Morphological and biochemical effects of carbon disulfide on rat liver

EXPERIMENTAL AND MOLECULAR PATHOLOGY 33, 333-344 (1980)

Morphological and Biochemical Effects of Carbon Disulfide on Rat Liver

M. TORRES, G.FELDMANN, M.A. PERRAULT, J. JARVISALO,' AND J. HAKIM~

Laboratoire d’lmmunologie et d’Ht!matologie and the Laboratoire d’tlistologie Embryologic, CytogtWtique of the Centre Hospitalo, Universitaire Xavier Bichat, Universiik Paris VII, and the Unite’ de Recherches de Physiopathologie Hkpatique, INSERM, Hbpital Beaujon, Clichy. France

Received April 17, 1980, and in revised form July 11, 1980

The early biochemical and ultrastructural effects on the liver of CS, administration in vivo to fasted rats were compared in animals treated and not treated with phenobarbital, to ascertain whether these effects differed quantitatively and/or qualitatively. Ultrastructural examination showed that in phenobarbital-treated rats, the main lesion induced by CS, was an increase in the number and size of the lysosomes containing cell debris from other cell organelles such as mitochondria. The endoplasmic reticulum was unchanged compared to that of rats treated only with phenobarbital. In particular, there was no decrease in the rough endoplasmic reticulum. By contrast, in rats not treated with phenobarbital, the main effect of CS, was a decrease in the number of ribosomes bound to the rough endoplasmic reticulum, with little or no change in other cell organelles. Cytochrome P-450 decreased more in phenobarbital-treated than in untreated rats, and cytochrome b5 decreased in phenobarbital-treated rats only. CS, lowered aniline hydroxylase activity, expressed per nanomole of cytochrome P-450 in untreated rats, but increased this activity in phenobarbital-treated rats. The opposite was observed for microsomal peroxidase and cytosolic glutathione reductase activities. Liver catalase diminished more in phenobarbital-treated than in untreated rats. The effects of CS, on liver endoplasmic reticulum as well as on the respective activities of aniline hydroxylase, microsomal peroxidase, and glutathione peroxidase show that untreated and phenobarbital-treated rats respond to CSp administration in qualitatively different ways. It is suggested that in untreated rats, the effects observed are mainly due to CS, itself, whereas in phenobarbital-treated animals, the effects are mainly caused by reactive CS, metabolites formed by the mixed function oxidase microsomal system.

INTRODUCTION

Bond et al. (1969) and Magos and Butler (1972) reported that in vivo administration of carbon disulfide (CS,) to fasted phenobarbital-treated rats leads to exten- sive centrilobular hepatic necrosis, whereas its administration to untreated rats only increases the amount of fat in the periportal zone and induces cell necrosis in a few hepatocytes. The mechanism by which CS, induces hepatic necrosis re- mains unexplained but is thought to result from the increased mixed-function-oxidase-catalyzed metabolism of CS, to reactive intermediates in phenobarbital- treated rats (Bond and DeMatteis, 1969; Gibson and Roberts, 1972; DeMatteis and Seawright, 1973; Dalvi et al. 1974, 1975). Since all previous morphological studies were performed by optical microscopy, one of our purposes here was to analyze by electron microscopy the early hepatocytic lesions induced by CS, administration to either untreated or phenobarbital-treated rats in order to detect the organelles primarily affected under these two experimental conditions.

’ Recipient of financial support from the Centre National de la Recherche Scientifique. Present address: Laboratory of Clinical Chemistry, IV Department of Medicine, Helsinki University Central Hospital, Unioninkatu 33, SF 00170 Helsinki, Finland.

’ TO whom correspondence should be sent at: Laboratoire Central d’Immunologie et d’Htmatologie, Hopital Bichat, 170 boulevard Ney, 75877 Paris Cedcx 18, France.

333 0014-4800/80/060333-12$2.00/O CopyriBht 0 I!%0 by Academic Press. Inc. All rights of reproduction in any Corn reserved.

334 TORRES ET AL.

CS, administration is also followed by a decrease in liver cytochrome P-450 detectable as the latter’s carbon monoxide complex. The drop is larger in phenobarbital-treated rats (Freundt and Dreher, 1%9; Bond and DeMatteis, 1969; Gib- son and Roberts, 1972; DeMatteis and Seawright, 1973; Dalvi et al., 1974; Mack et al., 1974). This probably results from the covalent binding to sulfhydryl cytochrome P-450 apoprotein groups of approximately half the sulfur atoms released during CS, metabolism (Dalvi et al., 1974, 1975; DeMatteis, 1974; Catignani and Neal, 1975; Neal et al., 1976; Savolainen et al., 1977). To ascertain whether the effects of CS, on liver proteins in untreated and phenobarbital-treated rats differed only quantitatively or both quantitatively and, qualitatively, we measured the activities of several microsomal and nonmicrosomal enzymes under both experimental conditions. The morphological and biochemical results obtained suggest that the differences in the effects of CS, in this respect are mainly qualitative.

MATERIALS AND METHODS

Treatment of Animals and Their Livers

Male Wistar rats (170-250 g body wt) were given a daily intraperitoneal dose of 80 mg/kg phenobarbital (phenobarbital-treated) or of 0.15 M NaCl (untreated) for 2 consecutive days. Twenty-four hours after the second injection, 30 ~1 of CS, in 0.5 ml olive oil was intraperitoneally injected into half the animals in the phenobarbital-treated and untreated groups. The other half received olive oil only. All animals were housed in fume cupboards until their decapitation 3 hr after the final injection. Animals were fasted for 24 hr before they were killed. Part of the liver was immediately excised and treated for morphological studies. The remain- der was cooled with ice and perfused in situ with 100 ml ice-cold 0.15 M NaCl through the vena cava in order to remove hemoglobin. Livers were then homo- genized in 4 vol of ice-cold 0.25 M sucrose. Cell debris and nuclei were separated by centrifugation at 600g for 20 min. Aliquots of the supematants were quickly pipetted into small plastic tubes, cooled at -80°C and stored until analysis. The remaining supernatants were further centrifuged twice at 10,OOOg for 20 min to avoid mitochondrial contamination of microsomal and cytosolic fractions. Cytosol was obtained from the postmitochondrial supematant by centrifugation at 90,OOOg for 45 min. Pellets were suspended in the same buffer. Aliquots of the microsomal and cytosolic fractions were quickly cooled to -80°C and stored until analysis.

Enzymatic Determinations

Microsomal NADPH-cytochrome c and NADH-dichlorophenol indophenol reductases were assayed according to Omura and Takesue (1970) and NADH-ferri- cyanide reductase, according to Mihara and Sato (1978). Microsomal aniline hydroxylase was assayed by the method of Kato and Gilette (1965) with minor modifications. Glutathione peroxidase was measured according to Tappel(1978), glutathione reductase according to Beutler (1%9), and catalase according to Luck (1965). Superoxide dismutase was determined as previously reported (Auclair et al., 1977) from chloroform-ethanol extracts, one unit of enzyme being taken as the amount of extract which inhibited by 50% the reduction of nitroblue tetrazolium. The peroxidase activity of microsomal cytochrome P-450 was assayed with cumene hydroperoxide as oxygen donor by following spectrophotometrically the Wurster blue radical generation from TMPD (N,N,N’,N’-tetramethyl-l-H- phenylenediammonium dichloride) as described by O’Brien and Rahimtula (1978).

CARBON DISULFIDE EFFECTS ON LIVER 335

Other Biochemical Measurements

Proteins were measured according to Lowry et al. (195 1). Cytochrome P-450 present in the. microsomal and 600g supernatant fractions was measured by the CO-binding difference spectrum of reduced microsomes (Omura and Sato, 1964b), and from the reduced-minus-oxidized difference spectrum of CO-bubbled supernatants (Matsubara et al., 1976) respectively. Cytochrome b5 was measured according to Omura and Sato (1964a). Microsomal thiobarbituric-reactive products were measured according to Ottolenghi (1959). All spectrophotometric measurements were made with an Aminco DW2a uv-vis spectrophotometer (Division of Travenol Laboratories, Inc., Silver Spring, Md.). All measurements were made in duplicate in the six animals of each of the four groups. Statistical significance of the results was determined by the Student t test.

Morphological Methods

The excised liver fragment was cut into two parts. One was fixed in Bouin’s fluid and embedded in paraffin. Five-micrometer-thick sections, stained with hemalun-eosin, were examined by optical microscopy. The other part of the liver was cut into l-mm3 blocks that were immediately fixed in a 2% glutaraldehyde solution buffered with 0.1 M phosphate buffer, pH 7.4, for 90 min at 4°C. After washing in phosphate buffer for 24 to 48 hr, blocks were postfixed for 60 min at 4°C in a 1.5% osmium tetroxide solution buffered with Verona1 buffer, pH 7.2. After dehydration with graded ethanols, blocks were embedded in epoxy resin. One-micrometer-thick sections, stained with toluidine blue, were cut from each block for orientation. Ultrathin sections stained with uranyl acetate and lead citrate were made from selected blocks and examined with a Siemens Elmiskop I A electron microscope.

RESULTS

Optical Microscopy

In rats given CS, only, hydropic degeneration was observed in the cytoplasm of a few hepatocytes exclusively located around the hepatic centrilobular veins. The increase of fat in the periportal zone previously observed by Bond et al. (1969) and by Magos and Butler (1972) was not seen.

In rats treated with phenobarbital before CS, liver lesions were similar to those already described (Bond et al., 1969; Magos and Butler, 1972). Cell necrosis was observed in many hepatocytes, mainly located around the centrilobular veins but also in single cells of the middle and periportal zones of the hepatic lobules. Most frequently hepatocyte necrosis was expressed as an acidophilic condensation of the cytoplasm either with homogeneous or granular aspects. Some intrasinusoidal eosinophilic bodies (Councilman-like bodies) were present. No morphological liver changes were observed in rats given olive oil, either alone or combined with phenobarbital.

Electron Microscopy

Rats given CS, only (Figs. I and 2). In this group, unlike the one given olive oil only, the most striking liver cell changes affected both the rough and smooth parts of the endoplasmic reticulum (ER). Cisternae of the rough endoplasmic reticulum (RER) were fragmented and dilated (Fig. 1). There was a striking drop in the number of ribosomes bound to RER membranes (Fig. 2). The smooth endoplasmic

336 TORRES ET AL.

FIG. 1. Rats given CS, only. Electron microscopic appearance of a part of a hepatocyte showing that after CS, administration, the most prominent changes affect the endoplasmic reticulum of the cell. The rough endoplasmic reticulum (arrows) is fragmented and dilated. There is also a dilatation of the smooth endoplasmic reticulum. A decrease in glycogen particles is visible (x9500).

FIG. 2. Rats given CS, only. At this high electron microscopic magnification, a striking decrease in the number of ribosomes bound to the membranes of the rough endoplasmic reticulum is visible (~29,000).


reticulum (SER) was dilated and vesiculated (Fig. 1). As a result of the changes in the ER, typical Golgi apparatuses were difficult to identify but when visible, were normal. In a few hepatocytes, lesions of the ER were accompanied by a slight increase in the number and volume of the lysosomes and by a decrease in glycogen particles (Fig. 1). No organelle fragments were seen in the lysosomes. The other organelles were normal or almost normal. The shape of some mitochondria was irregular; moderate dilatation of some mitochondrial cristae was observed. Peroxisomes and nuclei were unchanged. Small lipid droplets were sometimes visible. These were also seen in rats given olive oil only. No noticeable ultrastructural changes were observed in the plasma membrane or in the bile canaliculi.

Rats treated with phenobarbital and CS2 (Figs. 3 and 4). In this group, many hepatocytes exhibited ultrastructural changes usually limited to the cytoplasm (Fig. 3), in addition to the expected changes, i.e., proliferation of the SER (Burger and Herdson, 1966), which occurred in the group treated with phenobarbital only. The most important change affected the lysosomes. Many voluminous lysosomes (Fig. 4) were visible in the hepatocytes where SER proliferation was visible. Their appearance differed from one cell to another: some lysosomes were electron transparent and contained only membrane remnants, while others contained cytoplasmic organelles, such as mitochondria, modified in varying degrees. Others, again, usually very voluminous, contained a condensed cytoplasm, very electron dense. In these lysosomes, it was sometimes possible to identify mitochondria or ER fragments (Fig. 4). Condensation of whole hepatocytes affecting the cytoplasm and the nucleus was also sometimes observed. The RER, on the other hand, was normal (Fig. 3). Mitochondria were normal or displayed moderate changes similar to those observed in the hepatocytes of rats receiving CS, alone. No changes were observed in the Golgi apparatus. Owing to the SER proliferation, it was difficult to affirm that the number of glycogen particles had decreased. Peroxisomes were unchanged.

Biochemistry

Figure 5 shows the effects of CS, on microsomal proteins in the phenobarbital- treated group and the nonphenobarbital-treated group, respectively. Results are expressed as percentages of the values for each group’s controls, comprising one group which was given phenobarbital without C&, and another which received neither phenobarbital nor CS2, respectively. In line with previous reports (Bond and DeMatteis, 1969; Dalvi et al., 1975; JPvisalo et al., 1978) the decrease in liver microsomal cytochrome P-450 caused by CS, was larger in phenobarbital-treated rats than in untreated rats. Cytochrome b5 did not alter in untreated rats but, at variance with other reports (Bond and DeMatteis, 1969; Jarvisalo and Vainio, 1978), dropped by 20% in phenobarbital-treated rats. Results were similar when cytochrome P-450 and b5 were measured in the 600g supernatants (Fig. 5), sug- gesting that the loss in microsomes during preparation of the microsomal samples was not much affected by CS2 administration. The effects of CS, on the microsomal reductase activities of phenobarbital-treated and untreated rats were not significantly different (Fig. 6). Moreover, these effects were slight and insignifi- cant. CS, diminished equally the aniline hydroxylase activity in phenobarbital- treated and untreated rats, expressed per milligram of microsomal protein. How- ever, when expressed per nanomole of cytochrome P-450, this activity dropped by 47% in untreated rats and rose by 22% in phenobarbital-treated rats (Fig. 6).

338 TORRES ET AL.

FIG. 3. Rats treated with phenobarbital and CS,. A proliferation of the smooth endoplasmic reticulum is observed in the cytoplasm of this hepatocyte. The rough endoplasmic reticulum is unchanged (X9500).

FIG. 4. Electron microscopic appearance of a voluminous lysosome, observed in the hepatocytes of rats treated with phenobarbital and CS,. In this organelle, fragments of endoplasmic reticulum and mitochondria are still recognizable. (X 13,000).

CARBON DISULFIDE EFFECTS ON LIVER

@4Variations in % of the controls

o+ CYTOCHROME P-450 CYTOCHROME b5

339

FIG. 5. Variations in cytochrome P-450 and b5 after CS, administration. Cytochrome variations after CS, administration to untreated El and phenobarbital-treated •I rats are expressed as % of their respective controls. Control levels for untreated rats and phenobarbital-treated rats are on the left and right of each pair of columns, respectively. Concentrations measured in the microsomal fraction and the 6OOg supematant are respectively expressed in nmole-’ mg-’ microsomal protein and nmolel g-* wet wt liver. Asterisks indicate a statisticaiiy significant difference (at least P < 0.05) in the CS,- induced variation in untreated and phenobarbital-treated rats.

TMPD peroxidative activity of the microsomes was not modified by CS, in untreated rats but decreased by 77% in phenobarbital-treated rats when expressed per milligram of microsomal protein (Fig. 6). When expressed per nanomole of cytochrome P-450, the difference in the effects of CS, was more striking, sinee it increased microsomal TMPD peroxidative activity by 62% in untreated rats and lowered it by 34% in phenobarbital-treated rats (Fig. 6). CS, had no effect on

@& Variations in % of the Eontmls

t

25

/

25

50

75

a

I 1 * 4a.P +

1

c-4

WI Aniline Hydmxylase TMPD --pwolidmss

FIG. 6. Variations in microsomai enzyme activities after CS, administration. Variations in microsomal enzyme activities are indicated as for cytochromes (see legend to Fig. 5) except that control activities are expressed in nmol-’ min-’ microsomal protein. a Activities expressed in nmole-’ min-’ nmol cytochrome P-450. Asterisks: as in Fig. 5.

340 TORRES ET AL.

microsomal thiobarbituric-reactive products in either phenobarbital-treated or untreated rats. Figure 7 shows the effects of CS, administration to untreated and phenobarbital-treated rats on other liver proteins, expressed as a percentage of the values found in their respective controls. CS, caused a larger diminution of catalase in phenobarbital-treated animals than in untreated animals. The difference in the effect of CS, on glutathione reductase was more striking, since the latter’s activity rose by 17% in untreated rats, and fell by 32% in the phenobarbital-treated rats. Differences in the effects of CS, on the activities of glutathione peroxidase and superoxide dismutase in the cytosolic fractions from phenobarbital-treated and untreated rats were slight and not statistically significant.

DISCUSSION

Although some quantitative differences were observed in the liver, ultrastructural and biochemical changes occurring in the liver after CS, administration to untreated and phenobarbital-treated rats were mainly qualitative. An increase in the size and number of lysosomes was an ultrastructural change common to both groups. It was discrete in untreated rats and considerable in phenobarbital- treated rats. Changes in lysosomes are a very commonly observed lesion in the hepatocytes after administration of toxic agents (Ker, 1973). Their increase in number and size is explained by a mechanism of autophagy in which lysosomal enzymes damage the ‘organelles and proteins already modified by toxic agents. The voluminous electron-dense lysosomes observed in the phenobarbital-treated rats correspond to the acidophilic granular changes observed by optical microscopy in the cytoplasm of the hepatocytes (Ker, 1973). The detection of cytoplasmic organelles such as mitochondria, membrane remnants, and fragments of the endoplasmic reticulum suggests that the toxicity of liver CS, in phenobarbital- treated rats affected organelles other than the ER, even if no profound changes were observed in mitochondria, peroxisomes, nuclei, the Golgi apparatus or cytoplasmic membranes. With regard to the distribution of such toxicity, CS, lowered the cytochrome 65 content in microsomes and diminished the activity of cytosolic enzymes like glutathione reductase and peroxisomal enzymes like catalase in the phenobarbital-treated rats only (Fig.7). On the other hand, cytochrome P-450 fell in both groups of rats injected with CS, (Fig. 5). These findings imply that CS, toxicity was diffuse in the phenobarbital-treated rats and more localized in the

~_v.,iations in % of the cmtmls

FIG. 7. Variations in cytoplasmic enzyme activities after CS, administration. Variations in enzyme activities are indicated as for cytochromes (see legend to Fig. 3) except that control activities are expressed in IU per mg protein. a Activity in units per mg protein, one unit being the amount of enzyme that caused 50% inhibition of nitroblue tetrazolium reduction. Asterisks: same as in Fig. 5.


untreated rats. However, the diffuse toxicity seems to be selective, since changes in many other enzymes such as microsomal reductases, glutathione peroxidase, and superoxide dismutase were similar in both groups of C&-treated rats. The loss in the activities of glutathione reductase and catalase, enzymes known to possess sulfhydryl groups with important functions (Deisseroth and Dounce, 1970; Krohne-Ehrich et al., 1977), and the failure of CS, administration to phenobarbital-treated rats to affect other proteins suggest that, under the present experimental conditions, the reactive products formed by increased CS, metabolism (Bond and DeMatteis, 1969; DeMatteis and Seawright, 1973; Dalvi et al., 1975) lead to their binding to proteins with sulfhydryl groups in a manner similar to the well-demonstrated binding of the sulfur atom to cytochrome P-450 (Dalvi et al., 1974; DeMatteis, 1974; Catignani and Neal, 1975; Neal et al., 1976; Savolainen et al., 1977). The small amounts of these reactive products generated by microsomes in untreated rats might be completely neutralized by their binding to cytochrome P-450 because this is the site of their generation. In phenobarbital-treated rats, the generation of large amounts of C&-reactive products might exceed the binding capacity of cytochrome P-450 and allow their diffu- sion from the generation site. This could then lead to other cell protein lesions perhaps more crucial to cell survival.

Unexpectedly, the changes observed in the endoplasmic reticulum after CS, administration to phenobarbital-treated and untreated rats in relation to their respective controls predominated in the group given CS, without phenobarbital. These changes affected both the RER and SER. A decrease in the number of ribosomes bound to the membranes of the RER like that observed after CS, administration to untreated rats has been widely reported after exposure of animals to many different toxic and carcinogenic agents (Smuckler and Arcosay, 1969; Trump et al., 1973). The interpretation that ribosomal detachment from rough membranes (“degranulation”) underlies this decline has been supported by in vitro studies such as those with carbon tetrachloride (Arstila et al., 1972), ethionine (Kisilevsky and Weiler, 1974), and various carcinogenic compounds like dimethylnitrosamine and aflatoxin Bl (Williams and Rabin, 1971; Williams et al., 1973). Shires (1978), however, suggested that the disappearance of RER from the livers of animals ingesting compounds like carbon tetrachloride arises from the failure of polysome assembly on membranes of the ER rather than from detachment of active ribosomes from the membranes. Further studies are needed to explain the mechanism by which CS, induces “degranulation.” This degranulation on the other hand might explain the significant depression of the incorpora- tion of labeled leucine into protein observed by Bond and DeMatteis (1969) after CS, administration to untreated rats. Since most of the degranulations induced by toxic agents required the presence of NADPH, metabolism of the toxic agents by microsomal membranes was involved. With CS2, degranulation obviously occurred in untreated rats and not in phenobarbital-treated rats. This suggests that CS, itself and not one of its metabolites resulting from mixed-function oxidase activity is responsible for the degranulation effect, or at least that the factor responsible for the RER modification is not the same as that responsible for the decrease in cytochrome P-450, and for the other effects reported above in phenobarbital-treated rats. Other striking differences in the effects of CS, administration to untreated and phenobarbital-treated rats are those observed in microsomal aniline hydroxylase and TMPD peroxidase activities, when these activities

342 TORRES ET AL.

are expressed per nanomole of cytochrome P-450 (Fig. 6). CS, lowered aniline hydroxylase activity in untreated rats and increased it in phenobarbital-treated rats. This also suggests that the CS, molecule, and not one of its reactive metabolites, acts on a component of the mixed-function oxidase system and decreases the overall mixed-function oxidase activity with respect to aniline. By contrast, a C&-reactive metabolite might act on cytochrome P-450, inducing, as previously demonstrated, a decline in its CO-binding spectrum (Freundt and Dreher, 1969; Bond and DeMatteis, 1969; Gibson and Roberts, 1972; DeMatteis and Seawright, 1974; Mack et al., 1974) larger than the real loss of active cytochrome P-450 in metabolizing aniline. The effect of CS, administration to untreated and phenobarbital-treated rats on TMPD peroxidase activity is just the opposite of that observed on aniline hydroxylase activity. The binding to cytochrome P-450 of sulfur atoms (Kamataki and Neal, 1976) which are probably the reactive products of CS, metabolism by the microsomes, has been shown to inhibit drug hydroxyla- tion by cytochrome P-450 and cumene hydroperoxide (Yoshira and Neal, 1977). The exact mechanism by which CS, administration changes the activities of aniline hydroxylase and TMPD peroxidase activities in different ways in phenobarbital-treated and untreated rats is still not clear and needs further study. Our results, however, clearly show that CS, administration has opposite biochemical effects when administered to untreated and phenobarbital-treated rats. They also suggest that CS, itself and a CS,-reactive metabolite may be chiefly responsible for the effects observed in each group of animals, respectively.

The dilatation of the RER and the SER observed after CS, is commonly inter- preted as a consequence of water redistribution in which water and perhaps sodium penetrate the organelles, probably after changes in membrane structure (Trump et al., 1973). This dilatation explains, at least in part, the hydropic degeneration observed by optical microscopy.

On the other hand, the decrease in glycogen particles observed in the liver of rats after CS, is not clearly accounted for, although it might explain the greater toxicity of CS, in fasted than in fed rats (Magos and Butler, 1972).

In conclusion, the results of this study show that the effects of in vivo administration of CS, to fasted rats are strikingly different when the animals are pretreated or not pretreated with phenobarbital. Our results confirm and extend the significance of previous reports by others that in phenobarbital-treated rats, C& is a major toxic agent for the liver, inducing cell necrosis. As a result of the mixed- function oxidase metabolism of CS2, reactive metabolites (Bond and DeMatteis, 1969; Gibson and Roberts, 1972) which are probably sulfur atoms (Catignani and Neal, 1975; Dalvi et al., 1974, 1975; Neal et al., 1976) might diffuse from microsomes, thus altering several organelles and proteins possessing sulfhydryl groups important for their functioning and survival.

By contrast, unmetabolized CS, administered in vivo under our experimental conditions might lead to degranulation of the RER with no secondary toxic effects. The different effects of CS, on microsomal aniline hydroxylase and TMPD peroxidase activities in untreated and phenobarbital-treated rats, respectively, require further investigation.

ACKNOWLEDGMENTS

This work was supported by the Fondation pour la Recherche Medicale and by an Inserm grant (ATP 58.7890).


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Morphological and biochemical effects of carbon disulfide on rat liver

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