).

In the regional bifurcated lymph nodes, especially along the sinus ducts, considerable accumulations of similar cells were observed and free dust particles, which is in complete conformity with the increased radioactivity of the lymph nodes revealed by radiometrical investigation.

In the remaining organs no marked pathological lesions were detected. After 6 months the fibrotic lesions of the lungs which were already ap

parent at earlier stages had become more pronounced. Together with local enlargement of interalveolar septa and areas of emphysema, marked activation of histiocytic elements with formation of fine collagen fibres was observed, especially in the peribronchial and perivascular tissue. As far as the blood vessels were concerned acute thickening of the walls of some medium and small arteries was seen, due to hypertrophy of the middle layer and this sometimes caused obliteration of the lumen.

In a number of animals there was acute hyperplasia of the glands of the submucosal layer and proliferation of lymphoid cells in the peribronchiar tissue.


Chronic suppurative bronchitis with areas of perifocal pneumonia, obliteration of the lumen of small bronchi and development of bronchiectases were observed in almost all animals. In some animals in the inflamed areas there was flattening of the bronchial epithelium and its metaplasia into stratified squamous was observed (Fig. 7).

In one animal foci of squamous-cell keratinizing cancer of the lungs were observed with formation of typical "pearls" (Figs. 8 and 9).

Investigation of animals killed or dying 9-12 and 15 months after injection of 59Fe oxide revealed that the further development of the pathological process in the lungs took the form of an extension of the chronic suppurative process with formation of multiple bronchiectases, inflammation and fibrosis of the surrounding pulmonary tissue.

In a number of animals the pulmonary fibrosis had led to obliteration of the small bronchi, but in other places, to widening of their lumen, which together with acute hyperplasia of the submucosal glands and lesions of the alveolar epithelium, gave a picture resembling adenomatous growths (Fig. 10).

FIG. 8. Foci of squamous-cell keratinizing cancer of the lungs and formation of typical "pearls" 6 months after injection of 59Fe oxide (3-36 c).

The most characteristic lesions were observed in the bronchial epithelium. In 14 of 24 animals killed at these periods squamous metaplasia of the bronchial epithelium was observed.

In 8 animals multiple foci of squamous cell keratinizing bronchial cancer (Table 4) with outgrowths of epithelial complexes into surrounding tissues and formation of typical "pearls", and extensive areas of necrosis up to complete destruction of the tissue were observed (Figs. 11, 12 and 13). In


FIG. 9. The same area greatly enlarged. Mitoses of tumour cells are clearly visible.

FIG. 10. Pronounced hyperplasia of bronchial submucosal glands of adenoma growth type. Compression of lumen of small bronchi. At 12 months after injection

of 59Fe oxide (3-36 μο).


¥ \ j *

· *

% j «M

f ^ i I f

■fl JWPp "c**,

*»v lCi#? i*=

»'"

iäiw

FIG. 11. Complexes of epithelial cells, growing into the lung tissue. Particles of the injected material can be seen between the complexes. At 12 months after injection

of 59Fe oxide (27-5 μ^.

FIG. 12. Suppurative-necrotic liquefaction of an area of tumour. At 15 months after injection of 1-06 μοΙ59¥ε.


\+*L

rf:3Ìl^*É ■ ■ ' : ν * Λ -

- ^ »SiPPi

FIG. 13. The same preparation greatly enlarged. Fragments of keratinizing "pearls" can be seen in the necrotic area.

FIG. 14. Metastasis of a cancer tumour in a regional lymph node. The rat died 13 months after injection of 59Fe oxide (27-5 μο).


2 animals tumour métastases were observed : in one animal in the regional lymph nodes (Fig. 14), and in the other in the mesenteric lymph nodes.

In all cases the tumour consisted of large atypical epithelial cells with light eccentric nuclei in which mitoses were often observed.

TABLE 4. Distribution of Lung Cancer in the Different Animal Groups and Time of its Detection

Activity irjected

(μ<0

1-06 27-5

3-36 3-36 1-06 1-06

27-5 3-36

Time of tumour detection

(months after 5 9 Fe injection)

6 9 9

12 12 12 13 14

Notes

Killed Killed Killed Died Killed Killed Died Killed

One rat developed numerous encapsulated tumour nodes. In this animal the cells comprising the tumour were notable for their great polymorphism (cf. Fig. 15). The expectional variety of size, shape and staining characteristisc

FIG. 15. Area of tumour greatly magnified. Extreme polymorphism of the tumour cells can be seen. At 12 months after injection of 59Fe (1-06 μο).


of cells and nuclei indicates the low degree of differentiation of the cellular elements making up the tumour.

Another lung tissue damage, in the later periods following injection of 59Fe, was the deposition of calcium and formation of bony tissue in the muscle layer of the large blood vessels and in the lung tissue itself. This was observed in 7 of the 24 animals. Moreover, in 3 animals of this group formation of bone in testicular vessels was detected.

In the regional lymph nodes, especially along sinus ducts, clusters of large cells containing many separate particles were seen. Injuries of the trachea were also a frequent occurrence. Moderate sclerotic and degenerative lesions were observed in the other parenchymatous organs. In the brain marked thickening of blood-vessel walls and perivascular and pericellular oedema of brain tissue was detected.

Of the 15 animals which received three injections of 0-03 \LQ 59Fe oxide, two died. The cause of death of one animal on the 96th day after injection was pneumonia with haemorrhage, the histological picture of which was very similar to that shown in Fig. 6. In the second animal, which died 302 days after injection of 59Fe, bronchiectases and numerous abscesses in both lungs were observed.

In the early stages after 59Fe injection, histological lesions in the lungs of animals of this group differed from those described above only in being less pronounced. Nevertheless, even here at the 3rd, 6th and 8th months developing pneumosclerosis with local enlargement of interniveolar septa, areas of emphysema, thickening of small and medium blood-vessel walls, formation of fine collagen fibres and hyperplasia of lymphoid elements could be observed. However, the suppurative inflammatory lesions which could be detected in animals receiving higher doses of 59Fe at the 5th-6th month after injection were observed in this group only after 12-15 months.

The epithelium of the bronchial mucosa was remarkable, in the early stages, for the extreme height and size of its cells. Later, as the inflammatory process in the bronchi developed, flattening of the epithelium was observed, with transition into cubic, and in places, into stratified squamous ; however, in no case were atypical malignant growths observed.

Deposition of calcium and formation of osseous tissue was observed in pulmonary blood-vessels in 4 animals 12-15 months after 59Fe injection. As in previous groups, a number of animals showed acute hyperplasia of sub-mucosal glands and proliferation of lymphoid elements in the peribronchial tissue.

In the trachea, regional lymph nodes, spleen and other parenchymatous organs lesions identical with those described for earlier groups were also found.

In the animals of the control group, which also received an intratracheal injection of a stable iron oxide suspension, some sclerotic lesions of pulmonary tissue were detected 9-15 months after injection; these were within the range of age changes. Suppurative inflammatory phenomena were detected in the lungs of 3 animals, in one of which squamous metaplasia of the bronchial epithelium in a focus of inflammation was observed; but in no


instance was atypical cancerous growth of the bronchial epithelium or development of bony tissue in the vessels or parenchyma of the lungs detected.

Thus, prolonged deposition of 59Fe preparations in the lungs increased the frequency of diseases of the respiratory organs and gave rise to the development, in a number of animals, of malignant tumours of the bronchi.

Following intratracheal injection of rats with soluble 59Fe citrate and insoluble 58Fe oxide lesions of the lungs were found in all animals.

The histological lesions were of similar type in all the groups, but they developed to differing degrees according to the type of compound injected (soluble or insoluble) and its radioactivity. Whereas the animals which received 27-5, 3-36 and 1Ό6 \LC 59Fe oxide, after 6-15 months showed serious inflammatory lesions and squamous cell carcinoma of the bronchi, the animals receiving 0Ό3 \LC 59Fe oxide and 20 \LC 59Fe citrate showed similar inflammatory symptoms but to a lesser degree, and no concurring lesions.

In our experiments the development of bronchial tumours was observed at comparatively low total radiation doses (480-9790 rads), and this was associated with the high radiation dose received by small areas of tissue. The tumours developed at late stages against a background of external well-being. This confirms the fact that carcinogenic activity caused by ionizing irradiation can occur without overt radiation sickness.

The minimal amount of 59Fe producing lung cancer only exceeded the maximum permissible for a single injection (according to Morgan) by 10 times.

The importance of the chemical state of the radioactive substance, especially its solubility, is clearly seen in the experiments with intratracheal injection of 59Fe citrate. Injection of 20 μο of this material, which during the 15 months of the experiment gave a total dose to the lungs of 587 rads, in no case gave rise to tumours. This can be explained by the fact that most of the soluble 59Fe compound rapidly leaves the lungs. During the first few days after injection there is a high radiation dose (the radiation dose to the lungs during the first 10 days was 312 rads), but this dose declines quickly. However, as was indicated above, prolonged irradiation over some time is one of the factors causing the carcinogenic effect.

These injections of 0Ό3 μο 59Fe oxide, i.e. injection of the maximum permissible radiation dose, was not followed, in our experiments, by the development of malignant tumours. However, lesions of the pulmonary tissue, mainly of an inflammatory and sclerotic nature, were observed in these animals.

Our results provide evidence of the high radiotoxicity of different 59Fe compounds when administered through the respiratory tracts and can be used for the calculation or permissible levels of soluble and insoluble 59Fe compounds in inspired air.

TRS 11



1. Intratracheal injection of white rats with 20\LC of 59Fe citrate increased the frequency, by comparison with the controls, of the development of suppurative inflammatory lesions in the bronchi with pronounced sclerotic lesions in the peribronchial tissue.

2. Intratracheal injection of 27*5, 3*36 and 1Ό6 μc 59Fe oxide, which exceeds the maximum permissible dose for a single inhalation according to Morgan by respectively 250, 30 and 10 times, resulted in a higher mortality rate among the experimental animals, mainly caused by infections of the respiratory tract and the development of malignant tumours in the lungs.

3. Serious pathological lesions were observed in the lungs of animals which received 27-5, 3-36 and 1Ό6 \LQ, 59Fe oxide. Most characteristic was the development of chronic suppurative lesions of the bronchi with acute sclerotic changes in the peribronchial tissue, formation of bronchiectases and squa-mous metaplasia of the bronchial epithelium. In a number of animals ossification of connective tissue was observed.

4. In 8 animals of this group, against a background of serious inflammatory lesions, the development of squamous cell carcinoma of the bronchis was observed. Cancer of the bronchis was observed in animals from the 6th month after 59Fe injection at doses to the lung tissue of 480 to 9790 rads.

5. Three intratracheal injections of 0Ό3 fxc 59Fe oxide, i.e. at the level of the maximum permissible lung intake of 59Fe, gave rise to pronounced lesions of pulmonary tissue of a sclerotic and imflammatory nature.

6. The data obtained concerning the high carcinogenic activity of 59Fe oxide can be used as the basis for a more critical approach to the standardization of permissible atmospheric concentrations of insoluble 59Fe compounds.

REFERENCES

AVRUNINA G.A., Toxicology of Radioactive Substances (Materialy po toksikologii radio-aktivnykh veshchestv), vol. 2, pp. 14-26, Medgiz (1960).

BRYUS O., Advances in Cancer Study (Uspekhi v izuchenii raka), vol. 2, pp. 194-215, Moscow (1956).

BURYKINA L.N., Toxicology of Radioactive Substances (Materialy po toksikologii radio-aktivnykh veshchestv), vol. 1, pp. 115-130, Medgiz (1957).

CEMBER H., Second United Nations International Conference on the Peaceful Uses of Atomic Energy, p. 900 (1958).

CEMBER H., WATSON J. A. and SPRITZER A.A., Arch. Ind. Health, 19 (1), 14-24 (1959). F I N K E L M . P . et al., Second United Nations Conference on the Peaceful Uses of Atomic

Energy, p. 911 (1958). HORN H.A. and STEWART H., / . Nat. Cancer Inst., 12, 743-749 (1952). KOCHETKOVA T. A. and AVRUNINA G. A., Toxicology of Radioactive Substances (Materialy

po toksikologii radioaktivnykh veshchestv), vol. 2, pp. 153-170, Medgiz (1960). Lisco H. and FINKEL M., Feder. Proc, 8, 360-361 (1949). MARINELLI L.D. , / . Clin. Invest., 28 (6), 1271-1281 (1949).


MOSKALEV Yu. I., The time factor in injury by radioisotopes experimentally (O roli faktora vremeni pri porazhenii radioaktivnymi izotopami v eksperimente). Proceedings of a conference on radiation medicine, Moscow (1959).

POBEDINSKII M.N., Med. radiol, 5, 30-40 (1956). SHIMKIN M., Advances in Cancer Study (Uspekhi v izuchenii raka), vol. 3, pp. 62-112,

Moscow (1957). STREL'TSOVA V.N., The leucogenic effect of strontium-90 (O leikomogennom deistvii

strontsiya-90). Collected abstracts on radioactive strontium, Moscow (1959). STREL'TSOVA V. N. and MOSKALEV YU. I., Connections between the tumour effect of radio

active substances and their distribution in the body and physical properties (Zavisimost' opukholevogo deistivya radioaktivnykh veshchestv ot tipa raspredeleniya ikh v orga-nizme i fizicheskikh svoistv). Proceedings of X Session of Scientific Conference of AMN SSSR (1956).

WOOLLEY P. J. and WHERRY W.B., / . Med. Research, 25, 205-217 (1911).

MORPHOLOGICAL LESIONS IN THE ORGANS OF RABBITS DURING PROLONGED

ADMINISTRATION OF 59Fe

E. S. G A I D O V A

THERE are many reports in the literature concerned with the acute effect on the body of external and internal irradiation. However, not enough work has been done on the effect of small amounts of radioactive materials invested over a protracted period. Furthermore, morphological lesions in organs in the chronic experiment have received little study. Histological lesions following internal and external irradiation are described in the greatest detail by Bloom (1948), while Hempelman, Lisco and Hoffman (1954), give some interesting data. N.A.Kraevskii (1956) in his monograph Outlines of the Pathological Anatomy of Radiation Sickness devoted a whole chapter to "so-called internal irradiation". V. N. Strel'tsova (1952) describes lesions in the organs following various means of administration of 89Sr, 90Sr, 106Ru and 90Y, while L.N.Burykina (1957) used 89Sr, 134Cs and 106Ru for the same purpose, but their results are concerned chiefly with the acute poisoning. A.P.Novikova (1956) has studied morphological lesions in animal organs 5-46 months after administration of uranium salts and fission products ; and N.N.Litvinov (1957, 1958), lesions in bone tissue following prolonged 90Sr intoxication. V. V. Shikhodyrov (1956) has investigated lesions in the areolar tissue during chronic radiation sickness. Pathomorphological lesions following intake of radioactive thorium in small doses have been described by E.V.Erleksova(1956).

T. A. Kochetkova and G. A. Avrunina (1956,1957) have described the long-term effects of intratracheal injection of 24NaCl, Cr 3 2 P0 4 and colloidal 98Au, and A. S. Kaplanskii (1960) histological lesions during prolonged administration of 60Co. In the last 2-3 years many reports have appeared devoted to the long-term effects of previous acute radiation sickness (L. N. Bu-rykina et al, (1959), L.V.Mel'nichenko, 1956; A.P.Novikova, 1956, 1961; M.M.Aleksandrovskaya, 1958; M.Dmitrov and G.Markov, 1956; and many others). We have been unable to find any publications describing morphological lesions during prolonged administration of 59Fe.

The present paper describes part of a general investigation by this laboratory, begun in 1957, to study the effect of prolonged 59FeCl3 administration on experimental animals (rabbits).

Radioactive iron was administered orally every day apart from holidays in solution at a dose of 10 or 1 μ ο ^ .

158

Morphological Lesions in the Organs of Rabbits 159

The animals were divided into 4 groups: 2 experimental and 2 control (the animals of one control group received stable iron, the other rabbits received nothing). There were no differences in the morphological lesions observed in the rabbits of both control groups and so they were joined together as a single control group. The experimental and control animals were maintained under similar conditions.

The tissues of 97 rabbits, dying during the 2\ years, were examined macro-scopically and microscopically.

Methods. Air embolism was used to kill 56 rabbits, the rest dying. The material from killed and dying animals was fixed in 12 per cent neutral formalin, Orto Muller's fluid and 80° alcohol. It was then poured into celloidin and part of it, required for histo-chemical examination, poured into paraffin wax. The material was stained with haematoxylin-eosin, by Van Gieson's and Perles's method for iron. All organs were taken for histological examination.

E X P E R I M E N T A L R E S U L T S

In animals of the first group, which received 10 μc/kg 59FeCl3 (42 rabbits), no signs of intoxication were detected over a prolonged period. Their coat was downy, clean and they put on weight. Changes in the blood have been described by N. L. Beloborodova, E.K.Red'kina and V.L.Ponomareva in this volume. In some animals a fall in weight was observed, associated with secondary infection—generally pneumonia, from which the animals died.

At 1-4 months after commencement of administration of 59Fe neither macro- or microscopical lesions of organs could be seen in animals killed with no symptoms of intoxication. In animals which died, reduced feeding and bronchopneumonia was observed. Microscopic examination revealed foci of catarrhal bronchopneumonia in the lungs; extensive lymphocytic infiltration around the bronchi and blood vessels, and in places, foci of myeloid haemopoiesis; and protein dystrophy of the cell in the parenchymatous organs.

After 7-10 months increased fat deposition in the subcutaneous, retro-peritoneal and perinephric cellular tissue and in the mesentery could be seen macroscopically in all rabbits. Some animals suffered from pneumonia. In these animals the lungs showed many foci of consolidation. Microscopically, in the lungs, against a background of diffuse pneumosclerosis, catarrhal, with transition to purulent, pneumonia could be seen. Proliferati ve lesions of the bronchial epithelium were found, with mitoses, polypose growths of the bronchial mucosa and peculiar adenomatous growths of the bronchial muco-sal glands. Blood vessel walls in the lungs were loose and oedematous. There was interstitial oedema of the myocardium. Sclerotic and dystrophic lesions were found in the kidneys, dystrophic lesions of the parenchymal cells in the liver, and small round-cell infiltration around the interlobular vessels. In the spleen—congestion, oedema, desquamatous catarrh of the sinuses, and foci of myeloid haemopoiesis. The bone marrow was rich in cellular elements,


mature cells of the white and red series predominating. In some cases hyper-plasia of the epithelium and the reduction or complete disappearance of colloid in the thyroid gland was observed. In the stomach and small intestine superficial necroses of the mucosa were sometimes observed, and slight oedema of the submucosa.

After 11-14 months fatty change could be detected macroscopically in animal organs; and in those dying, the lungs showed many areas of consolidation.

In the lungs, the microscopic picture showed catarrhal desquamatous bronchopneumonia against a background of moderate diffuse fibrosis. Atypical growth of the bronchial epithelium, mitoses, in places polypose growths of the mucosal epithelium, signs of adenomatosis, and multiple foci of myeloid haemopoiesis were observed. In rabbit No. 2 there was a disparity between the morphological lesions and the gravity and duration of the disease. In the lungs large areas of liquefaction of the pulmonary tissue were seen, bacterial emboli were present in the vessels and bronchi and an almost

FIG. 1. Atrophy of gland cells of the stomach mucosa, sclerotic lesions of the stroma. Oedema of the submucosa. Haematoxylin-eosin stain. Administration of 10 acjkg

for 14 months. Photomicrograph (oc. 7, ob. 8)

complete absence of neutrophil leucocytes in the exudate. Moreover, there were septic emboli in the vessels of the myocardium and kidneys without a leucocytic reaction around them, probably indicating that the reactive capacity of the body has been reduced, as a result of which lethal infectious process of septicaemic type occur. In the liver, protein dystrophy of hepatic


cells was observed, sometimes cirrhosis; in the kidneys slight sclerosis, granular dystrophy of the epithelium of the convoluted tubules; in the spleen congestion, oedema, localized myeloid haemopoiesis and a slight reduction in the number of lymphatic follicles. Moderate atrophy of the mucous membrane and sclerosis of the stroma of the gastro-intestinal tract could be seen

FIG. 2. Interstitial oedema of the myocardium, and small haemorrhages along capillaries. Haemotoxylin-eosin stain. Administration of lOfxc/kg for 16 months.

Photomicrograph (oc. 10, ob. 40)

(Fig. 1). The bone marrow was active, rich in cellular elements, in which there was a small increase of megakaryocytes, and mature cells predominated over young cells. In one rabbit atrophy of the spermatogenic epithelium of the seminiferous tubules was seen, with sclerosis of the stroma of the testis. In the thyroid glands of 2 rabbits the follicles were small, the epithelium surrounding them high, almost completely filling the lumen, colloid absent.

After 15-16 months macroscopic features were adiposis and lungs with scattered foci of consolidation. Miscroscopically, in both dying and killed rabbits, interstitial oedema of the myocardium was observed (Fig. 2), and small haemorrhages along the capillaries. In the lungs, against a background of diffuse pneumosclerosis, there was focal catarrhal desquamatous broncho-pneumonia with liquefaction of pulmonary tissue and a relatively reduced neutrophil reaction (Fig. 3). Atypical growth of the bronchial epithelium was also noted (Fig. 4), adenomatous growths of the bronchial mucous glands (Fig. 5), desquamation and oedema of blood vessel walls (Fig. 6) and foci of myeloid haemopoiesis in the lung tissue; and in certain cases, a large number


FIG. 3. Bronchopneumonia with a very slight neutrophil reaction, oedema. Haema-toxylin-eosin stain. Administration of l(^c/kg for 15 months.


FIG. 4. Pneumonia. Bronchial epithelium atypical. Haemotoxylin-eosin stain. Administration of 10 μο/kg for 15 months.



' Α ' ί ί ΐ * * ^ %■ Ä K H ^ l

FIG. 5. Bronchopneumonia, adenomatosis, metaplasia of bronchial epithelium. Haematoxylin-eosin stain. Administration of l(^c/kg for 15 months.


FIG. 6. Lung: growth of internal membrane of arteriole, perivascular oedema. Haematoxylin-eosin stain. Administration of 10μΰ/1<£ for 16 months.



of giant cells (Fig. 7). In the liver congestion, oedema, granular and adipose dystrophy of cells, occasionally small localized necrosis (Fig. 8) and early cirrhosis were observed. In the kidneys of 2 rabbits, serous fluid, signs of protein dystrophy of the epithelium of the convoluted tubules and localised fibrosis were seen in some glomeruli. In the spleen the lymphoid tissue was reduced (Fig. 9), and in all the animals congestion, oedema and localized

FIG. 7. Giant-cell bronchopneumonia with a relatively reduced cellular reaction. Haematoxylin-eosin stain. Administration of l(^c/kg for 14 months.


mycloid haemopoiesis were observed. The bone marrow was active and rich in mature cellular elements. Slight atrophy of the mucous glands of the stomach, small and large intestine was observed. In the testes atrophy of the spermatogenic epithelium, tubules, absence of sperms and sclerosis of the stroma were seen (Fig. 10).

After 17-18 months in the lungs of all rabbits moderate diffuse sclerosis, and in dying animals, a superimposed suppurative bronchopneumonia with a marked leucocytic reaction and slight atypical growth of the bronchial epithelium were observed. Localized myeloid haemopoiesis, dystrophic lesions of hepatic cells and of the epithelium of the convoluted tubules in the kidneys; congestion, oedema, reduction in the number and size of lymphatic follicles in the spleen; atrophy of the spermatogenic epithelium of the seminiferous tubules, reduction or complete disappearance of sperms, slight atrophy of gastric mucous glands and oedema of the submucosa of the stomach and intestine.


FIG. 8. Fatty degeneration and focal necroses in the liver. Haematoxylin-eosin stain. Administration of 10 μ^1<^ for 17 months.


FIG. 9. Spleen, reduction of lymphoid elements. Loss of the pattern and deposition of an iron-containing pigment. Haematoxylin-eosin stain. Administration of ^ c / k g



After 21 months localized, in places confluent, bronchopneumonia with marked leucocytic infiltration could be seen in the lungs of dying rabbits. In all the animals of this group slight diffuse pneumosclerosis and degenerative lesions in the cells of the liver and renal convoluted tubules were observed. In the spleen of animals dying from pneumonia, some reduction of lymphoid tissue, with many plasma cells and localized myeloid haemopoiesis

FIG. 10. Destruction of tubules, atrophy of remaining spermatogenic epithelium. Sclerosis of stroma of testis. Haematoxylin-eosin stain. Administration of 10 μϋ/kg


was seen. In killed animals, hyaline degeneration of small artery walls in the spleen was observed, and in the testes atrophy of the spermatogenic epithelium of the seminiferous tubules, and hypertrophy of the stroma. Atrophy of mucous glands and oedema and sclerosis of the submucosa were seen in the stomach and intestine.

Thus, it can be seen that the most pronounced lesions in rabbits receiving the large dose (10 μ ΰ ^ ) of 59Fe are observed 10-17 months after commencement of administration. A significant number die from associated secondary infections. Secondary infection in the animals of this group was of the chronic type, with a weak neutrophil reaction in the foci of inflammation, suggesting that resistance had been reduced.

In almost all the animals of this group adiposis could be seen macro-scopically. Microscopical examination of the organs revealed oedema of the myocardium, diffuse and focal sclerosis of pulmonary tissue, atypical proliferation of the bronchial epithelium, and, in places, polypose growths of the bronchial mucosa, mitoses, appearance of abnormal cells, adenomatous


growths of the mucous glands of the bronchi, catarrhal-desquamatous bronchopneumonia with a weakly expressed leucocytic reaction, degeneration of blood vessel walls, a reduction in the amount of lymphoid tissue in the spleen, protein dystrophy of hepatic cells and of the epithelium of the renal convoluted tubules. In certain animals hyperplasia of the epithelium of the thyroid gland follicles occurred ; epithelium completely filled the lumen of the follicles, and the size of the latter was reduced. The colloid content was reduced or even completely absent. Atrophy of the spermatogenic epithelium of the gonads was observed. Atrophie lesions were seen in the epithelium of the mucous glands, with oedema and sclerosis of the submucosa of the stomach and intestine.

In killed animals of the second group, which received 1 μ^1<£ 59FeCl3 (20 rabbits), no morphological lesions were found 3-6 months after the commencement of administration. In animals which died, reduced feeding and lungs with many foci of consolidation could be observed macroscopic-ally. Microscopically, catarrhal desquamatous bronchopneumonia, congestion and protein dystrophy of liver cells and of the epithelium of the convoluted tubules in the kidneys could be observed. No lesions were found in other organs.

After 7-10 months no macroscopic lesions could be seen in animals killed without any clinical manifestations of intoxication.

Microscopic examination of the lungs revealed moderate diffuse pneumo-sclerosis, proliferation of lymphoid tissue, proliferation and slight atypical growth of the bronchial epithelium with mitoses and polypose growths, and local myeloid haemopoiesis. In the liver and kidneys granular dystrophy was observed ; in the spleen congestion, oedema, and multiple foci of myeloid haemopoiesis. The bone marrow was rich in mature cells; slight oedema of the submucosa of the stomach and small intestine occurred. In animals which died, suppurative pleurisy and suppurative bronchitis could be seen macro-scopically. Microscopically, the lungs showed catarrhal bronchopneumonia with extensive leucocytic infiltration. In other organs the lesions were similar to those found in the killed animals.

After 11-14 months adiposis, congestion of internal organs; and in dying rabbits, suppurative pleurisy and bronchopneumonia were observed. Microscopic examination of the lungs revealed catarrhal desquamatous bronchopneumonia against a background of slight diffuse sclerosis and numerous foci of myeloid haemopoiesis; in the liver and kidneys dystrophic lesions of the parenchymal cells and moderate sclerosis of the stroma were detected; in the spleen, multiple foci of myeloid haemopoiesis. The bone marrow contained mainly mature cells of the white and red series; no particular lesions were found in the endocrine glands. In the stomach vacuolization of mucous gland epithelium and oedema of the submucosa were observed.

After 16-18 months no lesions could be found macroscopically in killed rabbits apart from adiposis; catarrhal bronchopneumonia was observed in animals which died. In all the rabbits moderate diffuse pneumosclerosis occurred. The bronchial epithelium was hypertrophied but without atypical


growths, while hyperplasia of lymphoid tissue around the bronchi and localized myeloid haemopoiesis could be seen. In the liver and kidneys dystrophic lesions of the parenchymal cells and early cirrhosis were observed. In one animal necrotic nephrosis occurred in the kidneys. In the spleen of a rabbit dying from pneumonia slight reduction of the lymphoid tissue had occurred. In all animals there was oedema, congestion, and numerous foci of myeloid haemopoiesis in the spleen. Slight oedema of the submucosa of the stomach and small intestine were also noticed.

After 21 months all the remaining animals were killed; none showed any signs of intoxication; adiposis was observed in all. In the lungs moderate diffuse pneumosclerosis and hyperplasia of the bronchial epithelium were observed, and in one animal adenomatous growths of the bronchial mucous glands; there were foci of myeloid haemopoiesis. In the liver granular dystrophy of cells and round cell infiltrates around the blood vessels were observed; granular dystrophy of the epithelium of the convoluted tubules in the kidneys could also be seen. In the spleen oedema and numerous foci of myeloid haemopoiesis were seen. The bone marrow contained many mature cells. There was slight sclerosis of the stroma of the stomach and intestine and a reduction of the number of glands in the mucous membrane.

In rabbits, receiving the small dose of 59FeCl3 (1 μc/kg), inflammatory processes pursued a more acute course than in rabbits receiving the larger dose, where the inflammatory reaction was sluggish. Adiposis was observed 11 months after the beginning of the experiment. Histological examination of the lungs of both killed and dying rabbits revealed, apart from the usual bronchopneumonia, slight diffuse pneumosclerosis, moderate atypical overgrowth of the bronchial epithelium and degeneration and oedema of bloodvessel walls. In isolated cases, a slight reduction in the amount of lymphoid tissue in the spleen could be observed.

In the control group (35 rabbits) the majority of animals were clinically healthy throughout the experiment and only a few died from pneumonia. As is known, pneumonia frequently occurs spontaneously in rabbits and is a species characteristic of these animals.

After 1-4 months, in animals killed by air embolism there were no macro-or microscopic lesions in any organs.

After 7-10 months no macroscopic lesions could be seen. Microscopically it was possible to detect a weak protein dystrophy in the liver cells. No morphological lesions could be found in the haemopoietic organs or gastrointestinal tract.

After 11-14 months slight dystrophic lesions of the parenchymal cells in the liver and kidneys was detected. No macro- or microscopic lesions were observed in the haemopoietic organs, gonads or gastro-intestinal tract.

After 15-21 months no morphological lesions were detected in the organs of 13 killed clinically healthy rabbits.

In the very small number of rabbits dying from pneumonia the course differed greatly from the pneumonia observed in the experimental animals, and took an acute course with a vigourous leucocytic reaction.


Comparison of the lesions occurring in the control and experimental animals shows that pathomorphological changes in the latter differ considerably from those occurring in the controls. These changes are more clearly seen in rabbits receiving the larger dose (10 μc/kg) than in those receiving 1 μc/kg 59Fe. The most marked lesions are observed from 10 to 17 months. In the experimental animals which were killed with no signs of intoxication, diffuse pneumosclerosis in the lungs, atypical growth of the bronchial epithelium, and degeneration of blood vessel walls were seen ; in the liver and kidneys protein dystrophy occurred; in the spleen the amount of lymphoid tissue was reduced and numerous foci of myeloid haemopoiesis occurred, especially with pneumonias. A. S. Kaplanskii found similar lesions during prolonged administration of 60Co. In the thyroid gland we observed a reduction in the size of follicles, hyperplasia of the epithelium and a reduction or complete disappearance of colloid. In the stomach and intestine, moderately pronounced atrophy of the epithelium of the mucous glands occurred. In the gonads atrophy of the spermatogenic epithelium was observed in the later stages of the experiment. In the animals receiving the smaller dose, these changes were not so marked. In the control group, in animals killed at the same periods, apart from slight diffuse pneumosclerosis of the lungs and slight dystrophic lesions in the parenchymatous organs, no morphological abnormalities were found.

From the material set out above it can be seen that a considerable number of the experimental animals died from complications, the number increasing with increasing duration of intoxication.

The commonest cause of death was pneumonia. Pneumonia also occurred in the control animals but infrequently, and followed a more acute course accompanied by a vigorous leucocytic reaction. In the lungs of the experimental animals which died from pneumonia, extensive areas of tissue liquefaction with a considerably reduced leucocytic reaction were found. This was accompanied by proliferation of epithelium in the bronchi, mitoses and adenomatous growths in the mucous glands, and also by more pronounced dystrophic lesions in the parenchymatous organs (small areas of necroses in the liver, occasionally necrotic nephrosis), oedema of the myocardium. Frequently, a chronic septicaemia occurred. These results are in agreement with the literature. N. A. Kraevskii (1959) in his report to the Illrd All-Union Congress of Patho-anatomists indicated that chronic radiation sickness may not be manifested at all for a very long time, but different stresses, including an infectious process, immediately reveal significant abnormalities, because the defensive and regenerative capacities of the body are reduced. It must be pointed out that not all the changes observed are specific for 59Fe. Similar changes have been described by various workers using other radioactive substances and using different methods of administration and chronic exposure, and also at remote periods after a previous radiation sickness. A.P.Novikova (1959), analysing the long-term effects of uranium administration, reported that the direct cause of death of rabbits in these cases was most often pneumonia. The features of pneumonia and lesions in the lungs


have been described in detail by A. A. Pinus (1957). L. V. Mel'nichenko (1959) found, in the lungs of dogs a year after acute radiation sickness, adenomatous growths of the bronchial mucous glands, by description, similar to those found by us. F. L. Leites and L. S. Ruzer (1959) have found in rats which had inhaled radon, atypical epithelial growths in the lungs. G. A. Zedgenidze (1958), describing anatomical lesions of organs and systems in chronic radiation sickness, reported the possibility of development in the lungs of fibrosis and pneumosclerosis. V.B.Zairatyants (1960) describes the occurrence in the stomach and intestine of atrophie and sclerotic lesions at late stages in chronic radiation sickness. M.M.Aleksandrovskaya (1958), E. K. Dzhikidze (1959), P.N.Kiselev et al (1958), L.Ya.Ebert (1958), and O.I.Bazan (1958) have reported the lessened reactivity of the animal body as a consequence of radiation injury and described the course of inflammatory processes during infections. S.N.Sergeev (1957) and many others reported that a physical stress which occurs simultaneously with the course of a radiation injury, intensifies the dystrophic lesions occurring in the organs.


1. Prolonged administration of 59FeCl3 to rabbits produces pathological lesions in organs and, by lowering the body's resistance, predisposes to secondary infection.

2. In most rabbits the following were observed : (a) reduction of the amount of lymphoid tissue in the spleen and the ap

pearance of numerous foci of myeloid haemopoiesis; (b) different degrees of pneumosclerosis of the lungs, atypical growth of

the epithelium and adenomatosis of the bronchial mucous glands ; (c) oedema of the myocardium; (d) atrophie and sclerotic lesions of the mucosa of the gastro-intestinal

tract ; (e) dystrophic, and sometimes even necrotic lesions of the liver and kid

neys; (f) dystrophic and atrophie lesions of the gonads ; (g) general adiposis. 3. The degree of development of these pathological lesions is directly

proportional to the amount of 59Fe received.

REFERENCES

ALEKSANDROVSKAYA M.M., Tr. Inst, vysshei nerv, dey at. Moscow, 4, 126-137 (1958). BAZAN O.I., Staphylococcal pneumonia in irradiated animals (Stafilokokkovaya pnevmo-

niya u obluchennykh zhivotnykh). Proceedings of the 13th Scientific Conference of the Leningrad Medical Institute, Leningrad (1958).

BELOBORODOVA N. L., The chronic effect of radioactive ruthenium, caesium and strontium under experimental conditions (Materialy k khronicheskomu vozdeistviyu radioaktiv-


nykh ruteniya, tseziya i strontsiya v usloviyakh eksperimenta). Transactions of a meeting of institutes of hygiene on the problem of ionizing radiation, Moscow, pp. 28-30 (1955).

BLOOM W., Histopathology of Irradiation from External and Internal Sources, New York (1948).

B U R Y K I N A L . N . , The Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, Moscow (1957).

BURYKINA L.N. and ZAKUTINSKII D. I . et al., Med. radiol, 3, 3 (1959). DMITROV M. and MARKOV G., Izv. Inst. biol. Bolgar. Akad. nauk. (Sofia), 7, 231-278,

(1956). DzHiKiDZE E. K. and AKSENOVA L.S., Med. radiol., 4, 44 (1959). E B E R T L . Y A . , The effect of X-irradiation on the course of experimental streptococcal

pneumonia (Vliyanie rentgenovskogo oblucheniyana techenie eksperimental'noi strepto-kokkovoi pnevmonii). Proceedings of a Scientific Conference on the Chelyabinsk Institute to celebrate the 40th Anniversary of the Great October Socialist Revolution, Chelyabinsk (1958).

ERLEKSOVA E.V., Pathomorphological lesions in animals at long periods after small doses of radiothorium (Patomorfologicheskie izmeneniya u zhivotnykh v pozdnie sroki posle vozdeistviya radiotoriem v malykh dozakh). Abstracts of a Conference of the long-term effects of ionizing radiation, Moscow (1956).

HEMPELMAN L., LISCO G. and HOFFMAN D. , The Acute Radiation Syndrome (Ostryi luche-voi sindrom) (translatedfrom the English), Izd. IL, Moscow (1954).

IL'INA L.I. , Some aspects of protein metabolism in cell organoids of rat tissues in acute radiation sickness (Nekotorye storony belkovogo obmena v organoidakh kletok tkanei krys pri ostroi luchevoi bolezni). Dissertation, Moscow (1959).

KAPLANSKII A.S. , The Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, Moscow (1960).

K I S E L E V P . N . , RABINOVICH R .M. and M E T R I . D . , Med. radiol, 6, 3 (1938). KOCHETKOVA T. A. and AVRUNINA G. A., Lesions in the lungs and other organs following

intratracheal injection of certain radioactive isotopes (Ismeneniya v legkikh i drugikh organakh pri intratrakheal'nom wedenii nekotorykh radioaktivnykh izotopov). Proceedings of an All-Union conference on Medical Radiology, pp. 110-111, Moscow (1956).

KOCHETKOVA T. A. and AVRUNINA G. A., Toxicology of Radioactive Substances (Materialy po toksikologii radioaktivnykh veshchestv), vol. 2, pp. 153-170, Moscow (1957).

K R A E V S K I I N . A . , Outlines of the Pathological Anatomy of Radiation Sickness (Ocherki patologicheskoi anatomii luchevoi bolezni), Moscow (1956).

K R A E V S K I I N . A . , The chronic effect of low levels of radiation (K voprosu o khroniches-kom deistvii malykh urovnei radiatsii). Report ot the I l l rd All-Union Congress of Patho-anatomists, Kharkov (1959).

LEITIS F.L. and RUZER L.S., Arkh. patol, 1, 20 (1959). L I T V I N O V N . N . , Arkh. patol, 1, 25-30 (1957). L I T V I N O V N . N . , Med. radiol, 1, 41 (1958). MAKAROVA V. A., The pathomorphology of the central nervous system in the acutest form

of radiation sickness (Patomorfologiya tsentral'noi nervnoi sistemy pri ostreishei forme luchevoi bolezni). Dissertation, Leningrad (1959).

MEL'NICHENKO L. V., Lesions in animals at remote periods after radiation sickness (Izmeneniya v organizme zhivotnykh v otdalennye sroki posle perenesennoi luchevoi bolezni). Transactions of I l l rd All-Union Congress of Patho-anatomists, p. 487, Kharkov (1961).

M U D R E T S O V N . L , Pathoanatomical features of pulmonary complications of acute radiation sickness (Patologoanatomicheskaya kharakteristika legochnykh oslozhnenii ostroi luchevoi belezni). Proceedings of an All-Union Conference on Medical Radiology, Moscow (1956).

NOVIKOVA A. P., Morphological lesions in animals at remote periods after administration of certain radioactive substances (Morfologicheskie izmeneniya z zhivotnykh v otdalennye sroki posle vvedeniya radioaktivnykh veshchestv). Abstracts from a Conference on the long-term effects of ionizing radiation, Moscow (1956).

TRS 12


NOVIKOVA A.P., Long-term effects of single and multiple administration of uranium (Ot-dalennye posledstviya odnokratnogo i povtornogo otravleniya uranom). Transactions of I l lrd All-Union Congress of Patho-anatomists, Kharkov (1961).

PINUS A.A., Arkh. patol, 9, 27-35 (1957). Radioactive Decay and Medicine (translation from English), Izd. IL (1956). SERGEEV S.N., Heart changes in radiation sickness under various fatigue conditions (Iz-

meneniya serdtsa pri luchevoi bolezni v usloviyakh razlichnogo utomleniya organizma). Dissertation, Leningrad (1957).

SERGEEV S.N., Arkh. patol, 4, 29 (1960). SHIKHODYROV V. V., Lesions of areolar tissue in chronic radiation sickness (Izmenenie

rykhloi soedinitel'noi tkani pri khronicheskoi luchevoi bolezni). Abstracts of a Conference on the remote effects of ionizing radiation, pp. 29-32, Moscow (1956).

STREL'TSOVA V.N., The comparative morphology of radiation injury by some radioactive products of uranium (Sravnitel'naya morfologiya luchevogo porazheniya nekotorymi radioaktivnymi oskolkami urana). Dissertation, Moscow (1952).

STREL'TSOVA V.N. and BULDAKOV L. A., Med. radio!., 5, 37 (1958). ZAIRATYANTS V.B., Med. radiol, 1, 35 (1960). Z E D G E N I D Z E G . A . , Functional and anatomical lesions of organs and systems in chronic

radiation sickness (Funktional'nye i anatomicheskie izmeneniya organov i sistem pri khronicheskoi luchevoi bolezni). Proceedings of a scientific conference to celebrate the 40th Anniversary of the Central Research Institute of Roentgenology and Radiology, pp. 19-22, Leningrad (1958).

THE EFFECT OF THE CALCIUM D I S O D I U M S A L T O F

CYCLOHEXANE-DIAMINOTETRA-ACETICACID [CaNa2(CDTA)], THE CALCIUM DISODIUM SALT OF ETHYLENE-DIAMINOTETRA-

ACETIC ACID [CaNa2(EDTA)] AND PECTIN ON DISTRIBUTION AND EXCRETION OF 59Fe

A. A. RUBANOVSKAYA

THE TREATMENT of radiation injuries caused by the ingestion into the body of radioactive substances includes the use of preparations to accelerate the excretion of these isotopes.

Many workers have tested preparations, chiefly of the chelinating type, to accelerate the excretion of radioisotopes (Schubert, 1955; Hurch, 1949; Yu. I. Moskalev and L.Budko, 1958; D.I.Semenov and I.P.Tregubenko, 1958; Catsch and Melchinger, 1958; Yu. I. Moskalev, 1959).

The mechanism of action of these compounds is due to the ability to form, with cations, stable water-soluble complexes which are easily and rapidly evacuated in the urine. The compounds proposed have varying efficacy in respect of different isotopes; some of them may have only a limited application. Thus, for example, BAL is effective only with polonium and has no influence on other radioisotopes in the body.

As is known, one of the most effective chelinating compounds is ethylene-diaminotetra-acetic acid (EDTA), which facilitates excretion from the body of a whole series of radioactive metals: plutonium, yttrium, cerium, thorium, lead, zinc, etc. (D.I.Semenov, 1957; Schubert, 1955; Cohn, Gong and Fisch-ler, 1953; Foreman and Hamilton, 1951; and others). However, even this preparation is not a universal stimulus to excretion of radioisotopes. Moreover, at a long interval after intake, EDTA has only a small effect. Therefore, the trial of new means of increasing excretion of radioisotopes is of considerable interest.

A study has been made of the effect of the calcium-disodium salt of cyclo-hexane-diaminotetra-acetic acid on excretion and distribution of 59Fe. A 59Fe preparation in the form of iron citrate was used. It should be pointed out that solutions of 59Fe citrate contained a relatively large amount of stable carrier: from 1-5 to 2-5 mg of iron per ml of solution.

The experiments were carried out on rats weighing from 160 to 230 g. In TRS 12a

173


each variant of the experiment there were 10 rats: 5 experimental and 5 control.

The 59Fe solution was injected intraperitoneally 10 μc per rat. 0-3 ml per 30 mg of the compound was administered to each experimental rat intraperitoneally, intravenously or orally, at the same time 0-3 ml physiological saline was administered to each of the control animals. The number of injections of the compound varied: 1-2 in the course of a day or 2 injections per day for several days (3 and 10 days).

In order to discover the optimal conditions for use of the therapeutic preparation administration of the compound was begun at different periods after injection of 5 9Fe: after 1-2 minutes, 1 hour, 1\ hours, 2 days and 11 days.

The animals were killed 1, 4 or 11 days after the beginning of treatment (a day after the last injection of the preparation) and the radioactivity of excreta collected during the administration period of the preparation, and of the tissues and organs measured.

The experiments showed that a single intraperitoneal or intravenous injection of the preparation immediately after administration of the isotope (after 1-2 min) produces a sharp increase in excretion of 59Fe in the urine (Table 1).

TABLE 1. Excretion of59Fe in Rat Urine Following Injection of CaNa2 (CDTA) Immediately after S9fe Injection

G £

X

1

2

3

4

5

Num

ber

of a

nim

als i

n th

e exp

erim

ent

10

10

10

10

10

Path of injection and dose of CaNa2

(CDTA)

Intraperitoneal 30 mg



Intravenous 30 mg

Intravenous 30 mg

59Fe excreted in one day (% of amount injected)

control

11-8 ± 1-1

15-4 ± 1-5

15-6 ± 0-7

4-3 ± 0-4

11-5 ± 0-5

experimental

27-4 ± 2-2

32-3 ± 2-0

29-7 ± 2-0

74-6 ± 1-1

34-1 ± 2-2

Expe

rimen

tal

as p

erce

ntag

e of

con

trol

232-2

209-7

190-0

1734

296-5

Notes

Carrier— 0-07 mg/ml

From 11-5 to 15-6 per cent on average of the injected amount was excreted in the urine per day in the control rats. The experimental animals excreted from 27*4 to 34T per cent 59Fe.

Thus, excretion of the isotope in the urine increased by 2-3 times. The increase in the excretion of 59Fe from the body was accompanied by

its reduced deposition in the tissues and organs. The 59Fe content of the liver, spleen and kidneys declined markedly (Table 2). Thus, the liver of control rats a day after 59Fe injection contained from 29-2 to 43*1 per cent of the quantity administered, that of experimental animals from 15T to 20-7 per

TABL

E 2.

Con

tent

of59

Fe

of R

at T

issu

es a

nd O

rgan

s Fo

llow

ing

Inje

ctio

n of

CaN

a 2 (C

DTA

) 1-

2 m

in a

fter 5

9 Fe In

ject

ion

in W

hole

Org

an a

s a

Perc

enta

ge o

f To

tal R

ecei

ved

by th

e Bo

dy

No.

of

expe

rim

ent

1 2 3 4

Num

ber

of a

nim

als

in th

e ex

perim

ent

10

10

10

10

Gro

up o

f ra

ts

Expe

rimen

tal

Con

trol

Expe

rimen

tal (

as

perc

enta

ge o

f co

ntro

l)

Expe

rimen

tal

Con

trol

Expe

rimen

tal (

as

perc

enta

ge o

f co

ntro

l)

Expe

rimen

tal

Con

trol

Expe

rimen

tal (

as

perc

enta

ge o

f co

ntro

l)

Expe

rimen

tal

Con

trol

Expe

rimen

tal (

as

perc

enta

ge o

f co

ntro

l)

Bloo

d

4-0

± 0-

4 4-

5 ±

0-17

88

-8

4-1

± 0-

36

6-2+

1-3

66

-1

8-5

± 0-

53

40-9

± 2

-9

20-7

Live

r

15-1

± 1

-2

431

± 0-

37

35

20-7

± 0

-95

34-0

± 0

-95

60-8

19-8

± 1

-8

29-2

± 2

-1

67-3

3-5

± 0-

18

20-4

± 1

0

17-1

Sple

en

1-0 ±

0-0

9 1-5

4 ±

0-08

65

1-03

± 0-

2 1-6

7 ±

0-01

61

-6

1-07

± 0-

07

1-68

± 0-

26

63-6

0-33

± 0

-01

2-8

± 0-

36

11-7

Kid

neys

0-73

± 0

-02

1-34

± 0-

1 54

-4

1-1

± 0-

05

2-22

± 0

-01

50

0-95

± 0

-12

1-32

± 0-

17

71

0-62

± 0

-02

0-88

± 0

-07

70-4

Lung

s

0-15

±0-

01)

0-36

± 0

-04

>

41-6

J

0-27

± 0

-0Γ)

0-

28 ±

0-0

2 >

100

j

0-27

± 0

-03̂)

0-34

+ 0

-07

>

80

J

0-18

± 0

-02Ί

0-

81 ±

0-0

9 >

22

-1

J

Am

ount

of

car

rier

(mg/

ml)

1-5-

2-5

1-5-

2-5

1-5-

2-5

0-07

Not

es

Rat

s ki

lled

1 da

y af

ter 5

9 Fe

inje

ctio

n

Rat

s ki

lled

1 da

y af

ter 5

9 Fe

inje

ctio

n

Rat

s ki

lled

1 da

y af

ter 5

9 Fe

inj x

tion

Rat

s ki

lled

1 da

y af

ter 5

9 Fe

inje

ctio

n

Effect of Calcium Salts on Distribution and Excretion of "Fe 175


cent (from 35 to 67-5 per cent of the control); in the kidneys of control rats there was from 1*32 to 2-4 per cent; in experimental animals, from 0-73 to 1-1 per cent (54-71 per cent of the control).

It should, however, be realized that the amount of 59Fe excreted in the urine and its distribution in the body, as with other radioisotopes, depends on the quantity of carrier in the preparation. The results of experiment No. 4 illustrate this point. In this experiment the rats were injected with a 59Fe citrate solution containing 0-07 mg/ml of stable iron.

Excretion of 59Fe in the urine during one day under these conditions consisted of 4*3 per cent of the injected quantity in the control rats. The increase in excretion of 59Fe caused by the chelinating compound was significantly greater than in the previous experiments—on average by 17 times. Correspondingly, 59Fe content of the organs of the experimental animals was more reduced than when a 59Fe preparation with a greater amount of stable iron was used. The radioactivity of the blood, liver and spleen is reduced particularly sharply. Thus, when the amount of stable iron in the solution is small the chelinating compound is more effective.

The compound proved to have little effect when administered 11 days after the 59Fe injection. By this time the 59Fe was already firmly fixed in the tissues and excretion in the urine and faeces had become established at a low level. Administration of the compound for 3 and 10 days (30 mg each twice a day) at this period produces only a negligible increase of the absolute quantity of 59Fe in the urine: from 0-38 to 1*02 per cent of the amount injected, which cannot be of practical importance. Excretion of 59Fe in the faeces is unchanged.

Shortening the time interval between injection of 59Fe and administration of the preparation increases the efficacy of the latter. Administration (twice a day for 3 days) begun 2 days after 59Fe injection increases the absolute 59Fe content of the urine by a greater amount than injection begun after 11 days (from 1-9 to 3-8 per cent of the amount administered).

Intraperitoneal injection of the preparation, 1 and 3^ hr after 59Fe injection, increases excretion of 59Fe in the urine by 1^-2 times.

It is known that oral administration of chelinating compounds is less effective than intraperitoneal or intravenous injection. Therefore, the compound was administerd orally in considerably greater quantity (60 mg each 1 min before and \ hr after intraperitoneal injection of 59Fe). As can be seen from Table 3, these quantities of the preparation, when administered orally, had little effect on 59Fe evacuation.

Excretion of 59Fe in the urine under these conditions was 12-4 per cent of the amount administered in the control rats and 15-6 per cent in the experimental animals.

Thus, the therapeutic preparation is absorbed in small quantities from the gastro-intestinal tract.

Measurement of 59Fe in tissues and organs showed that, administration of the compound for 3 days (2 injections per day) commencing 11 days after 59Fe injection, has no effect on the figures obtained. However, more

TABL

E 3.

Exc

retio

n of

59Fe

in

Rat

Uri

ne a

nd F

aece

s Fo

llow

ing

Trea

tmen

t with

CaN

a 2 (C

DTA

) at

Var

ious

Inte

rval

s afte

r 59 Fe

Inje

ctio

n

No.

of

expe

rim

ent 6 7 8 9 10

11

Num

ber

of a

nim

als

in th

e ex

perim

ent

10

10

10

10

10

10

Met

hod

of a

dmin

istra

tion

and

dose

of

CaN

a 2

(CD

TA)

Intra

perit

onea

lly

30 m

g tw

ice

a da

y fo

r 3

days

D

itto

Ditt

o

Ditt

o D

itto

Ora

lly 6

0 m

g

Inte

rval

afte

r 59

Fe i

njec

tion

11 d

ays

2 da

ys

31 h

r

Ihr

Ihr

1 m

in b

efor

e an

d \

hr a

fter

intra

-pe

riton

eal

inje

ctio

n

Am

ount

exc

rete

d as

per

cent

age

of d

ose

adm

inist

ered

in th

e ur

ine

cont

rol

0-38

± 0

-03

1-9 ±

0-14

12

-6 ±

2-8

12-4

± 2

-0

14-0

± 1

-0

12-4

± 0

-53

expe

rimen

tal

1-02

± 0-

01

3-8

± 0-

9 21

-4 ±

1-0

24-5

± 2

.1

23-7

± 2

-8

15-6

± 0

-73

expe

rim

enta

l as

perc

enta

ge

of c

ontro

l

268-

4

200-

0 15

0-0

194-

3 16

9-3

125-

8

in th

e fa

eces

cont

rol

0-62

± 0

-06

expe

rimen

tal

0-87

± 0

-02

Not

es

Dur

ing

4 da

ys

Ditt

o D

urin

g 1

day

Ditt

o D

itto

Ditt

o



TABLE 4. Content of59Fe in Rat Tissues and Organs Following Treatment with in the Whole Organ as a per cent

No. of experiment

13

7a

Number of animals

in the experiment

10

10

10

10

Group of rats

Experimental Control Experimental (as

percentage of control)







Interval after 59Fe injection (days)

11

11

11

Dose and method of injection

of the compound

Intraperitoneally 30 mg twice a day for 3 days




prolonged injection of the preparation (for 10 days), even at these times has some effect (Table 4).

Treatment for 10 days beginning 2 days after intake of 59Fe results in a significant reduction in the amount of the isotope in tissues and organs.

All the data obtained by us showed that the compound is effective, at the short periods after 59Fe intake. A single injection of the compound immediately, and 1 and 3£ hr after intake of 59Fe sharply increased excretion of the isotope in the urine and reduced its deposition in the tissues and organs.

During the first few days after 59Fe intake prolonged, repeated administration of the preparation also has a considerable effect.

As was pointed out above, one of the most effective chelinating compounds in relation to a series of radioisotopes is EDTA. A comparison of the effectiveness of CDTA and EDTA in respect of 59Fe is of some interest. In the following series of experiments a comparison was made of the effects of two dosages of the calcium-disodium salts of both compounds : 30 mg per rat (0-3 ml 10 per cent solution) and 20 mg per rat (0-2 ml 10 per cent solution).

The rats were injected intraperitoneally with 59Fe solution and after l-2min one half of the animals received an intraperitoneal injection of CaNa2 (CDTA), and the other half CaNa2 (EDTA). The animals were killed a day later.

Effect of Calcium Salts on Distribution and Excretion of59Ee 179

CaNo2 (CDTA) Begun at Different Intervals after Injection of the Isotope of Total Received by the Body

Blood

9-67 + 0-70 10-2 ± 0-36

94-8

14-3 ± 0-79 15-0 + 0-95

95-0

13-4 ± 0-2 14-1 ± 0-8

95-0

12-8 ± 1-0 14-0 ± 0-33

91-0

Liver

9-1 ± 0-40 8-4 ± 0-60

108-3

17-3 ± 0-52 21-6 + 0-72

80-0

12-2 ± 1-6 18-4 ± 0-70

66-3

9-8 ± 0-70 12-8 ± 1-0

76-5

Spleen

0-73 ± 0-07 0-79 ± 0-08

92-4

0-86 + 0-05 0-76 ± 0-06

113-0

0-58 ± 0-10 1-04 ± 0-12

55-7

0-64 ± 0-04 1-1 ± 0-10

58-1

Kidneys

0-97 ± 0-07 0-94 ± 0-1

103-0

0-59 ± 0-05 0-9 ± 0-03

64-8

0-55 ± 0-01 1-10 + 0-07

50-0

0-66 ± 0-02 0-85 ± 0-06

77-0

Lungs

0-19 ± 001 0-15 ± 0-01

126-6

0-29 ± 0-03 0-26 ± 0-04

111-0

0-44 ± 0-07 0-61 ± 0-10

72-0

0-25 ± 0-01 0-38 65-7

Bone marrow

0-93 ± 0-14 1-05 ± 0-1

88-5

The rats which received 30 mg CaNa2 (CDTA) excreted in the urine during the day from 28-4 to 30-7 per cent of 59Fe administered, while rats receiving CaNa2 (EDTA) excreted approximately the same amount (from 30T to 30-8 per cent, cf. Table 5).

The radioactivity of the tissues and organs of animals of both groups in each experiment was also materially the same (Table 6).

Similar results were obtained when the preparations were used at the 20 mg dose.

Thus, at short periods after 59Fe injection CaNa2 (CDTA) proved as effective as CaNa2 (EDTA).

The purpose of the following series of experiments was to investigate the possibility of reducing the intake of 59Fe through the gastro-intestinal tract by means of adsorbents. A commercial preparation of pectin, obtained from sunflowers, was used as the adsorbent.

The pectins are a group of substances which contain a chain of galact-uronic acid radicals. The carboxyl groups of these radicals are free and can form compounds with metals.

As far as its physical properties are concerned pectin is a hydrophylic colloid with great adsorptive capacity.

Work by a number of writers has shown that pectin reduces absorption


of lead from the gastro-intestinal tract (Murer and Grandall, 1942; and others).

Our investigations have established that administration of pectin reduces absorption in the gastro-intestinal tract and deposition in the skeleton of radiostrontium. It might be assumed that the preparation would prove effective also in regard to other metals entering the gastro-intestinal tract.

TABLE 5. Effect ofCaNa2 (CDTA) and CaNa2 (EDTA) on Excretion of59Fe in Rat Urine

No. of experiment

1 2 3 4

Number of animals

in the experiment

10 10 10 10

Dose of compound

(mg)

30 30 20 20

59Fe content of urine in 1 day (percentage of amount administered)

CaNa2 (CDTA)

30-7 ± 0-77 28-4 ± 1-7 24-9 ± 0-5 29-7 ± 1-0

CaNa2 (EDTA)

30-8 ± 1-2 30-1 ± 1-7 24-1 ± 1-7 30-0 ± 0-8

The experiments of this series were carried out as follows. In each experiment 15-18 rats were used. Each animal received orally 20 μο 59FeCl3; 5-6 rats received orally 1-5 ml 2 per cent solution of pectin 1-2 min before administration of the 59Fe, and another 5-6 animals the same dose 1 hr before. The control rats were given physiological saline before receiving the 59Fe. The 59Fe solution contained a rather large amount of stable carrier: 0-67, 0-87 mg and only in one experiment 0Ό19 mg. The animals were killed 2-3 days after 59Fe administration and the 59Fe content of the blood, liver, spleen and lungs measured.

It can be seen from the results obtained that, when 59Fe enters the gastrointestinal tract, the amount retained in the body and its distribution depend on the amount of carrier in the preparation (Table 7).

When the quantity of carrier is large very little 59Fe is retained in the body. Thus, after 2-3 days from 1 to 1-7 per cent of the amount administered can be found in the liver and up to 5 per cent in the blood.

When the amount of carrier is small (cf. Table 6, experiment No. 3) retention in the liver is somewhat greater at these periods, while a considerable quantity of 59Fe can already be found in the blood (up to 21 per cent of the amount administered). The 59Fe content of the spleen and lungs in all the experiments at these periods was negligible—measured in hundredths and tenths of 1 per cent of the administered amount.

The results obtained (cf. Table 7) show that when the amount of stable iron in the 59FeCl3 solution is small the presence of pectin reduces the amount of radioactive iron which passes through the wall of the gastro-intestinal tract.

As can be seen from Table 7, the blood of rats, which received pectin before 59Fe with a low stable iron content, contained only half the amount of

TABL

E 6.

Effe

ct o

f CaN

a 2 (C

DTA

) an

d Ca

Na2 (

EDTA

) on

Dis

trib

utio

n of

59Fe

in

Rat

Tis

sues

and

Org

ans

a D

ay a

fter

Inje

ctio

n

No.

of

expe

rim

ent

1 2

Num

ber

of a

nim

als

10

10

Dos

e of

com

poun

d

CaN

a 2 (

CD

TA)

30 m

g C

aNa 2

(ED

TA)

30 m

g

CaN

a 2 (

CD

TA)

20 m

g C

aNa 2

(ED

TA)

20 m

g

Live

r Sp

leen

K

idne

ys

Lung

s Bl

ood

as p

erce

ntag

e of

am

ount

of 5

9 Fe re

ceiv

ed b

y th

e an

imal

25-1

± 0

-75

27-7

± 1

-2

19-2

± 0

-57

22-0

± 1

-4

1-29

± 0-

2

1-17

±0-1

1-1 ±

0-1

4

1-1 ±

0-16

1-07

± 0-

05

1-05

± 0-

6

1-0 ±

0-0

1-04

± 0-

05

0-45

± 0

-09

0-38

± 0

-07

0-6

± 0-

1

0-58

± 0

-02

2-6

± 0-

24

2-8

± 0-

16

2-8

± 0-

28

2-8

± 0-

19


TABL

E 7.

Con

tent

of59

Fe

of R

at T

issu

es a

nd O

rgan

s

No.

of

expe

ri

men

t

1 2 3

Num

ber

of a

nim

als

in t

he

expe

rim

ent

15

18

15

Am

ount

of

59F

e(m

g)

in a

dmin

iste

red

volu

me

of s

olut

ion

0-67

0-87

0-01

9

Gro

up o

f ra

ts

Exp

erim

enta

l 1)

Pec

tin a

t 1-

2 m

inut

es b

efor

e 5

9F

e ad

min

istr

atio

n 2)

Pec

tin

at 1

hr

befo

re

59F

e ad

min

istr

atio

n C

ontr

ol

Exp

erim

enta

l 1)

Pec

tin a

t 1-

2 m

inut

es b

efor

e 5

9F

e ad

min

istr

atio

n 2)

Pec

tin a

t 1

hr b

efor

e 5

9F

e ad

min

istr

atio

n C

ontr

ol

Exp

erim

enta

l 1)

Pec

tin a

t 1-

2 m

inut

es b

efor

e 5

9F

e ad

min

istr

atio

n 2)

Pec

tin a

t 1

hr

befo

re

59F

e ad

min

istr

atio

n C

ontr

ol

Org

ans

liver

sp

leen

lu

ngs

bloo

d

in w

hole

org

an a

s a

perc

enta

ge c

f to

tal r

ecei

ved

by th

e an

imal

0-9

± 0-

05

1-4

± 0-

05

1-3

± 0-

04

1-7

± 0-

3

1-0

± 0-

1

0-9

± 0-

1

1-5

± 0-

2

2-1

+ 0-

03

4-0

± 0-

4

0-05

± 0

-001

0-13

± 0

-02

0-21

±

0-00

8

0-07

± 0

-01

0-07

± 0

-01

0-06

± 0

-01

0-4

± 0-

01

0-4

± 0-

04

0-4

± 0-

03

0-06

±

0-00

2

0-09

±

0-00

3

0-12

±

0-00

7

0-15

±

0-00

2

0-07

±0-

011

0-06

± 0

-001

0-20

± 0

-03

0-21

± 0

-03

0-30

± 0

-02

2-0

± 0-

03

5-0

± 0-

4

4-4

± 0-

3

2-0

± 0-

2

1-7

± 0-

1

1-5

+ 0-

11

9-5

± 0-

4

21-0

±

1-4

Whe

n ki

lled

Aft

er

3 da

ys

Dit

to

Dit

to

Effect of Calcium Salts on Distribution and Excretion of59Fe 183

the isotope as did the blood of the control animals. Other investigations showed that the tissue content of 59Fe in the experimental rats was less than that of the control animals.

Little radioactive iron is found in tissues and organs after the oral administration of an 59FeCl3 solution containing a large quantity of stable iron. This is due to the low uptake of inorganic iron compounds in the gastrointestinal tract and the great dilution of the radioactive isotope in the administered solution.

Under these conditions pectin has virtually no effect on retention of 59Fe in the body. The 59Fe content of tissues and organs of rats receiving pectin is similar to that of the control animals.

Thus, the experiments have shown that when the amount of stable iron is low, pectin limits uptake of 59Fe by the gastro-intestinal tract.


1. CaNa2 (CDTA) accelerates excretion of 59Fe and reduces its deposition in tissues and organs.

2. CaNa2(CDTA) is effective in increasing excretion and reducing the amount of 59Fe deposited in the tissues if administered during very early periods after its intake.

3. CaNa2(CDTA) is as effective in respect of 59Fe as CaNa2(EDTA). 4. Pectin reduces uptake of 59Fe by the gastro-intestinal tract when the

orally administered solution contains little stable iron.

REFERENCES

CATSCH A. and MELCHINGER H., Strahlentherapie, 106 (4), 606 (1958). COHN J., GONG K. and FISCHLER A., Nucleonics, 11 (1), 56 (1953). FOREMAN H. and HAMILTON J., The Use of Chelating Agents for Accelerating Excretion of

Radioelements, AECD 3247 (1951). H U R C H J., / . Pharmac. exper. Therap., 103 (1951). MOSKALEV Yu.I . and BUDKO L., Med. radiol, 5 (1958). MOSKALEV Yu.L, Med. radiol, 1, 65 (1959). MURER H . H . and GRANDALL L., / . Nutrition, 23 (3), 249 (1942). SCHUBERT J., Ann. Review Nuclear Sci., 5 (1955). S E M E N O V D . I . and TREGUBENKO T.P., Biokhimiya, 23, 1 (1958).

POLYVINYLPYRROLIDONE AND EXPOSURE TO CERTAIN RADIOISOTOPES

A. A. RUBANOVSKAYA

POLYVINYLPYRROLIDONE (PVP) is a synthetic organic polymer with great ad-sorptive capacity. Schubert's investigations (1951) have shown that the preparation binds with various pharmacological poisons, toxins, stains, vitamins both in vitro and in vivo etc. The basis of this action lies in the formation of reversible absorptive compounds. When low-molecular fractions of PVP are used in vivo the compounds formed are rapidly excreted in the urine.

These properties of PVP led to the experimental investigation of the protective and antidotal effect of the preparation in different intoxications. Thus, Weidener (1953) has observed favourable results in treating fatal mercury bichloride poisoning with PVP (Periston N.). Hirsch and Voit (1954) have used the preparation to retard formation of kidney stones.

Burger and Lehman (1954) have successfully used Periston N. as a protective agent in exposure to ionizing radiation. These writers have found that when rats are injected with the preparation for 3 days after whole body irradiation with high X-ray doses, mortality falls sharply.

The mechanism of action of PVP, according to Burger and Lehman, consists in binding and accelerating the excretion of the poisonous products of tissue breakdown caused by irradiation. Schmidt-Matthiesen has shown that PVP retards the development of silicotic nodes in the tissue caused by injections of colloidal silicon dioxide; this effect is due to the abolition, by PVP, of the precipitating action of silicic acid on the tissue proteins.

It should be noted that other writers (Luchtrath, Friebel and Gravenitz, 1958) have not confirmed these results. Norman et al. (1953) have injected rats intravenously with 2 fractions of PVP with different molecular weights before and after intravenous injections of lead compounds. In these experiments no effect of the preparation on lead deposition in the bones and liver was detected. G. B. Berkengeim (1955) has demonstrated the capacity of PVP to prolong the effect of novocaine. This author showed, in experiments with animals, that intramuscular or intracutaneous injections of PVP slows down the rate of absorption of novocaine. Using aqueous solutions of PVP with novocaine in the clinic, the author succeeded in achieving prolonged local anaesthesia in patients. The anaesthetic effect lasted, according to the concentration of the solution, up to 9 days.

On the basis of the absorptive properties of PVP outlined above,- and among tests of various means for stimulating excretion of radioisotopes from

184

Polyvinylpyrrolidone and Exposure to Certain Radioisotopes 185

the body, some investigations were also carried out with polyvinylpyrrolidone-100 (molecular weight 10,000-20,000).

A study was made of the effect of an injection of PVP into the stomach on uptake and bone deposition of 90Sr.

The experiment was carried out on 15 rats (weight 150-160 g). All the animals received 10 \LC 90Sr orally. Eight rats (experimental) 1-2 min before ingestion of 90Sr received 1 ml 6 per cent aqueous solution of PVP (60 mg). The 7 control rats received up to 1 ml of water containing 90Sr.

TABLE 1. Content of90Sr in Rat Bones and Excreta as Percentages of Amount Administered

Group of rats

Control Experimental

Number of animals

7 8

90Sr content

in the bones

43-9 ± 2-4 32-2 ± 2-8

in the urine and faeces

45-4 ± 6-2 58-3 ± 0-97

The rats were housed in changeable cells where their excreta were collected. After 4 days the animals were killed and the 90Sr in the bones and excreta measured.

The 90Sr content of the bones of rats which had received PVP was less than that of the control animals (Table 1). On average, the bones of the experimental animals contained 73-3 per cent of the quantity of 90Sr found in the controls. Correspondingly, 28-4 per cent more 90Sr was excreted by the experimental animals compared with the controls.

Thus, administration of PVP into the stomach immediately before 90Sr reduced its uptake and deposition in the bones.

However, the degree of reduction of 90Sr deposition in the bones was considerably less than that obtained by the use of another absorbent investigated —pectin.*

The purpose of the following series of experiments was to investigate the possibility of using PVP to prolong the effect of a chelating compound administered to stimulate excretion of radioisotopes.

It is known that a chelating compound, injected intraperitoneally or intravenously, is not retained in the body and is very rapidly excreted.

Thus, Foreman and Hardy (1953) and others have shown that 2 hr after an intravenous injection of EDTA 60-70 per cent of the quantity injected is found excreted unchanged in the urine, and after 6 hr—90-95 per cent. The rapid excretion of the compound is a positive factor since a radioisotope bound in the complex is also removed. However, some of the injected compound is removed, apparently, without having reacted with the isotope. If the compound could be retained in the bloodstream, this would no doubt

* Cf. the article by A.A.Rubanovskaya: The effect of pectin on uptake of radiostron-tium experimentally from the gastro-intestinal tract. Gig. Truda iprof. zab., No. 4 (1961).

TABL

E 2.

Dist

ribut

ion

and

Excr

etio

n of

59Fe

in

Rat

s Fo

llowi

ng A

dmin

istra

tion

of C

aNa 2

(C

DTA

) an

d PV

P

No.

of

expe

rim

ent

1 2 3 4

Num

ber

of a

nim

als

10

10

10

10

Site

of

inje

ctio

n

Perit

onea

l

Perit

onea

l

Hea

rt

Hea

rt

Prep

arat

ion

inje

cted

CaN

a 2 (

CD

TA)

Ditt

o +

PVP

CaN

a 2 (

CD

TA)

Ditt

o +

PVP

CaN

a 2 (

CD

TA)

Ditt

o +

PVP

CaN

a 2 (

CD

TA)

Ditt

o +

PVP

Org

ans

liver

ki

dney

s sp

leen

lu

ngs

bloo

d ur

ine

59Fe

con

tent

as

perc

enta

ge o

f qua

ntity

inj

ecte

d

28-3

+ 1

-8

—

24-3

± Ό

-82

23-0

+ 0

-62

23-8

+ 0

-1

22-9

+ 1-1

20-8

±

1-18

19-2

+ 1

-6

0-7

± 0-

1 0-

54 ±

0-0

2

0-75

± 0

-03

C-66

+ 0

-03

1-34

+ 0-

1 1-4

5 +

0-29

1-35

± 0-

08

1-22

+ 0-

07

0-36

+ 0-

011

0-21

+ 0

-04

1-41

± 0-

1 1-2

1 ±

0-2

0-62

± 0

-1

0-74

+ 0

-2

0-37

+ 0

-04

0-3

+ 0-

001

0-33

± 0

-02

015

± 0-

003

0-37

± 0

-05

0-37

± 0

-026

0-33

± 0

-03

0-27

+ 0

-006

0-22

± 0

-02

0-17

+ 0

-004

—

11-2

+ 0

-78

11-6

zb 0

-62

8-5

+ 0-

85

11 +

1-1

6-3

± 0-

33

5-07

± 0

-06

40-7

± 2

-4

40-1

+ 1

-6

—

—

43-9

± 0

-5

32-6

+ 3

-1

45 +

2-5

45

± 4

-4

o Ci S- Ci Co

to

ri Toxicology of' Rodiouct itw Substanccs

Polyvinyìpyrrolidone and Exposure to Certain Radioisotopes 187

considerably increase its efficacy. At longer periods after intake of radio-isotopes, the retention of the compound in the blood at a high level would facilitate the greater mobilization of previously deposited isotope.

We have shown that cyclohexane-diaminotetra-acetic acid (CDTA) does not influence excretion of 90Sr but is very effective in regard to 59Fe, especially soon after intake.

Intravenous or intraperitoneal injection of this compound in rats, which have received 59Fe, increases excretion of the isotope in the urine and reduces deposition in the tissues and organs. Nevertheless, a significant proportion of the isotope still evades interaction with the compound, penetrates tissues and organs and is freed there.

An attempt was made to increase the efficacy of CDTA in rats which had received 59Fe by combining the compound with polyvinyìpyrrolidone.

The experiments were carried out as follows : all the experimental rats were injected intraperitoneally with 10 μο 59Fe citrate per rat.

At 1-2 min after 59Fe injection the experimental rats received a mixture of a solution of CaNa2 (CDTA) (the calcium-disodium salt of cyclohexane-diaminotetra-acetic acid) with a solution of polyvinyìpyrrolidone (CaNa2 (CDTA) + PVP). The control rats received only CaNa2 (CDTA) at the same periods.

The compound was used in a dose of 0-3 ml 10 per cent solution (30 mg), and PVP 0-5 ml 12 per cent solution (60 mg). The liquids were mixed in the syringe. The compound or mixture was injected intraperitoneally or into the heart.

The rats were killed 2 days later and 59Fe content of the liver, kidneys, spleen, lungs and urine (collected during the 2 days) determined.

The data obtained are presented in Table 2. As can be seen from these results the 59Fe content of the organs and urine of rats which received the mixture of compound and PVP did not differ from that of the control animals receiving the compound alone.

The effectiveness of the compound is not increased by the addition of polyvinyìpyrrolidone either by intraperitoneal or intracardiac injection.


1. When orally administered immediately before 90Sr polyvinylpyrro-lidone-100 reduces uptake of the 90Sr in the gastro-intestinal tract of rats.

2. The simultaneous intraperitoneal or intracardiac injection of a mixture of the compound CaNa2 (CDTA) and the preparation PVP does not influence excretion of 59Fe from the body.


REFERENCES

BERKENGEIM G.B., Polyvinylpyrrolidone, its properties and use in surgery (Polivinilpirro-lidon, ego svoistva i primenenie v khirurgii). Proceedings of the 200th Anniversary meeting of the Moscow Medical Institute, Moscow (1955).

BURGER H. and LEHMAN K., Naturwissenschaften, 41 , 8, 190 (1954). FOREMAN H. et al, Arch. Indust. Hygiene Occup. Med., 7 (2), 148 (1953). HIRSCH H. and V O I T E . , Klinische Wochenschrift, 651 (1954). LUCHTRATH H. et ai, Gewerbehygiene, 1 (16), 4 (1958). NORMAN S. et al., Arch. Indust. Hygiene Occup. Med., 7 (7), 217 (1953). SCHMIDT-MATTHIESEN, Arch. Gewerbepathol. und Gewerbehygiene, 16, 4 (1958). SCHUBERT R., Dtsch. Med. Wochenschrift, 47, 1487 (1951). WEIDENER J., Dtsch. Med. Wochenschrift, 35, 1192 (1953).

INDEX

Accumulation of 59Fe 15, 25, 35 Albumin/globulin ratio of rabbits receiving

59Fe 109 Anisocytosis, degree of, during prolonged

59Fe administration 68

Blood indices of rats after 59Fe oxide injection 100

Blood sugar of rabbits receiving 59Fe 107, 108

Blood vessel walls, oedema 163 Body radiation dose in rabbits 19 Bone marrow changes on 59Fe administra

tion 69 Bone marrow picture during pregnancy 86 Bronchi of rats after 59Fe injection 145 Bronchial epithelium of rabbits, atypical

growth 162 Bronchial mucous glands of rabbits, ade-

nomatous growths 163 Bronchopneumon ia 162-4

CaNa2(CDTA), effect on distribution and excretion of 59Fe 173

CaNa2(CDTA) and PVP mixture, effect on distribution and excretion of 59Fe 186

CaNa2(EDTA), effect on distribution and excretion of 59Fe 173

Carbohydrate metabolism, changes during prolonged 59Fe administration 105

Cardiac activity, effect of prolonged administration of 59FeCl3 114, 130

Colour index changes during and after pregnancy 188 changes during prolonged 59Fe ad

ministration 65

Distribution of 59Fe 13, 173

Electrical activity of the cerebral cortex of rabbits, effect of prolonged 59Fe administration 38

Electrocardiogram of rabbits, effect of prolonged internal administration of 59FeCl3 114

Erythropoiesis changes after bloodloss 78

changes during pregnancy 87 effect of 59Fe 53, 61

Evoked rhythm, changes after 59Fe administration 41-5

Excretion of 59Fe 16, 25, 173

Fatty degeneration in liver 165 Focal necroses in liver 165

Giant-cell bronchopneumonia 164

Haemoglobin level, changes after bloodloss 76

Haemopoiesis effect of 59Fe 53, 73 effect of pregnancy and parturition

during prolonged 59Fe administration 85

Haemorrhages along capillaries 161 Haemorrhagic inflammation of lungs after

59Fe oxide injection 147 Heart activity, effect of prolonged ad

ministration of 59FeCl3 114, 130

Leucocyte count after 59Fe oxide injection 99, 102

Leucocyte count, total, fluctuation after receiving 59Fe oxide 99

Leucocytes in peripheral blood after 59Fe injection 97

Leucopoiesis, effect of 59Fe 54 Lungs of rats, 59Fe content after injection

142, 144 Lymphocyte count

after bloodloss 81 after 59Fe oxide injection 102

Lymphocyte count, absolute, changes during and after pregnancy 91

Lymphocyte numbers, changes after 59Fe oxide injection 99

Lymphopoiesis, changes during and after pregnancy 91

Macrocytosis, changes after bloodloss 77 Maturation index of neutrophils, changes

during and after pregnancy 91

189

190 Index

Mean red cell diameter, changes during prolonged 59Fe administration 66

Morphological changes after 59Fe injection 140

Morphological lesions during prolonged 59Fe administration 158

Mucous membrane, atrophy of, during prolonged 59Fe administration 160

Myocardium, interstitial oedema of, during prolonged 59Fe administration 161

Neutrophil count, absolute, changes during and after pregnancy 91

Neutrophil count after 59Fe oxide injection 99, 102

Neutrophil leucopoiesis, changes during and after pregnancy 90

Pathological lesions after 59Fe injection 144

Pectin, effect on distribution and excretion of 59Fe 173

Peripheral blood changes after 59Fe injection 93

Peripheral blood picture during pregnancy 86

Pneumosclerosis after 59Fe citrate injection 146

Polyvinylpyrrolidone and exposure to certain radioisotopes 184

Red cell changes after bloodloss 75 Red cell count, changes during and after

pregnancy 87 Red cell sphericity index, changes during

and after pregnancy 88 Reticulocyte count, changes after bloodloss

77

Serum protein fractions, changes during prolonged 59Fe administration 105

Spermatogenic epithelium in testes, atrophy 166

Spleen, reduction of lymphoid elements 165

Stroma of gastro-intestinal tract, sclerosis 160

Stroma of testes, sclerosis 166

Thrombopoiesis, effect of 59Fe 54

White cell changes after bloodloss 81

MADE IN GREAT BRITAIN

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The Toxicology of Radioactive Substances. Volume 3.59