The time course of liver injury and [3H]thymidine incorporation in chlordecone-potentiated CHCl3 hepatotoxicity

The mechanism by which chlordecone (CD) potentiates CHCl3 hepatotoxicity and lethality remains unknown. We examined the time course of the hepatotoxicity by following serum enzymes, liver histopathology, hepatocellular regeneration, and tissue repair by morphometric analysis and [3H]thymidine (3H-T) incorporation into nuclear DNA. Male mice fed control, or CD (10 ppm), mirex (Mx. 10 ppm), or phenobarbital (PB. 225 ppm) diets for 15 days and receiving a single ip dose of 0.1 ml CHCl3/kg in corn oil vehicle were used. Liver damage was assessed by plasma alanine and aspartate transaminases and by histopathology at 4, 12, 24, 36, 48, 72, and 96 hr after CHCl3 administration. None of the dietary pretreatments caused plasma transaminase elevations, any liver necrosis, or any increase in 3H-T incorporation in nuclear DNA at any time. CHCl3 alone caused only limited hepatocellular necrosis without any increase in plasma transaminases. The same dose of CHCl3 given to CD-pretreated mice resulted in greatly increased liver injury. Plasma transaminases were elevated starting at 4 hr, reaching a maximum value at 12 hr and a decline starting at 48 hr. Centrilobular and midzonal necroses were evident at 12 hr onward. PB pretreatment caused some increase in CHCl3-induced necrosis and a moderate rise in transaminases at 24 hr, but Mx pretreatment caused neither effect. 3H-T incorporation was increased at 72 and 96 hr after CHCl3 alone. The same dose of CHCl3 caused only a modest increase in PB and Mx and a significant and maximal biphasic increase at 36 and 72 hr CD-pretreated mice. Morphometry of liver sections indicated that hepatocellular regeneration is stimulated at 72 hr after CHCl3 alone. The same dose of CHCl3 results in a greater stimulation of hepatocellular regeneration in CD-pretreated mice, and this event is pushed forward at 48 hr, continuing through 96 hr to compensate for greater hepatocellular necrosis associated with this treatment. Lesser stimulation of hepatocellular regeneration was observed in PB + CHCl3 and Mx + CHCl3 groups of mice consonance with much lesser hepatotoxicity. These results suggest that the critical decisive event in the recovery from limited hepatocellular injury is the hepatocellular regeneration and tissue repair, which appear to be stimulated in proportion to the injury.     

Protection of hepatotoxic and lethal effects of CCl4 by partial hepatectomy

CCl4 is a hepatotoxic haloalkane, capable of producing hepatocellular fatty degeneration and centrilobular necrosis. Previous reports indicate induction of liver regeneration after 36-48 hr of CCl4 treatment, which is considered as a secondary effect. The present investigation was undertaken to evaluate the primary effects of CCl4 on hepatic DNA synthesis and to correlate liver regeneration with CCl4 toxicity. These studies were conducted in normal and actively regenerating livers using male Sprague-Dawley rats undergoing sham operation (SH), or partial (70%) hepatectomy (PH). Incorporation of 3H-thymidine (3H-T) in hepatocellular nuclear DNA and autoradiographic analyses of liver sections served as indices for hepatocellular regeneration. Initial experiments established that peak regeneration occurs at 2 days post-PH (PH2) and liver regeneration phases out by 7 days post-PH (PH7). SH and PH rats were challenged with a single ip dose of either corn oil vehicle or CCl4 at either 0.1 ml/kg (to represent subtoxic dose) or 2.5 ml/kg (to represent toxic dose). The low dose of CCl4 was not toxic and did not alter 3H-T incorporation and percentage labelled cells at 6 or 24 hours after administration to SH, PH2 or PH7 groups, indicating that there was no interference with PH-stimulated hepatocellular regeneration. The high dose of CCl4 was significantly hepatotoxic and lethal in SH rats, while in PH2 rats both hepatotoxic and lethal effects were significantly decreased. 3H-T incorporation as well as percentage labelled cells, highly stimulated by PH, were significantly decreased by high dose of CCl4. However, hepatocellular regeneration in PH2 rats treated with high dose of CCl4 was still significantly higher than SH or PH7 groups by virtue of the stronger stimulatory effect of PH. In PH7 rats, where hepatocellular regeneration had returned to the SH level, the hepatotoxic and lethal effects of the large dose of CCl4 were also restored. These findings show that the progressive phase of a single high dose of CCl4 injury which normally culminates in hepatotoxic and lethal effects is significantly mitigated by previously stimulated hepatocellular regeneration. High dose of CCl4 suppresses hepatocellular regeneration at early time points after administration in contrast to the smaller subtoxic dose of CCl4. By virtue of the much stronger stimulatory effect, PH results in the protection against the hepatotoxic and lethal effects of CCl4 despite the obtunding effects of the high dose on hepatocellular regeneration.     

Role of hepatocellular regeneration in CCl4 autoprotection

The destruction of liver microsomal cytochromes P450 by a previously administered low dose of CCl4 has been widely accepted as the mechanism of CCl4 autoprotection. However, circumstantial evidence suggests that this mechanism cannot completely explain the phenomenon of autoprotection. The protective effect of a low dose of CCl4 (0.3 ml/kg, po) on the lethal effect of a subsequently administered high dose (5 ml/kg, po) was established in male Sprague Dawley rats. The protective dose permitted 100% survival, whereas only 15% survival was observed without it. Hepatotoxicity, measured by serum enzyme elevations (aspartate transaminase, alanine transaminase, and sorbitol dehydrogenase) and histopathological changes 24 hr after the treatment with high dose, was similar in both the groups, even though the protective dose had significantly decreased liver microsomal cytochromes P450 (to 62% of normal) and associated enzymes, aminopyrine demethylase and aniline hydroxylase. Rats pretreated with CoCl2 to decrease hepatic microsomal cytochrome P450 to 44% of normal levels did not show a significant protection from the hepatotoxicity of high dose of CCl4. Previous studies have established that hepatocellular regeneration is stimulated within 6 hr after the administration of a low dose of CCl4. Based on this observation, a premise that autoprotection results from augmented recovery from injury rather than decreased injury appears likely. Hence, the role of hepatocellular regeneration was evaluated by following 3H-thymidine incorporation in hepatocellular nuclear DNA, labelling index by autoradiography, and by morphometric estimation of mitotic index. After administration of the protective dose of CCl4, stimulated nuclear DNA synthesis measured by 3H-thymidine incorporation into nuclear DNA was increased and this remained high even after subsequent administration of high dose of CCl4. Forty-eight hr after the administration of a lethal dose of CCl4 alone (5 ml/kg, po), labelling index was slightly increased, but mitotic index was not increased. In the surviving rats (15%), both labelling index and mitotic index were significantly elevated after an additional 24 hr. In rats receiving the protective dose, a significantly greater elevation of labelling index as well as mitotic index occurred 48 hr after the administration of the same lethal dose of CCl4. These results suggest that hepatocellular regeneration stimulated by the protective dose, as a biological response recruited to overcome the accompanying limited injury, may augment and sustain tissue repair processes to permit tissue restoration even after the massive liver injury elicited by the subsequent large dose of CC14.     

Tolerance of aged Fischer 344 rats against chlordecone-amplified carbon tetrachloride toxicity

We have investigated the effects of chlordecone 1(CD)+CCl4 combination in adult (3 months), middle aged (14 months), and old aged (24 months) male Fischer 344 (F344) rats. After a non-toxic dietary regimen of CD (10 ppm) or normal powdered diet for 15 days, rats received a single non-toxic dose of CCl4 (100 microl/kg, i.p., 1:4 in corn oil) or corn oil (500 microl/kg, i.p.) alone on day 16. Liver injury was assessed by plasma ALT, AST, and histopathology during a time course of 0-96 h. Liver tissue repair was measured by [3H-CH3]-thymidine (3H-T) incorporation into hepatic nuclear DNA and proliferating cell nuclear antigen (PCNA) immunohistochemistry. Hepatomicrosomal CYP2E1 protein, enzyme activity, and covalent binding of 14CCl4-derived radiolabel were measured in normal and CD fed rats. Exposure to CCl4 alone caused modest liver injury only in 14- and 24-month-old rats but neither progression of injury nor mortality. The CD+CCl4 combination led to 100% mortality in 3-month-old rats by 72 h, whereas none of the 14- and 24-month-old rats died. Both 3- and 14-month-old rats exposed to CD+Cl4 had identical liver injury up to 36 h indicating that bioactivation-mediated CCl4 injury was the same in the two age groups. Thereafter, liver injury escalated only in 3-month-old while it declined in 14-month-old rats. In 24-month-old rats initial liver injury at 6 h was similar to the 3- and 14-month-old rats and thereafter did not develop to the level of the other two age groups, recovering from injury by 96 h as in the 14-month-old rats. Neither hepatomicrosomal CYP2E1 protein nor the associated p-nitrophenol hydroxylase activity or covalent binding of 14CCl4-derived radiolabel to liver tissue differed between the age groups or diet regimens 2 h after the administration of 14CCl4. Compensatory liver tissue repair (3H-T, PCNA) was prompt and robust soon after CCl4 liver injury in the 14- and 24-month-old rats. In stark contrast, in the 3-month-old rats it failed allowing unabated progression of liver injury. These findings suggest that stimulation of early onset and robust liver tissue repair rescue the 14- and 24-month-old F344 rats from the lethal effect of the CD+CCl4 combination.     

Acute hepatotoxicity and lethality of CCl4 in chlordecone-pretreated rats

In a subchronic dietary pretreatment protocol chlordecone (CD) is a powerful potentiator of CCl4 hepatotoxicity, as indicated by biochemical, hepatofunctional, histopathological, and lethality parameters. The purpose of this investigation is to further explore the CD + CCl4 interaction in an acute CD pretreatment protocol and to compare the two pretreatment protocols in terms of their effect upon quantitative histopathology, serum enzymes, and lethality. Groups of four male rats received one of the following four pretreatments: chlordecone (10 mg/kg; single po), mirex (10 mg/kg; single po), phenobarbital (PB) (80 mg/kg/day for 2 successive days; ip in 0.9% saline), or corn oil vehicle (1 ml/kg; single po). Twenty-four hours later, the rats were given a single ip injection of CCl4 (0.1 ml/kg). Twenty-four hours after CCl4 administration, serum enzymes (SGPT, SGOT, and ICD) were measured and the livers removed and fixed in 10% buffered formalin for histological evaluation. The LD50 were determined by the method of moving averages. CD + CCl4 was the most hepatotoxic combination, in terms of serum enzyme elevations and lethality followed by PB + CCl4. The PB + CCl4 combination caused a greater degree of hepatocyte necrosis. These findings indicate that the acute pretreatment with CD enhances hepatotoxicity and the lethality of CCl4 in a fashion qualitatively similar to the subchronic pretreatment protocol.     

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.     

Prolonged reduction of hepatocyte proliferative ability in rats after a single treatment with carbon tetrachloride.

Female Wistar rats were pretreated with I ml of carbon tetrachloride/kg of body weight or with olive oil. All the rats were given this dose of CCl4 20 or 40 days later. Liver regeneration as evaluated by 3H-thymidine incorporation into liver DNA and by the number of mitotic hepatocytes was markedly impaired in CCl4-pretreated rats when compared with olive oil-pretreated controls. DNA labelling reached only 83 and 59% and mitotic index 35 and 58% of control values, respectively, at 20-day and 40-day time intervals. The variables characteristic of liver damage did not parallel the changes in cell division. About 20% of hepatocytes were necrotic both in the CCl4-pretreated and in the control rats. The activity of serum alanine aminotransferase was higher in the CCl4-pretreated rats. Only serum aspartate aminotransferase activities were somewhat lower when compared to controls. Similarly, serum aminotransferases were much less affected by the pretreatment than the markers of regeneration when two low doses of CCl4 (0.125 ml/kg) were given to rats 20 days apart. The activities of microsomal enzymes aniline hydroxylase and pethidine demethylase were equal in control and in experimental rats 20 days after CCl4 pretreatment which indicated that the effects of CCl4 were not mediated by an overall decrease in cytochrome P-450 enzymes. In summary, a single pretreatment of rats with CCl4 induced changes in liver that lasted for 40 days and impaired liver regeneration when another dose of CCl4 was applied.     

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.     

Effects of carbon tetrachloride and azathioprine on diethylnitrosamine and N-2-fluorenylacetamide-induced hyperplastic liver nodule and hepatocellular carcinoma

Effects of carbon tetrachloride (CCl4) and azathioprine (AZP) on the evolution of hyperplastic liver nodules and foci and hepatocellular carcinoma (HCC) were tested in short- and long-term in vivo experiments. In diethylnitrosamine (DEN)-treated rats, which were fed a N-2-fluorenylacetamide (FAA)-containing diet and additionally treated with repeated CCl4 injections, gamma-glutamyl transpeptidase (gamma-GTP)-positive hyperplastic nodules were markedly developed in the 8th week of the experiment. However, their number and area in liver sections were remarkably small in DEN-treated rats fed a diet containing both FAA and AZP. Increased area of gamma-GTP-positive foci was also observed in the 12th week in DEN-injected rats fed a choline-devoid died alone or treated with repeated doses of CCl4 alone. Hepatocellular carcinoma in DEN-injected rats treated with both FAA and CCl4 was first detected in the 21st week, and the incidence up to the 36th week was very high. However, no hepatocellular carcinoma developed in DEN-injected rats treated with both FAA and AZP. The increased activity of liver aniline hydroxylase observed 12 h after the administration of FAA, AZP or DEN alone was not observed when AZP was administered simultaneously with FAA to DEN-injected rats. The mechanisms of the effects of CCl4 and AZP on hepatocarcinogenesis are discussed with special reference to drug interaction.     

Kupffer cells are associated with apoptosis, inflammation and fibrotic effects in hepatic fibrosis in rats

Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMN- and CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not α-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN- and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-α (TNF-α) and KC co-localized in the liver in the neighbor of HSCs. The double α-SMA- and collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for α-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver fibrosis models.     

Chlordecone-induced potentiation of carbon tetrachloride hepatotoxicity: a morphometric and biochemical study

The present study, conducted over a time course of 36 hr after CCl4 administration, describes sequential morphometric and biochemical changes which occur in livers of rats exposed to a combination of low levels of chlordecone (10 ppm for 15 days) and a single ip injection of CCl4 (0.1 ml/kg). Those changes were compared to hepatic alterations which occur in rats that received the same dose of chlordecone or CCl4 alone. Biochemical studies showed only trivial increases in levels of glutamic-pyruvic transaminase (GPT), glutamic-oxalacetic transaminase (GOT), and moderate but temporary increases in isocitrate dehydrogenase (ICD) after CCl4 alone. The combination of chlordecone and CCl4 resulted in significantly greater elevations of all three serum enzymes at all time intervals examined. Morphometric data showed no difference between normal diet controls and animals exposed to chlordecone alone as far as numerical density of hepatocytes or volume densities of hepatocytes with glycogen, lipid, dilated rough endoplasmic reticulum (RER), pyknosis, or mitoses. Morphometric analysis of livers from animals that received CCl4 alone showed decreases in numerical density, temporary decrease in percentage of hepatocytes containing glycogen, an increase in hepatocytes containing lipid, temporary increase in hepatocytes with dilated RER, and temporary increases in pyknotic nuclei. Soon after the initial hepatic injury was histologically evident between 4 and 6 hr, the number of mitoses increased dramatically and this progressed until complete recovery from CCl4 damage. From all indices of damage, complete recovery was evident by 36 hr after CCl4 administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of phenobarbital on hepatic injury induced by chronic carbon tetrachloride treatment

Rats were given 1 ml CCl4 per kg body weight subcutaneously 2 times a week, for 16 weeks. The effects of simultaneous phenobarbital (PB) treatment (0.05% in drinking water) on the hepatotoxicity of CCl4 was studied during 16 weeks of treatment. The retardation of growth, the increase in liver weight and mortality were greater in animals receiving both PB and CCl4 than those given CCl4 alone. Cirrhosis was apparent only in animals treated by PB + CCl4. The potentiating effect of PB on CCl4 hepatotoxicity was also seen in hexobarbital sleeping time, the rate of hexobarbital metabolism, and the cytochrome P-450 content in liver microsomes. The inducing effect of PB alone decreased with time both in vivo and in vitro, which suggests an adaptation or some kind of exhaustion of liver to the effects of PB.     

四氯化碳诱导肝纤维化早期大鼠肝窦内皮细胞及基底膜形态改变的动态研究

 目的：探讨四氯化碳诱导肝纤维化早期大鼠肝窦毛细血管化的形成过程。方法：清洁级雄性SD大鼠采用随机数字表法随机分为2组：正常对照组（N组,6只）和肝纤维化模型组（M组,32只）。M组大鼠腹腔注射50%四氯化碳蓖麻油混合液, N组大鼠腹腔注射生理盐水,剂量为2 mL/kg,每周2次,共4周。分别于造模第3天、1周、2周和4周处死大鼠,HE染色和Masson染色观察肝脏组织炎症及纤维化的改变,透射电镜观察肝窦内皮细胞（LSECs）窗孔与基底膜（BM）的改变,免疫组织化学检测LSECs表面标志物CD31及基底膜成分IV型胶原（Col IV）和层黏连蛋白（LN）的改变。结果：HE及Masson染色显示四氯化碳造模4周早期肝纤维化已形成。肝组织透射电镜显示四氯化碳造模第3天后开始出现LSECs窗孔直径变小及数目减少,随着造模时间的延长,LSECs失窗孔现象逐步严重,至第4周时局部内皮下可见连续的基底膜。免疫组化染色显示LSECs表面标志物CD31表达随着LSECs窗孔数目的减少而逐渐增强;基底膜成分Col IV于造模第2周时表达开始显著增强并随着造模时间延长表达逐渐增强,LN于造模第4周时表达开始显著增强。结论：肝纤维化早期大鼠局部肝组织可见典型的肝窦毛细血管化形成;肝窦壁内LN沉积是肝窦毛细血管化时形成连续基底膜的关键因素。     

Pentachlorophenol (but not phenobarbital) promotes intrahepatic biliary cysts induced by diethylnitrosamine to cholangio cystic neoplasms in B6C3F1 mice possibly due to oxidative stress

Administration of diethylnitrosamine (DEN) to B6C3F1 mice at low dose (20 ppm) in drinking water for long duration resulted in formation of multifocal cystic biliary lesions in the liver. To investigate the potential of the lesions to be promoted to neoplasias by chemicals, we examined the effects of 2 different types of hepatocarcinogenesis promoters, pentachlorophenol (PCP) and phenobarbital (PB) in B6C3F1 mice. Two weeks' exposure to PCP at a concentration of 600 ppm in the diet increased 8-oxodeoxyguanosine (8-oxodG) levels in liver nuclear DNA, and cell proliferation quantified by bromodeoxyuridine (BrdU) incorporation in epithelial cells of intrahepatic bile ducts as well as hepatocytes. In mice initiated with DEN at 20 ppm in the drinking water for the first 13 weeks followed, after a 4-week recovery interval, by PCP at a concentration of 600 ppm in the diet for 25 weeks, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinomas were present at statistically significant higher incidences. In contrast, neoplasia did not occur in animals treated with 500 ppm PB, and there were no elevations in 8-oxodG levels or increases in the proliferation of biliary epithelium, although proliferation was increased in hepatocytes. These findings suggest that oxidative stress due to PCP might exert a promoting action on the biliary cystic lesions produced by DEN.     

Retinol binding protein in plasma to evaluate the hepatotoxicity of rats treated with CCl4

Retinol binding protein (RBP) in plasma of rats treated with carbon tetrachloride (CCl4) was monitored to clarify if RBP is available for the evaluation of the drug-induced hepatotoxicity. Blood was withdrawn by heart puncture at 0 hr and 12 hr after i.p. administration of CCl4 (0.2 ml/kg) to rats. Lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) in plasma significantly increased at 12 hr after CCl4 administration, compared with the control, while RBP in plasma significantly decreased. On the other hand, albumin in plasma was unaffected at 12 hr after CCl4 administration. Thus RBP seems to monitor the different aspects in the drug-induced hepatotoxicity from LDH and ALT, and from the viewpoint of protein synthesis in the liver, to be more sensitively affected by the drug-induced hepatotoxicity than albumin.     

Expression of tumor necrosis factor-alpha and transforming growth factor-beta 1 in acute liver injury

Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1) have a number of in vitro functions that could be important in vivo in acute liver injury and repair. Therefore, we investigated these two cytokines in acute liver damage. Northern blots of RNA isolated from rats sacrificed at various time intervals after a single oral dose of CCl4 revealed that TNF-alpha mRNA levels were elevated within 6 hr of CCl4 administration and returned to control values by 24-32 hr. In contrast, TGF-beta 1 mRNA levels started to rise significantly at 24 hr, peaked at 48 hr, and approached baseline levels by 72 hr. Identical changes in TNF-alpha and TGF-beta 1 mRNA levels were also seen with D-galactosamine-induced hepatotoxicity. Immunohistochemical analysis using a TGF-beta 1 antibody demonstrated increased hepatic staining in CCl4-treated rats, at times corresponding to the increases in TGF-beta 1 gene expression. Therefore, there is a differential expression of these cytokines in acute CCl4 and galactosamine hepatotoxicity with an early rise in TNF-alpha, suggesting that this cytokine may affect inflammation and cell toxicity, while TGF-beta 1 peaks later, when it may regulate hepatocyte proliferation and extracellular matrix repair.     

Hepatocellular injury induced by chronic low-dose CCl4 in spontaneous and renal hypertensive rats: a correlation to the reversal of experimental rat hypertensive models.

Renal hypertension has been reduced to normal levels in the rat with subcutaneous injections of carbon tetrachloride (CCl4). Previous light microscopic evaluation of liver sections demonstrated varying degrees of hepatocellular and hepatic parenchymal injury secondary to CCl4 administration. In normotensive rat populations, saline injections have been shown not to cause hypertension, and the treatment of normal rats with subcutaneous CCl4 did not change their blood pressure over that of noninjected controls. In this study we compare the extent of blood pressure reduction with the degree of CCl4-induced liver injury in the renal-induced, spontaneous hypertensive rat (SHR), and normotensive rat. Statistically significant blood pressure reduction followed CCl4 injection of renal (p less than .01) and SHR (p less than .001) hypertensive animals. The morphological liver injury appeared most sensitively reflected in hepatocellular nuclear atypia. The SHR animals were the most resistant to CCl4 liver damage by chronic CCl4 treatment. There was no measurable effect of CCl4 treatment on the animals as assessed by their activity, rate of development and weight gain. The degree of blood pressure reduction due to CCl4 treatment in the SHR animals was similar to that previously reported in DOCA and renal-induced hypertensive animals.     

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.     

Enzyme markers of collagen synthesis in carbon tetrachloride-induced fibrosis and during colchicine modification of CCl4-induced liver injury

Serum galactosylhydroxylysyl glucosyltransferase (S-Glu-Gal-Hyl-Tase), liver galactosylhydroxylysyl glucosyltransferase (L-Glu-Gal-Hyl-Tase), liver hydroxylysyl galactosyltransferase (L-Gal-Hyl-Tase), and liver prolyl hydroxylase (L-PH) activities were measured in rats during the development of CCl4-induced cirrhosis (0.2 ml of 33% CCl4 in light mineral oil two times weekly for 10 weeks followed by 6 weeks of no treatment). Serum and liver markers of collagen synthesis increased in a time-dependent manner reaching maximum activity at 6 weeks (S-Glu-Gal-Hyl-Tase, two times; L-PH, two times). These enzyme levels returned to normal during the 4-week recovery period. In a separate 4-week experiment, colchicine (10 micrograms/rat/day) was administered with CCl4. Colchicine prevented the increase in S-Glu-Gal-Hyl-Tase, L-Glu-Gal-Hyl-Tase, and L-Gal-Hyl-Tase induced by CCl4 and resulted in a smaller increase in L-PH. These results demonstrate that S-Glu-Gal-Hyl-Tase elevation occurs following CCl4 because of increased liver collagen synthetic activity and the hepatocellular injury produced by CCl4.