Dose-dependent liver tissue repair in chloroform plus thioacetamide acute hepatotoxicity

The objective of this study was to test whether a binary mixture (BM) of chloroform (CHCl(3)) and thioacetamide (TA) causes a dose-dependent liver injury and an opposing tissue repair. Liver injury was assessed by plasma alanine aminotransferase (ALT) and histopathology. Tissue repair was measured by [(3)H-CH(3)]-thymidine ((3)H-T) incorporation into hepatonuclear DNA and PCNA over a time course of 0-72h. Male Sprague-Dawley (S-D) rats received six- and five-fold dose ranges of TA and CHCl(3), respectively. ALT levels and (3)H-T incorporation were in complete agreement with corresponding microscopic observations, and only ALT elevation and (3)H-T incorporation data are presented here. Liver injury observed after exposure to BM was no different than addition of injuries caused by individual compounds. Tissue repair was prompt and adequate, leading to recovery from injury and animal survival. Tissue repair is dose-dependent and plays central role in the hepatotoxic outcome.     

Dose-dependent liver regeneration in chloroform, trichloroethylene and allyl alcohol ternary mixture hepatotoxicity in rats

The present study was designed to examine the hypothesis that liver tissue repair induced after exposure to chloroform (CF) + trichloroethylene (TCE) + allyl alcohol (AA) ternary mixture (TM) is dose-dependent similar to that elicited by exposure to these compounds individually. Male Sprague Dawley (S-D) rats (250–300 g) were administered with fivefold dose range of CF (74–370 mg/kg, ip), and TCE (250–1250 mg/kg, ip) in corn oil and sevenfold dose range of AA (5–35 mg/kg, ip) in distilled water. Liver injury was assessed by plasma alanine amino transferase (ALT) activity and liver tissue repair was measured by ³ H-thymidine incorporation into hepatonuclear DNA. Blood and liver levels of parent compounds and two major metabolites of TCE [trichloroacetic acid (TCA) and trichloroethanol (TCOH)] were quantified by gas chromatography. Blood and liver CF and AA levels after TM were similar to CF alone or AA alone, respectively. However, the TCE levels in blood and liver were substantially decreased after TM in a dose-dependent fashion compared to TCE alone. Decreased plasma and liver TCE levels were consistent with decreased production of metabolites and elevated urinary excretion of TCE. The antagonistic interaction resulted in lower liver injury than the summation of injury caused by the individual components at all three-dose levels. On the other hand, tissue repair showed a dose-response leading to regression of injury. Although the liver injury was lower and progression was contained by timely tissue repair, 50% mortality occurred only with the high dose combination, which is several fold higher than environmental levels. The mortality could be due to the central nervous system toxicity. These findings suggest that exposure to TM results in lower initial liver injury owing to higher elimination of TCE, and the compensatory liver tissue repair stimulated in a dose-dependent manner mitigates progression of injury after exposure to TM.     

Liver regeneration: a critical toxicodynamic response in predictive toxicology

The objective of the present review is to discuss the importance tissue repair in the mixture risk assessment. Studies have revealed the existence of two stages of toxicity: an inflictive stage (stage I) and progressive or regressive stage (stage II). While much is known about mechanisms by which injury is inflicted (stage I), very little is known about the mechanisms that lead to progression or regression of injury. A wide variety of additional experimental evidence suggests that tissue repair impacts decisively on the final toxic outcome and any modulation in this response has profound impact in the final outcome of toxicity. We designed the present research to investigate the importance of tissue repair in the final acute hepatotoxic outcome upon exposures to mixture of toxicants comprising thioacetamide (TA), allyl alcohol (AA), chloroform (CHCl(3)) and trichloroethylene (TCE). Dose response studies with individual compounds, binary mixtures (BM), ternary (TM) and quaternary mixtures (QM) have been conducted. Results of CHCl(3) + AA BM [Anand, S.S., Murthy, S.N., Vishal, V.S., Mumtaz, M.M., Mehendale, H.M., 2003. Tissue repair plays pivotal role in final outcome of supra-additive liver injury after chloroform and allyl alcohol binary mixture. Food Chem. Toxicol. 41, 1123] and CHCl(3) + AA + TA +TCE QM [Soni, M.G., Ramaiah, S.K., Mumtaz, M.M., Clewell, H., Mehendale, H.M., 1999. Toxicant-inflicted injury and stimulated tissue repair are opposing toxicodynamic forces in predictive toxicology. Regul. Phramcol. Toxicol. 19, 165], and two representative individual compounds (TA and AA) [Mangipudy, R.S., Chanda, S., Mehendale, H.M., 1995a. Tissue repair response as a function of dose in thioacetamide hepatotoxicity. Environ. Health Perspect. 103, 260; Soni, M.G., Ramaiah, S.K., Mumtaz, M.M., Clewell, H., Mehendale, H.M., 1999. Toxicant-inflicted injury and stimulated tissue repair are opposing toxicodynamic forces in predictive toxicology. Regul. Phramcol. Toxicol. 19, 165] are described in this review. In addition, modulation of tissue repair in the outcome of hepatotoxicity and its implications in the risk assessment have been discussed. Male Sprague-Dawley (S-D) rats (250-300g) received a single i.p. injection of individual toxicants as well as mixtures. Liver injury was assessed by plasma alanine amino transferase (ALT) and histopathology. Tissue regeneration response was measured by [(3)H]-thymidine ((3)H-T) incorporation into hepatocellular nuclear DNA and PCNA. Only ALT and (3)H-T data have been presented in this review for the sake of simplicity. Studies with individual hepatotoxicants showed a dose-related increase in injury as well as tissue repair up to a threshold dose. Beyond this threshold, tissue repair was inhibited, and liver injury progressed leading to mortality. Since the highest dose of individual compounds resulted in mortality, this dose was not employed for mixture studies. While CHCl(3) + AA BM caused supra-additive liver injury, QM caused additive liver injury. Due to the prompt and robust compensatory tissue repair, all the rats exposed to BM survived. With QM, the rats receiving the highest dose combination experienced some mortality consequent to the progression of liver injury attendant to suppressed tissue repair. These findings suggest that liver tissue repair, the opposing biological response that restores tissue lost to injury, may play a critical and determining role in the outcome of liver injury regardless of the number of toxicants in the mixture or the mechanism of initiation of injury. These data suggest that inclusion of this response in risk assessment might help in fine-tuning the prediction of toxic outcomes.     

Extent and timeliness of tissue repair determines the dose-related hepatotoxicity of chloroform

As a part of mixture toxicity studies, the objective of the present investigation was to validate the hypothesis that the rate and extent of liver tissue repair response to a given dose determines the end result of toxicity (death or recovery), regardless of the mechanisms by which injury is inflicted, using a well-known environmental pollutant, chloroform (CHCl(3)). In future, the data will be used to compare with the results of mixtures containing CHCl(3) to aid in characterizing the safety of chemical mixtures and to construct a physiologically based pharmacokinetic (PBPK) model for dose, route, and species extrapolation. Hepatotoxicity and tissue repair were measured in male Sprague-Dawley rats (S-D) receiving a 10-fold dose range of CHCl(3) (74, 185, 370, and 740 mg/kg, IP) during a time course of 0 to 96 hours. Liver injury, as assessed by plasma alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) elevation, increased with dose over the 10-fold dose range. Because CHCl(3) is also known to cause kidney damage, blood urea nitrogen (BUN) and creatinine were measured to evaluate the kidney injury. With doses up to 370 mg/kg, liver injury increased in a dose-related fashion, which peaked at 24 hours and returned to normal after 48 hours, whereas at highest dose (740 mg/kg), the injury was progressive resulting in 90% mortality. Blood and liver CHCl(3) levels were quantified using gas chromatography (GC) over a time course of 30 to 360 minutes. The dose-related increase in the blood and liver CHCl(3) levels were consistent with dose-dependent liver injury. Tissue regeneration response, as measured by [(3)H]-thymidine incorporation into hepatocellular nuclear DNA peaked at 36 hours in rats treated with the lower two doses of CHCl(3) (74 and 185 mg/kg). Further increase in CHCl(3) dose to 370 mg/kg resulted in an earlier increase in [(3)H]-thymidine incorporation at 24 hours, which peaked at 36 hours. However, at the highest dose of CHCl(3) (740 mg/kg), tissue repair was delayed and attenuated, allowing for unrestrained progression of liver injury. The kidney injury markers after CHCl(3) administration were not different from controls. These results support the concept that in addition to the magnitude of tissue repair response, the time at which this response occurs is critical in restraining the progression of injury. Measuring tissue repair and injury as simultaneous biological responses to toxic agents might increase the usefulness of dose-response paradigms in predictive toxicology and risk assessment. Although the dosimetry of the present study was well beyond the environmental exposure levels of CHCl(3), a PBPK model will be developed in future based upon these data to evaluate the effects at environmental levels.     

Tissue repair plays pivotal role in final outcome of liver injury following chloroform and allyl alcohol binary mixture.

The objective of this study was to evaluate the interaction profile of chloroform (CHCl(3))+allyl alcohol (AA) binary mixture (BM)-induced acute hepatotoxic response. Plasma alanine aminotransferase (ALT) was measured to assess liver injury, and 3H-thymidine (3H-T) incorporation into hepatonuclear DNA was measured as an index of liver regeneration over a time course of 0-72 h. Male Sprague-Dawley (S-D) rats received single ip injection of 5-fold dose range of CHCl(3) (74, 185 and 370 mg/kg) in corn oil (maximum 0.5 ml/kg) and 7-fold dose range of AA (5, 20 and 35 mg/kg) in distilled water simultaneously. The doses for BM were selected from individual toxicity studies of CHCl(3) alone [Int. J. Toxicol. 22 (2003) 25], and AA alone [Reg. Pharmacol. Toxicol. 19 (1999) 165]. Since the highest dose of each treatment (CHCl(3)- 740 and AA- 50 mg/kg) yielded mortality due to the suppressed tissue repair followed by liver failure, this dose was omitted for BM. The levels of CHCl(3) (30-360 min) and AA (5-60 min) were quantified in blood and liver by gas chromatography (GC). The liver injury was more than additive after BM compared to CHCl(3) alone or AA alone at highest dose combination (370+35 mg/kg), which peaked at 24 h. The augmented liver injury observed with BM was consistent with the quantitation data. Though the liver injury was higher, the greater stimulation of tissue repair kept injury from progressing, and rescued the rats from hepatic failure and death. At lower dose combinations, the liver injury was no more than additive. Results of the present study suggest that liver tissue repair, in which liver tissue lost to injury is promptly replaced, plays a pivotal role in the final outcome of liver injury after exposure to BM of CHCl(3) and AA.     

Individual serum bile acids as early indicators of carbon tetrachloride- and chloroform-induced liver injury.

Individual serum bile acids (SBA) are emerging as potentially useful early indicators of liver injury. This study was undertaken to compare the usefulness of individual SBA with the routinely used assays for detecting the effects of the hepatotoxicants carbon tetrachloride (CCl4) and chloroform (CHCl3). Serum samples were assayed for liver injury by determination of alanine aminotransferase (ALT), aspartate amino-transferase (AST), alkaline phosphatase (ALP), bilirubin and total bile acid (by enzymatic kit). These results were compared with levels of individual SBA measured by high performance liquid chromatography (HPLC). Liver samples from CCl4-treated rats were taken for light and electron microscopic examination. The highest dose for each chemical caused increases in serum ALT and AST but not ALP. Chloroform at the highest dose increased bilirubin. Total SBA levels as assayed by the kit were elevated in response to CCl4 and CHCl3 at doses below which serum enzymes and bilirubin were increased. Some individual SBA were increased at a still lower dose for each of these two chlorinated solvents. At the lowest dose of CCl4 tested no consistent light microscopic or ultrastructural changes were found. At all the higher doses periacinar cells displayed typical accumulation of lipid droplets and degranulation and dilation of rough endoplasmic reticulum. The extent of the ultrastructural changes were dose-dependent. Thus individual SBA assayed by HPLC may be considered as a very sensitive indicator of liver injury induced by the classical hepatotoxicants carbon tetrachloride and chloroform.     

In vitro mutagenicity of water contaminants in complex mixtures

Trihalomethanes, Carbon tetrachloride and trichloroethylene were tested in single, binary and multi-complex mixtures using standard tester strains TA1535, TA1537, TA98 and TA100 of Salmonella typhimurium with and without addition of an in vitro metabolizing fraction S-9. Chloroform (CHCl3) was found to be mutagenic in all strains without S-9 activation. However, when tested with Bromoform (15%), which was nonmutagenic singly, the combined effect of the mixture was nonmutagenic. CCl4 was a direct mutagen (without S-9) in all strains except TA 1535. When combined with 85% CHCl3, only strains TA1535 and TA1537 were mutagenic. When tested with mammalian activation (S-9), CCl4 was mutagenic in all strains. However, when tested with CHCl3 (CHCl3 and CCl4-85:15), the mutagenic capability was lost. With or without S-9 Activation multi-complex mixture of CHCl3, CCl4 and TCE (85:8:7) was mutagenic for a narrow range of doses in all strains.     

Relationship between the carbon skeleton length of ketonic solvents and potentiation of chloroform-induced hepatotoxicity in rats

Previous studies have suggested that ketonic solvents potentiate the hepatotoxic action of CHCl3 in rats. In addition, the relative potentiating capacity of the ketones appeared to be related to the length of their carbon skeleton. To test this hypothesis CHCl3-induced liver injury was evaluated in male Sprague-Dawley rats pretreated (15 mmol/kg, p.o.) with acetone (Ac), 2-butanone (Bu), 2-pentanone (Pn), 2-hexanone (Hx) or 2-heptanone (Hp). After 18 h, a challenging dose of CHCl3, (0.50 or 0.75 ml/kg, i.p.) was given. Liver damage was evaluated 24 h after CHCl3 administration by determining elevations in plasma GPT and OCT activity. Neither Ac, Bu, Pn, Hx, Hp or the CHCl3 challenging dosages produced marked liver injury when given alone. However, each of the ketones potentiated CHCl3-induced liver damage. The severity of the potentiated hepatotoxic response was significantly (positively) correlated with the ketone carbon chain length. These observations suggest that carbon skeleton length may play a role in determining the relative potentiating capacity of ketonic solvents.     

Enhanced hepatotoxicity and toxic outcome of thioacetamide in streptozotocin-induced diabetic rats

Diabetes is known to potentiate thioacetamide (TA)-induced liver injury via enhanced bioactivation. Little attention has been given to the role of compensatory tissue repair on ultimate outcome of hepatic injury in diabetes. The objective of this study was to investigate the effect of diabetes on TA-induced liver injury and lethality and to investigate the underlying mechanisms. We hypothesized that hepatotoxicity of TA in diabetic rats would increase due to enhanced bioactivation-mediated liver injury and also due to compromised compensatory tissue repair, consequently making a nonlethal dose of TA lethal. On day 0, male Sprague-Dawley rats (250-300 g) were injected with streptozotocin (STZ, 60 mg/kg ip) to induce diabetes. On day 10 the STZ-induced diabetic rats and the nondiabetic rats received a single dose of TA (300 mg/kg ip). This normally nonlethal dose of TA caused 90% mortality in the STZ-induced diabetic rats. At various times (0-60 h) after TA administration, liver injury was assessed by plasma alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), and liver histopathology. Liver function was evaluated by plasma bilirubin. Cell proliferation and tissue repair were evaluated by [(3)H]thymidine ((3)H-T) incorporation and proliferating cell nuclear antigen (PCNA) assays. In the nondiabetic rat, liver necrosis peaked at 24 h and declined thereafter toward normal by 60 h. In the STZ-induced diabetic rat, however, liver necrosis was significantly increased from 12 h onward and progressed, culminating in liver failure and death. Liver tissue repair studies showed that, in the liver of nondiabetic rats, S-phase DNA synthesis was increased at 36 h and peaked at 48 h following TA administration. However, DNA synthesis was approximately 50% inhibited in the liver of diabetic rats. PCNA study showed a corresponding decrease of cell-cycle progression, indicating that the compensatory tissue repair was sluggish in the diabetic rats. Further investigation of tissue repair by employing equitoxic doses (300 mg TA/kg in the non-diabetic rats; 30 mg TA/kg in the diabetic rats) revealed that, despite equal injury up to 24 h following injection, the tissue repair response in the diabetic rats was much delayed. The compromised tissue repair prolonged liver injury in the diabetic rats. These studies suggest that the increased TA hepatotoxicity in the diabetic rat is due to combined effects of increased bioactivation-mediated liver injury of TA and compromised compensatory tissue repair.     

Stimulated Tissue Repair Prevents Lethality in Isopropanol-Induced Potentiation of Carbon Tetrachloride Hepatotoxicity

Published reports on the alcohol potentiation of CCl₄toxicity indicate that in spite of enhanced hepatotoxicity there is no increase in lethality. The objective of this study was to investigate the mechanism involved in animal survival despite significantly enhanced liver injury. Male Sprague–Dawley rats (175–225 g) were treated with isopropanol (ISOP, 2.5 ml/kg, 25% aqueous solution, po) 24 hr prior to CCl₄(1 ml/kg, ip) administration. Plasma enzymes (ALT and SDH), hepatic glycogen levels, and [³H]thymidine (³H-T) incorporation into hepatonuclear DNA were measured during a time course (0–96 hr) after CCl₄administration. Liver sections were examined for histopathology and cell cycle progression by proliferating cell nuclear antigen (PCNA) immunohistochemistry. Maximum injury was observed at 36 hr in both the groups as indicated by elevated plasma enzyme levels and by histopathology. The extent of injury in the ISOP + CCl₄group was higher than that in the H₂O + CCl₄group. Plasma enzyme activity returned to control levels by 60 hr, indicating recovery from injury in both groups. Maximum³H-T incorporation occurred at 48 hr in both groups (ISOP + CCl₄; vehicle + CCl₄), indicating maximum stimulation of S-phase synthesis. PCNA studies revealed a corresponding stimulation of cell cycle progression. The wave of S-phase synthesis and cell cycle progression returned to control levels in the H₂O + CCl₄group by 60 hr but continued up to 72 hr in the ISOP + CCl₄group. These findings support the hypothesis that in response to increased infliction of CCl₄injury by isopropanol, augmented stimulation of cell division and tissue repair restrain the progression of injury and restore hepatic structure and function, thereby allowing the rats to survive. Further, antimitotic intervention with colchicine (1 mg/kg, ip) led to decreased S-phase synthesis, followed by 60% lethality in the isopropanol-pretreated group in contrast to 40% lethality in the group receiving CCl₄alone (H₂O + CCl₄). These findings suggest that greater stimulation of tissue repair restrains the progression of ISOP-enhanced infliction of CCl₄liver injury and accounts for recovery from enhanced liver injury and animal survival. The findings are consistent with a two-stage model of toxicity wherein liver injury is linked by progression or regression of injury, which is governed by the extent of tissue repair to the final outcome.     

Effect of polybrominated biphenyls, beta-naphthoflavone and phenobarbital on arylhydrocarbon hydroxylase activities and chloroform-induced nephrotoxicity and hepatotoxicity in male C57BL/6J and DBA/2J mice

Administration of chloroform (CHCl3) to male C57/6J (C57) and DBA/2J (DBA) mice produced dose-dependent hepatic and renal damage. Hepatic arylhydrocarbon hydroxylase (AHH) activity was higher in C57 than DBA mice; in kidney, AHH activity was higher in DBA than in C57 mice. CHCl3 caused the same degree of liver damage in both strains of mice; however, nephrotoxicity of CHCl3 was greater in DBA than in C57 mice. Pretreatment of C57 and DBA mice with phenobarbital (PB) markedly increased hepatic AHH activity and hepatotoxicity of CHCl3 in both strains but did not affect renal AHH or nephrotoxicity of CHCl3. Similarly, beta-naphthoflavone (BNF) enhanced AHH activity and CHCl3 hepatotoxicity in C57 mice, but had little effect on nephrotoxicity. BNF did not affect hepatic AHH nor CHCl3-induced hepatic injury in male DBA mice. Pretreatment with polybrominated biphenyl (PBB) enhanced AHH activity in liver and CHCl3 hepatotoxicity in both strains. After PBB, nephrotoxicity of CHCl3 and renal AHH activity were increased in C57 mice whereas PBB did not alter nephrotoxicity or renal AHH in DBA mice. These results suggest that CHCl3-nephrotoxicity is independent of hepatotoxicity.     

The role of NF-kappaB signaling in impaired liver tissue repair in thioacetamide-treated type 1 diabetic rats

Previously we reported that an ordinarily nonlethal dose of thioacetamide (300 mg/kg) causes liver failure and 90% mortality in type 1 diabetic rats, primarily because of inhibited tissue repair. On the other hand, the diabetic rats receiving 30 mg thioacetamide/kg exhibited equal initial liver injury and delayed tissue repair compared to nondiabetic rats receiving 300 mg thioacetamide/kg, resulting in a delay in recovery from that liver injury and survival. These data indicate that impaired tissue repair in diabetes is a dose-dependent function of diabetes. The objective of the present study was to test the hypothesis that disrupted nuclear factor-kappaB (NF-kappaB)-regulated cyclin D1 signaling may explain dose-dependent impaired tissue repair in the thioacetamide-treated diabetic rats. Administration of 300 mg thioacetamide/kg to nondiabetic rats led to sustained NF-kappaB-regulated cyclin D1 signaling, explaining prompt compensatory tissue repair and survival. For the first time, we report that NF-kappaB-DNA binding is dependent on the dose of thioacetamide in the liver tissue of the diabetic rats. Administration of 300 mg thioacetamide/kg to diabetic rats inhibited NF-kappaB-regulated cyclin D1 signaling, explaining inhibited tissue repair, liver failure and death, whereas remarkably higher NF-kappaB-DNA binding but transient down regulation of cyclin D1 expression explains delayed tissue repair in the diabetic rats receiving 30 mg thioacetamide/kg. These data suggest that dose-dependent NF-kappaB-regulated cyclin D1 signaling explains inhibited versus delayed tissue repair observed in the diabetic rats receiving 300 and 30 mg thioacetamide/kg, respectively.     

Impact of repeated exposure on toxicity of perchloroethylene in Swiss Webster mice

The aim was to study the subchronic toxicity of perchloroethylene (Perc) by measuring injury and repair in liver and kidney in relation to disposition of Perc and its major metabolites. Male SW mice (25-29g) were given three dose levels of Perc (150, 500, and 1000 mg/kg day) via aqueous gavage for 30 days. Tissue injury was measured during the dosing regimen (0, 1, 7, 14, and 30 days) and over a time course of 24-96h after the last dose (30 days). Perc produced significant liver injury (ALT) after single day exposure to all three doses. Liver injury was mild to moderate and regressed following repeated exposure for 30 days. Subchronic Perc exposure induced neither kidney injury nor dysfunction during the entire time course as evidenced by normal renal histology and BUN. TCA was the major metabolite detected in blood, liver, and kidney. Traces of DCA were also detected in blood at initial time points after single day exposure. With single day exposure, metabolism of Perc to TCA was saturated with all three doses. AUC/dose ratio for TCA was significantly decreased with a concomitant increase in AUC/dose of Perc levels in liver and kidney after 30 days as compared to 1 day exposures, indicating inhibition of metabolism upon repeated exposure to Perc. Hepatic CYP2E1 expression and activity were unchanged indicating that CYP2E1 is not the critical enzyme inhibited. Hepatic CYP4A expression, measured as a marker of peroxisome proliferation was increased transiently only on day 7 with the high dose, but was unchanged at later time points. Liver tissue repair peaked at 7 days, with all three doses and was sustained after medium and high dose exposure for 14 days. These data indicate that subchronic Perc exposure via aqueous gavage does not induce nephrotoxicity and sustained hepatotoxicity suggesting adaptive hepatic repair mechanisms. Enzymes other than CYP2E1, involved in the metabolism of Perc may play a critical role in the metabolism of Perc upon subchronic exposure in SW mice. Liver injury decreased during repeated exposure due to inhibition of metabolism and possibly due to adaptive tissue repair mechanisms.     

Nonalcoholic steatohepatitic (NASH) mice are protected from higher hepatotoxicity of acetaminophen upon induction of PPARalpha with clofibrate

The objective was to investigate if the hepatotoxic sensitivity in nonalcoholic steatohepatitic mice to acetaminophen (APAP) is due to downregulation of nuclear receptor PPARalpha via lower cell division and tissue repair. Male Swiss Webster mice fed methionine and choline deficient diet for 31 days exhibited NASH. On the 32nd day, a marginally toxic dose of APAP (360 mg/kg, ip) yielded 70% mortality in steatohepatitic mice, while all non steatohepatitic mice receiving the same dose survived. (14)C-APAP covalent binding, CYP2E1 protein, and enzyme activity did not differ from the controls, obviating increased APAP bioactivation as the cause of amplified APAP hepatotoxicity. Liver injury progressed only in steatohepatitic livers between 6 and 24 h. Cell division and tissue repair assessed by (3)H-thymidine incorporation and PCNA were inhibited only in the steatohepatitic mice given APAP suggesting that higher sensitivity of NASH liver to APAP-induced hepatotoxicity was due to lower tissue repair. The hypothesis that impeded liver tissue repair in steatohepatitic mice was due to downregulation of PPARalpha was tested. PPARalpha was downregulated in NASH. To investigate whether downregulation of PPARalpha in NASH is the critical mechanism of compromised liver tissue repair, PPARalpha was induced in steatohepatitic mice with clofibrate (250 mg/kg for 3 days, ip) before injecting APAP. All clofibrate pretreated steatohepatitic mice receiving APAP exhibited lower liver injury, which did not progress and the mice survived. The protection was not due to lower bioactivation of APAP but due to higher liver tissue repair. These findings suggest that inadequate PPARalpha expression in steatohepatitic mice sensitizes them to APAP hepatotoxicity.     

Adaptive tolerance in mice upon subchronic exposure to chloroform: Increased exhalation and target tissue regeneration

The aims of the present study were to characterize the subchronic toxicity of chloroform by measuring tissue injury, repair, and distribution of chloroform and to assess the reasons for the development of tolerance to subchronic chloroform toxicity. Male Swiss Webster (SW) mice were given three dose levels of chloroform (150, 225, and 300 mg/kg/day) by gavage in aqueous vehicle for 30 days. Liver and kidney injury were measured by plasma ALT and BUN, respectively, and by histopathology. Tissue regeneration was assessed by (3)H-thymidine incorporation into hepato- and nephro-nuclear DNA and by proliferating cell nuclear antigen staining. In addition, GSH and CYP2E1 in liver and kidney were assessed at selected time points. The levels of chloroform were measured in blood, liver, and kidney during the dosing regimen (1, 7, 14, and 30 days). Kidney injury was evident after 1 day with all three doses and sustained until 7 days followed by complete recovery. Mild to moderate liver injury was observed from 1 to 14 days with all three dose levels followed by gradual decrease. Significantly higher regenerative response was evident in liver and kidney at 7 days, but the response was robust in kidney, preventing progression of injury beyond first week of exposure. While the kidney regeneration reached basal levels by 21 days, moderate liver regeneration with two higher doses sustained through the end of the dosing regimen and 3 days after that. Following repeated exposure for 7, 14, and 30 days, the blood and tissue levels of chloroform were substantially lower with all three dose levels compared to the levels observed with single exposure. Increased exhalation of (14)C-chloroform after repeated exposures explains the decreased chloroform levels in circulation and tissues. These results suggest that toxicokinetics and toxicodynamics (tissue regeneration) contribute to the tolerance observed in SW mice to subchronic chloroform toxicity. Neither bioactivation nor detoxification appears to play a decisive role in the development of this tolerance.     

Protective effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on chemical-induced hepatotoxicity

Protective effect of malotilate on the liver injuries induced by several hepatotoxins was studied in mice and rats. Malotilate suppressed the elevation of plasma glutamate pyruvate transaminase (p-GPT) activity induced by chloroform (CHCl3) in rats when the animals were treated with 25 mg/kg or more dose of malotilate at 6 hours prior to the treatment with CHCl3. The effect was observed even in the rats treated with malotilate 24 or 48 hours prior to the treatment with CHCl3. Malotilate, when orally administered 6 hours prior to treatment with hepatotoxins such as CHCl3, allyl alcohol, bromobenzene, dimethylnitrosamine or thioacetamide, suppressed the elevation of p-GPT activity, liver triglyceride content and/or the decrease of bromosulphalein clearance induced by these hepatotoxins in mice. Anethole trithione, which was used as a possible protective agent against chemical-induced hepatotoxicity, tended to normalize changes in the parameters induced by the most of these hepatotoxins, but enhanced the elevation of p-GPT activity induced by CHCl3. In a case of CHCl3-induced liver injury, the protective effect of malotilate was histopathologically confirmed. Malotilate and anethole trithione reduced p-nitroanisole 0-demethylation activity in rat liver 6 hours after the administration but increased or tended to increase the activity 48 hours after the administration. Malotilate showed a protective effect on the liver injury induced by CHCl3 even when the activity of drug metabolizing enzymes in the liver was increased, although anethole trithione enhanced the CHCl3-induced liver injury regardless of the activity of drug metabolizing enzymes.     

Elevation of individual serum bile acids on exposure to trichloroethylene or alpha-naphthylisothiocyanate

Rats were treated with trichloroethylene via intraperitoneal (ip) injection or inhalation, or with ip alpha-naphthylisothiocyanate (ANIT). Serum samples were assayed for indices of liver injury including alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (AP), and bilirubin. Liver from some rats was examined for histological appearance. These data were compared to levels of individual serum bile acids (SBA) determined by high-performance liquid chromatography. Trichloroethylene and ANIT, each at their highest dose only, caused elevations in ALT, but not SDH or AP. The highest dose of ANIT also caused elevated serum bilirubin and cholangitis in the liver. SBA were also elevated in response to both trichloroethylene and ANIT, but at doses below those at which other parameters of liver function were increased. For both chemicals, taurocholic acid was the most sensitive of the bile acids assayed, being elevated at the lowest doses tested of 10 mumols/kg for trichloroethylene and 5 mumols/kg for ANIT. As the doses were raised more of the individual bile acids showed increases. On exposure to trichloroethylene via inhalation taurocholic acid was one of two SBA to show elevation. Thus, both trichloroethylene and ANIT cause elevation in SBA at doses well below those which cause an increase in standard indicators of liver dysfunction. This suggests that SBA and perhaps taurocholic acid, in particular, may provide a sensitive tool for studying hepatobiliary effects of chemicals.     

Disrupted G1 to S phase clearance via cyclin signaling impairs liver tissue repair in thioacetamide-treated type 1 diabetic rats

Previously we reported that a nonlethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats because of irreversible acute liver injury owing to inhibited hepatic tissue repair, primarily due to blockage of G(0) to S phase progression of cell division cycle. On the other hand, DB rats receiving 30 mg TA/kg exhibited equal initial liver injury and delayed tissue repair compared to nondiabetic (NDB) rats receiving 300 mg TA/kg, resulting in a delay in recovery from liver injury and survival. The objective of the present study was to test the hypothesis that impaired cyclin-regulated progression of G(1) to S phase of the cell cycle may explain inhibited liver tissue repair, hepatic failure, and death, contrasted with delayed liver tissue repair but survival observed in the DB rats receiving 300 in contrast to 30 mg TA/kg. In the TA-treated NDB rats sustained MAPKs and cyclin expression resulted in higher phosphorylation of retinoblastoma (pRb), explaining prompt tissue repair and survival. In contrast, DB rats receiving the same dose of TA (300 mg/kg) exhibited suppressed MAPKs and cyclin expression that led to inhibition of pRb, inhibited tissue repair, and death. On the other hand, DB rats receiving 30 mg TA/kg exhibited delayed up regulation of MAPK signaling that delayed the expression of CD1 and pRb, explaining delayed stimulation of tissue repair observed in this group. In conclusion, the hepatotoxicant TA has a dose-dependent adverse effect on cyclin-regulated pRb signaling: the lower dose causes a recoverable delay, whereas the higher dose inhibits it with corresponding effect on the ultimate outcomes on hepatic tissue repair; this dose-dependent adverse effect is substantially shifted to the left of the dose response curve in diabetes.     

Hepatoprotection by dimethyl sulfoxide. III. Role of inhibition of the bioactivation and covalent bonding of chloroform

Dimethyl sulfoxide (DMSO) has previously been shown to have the ability to attenuate chloroform (CHCl(3))-induced liver injury in the naive rat even when administered 24 h after the toxicant. These studies were undertaken to determine if the protective action by late administration of DMSO is due to an inhibition of the bioactivation of CHCl(3). This was done by comparing the cytochrome P450 inhibitors, diallyl sulfide (DAS), and aminobenzotriazole (ABT) to DMSO for their protective efficacy when administered 24 h after CHCl(3) exposure. In addition, (14)CHCl(3) was utilized to measure the effect of DMSO and ABT on the covalent binding of CHCl(3) in the liver following their late administration. Male Sprague-Dawley rats (300-350 g) received 0.75 ml/kg CHCl(3) po. Twenty-four hours later, they received ip injection of 2 ml/kg DMSO, 100 mg/kg DAS, or 30 mg/kg ABT. Plasma ALT activities and quantitation of liver injury by light microscopy at 48 h after CHCl(3) dosing indicated that all three treatments were equally effective at protecting the liver. A detailed study of the time course of injury development indicated that the protective action of DMSO was occurring within 10 h of its administration. Therefore, in the radiolabel studies, rats were killed 24-34 h after receiving 0.75 ml/kg CHCl(3) (30 microCi/kg (14)CHCl(3)) po. Treatment with ABT at 24 h after (14)CHCl(3) dosing decreased the covalent binding of (14)C to hepatic protein by 35% and reduced the amount of (14)C in the blood by 50% by 10 h after its administration. DMSO treatment did not significantly affect any of these parameters. The lack of effect by late administration of DMSO on the covalent binding of CHCl(3) would indicate that DMSO may offer protection by mechanisms other than inhibition of the bioactivation of CHCl(3). These studies also indicate that specific cytochrome P450 inhibitors may be of benefit in clinical situations to help treat the delayed onset hepatitis that can result following poisoning with an organohalogen, even if the antidotes are administered a number of hours after the initial exposure.     

Disposition and excretion of intravenous 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) in rats

Polybrominated dibenzodioxins and dibenzofurans are of toxicologic interest due to potential occupational and environmental exposure and because of their structural similarity to the highly toxic chlorinated analogues. The excretion and terminal tissue distribution of [3H]TBDD was studied in male F344 rats for 56 days following single iv doses of .001 or 0.1 mumol/kg. The major tissue depots of radioactivity were liver, adipose tissue, and skin, and tissue distribution was dose-dependent. At 56 days, liver concentrations in the high dose group were disproportionately increased compared to those of the low dose group. Liver:adipose tissue concentration ratios were 0.2 and 2.6 at the low and high doses, respectively. Elimination of radioactivity in the feces, the major route of excretion, and urine was also nonlinear with respect to dose. By Day 56, feces accounted for approximately 50% of the administered dose at the low dose versus 70% at the high dose. Based on fecal excretion, the apparent terminal whole body half-life was estimated to be 18 days for both dose groups. The time-dependent pattern of tissue disposition was characterized at the low dose over a 56-day period. Blood levels of radioactivity declined rapidly with 2% remaining in the blood by 24 hr. Radioactivity levels in the liver peaked by 7 hr and then gradually declined concomitant with a slow accumulation in adipose tissue. The terminal excretion half-life of radioactivity in adipose tissue was estimated to be 60 days. Liver:adipose tissue concentration ratios declined with time. Thus, the overall disposition of TBDD appears similar to that observed for the chlorinated analogue, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results of these studies are consistent with the hypothesis that TBDD, like TCDD, induces a binding species in the liver which accounts for higher liver:adipose tissue concentration ratios at the high dose. The dose-dependent tissue disposition and excretion kinetics of these compounds suggest important considerations for extrapolations from high to low doses.