Chlordecone-induced potentiation of carbon tetrachloride hepatotoxicity: a light and electron microscopic study

Previous studies have shown that a chlorinated pesticide, chlordecone (Kepone), greatly potentiates carbon tetrachloride (CCl4) hepatotoxicity and lethality (Curtis, L.R., Williams, W.L., and Mehendale, H.M. (1979). Toxicol. Appl. Pharmacol. 51, 283-293; Curtis, L.R., and Mehendale, H.M. (1980). Drug Metab. Dispos. 8, 23-27). The present study describes sequential morphologic changes which occurred in livers of rats given a "nontoxic" level of chlordecone (10 ppm for 15 days) followed by a single injection of CCl4 (0.1 ml/kg). The hepatic alterations were examined 1 to 36 hr after exposure of the rats to CCl4. Those changes were compared to hepatic alterations which occurred in rats that received the same dose of chlordecone (10 ppm for 15 days) or a single injection of CClr (0.1 ml/kg) alone. The only change noted in livers from rats that received chlordecone alone was focal increase in smooth endoplasmic reticulum (SER) of hepatocytes at 24 hr and continuing throughout the time course of the experiment. Livers from animals that received CCl4 alone showed morphologic changes at 6 hr consisting of glycogen loss, increase in SER, and dilatation of rough endoplasmic reticulum (RER) in pericentral hepatocytes. Accumulation of small lipid droplets was also noted in midzonal hepatocytes. After 6 hr, there was no further increase in severity of injury. At 12 hr recovery was noticeable and, by 36 hr, livers from the CCl4 group appeared normal. Prior administration of chlordecone greatly potentiated pathologic changes in livers of animals that received CCl4. By 4 hr, there was total loss of glycogen in hepatocytes throughout the entire lobule. Small lipid droplets were present in pericentral, midzonal and periportal hepatocytes. Hepatocytes with extremely dilated RER were randomly scattered throughout the entire lobule. At 6 hr, there was further accumulation of lipid in the form of large droplets in hepatocytes. Focal, necrotic cells surrounded by polymorphonuclear leukocytes were randomly distributed throughout the lobule. The number of necrotic foci had progressively increased at the 12- and 24-hr intervals. By 36 hr, confluent areas of necrosis in pericentral and midzonal areas were observed in livers of some animals. This study indicates that although the combination of chlordecone and CCl4 produces much greater hepatic injury resembling damage due to a massive dose of CCl4, histologically, some differences in the progression and distribution of hepatocellular damage within the lobular architecture of the liver are evident.     

Further studies on the late preventive effects of the anticalmodulin trifluoperazine on carbon tetrachloride-induced liver necrosis

Trifluoperazine (TFP) (50 mg/kg ip) administration to rats 6 or 10 hr after CCl4 (1 ml/kg ip in olive oil) significantly prevented liver necrosis but not fatty liver caused by the hepatotoxin at 24 hr as evidenced by either histology or electron microscopy. TFP given 6 hr after CCl4 significantly decreased the CCl4-induced increases in liver calcium content. TFP raised four to five times the liver glycogen content in control rats but was unable to modify decreased glycogen content of CCl4 poisoned animals. TFP administration increased phospholipid and protein synthesis as evidenced by studies on 32P incorporation into microsomal phospholipid and by experiments on [14C]leucine incorporation in microsomal protein fractions from control rat livers. No significant changes were observed in microsomal phospholipid degradation as studied by decay of label from 32P-prelabeled microsomal lipids or in increased protein degradation as evidenced by decay of label from [14C-guanidino]arginine-prelabeled microsomal proteins found in livers of control rats after TFP treatment. Electron microscopy observations of liver from control animals treated with TFP evidenced accumulation of glycogen in areas close to smooth endoplasmic reticulum (SER); large Golgi areas with an abundant number of lysosomes, and minor dilatation effects on the rough endoplasmic reticulum (RER) and nuclear membrane. Results suggest that TFP preventive effects might be due to the anticalmodulin actions of this drug.     

Protective effect of tryptophan and cysteine against carbon tetrachloride-induced liver injury

The effects of the administration of tryptophan and/or cysteine on carbon tetrachloride (CCl4)-induced hepatic injury were investigated. Rats received CCl4 (1 ml/kg ip) followed 6 hr later by tryptophan (300 mg/kg) and/or cysteine (950 mg/kg) via stomach tube and rats were killed after 24 hr. Treatment with tryptophan, cysteine, or both reduced the degree of hepatic necrosis observed histologically. While CCl4 caused polyribosomal disaggregation and decreased [14C]leucine incorporation into liver proteins in vitro and in vivo, treatment with tryptophan, cysteine, or both caused a shift in polyribosomes toward heavier aggregation and protein synthesis was increased. Serum activities of lactic dehydrogenase (LDH), glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and gamma-glutamyl-transpeptidase were markedly increased after CCl4 alone but after subsequent treatment with cysteine or with tryptophan and cysteine appreciable decreases occurred. Glutathione concentration decreased but total amount remained constant in the livers of CCl4-treated rats while subsequent treatment with cysteine alone or together with tryptophan elevated both levels of glutathione. Using isolated hepatocytes, CCl4 caused decreases in cell viability, in release of LDH, and in [14C]leucine incorporation into protein. Treatment with CCl4 and tryptophan and/or cysteine revealed that cysteine alone or with tryptophan improved cell viability and decreased LDH release of the cells, while tryptophan alone or with cysteine improved protein synthesis. Upon cytologic evaluation, the isolated hepatocytes revealed membrane distortions after CCl4 alone but these were less marked after CCl4 plus tryptophan, cysteine, or both (most improvement). Thus, tryptophan and cysteine act in a beneficial manner against CCl4-induced hepatic injury in the rat.     

Isopropanol and chlordecone potentiation of carbon tetrachloride liver injury: retention of potentiating action in hepatocyte suspensions prepared from rats given isopropanol or chlordecone

Administration of isopropanol (2.5 ml/kg, po) or chlordecone (15.2 mg/kg, po) potentiated the release of glutamic oxaloacetic transaminase (GOT) into serum 17- or 7-fold, respectively, in rats exposed subsequently to 30 microliter CCl4/kg, po. Hepatocytes isolated from isopropanol-treated rats, incubated with low concentrations of CCl4 (0.3 or 0.9 mM), did not have significant increase in the amount of GOT released after 30 min compared to control cells exposed to CCl4. However, at 3 hr cells from isopropanol-treated rats released 10- or 3-fold more GOT when exposed to 0.3 or 0.9 mM CCl4, respectively, than control cells exposed to CCl4. By hour 5 of incubation this differential of GOT release was not observed. The same dose and time-dependent pattern of potentiated GOT release upon exposure of CCl4 was seen in hepatocytes obtained from chlordecone-treated rats. These results indicate that the potentiation by isopropanol or chlordecone of CCl4-induced release of GOT from liver is retained through the procedures of cell isolation.     

Rubratoxin B hepatotoxicity: An electron microscopic study

Rubratoxin B, the principal toxin produced by Penicillium rubrum, causes hepatic damage. The present study describes ultrastructural changes in rat livers at intervals of 3, 6, 24, and 72 hr following administration of 0.36 mg/kg rubratoxin B. Three hours after treatment, minimal ultrastructural changes including loss of glycogen and dilatation of rough endoplasmic reticulum (RER) were observed in groups of hepatocytes. These groups of altered cells showed a random, multifocal pattern of distribution throughout the liver by light and electron microscopy. At 6 hr, hepatocytes in the focal areas affected by rubratoxin showed more severe ultrastructural changes. There was increased vesiculation of RER with loss of ribosomes from the membranes and disaggregation of polyribosome complexes. Mitochondria contained inclusions of cytoplasmic material and intramatrix myelin figures. Increased numbers of lipid droplets were observed in some cells. Sinusoids appeared to be slightly congested. At the 24-hr interval, hepatocytes in the affected areas showed progressive degenerative changes. Mitochondria were extremely swollen and showed loss of cristae and increased numbers of myelin figures in their matrix. Accumulation of cytoplasmic lipid was more evident, and autophagic vacuoles were occasionally seen. Nuclear changes in these hepatocytes included clumping and margination of chromatin. Sinusoids at the 24-hr interval were severely congested with blood cells, platelets, and fibrin. Endothelial cells lining the sinusoids were fragmented and, in some areas, completely absent. By 72 hr, most hepatocytes in the focal, involved areas were completely necrotic. Cytoplasmic organelles showed irreversible, degenerative changes and cell membranes were frequently ruptured. Sinusoids showed complete loss of lining endothelium, and the lumens were occluded with blood cells, fibrin, and cellular debris.     

Rapid alterations induced by insulin in hepatocyte ultrastructure and glycogen levels

The speed with which insulin alters hepatocyte ultrastructure and glycogen levels in insulin-deficient rats has been studied. Insulin deficiency was induced with alloxan, followed by insulin treatment with regular and NPH insulin. Rats were killed at various times after the insulin injection, blood samples were obtained, plasma glucose levels were determined, and liver samples were prepared for electron microscopy and glycogen determinations. Plasma glucose levels in insulin-deficient rats declined to normal values by 4 hours post insulin, returning to insulin-deficient levels by 8 hours post insulin. Hepatic glycogen was considerably reduced in the insulin-deficient rats. By 1 hour post insulin hepatic glycogen increased, reached maximal levels by 8 hours, then declined to insulin-deficient levels by 36 hours. The ultrastructural appearance of both centrilobular and periportal hepatocytes from insulin-deficient rats showed abundant vesicular smooth endoplasmic reticulum (SER), decreased rough endoplasmic reticulum (RER), and enlarged RER intracisternal spaces. One-half hour post insulin, centrilobular hepatocytes were unchanged. In periportal hepatocytes, however, vesicular SER was no longer visible, the RER intracisternal spaces appeared normal, and the amount of RER had increased. By 1 hour post insulin the centrilobular hepatocytes showed similar ultrastructural changes. These changes became more pronounced in the next few hours and remained through 24 hours. By 36 hours both centrilobular and periportal hepatocytes appeared similar to those in the insulin-deficient rat. These results demonstrate the rapid and lobular-specific effects insulin has on the hepatocyte.     

Abnormal hepatic lipid accumulation following treatment of diabetic rats with insulin and a high-carbohydrate,fat-free diet

This study correlates the morphological and biochemical events during the accumulation of hepatic lipids in diabetic rats in response to insulin treatment and a high-carbohydrate, fat-free diet. Alloxan-diabetic rats were fed a high-carbohydrate, fat-free diet and treated with insulin for 12, 36, or 60 hr or 4.5 or 6.5 days. Samples of livers were obtained for determination of malic enzyme activity and the histochemical demonstration of lipids. An increased accumulation of hepatic lipids, although delayed, was observed following insulin treatment of diabetic rats fed the special diet. Small lipid droplets were visible after 36 hr of treatment, which later increased and coalesced into larger droplets present in all hepatocytes. Maximal accumulation was observed at 4.5 days of treatment. These changes were paralleled by an increase in the activity of hepatic malic enzyme. By 6.5 days of treatment, the lipid content of the hepatocytes had decreased and a periportal pattern was discernible. In contrast, malic enzyme activity continued to increase through 6.5 days of treatment. By comparison, no hepatic lipid accumulation occurred in regular chow-fed diabetic rats receiving insulin treatment or in diabetic rats placed on the special diet alone. These results suggest that the combination of insulin treatment and a high-carbohydrate, fat-free diet caused an imbalance in the production and mobilization of hepatic lipids.     

Electron microscopical investigation on aldrin-induced hepatocyte pathology in Rana catesbeiana, with special emphasis on peroxisomes.

We examined the effect of aldrin on hepatocyte ultrastructure in liver of Rana catesbeiana. The frogs were experimentally exposed to chemical substance and liver fragments processed for routine transmission electron microscopy. Hepatic peroxisomes were visualized after incubation with alkaline 3,3'-diaminobezidine (DAB) method. Ultrastructural analysis revealed progressive hepatocyte changes induced by this drug. After 2-weeks, in the hepatocytes the nuclear envelop and the cisternae of both smooth and rough endoplasmic reticulum (SER und RER, respectively) were unusually enlarged. Reduction of glycogen granules associated with an increased frequency of lysosomes was observed. Normal appearing peroxisomes were present in clusters. Lipid droplets were also visualuzed. After 4-weeks, there was a new increase of glcogen associated with a great number of mitochondria and peroxisomes. Moreover, SER und RER were still dilated. Intracellular lipid inclusions became more abundant. These results suggest that the aldrin 250 induces ultrastructural changes in the hepatocyte of Rana catesbeiana.     

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 isoforms expression and on glycogen content.

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers were assessed by immunohistochemistry. Additionally, effects on glycogen content within the hepatocytes were examined. In the livers AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ led to a perivenous necrosis of single cells only, whereas CCl4 and TAA caused complete necrosis of the centrilobular parenchyma. Treatment with each of the four cytotoxins led to necrosis and fatty degeneration of single or groups of hepatocytes within the intrasplenic transplants. This effect was most pronounced with CCl4 and TAA. The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 in the periportal areas was even decreased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. TAA and, more pronounced, CCl4 administration caused a strong reduction in the expression of all three P450 isoforms. Spleens of control rats displayed almost no P450 isoforms expression, independent of the treatment with the cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants. AAL administration slightly reduced the P450 2B1 and 3A2 expression in the transplants. BBZ and, much more pronounced, CCl4 and TAA treatment diminished the staining for all three P450 isoforms. AAL administration led to a marked decrease in the glycogen content of the hepatocytes of the periportal zones of the liver lobules, whereas after BBZ, CCl4 and TAA treatment a strong perivenous reduction in the glycogen content was seen. Similarly, within the intrasplenic transplants a remarkable decline in the glycogen content of the hepatocytes was caused by the treatment with each of the four cytotoxins. Especially after AAL and BBZ treatment the glycogen depletion within both livers and transplants was much more pronounced than the effects on morphology or P450 isoforms expression. It can be concluded that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA seen in normal orthotopic liver are exerted in a similar way also in intrasplenic liver cell transplants.     

Acute toxic effects of paraquat on ultrastructure of rat liver

Paraquat was administrated to pathogen-free male rats orally, and the livers were studied by light and electron microscope at intervals of 6 hours to 5 days. Congestion and hepatocellular injury (degeneration and/or fatty metamorphosis) were seen by light microscope. Electron microscope showed that degranulation of RER, proliferation of SER, decreasing of glycogen particles and mitochondrial swelling occurred in the cytoplasm of the hepatocytes within 2 layers around the central vein at 6 hours. After 12 hours the liver cells throughout the centrolobular area were injured. Degranulation of RER, proliferation of SER, and decreasing of glycogen particles became prominent, and mitochondria showed swelling and transformation. In the midzonal and periportal areas, numerous lipid droplets were seen in the cytoplasm of the hepatocytes. From the result of ultrastructural findings, it is considered that detoxication and biotransformation of paraquat occur in the hepatocytes within 2 layers around the central vein at an early stage, and spread to the hepatocytes throughout centrolobular area later.     

Interaction of hypoxia and carbon tetrachloride toxicity in hepatocyte monolayers

The toxicity of carbon tetrachloride (CCl4) in monolayer cultures of primary hepatocytes was investigated at oxygen concentrations that prevail in the liver under conditions that range from normoxia to hypoxia: 0.5, 1, 2, and 20% O2. CCl4 was administered in the vapor phase at concentrations that produce aqueous concentrations at 37 degrees C of 0.4, 2.0, and 4.0 mM. Damage was assayed by leakage of aspartate transaminase and the inclusion of Trypan Blue immediately after the 2-hr incubation and after an additional 6-hr incubation in 20% O2. Only in the case of 0.5% O2 and 4 mM CCl4 were the monolayers damaged (18%) immediately after the 2-hr exposure; all other exposed cells were undamaged at that time point and the dose response of cell death as a function of CCl4 and oxygen concentration was not evident until the 6-hr time point. The monolayers exposed to 4 mM CCl4 and 1, 2, or 20% O2 exhibited little immediate damage but were all 100% dead 6 hr later. The monolayers exposed to 2 mM CCl4 and 0.5, 1, 2, or 20% O2 were 53, 48, 40, and 22 +/- 2% dead after 6 hr, respectively. These results suggest that effects of CCl4 exposure, for example alterations in the function or synthesis of essential proteins, require several hours to affect cell viability.     

Effects of glucagon on hepatic glycogen and smooth endoplasmic reticulum

The action of glucagon on hepatic glycogen and smooth endoplasmic reticulum (SER) was studied in samples of liver taken sequentially from anesthetized rats. The physiological state of each animal was assessed by continuously monitoring aortic blood pressure and blood lactate/pyruvate ratios. High hepatic glycogen levels were established by using 10–12 hr fasted control-fed rats infused continuously with glucose. In rats receiving glucose only, hepatic glycogen levels remained above 5.0% during the 4-hr period of glucose administration. Centrilobular hepatocytes displayed an abundance of glycogen which often appeared dispersed with elements of SER between the glycogen particles. Periportal cells had dense clumps of glycogen with few vesicles of SER restricted to the periphery of the glycogen masses. The addition of glucagon to the glucose infusate caused a marked stimulation of glycogenolysis. In these rats, the hepatic glycogen level (x?±SE) was 6.71±.15% 1 hr after glucose and declined after initiation of glucagon infusion as follows: 5.86±.29% (15 min), 4.89±.26% (1 hr), 2.16±.40% (2 hr), and 1.66±.29% (3 hr). The fine structure of hepatocytes showed a dramatic response to the administration of glucagon. The glycogen regions of the cells were noticeably decreased in size and number of glycogen granules 3 hr after initiation of glucagon infusion, and SER was abundant in both periportal and centrilobular hepatocytes. The interpretation offered is that glucagon induces the formation of new SER membranes which participate in glycogen breakdown and/or glucose release from hepatocytes.     

Further studies on the mechanism of the late protective effects of phenylmethylsulfonyl fluoride on carbon tetrachloride-induced liver necrosis

We previously reported that phenylmethylsulfonyl fluoride (PMSF) administration to rats (100 mg/kg, ip in olive oil) as late as 6 or 10 hr after CCl4 (1 ml/kg, ip as a 20% v/v solution in olive oil) can partially prevent the necrogenic response to the hepatotoxin at 24 hr. Here we confirm that observation by electron microscopy and provide further evidence that only in these circumstances were nuclear clumping of chromatin, slight dilatation of the endoplasmic reticulum, myelin figures and lipid droplets in the cytoplasm, large numbers of lysosomes and peroxisomes, glycogen, and slightly swollen mitochondria observable in the protected animals. A very minor part of the late protective effects of PMSF might be due to the effects of this drug on decreasing the intensity of covalent binding of CCl4-reactive metabolites or the intensity of CCl4-induced lipid peroxidation still occurring 6 or 10 hr after CCl4. PMSF administration did not prevent CCl4-induced decreases in cytochrome P450 content or glucose-6-phosphatase activity but partially prevented CCl4-induced calcium accumulation in liver. PMSF treatment increased glutathione and glycogen content in CCl4-poisoned animals, but did not markedly modify protein/phospholipid synthesis or degradation processes. Results suggest that the late protective effects of PMSF administration in CCl4-induced liver necrosis might be due to a favorable modulation of the calcium-calmodulin system similar to that previously described for other drugs.     

Prevention of carbon tetrachloride-induced liver necrosis by the chelator alizarin sodium sulfonate.

The administration of the calcium chelator alizarin sodium sulfonate (ASR) (100 mg/kg ip in saline) 30 min before or 6 or 10 hr after CCl4 (1 ml/kg ip as a 20% v/v solution in olive oil) partially prevents the necrogenic effect of the hepatotoxin at 24 hr, but prevention of CCl4 fat accumulation was not observed. Protective action cannot be attributed to potential decreasing effects of ASR on CCl4 levels reaching the liver, on the covalent binding of CCl4-reactive metabolites to cellular components, or on CCl4-induced lipid peroxidation because ASR does not modify these parameters significantly. ASR administration increases GSH levels in livers of both control and CCl4-poisoned animals and decreases the calcium content of intoxicated animals at 24 hr of poisoning. ASR significantly lowers the body temperature of CCl4-treated animals at different times of the intoxication process. Present and previous results from our laboratory on the preventive effects of another very specific calcium chelator, calcion, and several anticalmodulins suggest that the beneficial effects of ASR might be associated with its calcium chelating ability. Other protective effects of ASR, such as lowering body temperature or increasing GSH content in liver, cannot be excluded.     

Lobular and cellular patterns of early hepatic glycogen deposition in the rat as observed by light and electron microscopic radioautography after injection of 3H-galactose

Very low hepatic glycogen levels are achieved by overnight fasting of adrenalectomized (ADX) rats. Subsequent injection of dexamethasone (DEX), a synthetic glucocorticoid, stimulates marked increases in glycogen synthesis. Using this system and injecting ³H-galactose as a glycogen precursor 1 hr prior to sacrifice, the intralobular and intracellular patterns of labeled glycogen deposition were studied by light (LM) and electron (EM) microscopic radioautography. LM radioautography revealed that 1 hr after DEX treatment, labeling patterns for both periportal and centrilobular hepatocytes resembled those in rats with no DEX treatment: 18% of the hepatocytes were unlabeled, and 82% showed light labeling. Two hours after treatment with DEX, 14% of the hepatocytes remained unlabeled, and 78% were lightly labeled; however, 8% of the cells, located randomly throughout the lobule, were intensely labeled. An increased number of heavily labeled cells (26%) appeared 3 hr after DEX treatment; and by 5 hr 91% of the hepatocytes were intensely labeled. Label over the periportal cells at this time was aggregated, whereas centrilobular cells displayed dispersed label. EM radioautographs showed that 2 to 3 hr after DEX injection initial labeling of hepatocytes, regardless of their intralobular location, occurred over foci of smooth endoplasmic reticulum (SER) and small electron-dense particles of presumptive glycogen, and in areas of SER and distinct glycogen particles. After 5 hrs of treatment with DEX, the intracellular distribution of label reflected the glycogen patterns characteristic of periportal or centrilobular regions.     

Hepatic toxicity of nickel chloride in rats

Enhanced lipid peroxidation was observed in livers of rats killed 24 hr after sc injection of nickel chloride (NiCl2) (750 mumol per kg), as evidenced by 13-fold increase of conjugated dienes in microsomal lipids and 4-fold increase of thiobarbituric acid (TBA) chromogens in hepatic cytosol. Histologic examination of livers from rats killed one to three days after NiCl2 injection (500 mumol per kg) showed microvesicular fatty metamorphosis, mild hydropic degeneration, and foci of inflammation. Microvesicular steatosis of hepatocytes was confirmed by electron microscopy. Dose-related increases of serum aspartate aminotransferase (ALT) activity (up to 7-fold vs controls) and alanine aminotransferase (ALT) activity (up to 3-fold vs controls) were observed 24 hr after injection of NiCl2 (125 to 750 mumol per kg); diminished serum alkaline phosphatase activity (up to 72 percent reduction vs controls) was seen at NiCl2 dosages from 375 to 750 mumol per kg. Diethyldithiocarbamate did not influence the effects of NiCl2 on TBA-chromogens in liver homogenates or on serum AST and ALT activities but acted synergistically with NiCl2 to diminish serum alkaline phosphatase activity and to increase serum bilirubin concentration. This study demonstrates that parenteral administration of NiCl2 to rats produces acute hepatic toxicity, with enhanced lipid peroxidation, microvesicular steatosis, and increased serum AST and ALT activities.     

Glycogen metabolism in cultured chick hepatocytes: A morphological study

Ultrastructural and autoradiographic observations of cultured chick hepatocytes under the following conditins are described: Induction of glycogen synthesis with glucose alone and glucose plus insulin, and glucagon-induced glucogen glucogen breakdown. Profiles of hepatocytes cultured in medium containig 10 mM glucose showed typical cellular organelles and occasionally a few glycogen granules. After incubation of hepatocytes with ³H-glucose, silver grains were found over these sparse glycogen granules, indicating a low level of glycogen synthesis by a few cells. After addition of 75 mM glucose for 1 hr about 3% of the profiles of cells showed glycogen, and by 24 hr half of the hepatocytes had glycogen. Addition ofinsulin plus glucose induced glycogen synthesis in 82% of the cells after 6 hr. and by 24 hr almost every cellular profile showed glyocgen particles. Morphologically, glycogen accumulation was similar whether the cells were stimulated by high glucose or by glucose plus insulin: glycogen granules appeared in restricted regions of the cytoplasm, which were rich in smooth endoplasmic reticulum (SER), and peroxisomes were found close to the newly deposited glycogen particles. At maximum glycogen accumulation the association of SER and peroxisomes with glycogen was less obvious. Glycogenolysis induced by incubation of glycogen-rich hepatocytes with glucagon resulted in proliferation of SER in the glycogen regions of the cells. These observations are compatible with the concept of regions in the hepatocyte cytoplasm specialized for glycogen metabolism. Possible roles for SER and peroxisomes found near glycogen particles and other organelles in hepatic glycogen metabolism are discussed.     

Pivotal role of hepatocellular regeneration in the ultimate hepatotoxicity of CCl4 in chlordecone-, mirex-, or phenobarbital-pretreated rats.

Our earlier histomorphometric and biochemical studies suggested that the progressive phase of the interactive toxicity of chlordecone (CD) + CCl4 involves suppression of hepatocellular regeneration. The objective of the present work was to correlate hepatocellular regeneration with CCl4 (100 microliters/kg)-induced hepatotoxicity in rats maintained for 15 days on a normal (N) diet, relative to the regenerative response in rats maintained on a diet containing either 10 ppm CD, 225 ppm phenobarbital (PB), or 10 ppm mirex (M). Hepatocellular regeneration was assessed by measuring DNA and 3H-thymidine (3H-T) incorporation, followed by autoradiographic analysis of liver sections. Hepatotoxicity was assessed by measuring plasma transaminases (aspartate and alanine) followed by histopathological observations of liver sections for necrotic, swollen, and lipid-laden cells. Lethality studies were also carried out to assess the consequence of hepatotoxicity on animal survival. Dietary 10 ppm CD potentiated the hepatotoxicity of CCl4 to a greater extent than PB or M, as evidenced by elevations in plasma enzymes. Although the serum enzymes were significantly elevated in PB rats in contrast to the slight elevations in N and M rats, they returned to normal levels by 96 hr. However, serum enzyme elevations in CD rats were progressive with time until death of the animals. Actual liver injury by CCl4 was greater in PB- than in CD-pretreated rats, as evidenced by histopathological observations. A 100% mortality occurred in CD-pretreated rats at 60 hr after CCl4 administration, whereas no mortality occurred in either N-, M-, or PB-pretreated rats, indicating recovery from liver injury. Hepatocellular nuclear DNA levels were significantly decreased starting at 6 hr after CCl4 administration to CD-pretreated rats, but not in M- or PB-pretreated rats. 3H-T incorporation into nuclear DNA as well as percentage of labeled cells showed a biphasic increase in N rats: 1 at 1-2 hr, and the other at 36-48 hr after CCl4 administration. However, only 1 peak of 3H-T incorporation at 36-48 hr was observed in the CD + CCl4 combination, which was also significantly lower when compared to that observed after the M or PB + CCl4 combination treatments. These findings suggest that there is recovery in N-, PB-, or M-pretreated rats from CCl4-induced injury by virtue of the stimulated hepatocellular regeneration and tissue repair.(ABSTRACT TRUNCATED AT 400 WORDS)     

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short-and longterm stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of ³H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after adminstration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

Animal model of margosa oil ingestion with Reye-like syndrome. Pathogenesis of microvesicular fatty liver

The aetiology and pathogenesis of Reye's syndrome (RS) are incompletely understood. A number of environmental toxins and biological agents, including viruses, have been postulated to cause RS, either acting alone or synergistically. Most investigations have suggested that the primary insult is in the liver mitochondria, leading to a complex biochemical catastrophe, with death from encephalopathy. Margosa oil (MO), a long-chain fatty acid compound, has been shown to cause a Reye-like syndrome with death from hepatoencephalopathy, in children in Malaysia and India. The present time-course study performed in MO-administered mice showed the development of hepatic lesions with many features of RS. MO acts rapidly, within 30 min, on the nuclei of hepatocytes inducing mitoses and binucleated cells. This is followed by mitochondrial injury, with swelling, rarefaction of matrix, loss of dense bodies, pleomorphism, and loss of ribosomes starting at 60 min. There is loss of liver glycogen, and proliferation and hypertrophy of the endoplasmic reticulum (ER), followed by the presence of lipid droplets in the hyaloplasm, and globules within dilated cisterns of the ER. Additional fatty acids from lipolysis of body adipocytes, and fat globules from intestinal MO ingestion further aggravate the liver fatty change. There is evidence of fat globule ingestion by endocytosis into hepatocytes at the level of the sinusoids. The development of microvesicular liver steatosis and glycogen depletion due to involvement of liver cell organelles occur rapidly as in RS.