Irreversible Fixation of Increased Level of Muscle Type ... · Irreversible Fixation of Increased Level of Muscle Type Aldolase1 Activity Appearing in Rat Liver in the Early Stage

[CANCER RESEARCH 30, 743-752, March 1970]

Irreversible Fixation of Increased Level of Muscle TypeAldolase1 Activity Appearing in Rat Liver in the EarlyStage of Hepatocarcinogenesis2

Hideya Endo. Masao Eguchi, and Susumu Yanagi

Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka, Japan

SUMMARY

With the use of antimuscle aldolase sera, muscle typealdolase activity in the liver of adult rats was determinedduring a 60-day interval after stopping the administrationof a diet containing 0.06% 3'-methyl-4-dimethylamino-

azobenzene for 60 days.The muscle type aldolase in the livers of rats thus

treated was found to be elevated when compared to normal liver, although no appreciable differences were observed in total aldolase activity, mitotic index, histologi-cal architecture, and soluble protein, as well as DNAcontent.

The appearance of this enzymic change was alreadyobservable by administrating the azo dye only for 15 days.

The increased level of muscle type aldolase activity observed in the liver of rats fed azo dye for 60 days wasmaintained during a 300-day observation period.

A similar enzymic alteration could be induced by another hepatocarcinogen, /V,7V'-2,7-fluorenylenebisaceta-mide, whereas a noncarcinogen, 2-methyl-4-dimethylami-noazobenzene, failed to induce this change.

INTRODUCTION

In 1949, Clayton and Bauman (4) observed a highincidence of hepatoma in rats when they administereda submanifestational dose of 3'-Me-DAB,4 which was

followed by no carcinogen and then a subsequent administration of the same dose after a 12-week interval.

Similar findings were also obtained by Nakahara andFukuoka (16) using 2 different carcinogens affecting thesame target organ; 20-methylcholanthrene and 4-nitro-quinoline 1-oxide. In this case, the administration of submanifestational doses of the respective carcinogen at 200-day intervals resulted in skin cancer production in mice.

1In this paper we tentatively defined the liver enzyme neutralizablewith antimuscle aldolase sera as "muscle type aldolase," distinguished

from muscle aldolase, since the identity of both enzymes is not yet conclusively determined.

â€¢This work was supported by a grant-in-aid for scientific research

from the Ministry of Education, Japan.' Present address: Department of Orthopaedy, Faculty of Medicine,

Kyushu University, Fukuoku, Japan.'The abbreviations used are: 3'-Me-DAB, 3'-methyl-4-dimethvl-

aminoazobenzene; 2-Me-DAB, 2-methyl-4-dimethylaminoazobenzene:2,7-FAA, /V,yV'-2,7-fluorenylenebisacetamide.

Received May 19, 1969; accepted August 19, 1969.

The results in both groups seem to suggest that the firstexposure to the carcinogenic stimuli causes an irreversiblechange in the cell, and that this change is maintained inthe cell during the long subsequent period during which acarcinogen-free diet is administered.

The present work was undertaken to see whether suchan irreversible change involved in the transformation ofthe cells to the malignant state does indeed exist and, ifso, how might it be expressed biochemically. We have investigated the aldolase isozyme pattern during carcino-genesis. Aldolase in the liver has been found to be composed of 2 isozymes, liver type aldolase (EC 4.1.2.7) andmuscle type aldolase. The latter was shown to be at leastimmunologically identical with muscle aldolase (EC 4.1.2.13) (3, 18), which differs from liver type aldolase in substrate specificity (3, 7, 9, 21-23) and in kinetic (26), andimmunological properties (3). Especially noteworthy arethe findings of Schapira et al. (24), Sugimura et al. (27),and Adelman et al. (1), who found that the activity of livertype aldolase is much higher than that of muscle type aldolase in normal liver and that the converse is true inpoorly differentiated malignant liver tumor. These findings led us to presume that the process of hepatocarcino-genesis might eventually be analyzed quantitatively byusing the aldolase isozyme pattern.

In analyzing biochemical changes occurring duringchemical carcinogenesis, it seems especially desirable toeliminate the problem of nonspecific toxic effects of thecarcinogen on the target organ. Otherwise, the possibilitycannot be excluded that the results obtained may be amanifestation of the toxic effect of the carcinogen andmay be unrelated to transformation of the cells. The toxiceffect of carcinogens, similar to the pharmacological effectof drugs in general, disappears after the administration ofcarcinogens ceased. With this point of view, attention hasbeen given in this study to the performance of the biochemical analysis on the target organ of experimental animals at a sufficient time interval after stopping the administration of the carcinogen so that only irreversible changeswould be observed.

MATERIALS AND METHODS

Animals. Male albino rats (Wistar strain, 150 to 200 g)were separated into 5 groups, housed in stainless steelcages (5 rats/cage), and fed diet and water ad libitum.

MARCH 1970 743

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Hideya Endo, Masao Eguchi, and Susumu Yanagi

The basal diet, CE-2 from Nippon Clea Company,Ltd., (Osaka, Japan), consisted of the following parts per100; water, 6.0; crude carbohydrate, 56.0; crude protein,24.0; crude fat, 3.5; crude cellulose, 4.5; ash, 6.0. Vitamincontents of this diet were: vitamin A, 10; D3, 2 i.u.; E, 10;Bi, 5.6; B2, 10.0; B6, 4; B,2, 0.02; calcium pantothenate,26; niacin, 44; folie acid, 0.8; and choline chloride, 400^g/g, respectively. The animals of the first group (control) were maintained on the basal diet and water andsacrificed at the same time as in the second group. Thosein the second group were transferred to the basal dietcontaining 3'-Me-DAB at a concentration of 0.06% for 15,

30, 45, and 60 days. Among these animals, those fed theazo dye diet for 15, 30, and 45 days were sacrificed on the60th day after stopping the adminstration of the azo dyediet. Those fed azo dye for 60 days were sacrificed at 60,90, 210, and 300 days after transferring to the basal diet.Animals in the third group were transferred to the sameazo dye diet and maintained for 180 days and then transferred to the basal diet. These animals were sacrificedwhen tumor development was determined by palpation inthe liver region or when the animal showed severe general emaciation. Animals in the fourth and fifth groupswere those fed for 60 days with a diet containing 0.06%2-Me-DAB and 0.025% 2,7-FAA, respectively, and weresacrificed at the 60th day after the respective diet wasstopped.

Each rat was decapitated, and the liver and tumor aswell as tumor-free area adjacent to it were subjected tofurther experiment. Liver tissue and tumor tissue wereweighed, cut in pieces with scissors, and homogenizedwith 9 volumes of cold 0.02 M Tris buffer, pH 7.4, containing 80 mM KC1 and 5 TOMMgCh, in a Potter-Elvehjem homogenizer. Each homogenate was centri-fuged at 105,000 X g for 1 hour. The supernatantobtained was used as the crude enzyme extract for thealdolase assay and protein estimation.

Enzyme Assay. The aldolase activities were assayed byspectrophotometric measurement of the decrease at 340m^i, with the use of a-glycerophosphate dehydrogenase,according to slight modification of the method of Racker(19) and Blostein and Rutter (3). Tris-HCl buffer (0.12M, pH 7.5) was used instead of glycylglycine buffer (3),and all assays were carried out at 30Â°.The reaction wasinitiated by the addition of fruciÃ³se-1,6-di-P (final concentration, 2 mM) or fructose-1-P (final concentration, 10HIM).

For the differential assay of muscle type and liver typealdolase activities, 0.1 ml of the antisera described below(twice diluted) was added to the enzyme extract, and, 10min after incubation at 30Â°,the activity remaining wasmeasured in the same manner as described above andwithout centrifugation for removal of the antigen-antibody complex. The amount of antisera added is enoughto neutralize muscle type isozyme in the enzyme extract,since more addition of antisera did not affect the remaining activity. The value obtained by subtracting the activity of antisera alone from the activity remaining was takenas liver type aldolase. The muscle type aldolase activity

was calculated as the difference between the total activity (without addition of antisera) and the liver type aldolase activity.

Differential assay of muscle type and liver type aldolase by measurement of the ratio of cleaving velocity offructose-1,6-di-P to that of fructose-1-P was also carriedout according to the method of Schapira (23, 24) andRutterÃ al. (21, 22).

Protein Estimation. Protein content of the supernatantof tissues was estimated by the method of Lowry et al.(10), with bovine albumin powder (Armour) as the colorimetrie standard.

DNA Estimation. DNA content of tissues was determined by the diphenylamine method (25), with calf thymus DNA as the standard.

Preparation and Immunological Property of AntimuscleAldolase Sera. Crystalline muscle aldolase was preparedfrom rat muscle according to the method of Warburg andChristian (29) and Taylor el al. (28). Antisera were obtained in hens as follows. Ten mg crystalline muscle aldolase in Freund's complete adjuvant were injected s.c. or

i.m. After 2 weeks, 7 mg of the enzyme in 0.9% NaClsolution were injected i.v. as a booster 3 times every otherday. The immune sera were obtained 1 week after thelast injection and were stored at â€”15Â°until used. Neutralization analysis and quantitative precipitation reaction(8) revealed that 1 ml antisera neutralized 0.12 unit crystalline muscle aldolase completely and precipitated 0.32mg of the enzyme. Double diffusion analysis in agar gel(8) showed that the antisera produced a single precipitation line against crystalline enzyme and also muscle extract as well as liver extract, and these lines fused witheach other. The antisera described above were not obtained in each case but only in one-third of the immunized hens.

Materials. 3'-Me-DAB and 2,7-FAA were purchasedfrom Tokyo Kasei Company (Tokyo, Japan); the formerwas recrystallized from benzene and the latter from alcohol. Their melting points were 119Â°and 293Â°,respectively.2-Me-DAB was synthesized in our laboratory and was re-crystallized from benzene. Its melting point was 67Â°.Thecarcinogens were sent to Nippon Clea Company, wherethe rat laboratory chows containing 0.06% 3'-Me-DAB,0.025% 2,7-FAA, and 0.06% 2-Me-DAB were prepared.

Fructose-1,6-di-P and a-glycerophosphate denydro-genase-triose phosphate isomerase mixture were purchased from C. F. Boehringer and Soehne (Mannheim,Germany). NAD was obtained from the Sigma ChemicalCompany (St. Louis, Mo.) and was reduced by sodiumdithionite to prepare NADH (2) shortly before the enzyme assay.

RESULTS

Inhibition by Antimuscle Aldolase Sera for the Activityof Crude Liver Enzyme. For determination of muscle typeand liver type isozyme activities in the livers of control andrats treated with azo dye and in liver cancer, differential

744 CANCER RESEARCH VOL. 30



Fixation of Muscle Type Aldolase in Hepatocarcinogenesis

assays were carried out primarily with antimuscle aldol-ase sera as described in "Materials and Methods." Forthis purpose, it is an absolute requirement that the anti-sera neutralize only the muscle type isozyme and notcross-react with the liver type isozyme. The specificity ofantisera was scrutinized by double diffusion analysis, thequantitative precipitation test, and the neutralization ofenzyme activity. These results, however, do not tell us thequantitative relationship between the antisera and the enzyme in the crude extract. In order to determine this, theequal volumes of the crude liver enzyme and the crystalline muscle aldolase solution were mixed, and the fructose-1,6-di-P-cleaving activities of the mixture were determined before and after the treatment of antimusclealdolase sera. As shown in Table 1, the mixture of crudeliver extract and crystalline muscle aldolase was found toshow the same frustose-1,6-di-P-cleaving activity as thesum of the two premixed components, before and afterthe treatment with antisera. These results indicate clearlythat the substance mentioned above and immunologicallyidentical with muscle aldolase is neutralized certainly byantimuscle aldolase sera in the same manner as crystallinemuscle aldolase and that the remaining activity is neveraffected by antisera in the experimental condition used.

Examination of the Change in HistolÃ³gica!Architecture.In this study, biochemical analysis was attempted on thelivers of rats at a sufficient time interval after the stoppingof the administration of 3'-Me-DAB to avoid the non

specific toxic effects of the carcinogen. We thereforeneeded to know the time interval required for the reversal of the toxic effects of 3'-Me-DAB. For this purpose,

histological and cytological examinations were performedon the livers at various time intervals after azo dye administration was stopped.

In comparison with normal liver (Fig. 1), severe damage was observed in the specimen prepared from the livers of rats fed azo dye for 60 days as shown in Fig. 2. This

Table 1Differential assay of muscle type and liver type aldolase

with antimuscle aldolase sera

Activity foundwithoutantisera(total activ

ity)"Activity

remainingafterneutralizationwithantisera

(livertypeactivity)"1.Crudeliver0.0600.0522Muscle

1 +2aldolase(expected)0.062

0.1220

0.0523.Mixtureof

1and2(observed)0.1230.050

Activity neutralizedwith antisera (muscletype activity)"

0.008 0.062 0.070 0.073

" Aldolase activity was expressed as the decrease of absorbance at340 m>i/min at 30Â°(3). Fructose-1,6-di-P (2 HIM)was used as substrate.

damage was still observed in the specimens prepared 14days after the cessation of dye feeding (Fig. 3), but considerable recovery was seen in the specimen at 30 daysafter the cessation of dye treatment (Fig. 4). The specimen prepared at 60 days after the end of dye treatment(Fig. 5) was practically indistinguishable from that ofnormal liver.

The percentage distribution of the 5 types of cells composing liver tissues was measured according to the classification of Daoust et al. (5). As shown in Table 2, a remarkable decrease of parenchymal cells as well as aconcomitant increase in bile duct and connective tissuecells was observed immediately after administration ofazo dye for 60 days. These changes also seem to be reversed during the recovery period after the stopping ofazo dye feeding, and the percentage distribution of thecells in the liver measured at the 60th to 90th day afterthe termination of dye feeding was found to be practicallythe same as that of normal liver.

Examination of Change in Mitotic Rate. As shown inTable 2, the mitotic rate which is normally 0 to 3/4000parenchymal cells in liver was found to be elevated to 12to 20/4000 cells immediately after the cessation of 60 daysof azo dye administration. This change, however, seemsto be reversed during the interval after the feeding of azodye was stopped. Thus the mitotic rate in the liver wasfound to be practically at the same level as that of normalliver when measured at 60th to 90th day after the end ofdye feeding.

Examination of Change in DNA and Soluble ProteinContent. The content of DNA and soluble protein per wetweight of liver were measured in normal liver and in liverfrom rats 60 days subsequent to the end of azo dye administration. As shown in Table 2, no appreciable difference could be seen in the livers of rats fed with azo dyefor 60 days and then with normal diet for 60 days as compared with that of normal rats.

These results indicate that the nonspecific toxic effectof 3'-Me-DAB can be eliminated by the feeding of a nor

mal diet for 60 days after 2 months of azo dye administration.

Total Aldolase and Muscle Type Aldolase Activity in theLivers and Liver Cancers of Rats Fed 3'-Methyl-DAB. Theliver tissues of rats fed azo dye for 15, 30, 45, and 60 dayswere assayed for total aldolase and muscle type aldolaseactivity on the 60th day after stopping the azo dye administration. The total aldolase and muscle type aldolase activity were assayed with fructose-1,6-di-P as substrateand were compared with the activity found in normalliver, azo dye-induced liver cancer, and a tumor-free areaadjacent to the cancer. Both activities in normal liverwere fairly constant during a period of 180 days of feedingthe standard diet.

With respect to the total activity, no appreciable difference could be seen in the various tissues tested, asshown in Table 3. On the contrary, the muscle type aldolase activity was found to be greatly elevated in liver cancer as compared with normal liver, and the increase inmuscle type aldolase was already seen in the livers of rats

MARCH 1970 745




Table 2Changes in histolÃ³gica! architecture and milolic rale and in content of DNA and soluble protein in liver of rats fed on

i'-Me-DAB diet for 60 days and further on basal diet for carious periods

Days of feeding on Distribution of cell Content (mg/g wet liver)of

3'-Me-DAB Basal die(diet"Basal

diet6060606060only(control)0143060

90150164.626.544.054.062.058.8II27.932.036.031.029.830.51112.725.011.08.02.63.7IV2.010.57.05.03.53.1V2.86.02.02.02.13.9Mitotic

rate'0312

205-122-80-603DNA"2.32

Â±0.16(6)'2.35

Â±0.11(6VProtein"95.5

Â±14(35)'92.6

Â±11(35)'.5.0

" 3'-Me-DAB diet contained 0.06% 3'-Me-DAB in basal diet.'Cell types composing liver tissue: I, parenchymal cells; II. littoral cells; III, cells of bile ducts; IV, cells of connective tissue: V, cells of blood

vessel walls.' Mitotic rate was expressed as the no. of mitotic figures per 4000 parenchymal cells.* Data were expressed as the mean value Â±S.D.' No. of rats assayed.

Table 3Changes in total and muscle type isozrme activities of liver aldolase of

rats fed on the diet containing carcinogenic and noncarcinogeniccompounds and fed afterward on basal diet

Data are expressed as the mean value Â±S.D.

Days or Teedingon3'-Me-DAB

diet*BasaldietBasal

dietonly(control)1530456060

(interrupted)'60606060606060

9060150210300Liver

cancerTumor-freeareaadjacent

tocancerBasal

dietonly(control)3'-Me-DABdiet

602-Me-DABdiet

602,7-FAA'diet

60Basaldiet

60Basaldiet

60Basaldiet

60No.

ofrats25589144434949101310Aldolase

activity (pmolefructose- 1.6-di-P cleaved/

min/mg protein at30Â°)Total0.094

Â±0.0140.088

Â±0.0130.104Â±0.0140.113Â±0.0170.092Â±0.0150.109Â±0.0070.128

Â±0.0090.112Â±0.0040.108Â±0.0120.090Â±0.0290.080Â±0.0220.095

Â±0.00760.091

Â±0.00920.093

Â±0.01340.097

Â±0.0102Muscle

type0.0115

Â±0.00270.0172'Â±0.00110.0169*Â±0.00380.0197'Â±0.00510.0203*Â±0.00340.0209*Â±0.00300.0242*

Â±0.00360.0212*Â±0.00300.0204*Â±0.00120.0567'Â±0.03120.0244*Â±0.00920.0125

Â±0.00320.0174''

Â±0.00270.0119

Â±0.00270.0174"

Â±0.0045

' 3'-Me-DAB diet and 2-Me-DAB diet contained 0.06% 3'-Me-DABor 2-Me-DAB, respectively, in basal diet.

*Differences from the control value were statistically significant

(p < 0.001).' Rats were fed on 3'-Me-DAB diet for 30 days, basal diet for 30

days, and 3'-Me-DAB diet for 30 days." Differences in muscle type aldolase activities between the 3'-Me-

DAB group and their control, and the 2,7-FAA group and their controlwere statistically significant (p < 0.01 and p < 0.02, respectively).

'2,7-FAA diet contained 0.025% 2,7-FAA in basal diet.

fed azo dye for only 15 days. A similar increase was alsoobserved in the group fed azo dye for 30 days. The liversof rats fed azo dye for 45 and 60 days showed slight increases of muscle type aldolase as compared with that ofanimals given the carcinogen for 15 or 30 days. The interrupted feeding experiment in which the administration ofazo dye for 30 days was followed by 60-day intervals ofno dye feeding revealed the same activity of muscle typealdolase as that observed in animals given prolongedtreatment with the carcinogen for 60 days.

Moreover, tumor-free areas of the liver adjacent to thecancer showed more increased activity of muscle typealdolase than those of the rats given azo dye for 60 daysor with the interrupted feeding schedule, although theactivity was about one-half of that of liver cancer.

Statistically, the observed elevation of the muscle typealdolase activity mentioned above showed a significantincrease (p = 0.001) as compared with control value.

Total and Muscle Type Aldolase Activities of the LiverMeasured at Various Time Intervalsafter Stopping Administration of Azo Dye Fed for 60 Days. Next, an attemptwas made to see how the elevated activity of muscle typealdolase of the liver observed at the 60th day after stopping of 60 days of azo dye feeding was influenced by further administration of basal diet. As shown in Table 3,the results indicate clearly that the elevated muscle typealdolase activity was fixed irreversibly in the liver for aslong as 300 days after 60 days of administration of 3'-Me-DAB. Total activity varied only slightly.

Effect of the Noncarcinogenic Azo Dye, 2-Me-DAB, andAnother Hepatocarcinogen, 2,7-FAA, on Total Aldolaseand Muscle Type Aldolase Activities of Rat Liver. It seemsnecessary to determine whether the observed increase ofmuscle type aldolase in the liver after short-term administration of carcinogenic azo dye is specific for hepatocar-cinogenesis. For this purpose, 2-Me-DAB and 2,7-FAA




Fixation of Muscle Type Aldolase in Hepatocarcinogenesis

were chosen. 2-Me-DAB is known to bind with protein(12) despite its inability to produce cancer (11). Thus theeffect of 2-Me-DAB was examined with the same experimental procedure as that of 3'-Me-DAB. 2,7-FAA is

known to be a potent hepatocarcinogen, although thiscompound induces malignant tumors in various organsother than liver in low frequency (13-15).

The results are shown in Table 3. Compared with normal liver, no appreciable increase of muscle type aldolasewas seen in the livers when 60 days of 2-Me-DAB administration were followed by the subsequent administrationof standard diet for another 60 days.

On the other hand, an increase of muscle type aldolaseactivity in the liver was observed in the 2,7-FAA-fed animals. The degree of this increase was found to be of thesame order as that observed with 3'-Me-DAB-fed ani

mals.Relation between Muscle Type Aldolase Activities Meas

ured by Antisera and Fructose-1,6-di-P: Fructose-1-P Ratio. The activities of aldolase isozymes in crude liver extract have been determined by measurement of the ratioof cleaving velocity of the substrate fructose-1,6-di-Pto that of the substrate fructose-1-P (21-24). The fructose-1,6-di-P .-fructose-1-P ratio, however, changes not linearly but hyperbolically, responding to percentage of increase of muscle type aldolase activity, and varies slightlyeven by the considerable change of the muscle type aldolase content, especially in its low level. This property isnot considered to be adequate for the purpose of the present study in which only slight increases of muscle typealdolase activity were observed. On the other hand,changes in the muscle type aldolase activity measured bythe antisera method are expressed linearly. This is whythe antisera method was used in this experiment.

To confirm further the reliability of the results obtainedin the present study, an attempt was made to see whetherthe same result may be obtained by both the antisera andthe fructose-1,6-di-P:fructose-1-P ratio methods. Forthis purpose, a theoretical relation between the ratio ofmuscle type aldolase activity to the total activity and thefructose-1,6-di-P:fructose-1-P ratio was calculated. Inthis case, the following experimental values were used;fructose-1,6-di-P:fructose-1-P ratio is 1 for liver typeisozyme (18, 20) and 50 for muscle type isozyme (unpublished data), just as for muscle aldolase (18, 20). Considering now the mixture of muscle type and liver type isozymes showing the different activities against the 2substrates fructose-1,6-di-P and fructose-1-P, wheremuscle type aldolase activity for fructose-1-P is a and forfructose-1,6-di-P is 50 a and where liver type aldolase forfructose-1-P is b and for fructose-1,6-di-P is b, the fructose-1,6-di-P: fructose-1-P ratio is expressed as (50 a +b)/(a + b)', thus the following equation can be given,

b = a (50 - R)/(R - 1) (A)

where R denotes fructose-1,6-di-P:fructose-l-P ratio.Since the percentage, />, of muscle type activity to totalactivity is shown to be

P = 50a/(50a + b) X 100 (B)

the following equation can be derived from EquationsA and B:

P = (l -- \/R) X 100 (C)

This implies that the percentage of muscle type aldolasein total aldolase can be calculated directly from the fructose-1,6-di-P: fructose-1-P ratio. Indicating P (%) on theordinate and \/FDP:F/P on the abscissa. Formula Ccan be expressed in a line as shown in Chart 1. Next,fructose-1,6-di-P:fructose-1-P ratio and the percentageof muscle type aldolase in the total aldolase were measured on normal liver, liver of rats fed azo dye for 60 days,azo dye-induced liver cancer, and tumor-free areas adjacent to liver cancer, and all values obtained on each tissue sample were plotted. As shown in Chart 1, the measured values were found to coincide well with the theoretical curve in the cases showing low-level muscle typealdolase content, whereas the deviation from ideality,although small, increases with appearance of muscletype aldolase, presumably because of the use of an insuf-

(P)KM

90

70

60*< 40UJCL

lÂ»

Â§10

0 0.1 02 0.3 04 0.5 0.6 07 08 0.9 VO

1FDP/ FI P

Chart l. Relation between muscle type aldolase activities respectivelymeasured by antisera and by fructose-1,6-di-P:fructose-1-P (FDP/FIP)ratio. The line showed with the formula

50

49 JX 100

FDP. FÃ•P

indicates the theoretical relation (see text). Each point shows the experimental value measured on the liver tissue of normal rat ( O). of fatfed 3'-Me-DAB diet for 60 days (â€¢),adjacent to liver cancer (X), and

on liver cancer (O). Ordinate, percentage (P) of muscle type aldolaseactivity measured by antisera method to total aldolase; abscissa, inverseratio of aldolase activities with fructose-1,6-di-P and fructose-1-P. respectively, as substrate.

MARCH 1970 747




ficient amount of fructose-1-P, as discussed below. Thisimplies that, at least on the liver tissues in the early stageof hepatocarcinogenesis, the similar results can be obtained even in the present experimental condition by either the use of the fructose-1,6-di-P: fructose-1-P ratioor the antimuscle aldolase sera, with respect to the percentage of muscle type aldolase in the total activity.

DISCUSSION

This study provides evidence that an enzymic changeappears in the liver in the early stage of azo dye hepatocarcinogenesis, at which time no appreciable morphological change can be seen. This early event is expressed asan increase of muscle type aldolase activity and is fixedirreversibly for at least 300 days. Moreover, it was alsodemonstrated that the increase of muscle type aldolasewas seen by feeding the other hepatocarcinogen, 2,7-FAA, whereas the noncarcinogen, 2-Me-DAB, failed toinduce this enzymic change, indicating the carcinogen-specific property of this event. Thus it seems likely thatthe carcinogenic stimuli, directing the cell to cancer formation, is fixed in the cell as an irreversible change.

In the present experiment, the muscle type aldolase activity of normal rat liver was shown to be about 13% inaverage. This value is considerably higher than that reported by Rutter et al. (3), 7%, and is rather near to thatof normal human liver (17). The discrepancy of both values may be mainly due to the difference of animals species and strains.

It is well known that 3'-Me-DAB induces not only hep-

atoma but also cholangiocarcinoma in rat liver. In thepresent study, however, both malignant tumors were subjected to the experiment unclassified. According to therecent study of Adelman et al. (1), the fructose-1,6-di-P: fructose-1-P activity ratio of 3'-Me-DAB-induced hep-

atoma was about 2, and that of cholangiocarcinoma wasabout 20. It therefore seems likely that the fluctuation ofthe muscle type aldolase activities of 3'-Me-DAB-induced

malignant tumors observed in the present work may bedue to the unseparate handling of both tumors. It must bepointed out, however, that the fructose-1,6-di-P:fructose-1-P ratio, 2, when expressed with percentage ofmuscle type aldolase to total aldolase activity, is 51.0%.Thus, the muscle type aldolase activity of 3'-Me-DAB-

induced hepatoma is much higher than those of normalliver as well as the liver of rats fed 3'-Me-DAB for 60

days and then fed normal diet for further 60 days.As illustrated in Chart 1, the deviation of experimental

values from theoretical line, although small, increaseswith appearance of muscle type aldolase in tissues. Thisis partly due to the fluctuation in the values of azo dye-induced liver tumors as described above. However, themain cause of the deviation seems to be the use of 10 mwfructose-1-P, since this concentration is very close to theKm value for rat muscle aldolase, whereas the use of 2mM fructose-1,6-di-P in this experiment is sufficient tosaturate either muscle type or liver type aldolase of rat.

Thus, a higher maximal velocity with fructose-1-P wouldresult in a higher fructose-1-P:fructose-1,6-di-P ratiofor any given percentage of muscle type aldolase, in better agreement with the theoretical line. In this sense, theuse of a large quantity of fructose-1-P is needed at leastfor the determination of fructose-1,6-di-P:fructose-1-Pratio in tissue extracts showing high activity of muscle typealdolase.

In the present study, we did not measure the actual concentration of aldolase isozymes, but merely their activities. It should, however, be mentioned that the actualconcentration of muscle type aldolase in tissues containing more than one molecular species of aldolase is exaggerated greatly by the high turnover of muscle type iso-zyme relative to liver type aldolase. In this respect, weare studying now whether the observed increase of muscle type aldolase activity appearing in rat liver in the earlystage of hepatocarcinogenesis is actually caused by its netsynthesis.

Recently, Penhoet et al. (18) demonstrated that a molecule of aldolase is composed of a tetramer, like that oflactic dehydrogenase, and, in addition to muscle type andliver type isozymes, 3 kinds of hybrids exist in the liver,which consist of subunits of both isozymes. In the presentstudy, it seems to be very important to solve whether theinhibition by antimuscle aldolase sera for the activity ofeach hybrid is parallel to the content of muscle type sub-units. The antimuscle aldolase sera were shown to inhibitthe activity of muscle type aldolase completely and, in decreasing fashion, the 3 hybrids, but they had no effect onthe liver type isozyme (18). The same results were alsoobtained in our laboratory with hybrids that were reconstituted artificially and isolated by isoelectric focusingelectrophoresis (unpublished data). These results implythat the antimuscle aldolase sera block the activity of muscle type subunit of each hybrid selectively without interfering with liver type subunit, and, thus, the muscle typealdolase activity measured by the use of antisera is thesum of the activities of pure muscle type isozyme andmuscle type subunit in each hybrid.

Regarding the noncarcinogen, 2-Me-DAB, Warwickreported recently that this azo dye produced hepatomasin partially hepatectomized rats (30). For confirmation ofwhether the increase in muscle type aldolase activity observed in precancerous liver has any correlation to hepatocarcinogenesis, it may be necessary to assay muscle typealdolase activity in the liver of rats partially hepatectomized during administration of 2-Me-DAB diet.

In 1948, Druckrey and Kupfmiiller (6) advocated a"summation theory" based on their findings that the total

dose necessary for producing cancer is practically constant when the daily doses are varied. From this point ofview, an attempt was made in the present experiment todetermine whether the increase of muscle type aldolaseis summed up (Table 3). From the obtained results wecould not, however, reach any conclusion, since statistically significant differences could not be gained in therespective increased level of muscle type aldolase activityinduced by the administration of azo dye for 15, 30, 45,




Fixation of Muscle Type Aldolaxe in Hepatocarcinogenesis

and 60 days, including interrupted feeding. In this connection, it is noteworthy that the increased level of muscle type aldolase in adjacent liver tissue of the tumor isat most 30% of total activity, in spite of the long-termfeeding of azo dye. This led us to consider that the summation of the observed irreversible change, if existing,might occur within a definite limit, and the increase beyond the limit might be due to the growth of the cellwhich acquired autonomy, that is, of the cancerized cell.

ACKNOWLEDGMENTS

We extend our sincere gratitude to Dr. H. Otsuka, Department ofPathology, Faculty of Medicine, Tokushima University, and Dr. N.Takizawa, Department of Pathology, Faculty of Medicine, Chiba University, for their help in studying histolÃ³gica!specimens. We are alsoindebted to Dr. T. Kamiya and Dr. T. BabÃ in our Institute for their invariable advice during the study. Special thanks are also due Dr. HarryV. Gelboin, National Cancer Institute, Bethesda, Md., for his kind reviewing and correcting of the manuscript.

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methylaminoazobenzene and its C-Monomethyl Derivatives. Cancer Res., 9: 336-343, 1949.

13. Morris, H. P., Wagner. B. P., Ray. F. E., Snell. K. C., andStewart, H. L. Comparative Study of Cancer and Other Lesions ofRats Fed /V,jV'-2,7-Fluorenylenebisacetamide or /V-2-Fluorenyl-acetamide. Nati. Cancer Inst. Monograph. 5: 1-53, 1961.

14. Morris, H. P., Wagner, B. P.. Ray, F. E., Stewart, H. L., and Snell,K. C. Comparative Carcinogenic Effects of /V,A"-2,7-Fluorenylene-bisacetamide by Intraperitoneal and Oral Routes of Administra-tration to Rats, with Particular Reference to Gastric Carcinoma.J. Nati. Cancer Inst., 29: 977 1011, 1962.

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enebisacetamide Combined with Traumatic Ulcers of the GlandularStomach of Buffalo Rats. J. Nati. Cancer Inst., 35: 829 840, 1965.

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in Primary Liver Cancer. Nature, 200: 995-997, 1963.25. Schneider, W. C. Determination of Nucleic Acids in Tissues by

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properties of Native and Carboxypeptidase-treated Aldolases ofRabbit Muscle and Liver. J. Biol. Chem., 240: 1327-1337, 1965.

27. Sugimura, T., Matsushima, T., Kawachi, T., Hirata, Y.. andKawabe, S. Molecular Species of Aldolases and Hexokinases inExperimental Hepatomas. In: O. Hayaishi and H. P. Morris (eds.).Biological and Biochemical Evaluation of Malignancy in Experimental Hepatomas, Gann Monograph 1, pp. 143 149. Tokyo,Japan: Japanese Cancer Association, 1966.

28. Taylor, J. F., Green, A. A., and Cori, G. T. Crystalline Aldolase.J. Biol. Chem., 173: 591 604, 1948.

29. Warburg, O., and Christian, W. Isolierung und Kristallisation desGÃ¤rungsfermentsZymohexose. Biochem. Z., 314: 149-176, 1943.

30. Warwick, G. P. The Covalent Binding of Metabolites of 4-Dimeth-ylamino-2-methylazobenzene to Rat-Liver Nucleic Acids, and theCarcinogenicity of the Compound in Partially HepatectomizedRats. Biochem. J., 104: 6p, 1968.

MARCH 1970 749



Hideva Endo, Masao Eguchi, and Susumu Yanagi

Fig. 1. Section of normal rat liver. H & E, X 50 (A); X 125 (ÃŸ).Fig. 2. Section of rat liver prepared immediately after stopping 60-day administration of diet containing 0.06% 3'-Me-DAB. Severe damages

caused by the azo dye are seen in the specimen. H & E, X 50 (A(; X 125 (B).Fig. 3. Section of rat liver prepared at 14-day intervals after stopping 60-day administration of 3'-Me-DAB diet. The damages by the azo dye

are still retained in the liver. H & E, X 50 (A); X 125 (B).Fig. 4. Section of rat liver prepared at 30-day intervals after stopping the 60-day administration of 3'-Me-DAB diet. Considerable recovery

from the damages can be seen in the specimen. H & E, X 125.Fig. 5. Section of rat liver prepared at 60-day intervals after stopping the 60-day administration of 3'-Me-DAB diet. This specimen is prac

tically indistinguishable from that of normal liver (Fig. 1). H & E, X 50 (A); X 125 (B).




Fixation of Muscle Type A Â¡dolasein Hepatocarcinogenesis

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1970;30:743-752. Cancer Res Hideya Endo, Masao Eguchi and Susumu Yanagi HepatocarcinogenesisActivity Appearing in Rat Liver in the Early Stage of Irreversible Fixation of Increased Level of Muscle Type Aldolase

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Irreversible Fixation of Increased Level of Muscle Type ... · Irreversible Fixation of Increased Level of Muscle Type Aldolase1 Activity Appearing in Rat Liver in the Early Stage