Fine structural changes induced in rat hepatocytes by single doses of 3'-methyl-4-dimethylaminoazobenzene

Chem.-Biol. Interactions, 11 (1975) 277-289

277

0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

FINE STRUCTURAL CHANGES INDUCED IN RAT HEPATOCYTES SINGLE DOSES OF 3’-METHYJ,4DIMETHYLAMlNOAZOBENZENE

BOJAN FLAKS

AND

ENG-CHUAN

BY

TEH’

Department of Pathology, University of Bristol, The Medical School, Bristol (Great Britain) (Received October 14th, 1974) (Revision received March 13th, 1975) (Accepted March 14th. 1975)

SUMMARY

Male Leeds rats were given single oral doses of 150 or 300 mg/kg body weight of 3’-methyl-4-dimethylaminoazobenzene (3’-MeDAB). They were sacrificed 24 or 48 h after treatment and their hepatic tissues examined by electron microscopy. The development was observed of an unusual cytoplasmic change, which consisted of large perinuclear areas of decreased hyaloplasmic density, devoid of glycogen or organized structures, which displaced the organelles to the cell periphery. This arose by the formation of large glycogen lakes, which coalesced and then lost their glycogen content, and this was accompanied by nuclear irregularity and shrinkage. Other changes, affecting the endoplasmic reticulum and the cell surfact:, appeared to be similar to those induced by chronic azo dye feeding. It was concluded that the acute lesion observed may limit any specific responses of the hepatocytes to the presence of 3’.MeDAB.

INTRODUCTION

The hepatocarcinogenicity of some azo dyes, including 3’.MeDAB is well known’-5. Their chronic administration to the rat results in hepatic cell necrosis, “oval cell” or bile duct cell proliferation and nodular regenerative hyperplasia in the livers-15. At the level of fine structure the changes induced in the rat hepatocyte by chronic exposure include proliferation of the agranular endoplasmic reticulum*6-22, disorganization and loss of the granular endoplasmic reticulum1~-~9~22~23, glycogen * Present address: Department of Pathology, Faculty of Medicine, University of Malaya,

Abbreviation:3’-MeDAB, 3’-methyl-4-dimethylaminoazobenzene.

Malaysia

278 and lipid accumuladepletion16J7Je, increased numbers of lysosolnes16~1s~l@~22 tionl7-lB, as well as alterations of the mitochondria16J7Jss22, Golgi apparatusl**20 and, occasionally, the nucleus 17Js. Many of these alterations are also present in the resulting liver adenomata and hepatocellular carcinomatal*J9*2*. On the other hand, the early liver changes induced by acute intoxication with 3’-MeDAB or related compounds have received relatively little attention. However, the earliest histological changes observed during azo dye feeding experiments include glycogen depletion and the appearance of eosinophilic or hyaline cytoplasmic inclusions which may eventually encircle the nuclei‘sJOJ1J6. These inclusions have been attributed to hypertrophy of the agranular endoplasmic reticuluml6. Single doses of azo dyes have been reported to give rise, in the rat hepatocyte, to “hydropic degeneration”‘, cellular swelling19 or progressive “vacuolation”25. In many respects the fine structural appearance of these hepatocyteslgJ1*26*27 resembles that observed in rats unc?ergoing chronic azo dye treatment, but Arcasoy et al.26 described, in addition, a previously unreported alteration which consisted of large clear cytoplasmic areas containing unidentified small dark granules. Hitherto the nature of this change has not been investigated further. It is also interesting that these workers did not observe the severe histological changes recorded by other investigators7J9325. Thus, the hepatic cell changes induced by a single dose of 3’-MeDAB do not appear to have been characterized unequivocally. The present investigation was carried out with the aim of defining them and of studying the pathogenesis of the unusual clear cytoplasmic areas reported previously26. MATERIALS AND METHODS

Animals The animals used in this study were adult male rats of the Leeds albino strain, bred in this laboratory and maintained on Oxoid 418 and water ad libitrrm. Carcinogen 3’-MeDAB (Koch-Light, Colnbrook, England) was suspended in olive oil to a concentration of 0.5 g/ml and was administered orally by means of a stomach tube. Experimental treatment Two groups of ra ts, each consisting of 6 animals, were used. Each rat of the first group received a single oral dose of 150 mg/kg body weight of 3’-MeDAB; in the second group each rat was similarly given 300 mg/kg. Three rats from each group were killed by stunning after 24 h and the remaining three after 48 h. Samples of hepatic tissue were immediately removed for processing for electron microscopy and high resolution light microscopy, as well as routine histology.

Since 3’-MeDAB treatment is followed by a reduction in food consumption, which could itself alter hepatic cell ultrastructure, fasted rats were used as controls.

279 Three rats were killed after 24 h of complete fasting and a further three after 48 h. As no strict quantitative investigation was being carried out, pair-feeding was not used. A number of untreated, non-fasted rats were also killed and examined. Treatment of tissues

The hepatic tissue was divided into 1 mms blocks in ice-cold 4% glutaraldehyde, in 0.067 M cacodylate buffer, pH 7.2 (refs. 28, 29). Fixation was continued for 4 h at o-4” and the fixed tissue was then washed for not less than 16 h at O-4’ in 0.1 M sodium cacodylate in 0.25 M sucrose, before being postfixed in phosphate buffered 1% osmium tetroxides, at O-4” for a further 2 h. The tissue was dehydrated in a graded ethanol series at room temperature and embedded in Epon 812, essentially by the method of Luftsl. For electron microscopy thin sections were cut on a Sorvall MT-I Porter-Blum ultramicrotome, using a diamond knife. They were mounted on copper grids and double stained with saturated uranyl acetate in 507: ethanol and lead tartrates2, before examination in a Philips EM 300 electron microscope. For high resolution light microscopy, thicker sections (approximately 0.25 to 0.5 ,u) were cut from the same material by the same method. These sections were stained with 1% methylene blue in I % borax and examined by Nomarski differential interference microscopy, to increase optical contrast. RESULTS

Fasted controls Light microscop)J. No difference between the livers of normal and fasted rats was

seen, although PAS-staining did demonstrate progressive glycogen depletion. High resolution light microscopy showed that there was a generalized loss of the dark glycogen areas of normal hepatocytes (Fig. I) and some lipid accumulation, but apparently no other change. Electron microscopy. The fine structure of the hepatocytes of fasted rats showed only minimal changes, even after 48 h. These consisted of progressive glycogen depletion, the remaining glycogen being interspersed with a mildly hypertrophic agranular endoplasmic reticulum. Groups of large lipid droplets were sometimes present in the peripheral cytoplasm. The granular endoplasmic reticulum was essentially normal, the majority of its cisternae being present in parallel arrays. All other organelles appeared to be normal. 3’-MeDAB Light microscopy. The histological

changes induced by single doses of 3’MeDAB in the rat liver have been described in a previous papers5 and consist of vacuolation and nuclear shrinkage, which are dose-related and become more severe with time. High resolution light microscopy (Figs. I and 2) showed that this vacuolation corresponds to a displacement of cytoplasmic organelles towards the hepatic cell periphery by pale areas surrounding the nucleus, and demonstrated the loss of normal dark-staining glycogen areas.

280

281

282

283 glycogen rosettes appeared to become less closely packed. In some cells (Fig. 4) there appeared to be little glycogen, the ground cytoplasm being extremely pale. In the extreme examples (Fig. 5) the nucleus came to be surrounded by a large clear area of apparently structureless, pale cytoplasm, while the other organelles were closely packed near the cell surface. Detailed examination of the large juxtanuclear glycogen areas of the hepatocytes revealed that, in comparison to those of normal rat hepatic cells (Fig. 6), many of the glycogen rosettes were pale in appearance (Fig. 7). As the change became more severe, the glycogen became increasingly difficult to visualize (Fig. 8), only a few dark granules remaining. Finally, the glycogen disappeared entirely leaving areas of electron-lucent cytoplasm with a faintly flocculent texture (Fig. 9). These areas were not invaded by the agranular endoplasmic reticulum within the duration of the experiment. Lipid accumulation was evident only in some hepatocytes (Fig. 4). The mitochondria and microbodies retained an essentially normal morphology and there was no significant increase in lysosomes or autophagic vacuoles. However, the Golgi zones were abnormally small and frequently empty in appearance. The granular endoplasmic reticulum was relatively normal in its morphology in those hepatocytes which showed minimal damage but otherwise progressively decreased in quantity and became disorganized, losing its normal parallel stacking. Typically this organelle was present as single short cisternae either lying free or encircling the mitochondria, and occasionally showing focal loss of ribosomes. The nuclei of the hepatocytes showing the most extreme changes were shrunken and frequently irregular in outline (Fig. 5), while the nucleoli sometimes exhibited microsegregation. The peripheral cytoplasm of the 3’-MeDAB-intoxicated rat hepatocytes was found to contain moderately increased quantities of the agranular endoplasmic reticulum. The cell surface was generally normal in appearance, but the bile canaliculi were frequently distorted in form and increased numbers of intercellular “i?egs” were observed. DWUSSION

The present investigation revealed that acute doses of 3’-MeDAB can induce a marked change in the fine structural organization of the rat hepatocyte. The most conspicuous and consistent alteration was the peripheral displacement of cytoplasmic organelles by grossly enlarged glycogen areas, which in turn became totally depleted of glycogen. The most severely affected cells contained shrunken, irregular nuclei,

Fig. 4. Rat liver 24 h after a dose of 300 mg/kg of 3’-MeDAB. Note the pale cytoplasmic areas which now contain little glycogen (*). The nuclei are shrunken in size and some cytoplasmiclipid is present. Magnification j< 5000. Fig. 5. Rat liver 48 h after a dose of 300 mg/kg of 3’-MeDAB. The nucleus (N) is both shrunken and irregular and is surrounded by a large area of cytoplasm which contains no organized structures and exhibits reduced hyaloplasmic density. The organelles are closely packed around the hepatic cell periphery. Magnification x 5000.

284

285 encircled by large pale areas of cytoplasm which showed reduced density of the hyaloplasm. This corresponds well with the glycogen depletion an4 cytoplasmic “vacuolation” or hydropic degeneration observed in light microscope studies;s’j, and is quite distinct from the pattern of glycogen depletion seen during fasting. In addition,

it is

clearly similar to the alteration described by Arcasoy rt a/.“‘. Jt seems highly likely that the small dense particles, observed by these investigators within the pale cytoplasmic areas, were in fact the remnants of glycogen, the quantity being insufficient, however, to give positive PAS-staining.

The main difference between the results obtained in this

previous study”6 and those described here is quantitative,

the most probable reasons

for this difference are the use of different strains of rat and the avoidance, in the present study, of fasting before treatment. The nature of the acute change induced by 3’-MeDAB

is not clear. It does not

resemble any of the lethal changes induced by various other hep;ttotoxins”:~-38, nor do the pale areas of cytoplasm contain any obvious structures, such as those of alcoholic hyaline:s”. However, in view of the effects of cortisone and cyclic AMP40~4l on liver glycogen and excess fructose on the density of the hyaloplasm-lZ, it is possible that the change described here m:ly be associated with alterations and glycogenolysis.

in both cellular hydration

It is of some interest that acute DL-ethionine

intoxication

also

produces similar, but much less marked. tine structural changes-‘“. The proliferation

of the agranular endoplasmic reticulum observed in the pre-

sent study was much less marked than that found by Arcasoy et NI.~~. Hypertrophy this organelle appears to be associated with the induction

of

of liver drug-metabolizing

enzymesll-4T

and is a response which is elicited by many agents, both carcinogeni& 3’_MeDABl6.19-22. The relative lack Is--x, and non-carcinogenic 15--47,58--G.includillg

of proliferation

obtained in the present experiment may be due to the other hepato-

cellular changes which may have inhibited this response. This view is supported by the fact that similar differences exist between the acute and chronic effect\ of carbon tetrachloride

on the induction

of agranular

endoplasmic

reticulum

hypertrophy

in

rat liver celW. The decrease in quantity and disorganization

of the granular endoplasmic reti-

culum resembles that recorded by Arcasoy et ~1.“~ and also, in general, that produced by chronic azo dye fcedinglG-lg*““*““. However, the segregation of organelles in the peripheral

cytoplasm renders such comparison

difficult.

The atrophy

of the GoI@

zones, together with the granular endoplasmic reticulum changes, and the absence of any significant increase in lysosomes or autophagic vacuoles may be related to the decline in protein synthesis induced by 3’-MeDAB”G*“7.

Hawkins et al.“” have pointed

Fig. 6. Glycogcn areas (*) of normal rat hcpatocytes. showing dense, closely packed rosettes of gly. 22 500. Fig. 7. Glycoyen area (*) in a hepatocyte from a rat which had received I50 mglkg of 3’-MeDAB 4X h previously. Note the decreasing density of much of the glycogen. Magnification I~>22 500. Fig. 8. Rat liver 48 h after a dose of 150 mglkg of 3’.MeDAB. Very little glycogen remains in the pale perinuclear areas (*). Magnification ., 22 500. Fig. 9. Rat liver 24 h after a dose of 300 mglkg of 3’-MeDAB. A large clear cytoplasmic area is shown which is devoid of glycogen and exhibits reduced density of the hyaloplasm. Magnification Y. 22 500. CO~~II.Magnification

286 out that lysosomes are probably not involved in the events leading to cell death, and their increase is therefore not necessarily an indication of the severity of cell injury. Previous worker@ had found that, in contrast to the present study, a single dose of 3’-MeDAB led to a marked increase in autophagic activity. Since fasting may modify or potentiatess the action of hepatotoxic drugs the different results obtained in the present experiment may be attributable to avoidance of fasting in treated rats. The increased abundance of intercellular “pegs”, together with the bile canalicular changes represents, in a general sense, an increase in irregularity of the cell surface, an alteration which is also frequently found during chronic feeding of carcinoge&s*=* Nuclear microsegregation was neither pronounced nor widespread in occurrence but the degree of nuclear shrinkage and irregularity appeared to be directly related to the extent of the cytoplasmic change. This supports the view that the presence of clear cytoplasmic areas following 3’-MeDAB treatment represents direct toxic damage to the hepatocyte, whereas some of the other changes many be due to more specific effects of the carcinogen, since they are also induced by chronic treatment. CONCLUSlONS

It has been shown, in this study, that acute doses of 3’-MeDAB can induce unusual ultrastructural changes in rat hepatocytes. These appear to be related to toxic damage caused by the azo dye, rather than to its carcinogenic effects, and may indeed tend to mask any changes due to the latter. The substantial differences between the present findings and those of some other workers appear to be due to relatively small differences in experimental conditions. It thus appears that any single study of the acute fine structural effects of an agent should not be regarded as definitive. In addition, since such morphological differences may be expected to reflect equally significant biochemical differences, the importance of monitoring biochemical studies of the acute effects of carcinogens by electron microscopy is clearly indicated. ACKNOWLEDGEMENTS

We wish to thank the Cancer Research Campaign for financial support of this work and Miss S. A. Bigwood for her valuable technical assistance.

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