Ultrastructural pharmacopathology: I. Comparative morphology of the livers of the normal street dog and purebred beagle. A base-line study

EXPERIMENTAL AND MOLECULAR PATHOLOGY 5, 195-224 (1966)

Ultrastructural Pharmacopathology

I. Comparative Morphology of the Livers of the Normal Street

Dog and Purebred Beag!e. A Base-Line Study

ROBERT J. STEIN,~ WARD R. RICHTER,~ AND GISSUR BRYNJOLFSSON~

Department of Pathology, Abbott Laboratories, North Chicago, Illinois; aad Department of Pathology, S&itch School of Medicine, Loyola University, Chicago, Zllinois

Received February 25, 1963

The use of electron microscopy in the study of hepatic ultrastructure is well docu- mented in numerous detailed accounts and reviews describing pertinent changes in several animal species and man. The references are too numerous to cite more than a few (Fawcett, 195.5, 1961; Novikoff and Essner, 1960; Novikoff and Shin, 1964; Rouiller, 1957; Schaffner and Popper, 1961; Steiner and Carruthers, J. S., 1961; Trump et al., 1962). The amount of electron microscopy done on dog Iiver is extremely small (Steiner and Carruthers, 1961; Hift and Strawitz, 1961; Matsumoto, 1960; Tajima and Motohashi, 1958), although the dog is an animal of prime im- portance as an experimental animal. We are not aware of any detailed reports on the electron microscopy of normal base-line liver for this species. The utilization of electron microscopy in the study of drug toxicity as related to drug safety evaluation is new and there is a paucity of publication (Herdson et al., 1964a,b). Most toxicity studies have been done on hepato-carcinogens and hepato-toxic agents of no known therapeutic use. Frequently with the use of conventional light microscopy, it is difficult and more often impossible to see any morphologic changes which can be attributed to drug intolerance. In experimental chronic drug administration with clinically toxic or lethal dosage levels, histologic sections often appear “normal” when examined in the usual manner. Electron microscopy offers the possibility for more critical evaluation of such clinically toxic drugs.

During the past five years members of this Laboratory have been investigating the histotoxic effects of drugs of different chemical constitution by means of conventional light microscopy and electron microscopy. A number of ultrastructural changes have been observed which could not be explained on the basis of drug toxicity. It has been noted that even in control animals under no medication, distinct changes of the intracellular organelles were seen. To determine whether these findings were the result of intraspecies difference or were due to some other unknown factors, a study was undertaken to establish a base line of canine liver ultrastructure and to determine if any aIteration could be noted between common street dogs and purebred beagles.

1 From the Department of Pathology, Abbott Laboratories, North Chicago, Illinois. 2 From the Department of Pathology, Stritch School of Medicine, Loyola University, Chicago,

Illinois.

195

196 ROBERT J. STEIN ET AL.

MATERIALS AND METHODS

Percutaneous liver biopsies were obtained from 32 adult street dogs of beagle type and from 14 registered beagle dogs. In addition, liver tissue was obtained from 12 control rats from our colony. The street dogs were held in quarantine for approximately 4 weeks, during which time they were observed and acclimated to a kennel environment and diet. The registered beagles had been raised in a kennel and were placed directly in the laboratory animal quarters without quarantine. Both groups of dogs were treated for internal parasites, dipped in chlordane for external parasites, vaccinated against canine distemper, infectious canine hepatitis, leptospirosis and rabies, and checked for freedom from microfilaria prior to transfer to the laboratory quarters.

A general physical examination and a battery of clinical-laboratory tests were conducted after the dogs arrived in the laboratory. The procedures included the following:

1. Hematology-Hemoglobin, hematocrit, total and differential leucocyte counts. 2. Blood chemistry-Glucose, urea nitrogen, electrolytes (Na, K, Cl) bilirubin,

BSP, SGPT, SGOT, ICD, alkaline phosphatase, PSP. 3. Urinalysis (Chemical, microscopic).

These laboratory tests revealed no significant differences between the street dogs and the beagles; all were within normal limits.

Needle biopsies were obtained from the liver during Nembutal@ anesthesia. A part of the tissue was fixed in buffered lO$ formalin for light microscopy and the re- maining tissue was placed in a pool of 1% osmium tetroxide [Palade (1952) as modified by Caulfield (1957) ] within 10 seconds of removal and cut into 051.0 mm pieces which were then transferred to a vial of fixative for 30 to 45 minutes of fixation. Dehydration was in a graded series of ethanol and embedment was in Epon 812 (Luft, 1961) or Vestopal W.

The embedded blocks were trimmed and studied by light microscopy for selection of suitable areas. Mid-zonal areas were selected for electron microscopy in all animals and portal areas and central lobular areas were examined in selected animals from each of the two groups. Ultrathin sections were cut, stained with uranyl acetate or lead citrate (Reynolds, 1963) and were examined with RCA EMU-SE or 3G electron microscopes.

RESULTS

The liver function studies are presented in Table I and all values are within normal limits for the dog, although one street dog had an alkaline phosphatase of 15.5 units. These animals also were within normal limits as determined by general physical examination. In addition, there was no significant variation between the two groups of animals, street dogs and beagles.

The liver of all animals was studied by light microscopy utilizing 7-p paraffin sections stained with hematoxylin and eosin and 1-u Epon sections stained with toluidine blue. In all animals of both groups the liver architecture was considered normal with the exception of the presence of intranuclear crystals (Fig. 6) in the hepatocytes of ten street dogs and fibrosis in one street dog. Crystals such as these

ULTRASTRUCTURAL PHARMACOPATHOLOGY 197

are regularly observed as random findings in most populations of dogs and have been described in detail by Thompson et al. (1959a,b). They occur most frequently in hepatic nuclei but have also been described in the nuclei of renal tubular epithelium. Intranuclear crystals were not seen in the sections of beagle liver, This histologic picture of the liver was normal in all other respects.

TABLE I

LIVER FUNCTION DATA FROM STREET DOGS AND BEAGLES

Dogs

BSP SGOT

% units

SGPT

units

ICD

units

Alk. P.

units

Mean of 32

street dogs

Range of 32

street dogs

Mean of 14

beagles

Range of 14

beagles

1.04 22.72 27.95 82 s4 2.95

l-3.4 15-38 14-59 1-185 O-15.5

2.56 24.25 31.08 34.7 4.625

l-4.5 17-38 25-40 1-145 I-6.5

Electron microscopy was utilized to establish the ultrastructure of the liver of the beagle and the control rats from our colony in order to compare the canine liver with that of the classic picture described for the rat. Hepatic ultrastructure of our series of rats was no different than that already described and the registered beagles were similar to the rat. Only minor variations existed. The registered beagles were remarkably uniform and were considered normal in all respects except for two unusual mitochondria to be described later. Normal canine liver ultrastructure is illustrated in Figs. l-5 in a series of pictures selected from the beagles as well as some of the normal street dogs.

The pattern of glycogen distribution was the most striking difference between rats and dogs. None of the dogs had large deposits of glycogen as seen in a well-fed rat. The glycogen was found in small clusters or was spread thinly in the cytoplasm (Figs. 1 and 2). This is unlike the concentrated masses devoid of mitochondria and rough endoplasmic reticulum which are observed in rats. Large areas of smooth endoplasmic reticulum devoid of glycogen were present (Fig. 3) _ Such an arrangement is more often seen in rats on a minimal diet or following a short period of fasting. The findings in the dog may be related to eating habits and the relationship of feeding time to the time of obtaining biopsies. The pattern of glycogen deposition was relatively constant from the periphery to the center of hepatic lobules and little variation occurred from animal to animal.

Collagen in small amounts was a common finding in the space of Desse in the rat and was also present in the beagles. However, collagen bundles were more numerous and slightly larger in some dogs. A subtle difference exists in the sinusoidal epithelium of the rat and beagle. It is relatively common to see the endothelium of the rat sinusoid as a very thin crisp structure no more than a few hundred angstrom units thick. It may extend for the distance of many hepatic cells without any appre- ciable thickening. In contrast, the canine sinusoidal epithelium was rarely observed as such a narrow cell. In addition the space of Desse was smaller as a result of less


FIG. 1. Normal liver cells from a registered beagle. Nuclei (N), mitocbondria (M), microbodies (B), canaliculi (C), glycogen (gl), and rough endoplasmic reticulum (R) are indicated. X 12,500.

199


FIG. 2. Normal bile canaliculus (C) and Golgi complex (G). Street dog. x 20,000.

FIG. 3. A large area of smooth endoplasmic reticulum (S) is located to the left in a cell with

ittle glycogen. The nucleus (N) is at the right. Normal beagle. X 25,000.

201


FIG. 4. Mitochondria normally appear in a variety of sizes and shapes although most appear

round as those to the right. Street dog. X 15,000.

FIG. 5. Microbodies or cytosomes contain a dense nucleoid. When sections are sufficiently thin

and in the correct plane, the crystalline nature is readily visible (arrow). Street dog. X 100,000.

203


space between the tips of microvilli and the endothelium. This may be related to fixation although fixation and processing of the tissue was similar for both species.

Mitochondria occurred as round, oval, or elongated structures (Fig. 4). A few long and unusually shaped mitochondria were always present and were considered normal. Two abnormal mitochondria were seen in one beagle. They were in adjacent cells and contained excessive cristae (Figs. 12 and 13). In one, the cristae were exceptionally long and extended the length of the mitochondrion which was also enlarged (Fig. 13).

The dense nucleoid of the microbodies was quite prominent and its crystalline nature was remarkably distinct in many dogs (Fig, 5). In these cases, it was possible to see the distinct periodicity of the nucleoid. The longitudinal banding was made up of dark and light lines with no distinct sequential pattern. A 35 A spacing existed in the crystalline structure.

In contrast to the beagles, the street dogs were not a uniform group of animals with respect to hepatic ultrastructure. Variation within normal limits was far greater than that observed in the beagles, and distinctly abnorma1 structures were also found. The glycogen deposits were more variable, extending from no significant amount to moderate deposition. The size and structure of the various components of the smooth endoplasmic reticulum and Golgi complex varied from animal to animal. The number and size of fat droplets and lysosomes were not as constant in the street dogs. However, all of these differences were within normal limits and could reflect individual variation in this heterogeneous group of dogs.

Of far more significance was the presence of ultrastructural abnormalities or lesions in the street dog liver. Only 9 of the 32 street dogs were free of intracellular lesions of the hepatocyte. These included the presence of nuclear crystals, crystaloids or filaments in the mitochondria, abnormal cristae, myelin figures associated with abnormalities of the endoplasmic reticulum, vesiculated endoplasmic reticulum, unusual or excessive lipid droplets, and excess microbodies (Table II).

TABLE II A COMPARISCN OF ULTRASTRUCTURAL LESIONS OBSERVED IN STREET DOG AND BEAGLE LIVER

Snecific lesions 32 Street dogs 14 Beagles

No. involved No. involved

Nucleus

Nuclear crystals

Mitochondria

Crystaloids Abnormal cristae

Endoplasmic reticulum

Vesiculated smooth ER Disorganized smooth ER Myelin figures

Other Excess microbodies Excess lipid

Normal

10 0

12 0 2 1

1 0 5 0 5 0

1 0 6 0

9 13


The nuclear crystals were observed by light microscopy (Figs. 6 and 7). They were moderately electron dense and did not have an unusual affinity for uranyl acetate or lead citrate stains. Nuclear changes were limited to a distortion of the nuclear shape, which was often considerable. Some nuclei were greatly elongated. None of the crystals seen with the electron microscope were as large or the nucleus as distorted as many seen by light microscopy in dogs from previous studies.

The most common intracellular lesion was the presence of crystalloids consisting of bundles of filaments or lamellae in the mitochondrial matrix of twelve of the dogs (Figs. 8-l 1). The frequency of abnormal mitochondria varied from a minimum of one per 100 cells to several in every cell. Because it took considerable searching to find them in some dogs, it is quite possible that they were also present in others but not observed. Additional searching of many sections would undoubtedly increase the incidence. Frequency did not vary from block to block. They were observed in the same frequency in one dog in tissues taken from superficial and deep areas of each liver lobe. When only a few abnormal mitochondria were seen, they were found in a small group of two or more adjacent cells. These cell groups were widely scattered but could not be related to any specific part of the hepatic lobule. They were observed in centro-lobular, midzonal and portal areas of the lobule.

In four animals with mitochondrial filaments, liver biopsies or post-mortem tissues were examined 6 months later. Mitochondria with filaments were still present at this time in approximately the same numbers as in the base-line tissues. These abnormal mitochondria appear with the same frequency in all of our street dogs used for research in other toxicity projects or for drug safety studies. In the past year liver tissue from 84 dogs from various experiments other than this series was examined and mitochondria with filaments were present in one-third of these animals. Many of these dogs were housed in the laboratory under ideal conditions for as long as 2 years. The presence or absence of this mitochondrial abnormality could not be related to experimental procedure in any animal.

These abnormal mitochondria contained filaments which were located in the matrix and could be seen in cross section or longitudinal section. Both longitudinal and cross sections were present in some mitochondria (Fig. IO). Longitudinal sections were much more common and it may be possible that both lamellae and filaments were present. In cross section the filaments were 60 A in diameter with 110 A center- to-center spacing.

The filaments were present in bundles or packs ranging in length from a few hundred angstrom units long to several microns long. In these instances the mitochondria were also elongated (Fig. 9). When long, they often filled a mitochondrion and when short they occupied a small area or existed as multiple bundles (Fig. 8). The tila- ments often had a beaded structure which made them appear coiled (Fig. 11). The density of the mitochondrial matrix was normal when there was no enlargement of the mitochondria but was lowered in some giant mitochondria with scattered filament groups. Mitochondrial size varied from normal to much enlarged and some degree of enlargement was quite common. A few were as large as nuclei and contained dense bodies of various sizes in addition to filaments (Fig. 9). The limiting membrane was always double and normal. Cristae were present, although folded near the limiting membrane by the filaments. Even the largest abnormal mitochondria con-


FIG. 6. Oil immersion light micrograph of a nuclear crystal (arrow). Street dog. Toluidine blue

stain of epon section.

Fro. 7. Electron micrograph of a nuclear crystal similar to that seen in Figure 6. Street dog.

X 16,000.

207


FIG. 8. Mitochondrion containing numerous crystalloids consisting of short bundles of filaments or plates, Note cristae, at the periphery (arrows). Street dog. X 42,000.

FIG. 9. A large mitochondrion containing long bundles of crystalloid material, electron-dense

bodies (B) and a vesicle (V). Other mitochondria in the same cell are normal (M). Street dog.

x 13,500.

209


FIG. 10. Mitochondrial crystalloid illustrating cross section (X) and longitudinal sections of the

filaments comprising them. Street dog. X 60,000. FIG. 11. Longitudinal section of mitochondrial crystalloid. Note the beaded appearance of the

filaments suggesting that the filaments are helical (arrow). Street dog. X 60,000.

211


FIG. 12. A mitochondrion in which there are excessive cristae some of which appear detached

from the periphery. Registered beagle. X 55,000.

FIG. 13. A large mitochondrion with numerous long cristae (arrow) in an abnormal pattern.

Registered beagle. X 25,000.

213

214 ROBERT J. STEIN ET AI..

FIG. 14. A large complex myelin figure within the cytoplasm. Note that the membranes are continuous

with the endoplasmic reticulum (ER) intermixed with dark glycogen granules. The nucleus (N) is at the

bottom of the picture. Street dog. X 22,000.

. : ..,_ .

215


FIG. 15. Myelin figures were found in most cells of this dog as illustrated (arrows). Large masses of dis-

organized endoplasmic reticulum (ER) were also found. The outer layers of the myelin figures often enclosed

glycogen granules (see insert). Street dog. X 7,500; insert X 48,000.


FIG. 16. A cell containing many small myeIin figures. Street dog. X 12,000.

FIG. 17. A high magnification of the disorganized membranes of endoplasmic reticulum found

in cells with myelin figures. Street dog. X 35,000.

219


tained cristae, but there was no evidence of excessive formation of cristae in these cases. Normal electron-dense bodies were present in most cases.

Another mitochondrial abnormality was observed in a single mitochondrion in each of two street dogs and was similar to that seen in one of the beagles (Fig. 12). In these sections there were excessive cristae, with an apparent detachment of cristae from the wall and the formation of a small sheet of cristae free in the matrix. The size and arrangement of these membranes was unlike that of the previously described filaments and were found in dogs in which no filaments were observed.

Myelin figures were present in the hepatic cell cytoplasm of five dogs (Figs. 14-16). In all animals they were associated with a disorganization of the smooth endoplasmic reticulum. This disorganization consisted of the disappearance of vesicles and tubules, with the formation of a loose arrangement of membranes interspersed with single glycogen particles (Fig. 17). The arrangement resembled a loose bundle of string. The rough endoplasmic reticulum was reduced in amount, did not exist as stacked arrays, and only occasionally was in long formed tubules.

This arrangement of endoplasmic reticulum was never present unless myelin figures were also present in the hepatocytes. Both did not necessarily exist in the same cell but both were present in the same liver. In each case, it was possible to predict that myelin figures would be present because disorganized endoplasmic reticulum was seen in the first cells to be examined.

The myelin figures were not numerous in three of the five dogs but very common in two of them. In these latter two, at least 50% of the cells contained excessive membranous whorls. The membrane masses were single or multiple, small or large and often filled much of the cell. All were smooth-surfaced membranes and were continuous with vesicles and membranes of the smooth endoplasmic reticulum (Fig. 14). Glycogen particles were often located within the outer layers of some whorls (Fig. 15 insert) and mitochondria or other cytoplasmic components were found in the core of a few.

Excessive lipid was present in 6 street dogs. In one dog the membrane-bound lipid was made up of many tiny droplets and vacuoles (Fig. 20). Lipid was usually in the form of single homogeneous droplets of various sizes. These were scattered at random within the cytoplasm.

There was extensive vesiculation of the endoplasmic reticulum in one street dog (Fig. 19). Most cells were filled with vesicles although a few were normal. Many cells in this animal also contained numerous microbodies (Fig. 18).

Lysosomes were not unusual or significantly varied from those seen in beagles. The Golgi complex was similar in its various forms in both groups. There were no significant differences in the sinusoids, space of Desse microvilli, pinocytotic vesicles, bile canaliculi, or portal structures.

DISCUSSION

It is stated in the charter of the Commission on Drug Safety (1964) that “the basic purpose of the Commission is to broaden scientific knowledge of the predict- ability of action of drugs in human beings. Thus, the therapeutic advances of the future will be made safer for both patient and physician without delaying the avail- ability of new drugs and without discouraging imaginative research on new and

FIG. 18. A hepatic cell with excessive microbodies. Street dog. X 15,000.

FIG. 19. Vesiculation of the endoplasmic reticulum. Street dog. X 28,000.

221


better agents to that disease.” It has been further stated in the Commission’s report, “. . . that refinement of current experimental approaches and development of novel basic procedures would help materially in insuring rational use of new agents (drugs) in man.” It is with these concepts in mind that this study has been undertaken. It is an accepted fact, that with the application of electron microscopy many

FIG. 20. Fat droplets (arrows) with multiple vesicles. Street dog. X 16,000.

new discoveries have been made relative to the ultrastructural morphology of cells and tissues. The application of this technique in the study of drug toxicity and pharmacologic activity is new and its future potential is great. As Lowry (1963) states, “. . . electron microscopy is a tool of necessity for the cell pharmacologist.” However, before one can state with certainty that a given subcellular change reflects a reaction of injury, be it chemical or physical, a detailed study of the organelles under “normal” conditions is imperative.

Numerous publications stressing submicroscopic cellular change under different physiochemical stress situations have been reviewed by David (1964). Although these reports cover many cytoplasmic lesions associated with experimental cell injury, few of them emphasize the possible adverse effect of variations in the experimental animal on interpretation. The existence of similar lesions in pre-experimental animals as a result of previous stress situations is a definite possibility, especially in species such as the dog where animal sources are not as standardized as in the rat.

In our studies nontreated purebred beagles showed no hepatic ultrastructural


lesions. The livers of the untreated street dogs, however, exhibited changes which have been associated with various diseases as reported in the literature. Mitochondrial crystaloids or filaments which were very common in our dogs have been reported in human liver associated with viral hepatitis and carcinoma of the common bile duct (Jezequell, 1959)) extra hepatic biliary obstruction (Ekholm and Edlund, 1960)) and diabetes mellitus (Laguens and Bianchi, 1963); in the rat liver associated with protein deficiency (Svoboda and Higginson, 1964) and copper intoxication (Barka et al., 1964); and in the pig in lead poisoning (Watrach, 1964). Concentric arrange- ments of the endoplasmic reticulum (myelin figures) are considered to be a local degenerative process of the cell (David, 1964). They are reported to have been induced in rat liver by high doses of compounds of therapeutic value (Herdson et al., 1964a, b). Myelin figures were also observed in the livers of our untreated “normal”

street dogs. Hence, in the evaluation of drug toxicity by the use of electron microscopy one must have rigid controls to be able to come to a logical decision.

These rigid controls must consist of a population of experimental animals in which the tissue ultrastructure is well established and which has a uniform known history. Purebred beagles from a single, well-managed kennel can provide such a population. Street dogs, even though well kept and clinically normal, do not provide a sufficiently uniform population. Kennel-raised dogs from many sources may not be satisfactory because they also could have a varied environmental or genetic background. It is important, therefore, that the investigator establish the base-line ultrastructure for his population of animals.

SUMMARY

The normal ultrastructure of dog liver was studied to provide base-line information necessary

for adequate evaluation of chronic toxicity experiments. Thirty-two clinically normal beagle-type

street dogs were compared to 14 clinically normal registered beagles. The dogs, in turn, were

compared to the classic picture of liver ultrastructure as established for the laboratory rat. The registered beagle liver ultrastructure was similar to that of the laboratory rat, while 23 of the

street dogs had abnormalities in ultrastructural lesions in the liver. Street dog liver was not

uniform or “normal” when studied by electron microscopy. This would eliminate this source of

dogs as an adequate animal for ultrastructural chronic toxicity work.

ACKNOWLEDGMENT

The authors gratefully acknowledge the assistance of M. .4. Bischoff, R. A. Churchill, J. L.

Langdon, S. M. Maize, E. J. Rdzok and H. U’. Thomas; all of whom contributed to the project.

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Documents

Ultrastructural pharmacopathology: I. Comparative morphology of the livers of the normal street dog and purebred beagle. A base-line study