Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes.

Increase dincidences of hepatotoxicity have been observed in diabetic patients receiving drug therapies. Neither the mechanisms nor the predisposing factors underlying hepatotoxicity in diabetics are clearly understood. Animal studies designed to examine the mechanisms of diabetes-modulated hepatotoxicity have traditionally focused only on bioactivation/detoxification of drugs and toxicants. It is becoming clear that once injury is initiated, additional events determine the final outcome of liver injury. Foremost among them are two leading mechanisms: first, biochemical mechanisms that lead to progression or regression of injury; and second, whether or not timely and adequate liver tissue repair occurs to mitigate injury and restore liver function. The liver has a remarkable ability to repair and restore its structure and function after physical or chemical-induced damage. The dynamic interaction between biotransformation-based liver injury and compensatory tissue repair plays a pivotal role in determining the ultimate outcome of hepatotoxicity initiated by drugs or toxicants. In this review, mechanisms underlying altered hepatotoxicity in diabetes with emphasis on both altered bioactivation and liver tissue repair are discussed. Animal models of both marked sensitivity (diabetic rats) and equally marked protection (diabetic mice) from drug-induced hepatotoxicity are described. These examples represent a remarkable species difference. Availability of the rodent diabetic models offers a unique opportunity to uncover mechanisms of clinical interest in averting human diabetic sensitivity to drug-induced hepatotoxicities. While the rat diabetic models appear to be suitable, the diabetic mouse models might not be suitable in preclinical testing for potential hepatotoxic effects of drugs or toxicants, because regardless of type 1 or type2 diabetes, mice are resistant to acute drug-or toxicant-induced toxicities.

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.

Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats.

Earlier studies have shown highly exaggerated mechanism-based liver injury of thioacetamide (TA) in rats following moderate diet restriction (DR) and in diabetes. The objective of the present study was to investigate the mechanism of higher liver injury of TA in DR rats. Since both DR and diabetes induce CYP2E1, we hypothesized that hepatic CYP2E1 plays a major role in the bioactivation-based liver injury of TA. When male Sprague-Dawley rats (250-275 g) were maintained on diet restriction (DR, 35% of ad libitum fed rats, 21 days) the total hepatic microsomal cytochrome P450 (CYP450) was increased 2-fold along with a 4.6-fold increase in CYP2E1 protein, which corresponded with a 3-fold increase in CYP2E1 activity as measured by chlorzoxazone hydroxylation. To further test the involvement of CYP2E1, 24 and 18 h after pretreatment with pyridine (PYR) and isoniazid (INZ), specific inducers of CYP2E1, male Sprague-Dawley rats received a single administration of 50 mg of TA/kg (i.p.). TA liver injury was >2.5- and >3-fold higher at 24 h in PYR + TA and INZ + TA groups, respectively, compared with the rats receiving TA alone. Pyridine pretreatment resulted in significantly increased total CYP450 content accompanied by a 2.2-fold increase in CYP2E1 protein and 2-fold increase in enzyme activity concordant with increased liver injury of TA, suggesting mechanism-based bioactivation of TA by CYP2E1. Hepatic injury of TA in DR rats pretreated with diallyl sulfide (DAS), a well known irreversible in vivo inhibitor of CYP2E1, was significantly decreased (60%) at 24 h. CCl(4) (4 ml/kg i.p.), a known substrate of CYP2E1, caused lower liver injury and higher animal survival confirming inhibition of CYP2E1 by DAS pretreatment. The role of flavin-containing monooxygenase (FMO) in TA bioactivation implicated by previous in vitro studies, and consequent increased TA-induced liver injury in DR rats was tested in vivo with a relatively selective inhibitor of FMO, indole-3-carbinol, and then treated with 50 mg of TA/kg. FMO activity and alanine aminotransferase levels measured at different time points revealed that TA liver injury was not decreased although FMO activity was significantly decreased, suggesting that hepatic FMO is unlikely to bioactivate TA. These findings suggest induction of CYP2E1 as the primary mechanism of increased bioactivation-based liver injury of TA in DR rats.

Diallyl sulfide inhibition of CYP2E1 does not rescue diabetic rats from thioacetamide-induced mortality.

Previously we have shown that hepatotoxicity of thioacetamide (TA) was increased in streptozotocin (STZ)-induced diabetic (DB) rats due to combined effects of enhanced bioactivation-based liver injury of TA and compromised liver tissue repair response. We have also shown that TA is primarily bioactivated by hepatic CYP2E1. The present study was done to further investigate the importance of liver tissue repair in determining the final outcome of hepatotoxicity. STZ-induced DB rats were pretreated with a CYP2E1 inhibitor, diallyl sulfide (DAS), to decrease the bioactivation-based liver injury of TA. The treatments were as follows: DB/DAS/TA, DB/corn oil/TA, and DB/DAS/saline. Nondiabetic (non-DB) rats received the same treatments as controls. A dose of TA (300 mg/kg ip), which was nonlethal in non-DB rats, caused 92 and 90% mortality in DB/DAS/TA and DB/corn oil/TA groups, respectively. At various times (0--60 h) after treatment, liver injury was assessed by plasma alanine aminotransferase and histopathology. Cell proliferation was evaluated by [(3)H]thymidine incorporation and immunohistochemical staining of proliferating cell nuclear antigen (PCNA). In the DB/DAS/TA rats, DAS pretreatment markedly reduced the CYP2E1-dependent liver injury of TA compared to that in DB/corn oil/TA rats. However, subsequent hepatic DNA synthesis in both DB groups was inhibited approximately 50%. PCNA analysis showed a corresponding decrease in cell-cycle progression. This compromised tissue repair response in DB rats was insufficient to compensate for cell loss, resulting in progression of liver injury and culminating in high mortality in both DB groups. Furthermore, non-DB rats were pretreated with a CYP2E1 inducer, isoniazid, to increase the bioactivation-based TA liver injury equal to the liver injury observed in DB/DAS/TA rats. Despite equal injury up to 36 h following TA treatment, the tissue repair response in the non-DB rats was highly stimulated to compensate for liver injury and led to 70% survival in this group. These studies underscore the importance of adequate and timely tissue repair in compensating for liver injury and protecting from lethality.

PPAR-alpha: a key to the mechanism of hepatoprotection by clofibrate.

The article highlighted in this issue is "Peroxisome Proliferator-Activated Receptor Alpha-Null Mice Lack Resistance to Acetaminophen Hepatotoxicity Following Clofibrate Exposure" by Chuan Chen, Gayle E. Hennig, Herbert E. Whiteley, J Christopher Corton, and José E. Manautou (pp. 338-344).

Potentiation of thioacetamide liver injury in diabetic rats is due to induced CYP2E1.

Thioacetamide (TA)-induced hepatotoxicity is potentiated in streptozotocin (STZ)-induced diabetic rats. The relative roles of CYP2E1 and FMO1 in the mechanism of TA-associated liver injury were investigated. In the STZ-induced diabetic rat, hepatic CYP2E1 protein concentration and p-nitrophenol hydroxylation were induced 8- and 5.6-fold, respectively. Pretreatment with the CYP2E1 inducer, isoniazid (INH, 250 mg/kg, i.p.) before TA (300 mg/kg, i.p.) administration significantly increased TA-associated liver injury as assessed by plasma alanine aminotransferase (ALT). Hepatic CYP2E1 expression and p-nitrophenol hydroxylation were induced 2.2- and 2. 5-fold in the INH-pretreated rat, respectively. Inhibition of CYP2E1 by diallyl sulfide (DAS, 200 mg/kg, p.o., two doses) before TA administration, decreased plasma ALT activity by 60% in the nondiabetic rat and by 75% in the diabetic rat. Abolition of microsomal p-nitrophenol hydroxylation and CCl(4)-induced liver injury confirmed that hepatic CYP2E1 was highly inhibited by DAS. Hepatic flavin-containing monooxygenase (FMO) form 1 expression and methimazole-dependent oxidation of thiocholine were induced 2.5- and 1.8-fold in the diabetic rat, respectively. Dietary administration of 0.25% indole-3-carbinol (I3C) for 10 days inhibited FMO1 expression and enzyme activity in both nondiabetic and diabetic rats. Paradoxically, TA-induced liver injury was increased in these I3C-pretreated rats. These findings indicate that hepatic CYP2E1 appears to be primarily involved in bioactivation of TA. In the STZ-induced diabetic rat, diabetes-induced CYP2E1 appears to be responsible for the potentiated liver injury; Even though hepatic FMO1 is induced in the diabetic rat, it is unlikely to mediate the potentiated TA hepatotoxicity.

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 pulmonary cell hyperplasia underlies resistance to alpha-naphthylthiourea.

The rodenticide alpha-naphthylthiourea (ANTU) causes pulmonary edema and pleural effusion that leads to death via pulmonary insufficiency. Rats become resistant to the lethal effect of ANTU if they are first exposed to a small, nonlethal dose of ANTU. Young rats are also resistant to ANTU. The mechanism by which rats develop resistance by a prior, small dose exposure has yet to be determined. Growth factor induced-pulmonary hyperplasia has been demonstrated to attenuate ANTU-induced lung leak. We hypothesized that a small dose of ANTU protects against a large dose through pulmonary cell hyperplasia induced by the protective dose. Furthermore, we hypothesized that this hyperplasia is associated with altered transcription of growth factors. Male Sprague-Dawley rats (175-225 g) were treated with a low dose of ANTU (5 mg ANTU/kg; ANTU(L)) 24 h before challenge with a 100% lethal dose of ANTU (70 mg ANTU/kg; ANTU(H)) resulting in 100% protection against the lethal effect of ANTU(H). ANTU(L) protection against ANTU(H) lasted for 5 days, slowly phased out, all being lost by day 20. Injury was assessed by estimating pulmonary vascular permeability and through histopathological examination. ANTU(H) alone resulted in an increase in pulmonary edema leading to animal death. However, injury was prevented if the rats were first treated with ANTU(L). There was a stimulation of pulmonary cell hyperplasia in the lungs of ANTU(L) treated rats as measured by [3H]-thymidine and bromodeoxyuridine incorporation. Treatment with the antimitotic agent colchicine abolished ANTU(L)-induced resistance to ANTU(H). ANTU resistant rats were also resistant to the lethal effect of paraquat. Paraquat is not taken up by pneumocytes if they are undergoing hyperplasia. ANTU(L) administration resulted in an up regulation of gene transcription for keratinocyte growth factor, transforming growth factor-beta, keratinocyte growth factor receptor and epidermal growth factor receptor as determined through reverse transcription-polymerase chain reaction. A significant increase in transforming growth factor-alpha was not observed. These findings collectively suggest that ANTU(L)-induced pulmonary cell hyperplasia underlies resistance to ANTU(H). Furthermore, the stimulation of hyperplasia may be due to altered growth factor and growth factor receptor expressions.

Differential protooncogene expression in Sprague Dawley and Fischer 344 rats during 1,2-dichlorobenzene-induced hepatocellular regeneration.

Significant differences in hepatotoxic injury of 1,2-dichlorobenzene (o-DCB) have been reported (Gunawardhana, L., Sipes, I.G., 1991. Dichlorobenzene hepatotoxicity strain differences and structure activity relationships. Adv. Exp. Med. Biol. 283, 731-734; Stine, E.R., Gunawardhana, L., Sipes, I.G., 1991. The acute hepatotoxicity of the isomers of dichlorobenzene in Fischer 344 and Sprague-Dawley rats: isomer specific and strain-specific differential toxicity. Toxicol. Appl. Pharmacol. 109, 472-481; Valentovic, M.A., Ball, J. G., Anestis, D., Madan E., 1993a. Acute hepatic and renal toxicity of dichlorobenzene isomers in Fischer 344 rats. J. Appl. Toxicol. 13, 1-7; Kulkarni, S.G., Duong, H., Gomila, R., Mehendale, H.M., 1996. Strain differences in tissue repair response to 1,2-dichlorobenzene. Arch. Toxicol. 70, 714-723. Kulkarni, S.G., Warbritton, A., Bucci, T., Mehendale, H.M., 1997. Antimitotic intervention with colchicine alters the outcome of o-DCB-induced hepatotoxicity in Fischer 344 rats. Toxicology. 120, 79-88). Although, hepatotoxic injury of o-DCB is greater in Fischer 344 (F344) when compared with Sprague Dawley (S-D) rats, this interstrain difference does not transcend into any difference in lethal effects of o-DCB. Interstrain difference in compensatory tissue repair has been suggested as the underlying mechanism for the lack of strain differences in lethality (Kulkarni et al., 1996; Kulkarni et al., 1997, see these refs. above). However, the mechanism(s) for this interstrain difference in tissue repair is (are) not currently understood. The objectives of the present study were (1) to investigate if the differences in compensatory tissue repair are reflected in differential protooncogene expression in S-D versus F344 rat livers and (2) to investigate if changes in protooncogene expression could explain the decrease and delay in tissue repair response beyond a threshold of 0.6 ml o-DCB/kg. Male S-D and F344 rats (8/9 weeks old) were administered either 0.6 or 1.2 ml o-DCB/kg and changes in expression of protooncogenes c-myc (immediate early) and Ha-ras (delayed early) were examined over a time course. Findings of this study indicate that the timing and extent of c-myc and Ha-ras expression varies in the two strains following administration of o-DCB. Thus, the timing and extent of compensatory liver regeneration that ensues following o-DCB administration in S-D and F344 rats is temporally concordant with the protooncogene expression in the two strains.

Toxicant-inflicted injury and stimulated tissue repair are opposing toxicodynamic forces in predictive toxicology.

These studies were designed to investigate the dose response for liver injury and tissue repair induced by exposure to four structurally and mechanistically dissimilar hepatotoxicants, individually and as mixtures. The objective was to illuminate the impact of the extent and timeliness of tissue repair on the ultimate outcome of toxicity. Dose-response relationships for trichloroethylene (TCE), allyl alcohol (AA), thioacetamide (TA), and chloroform alone or as mixtures were studied. Male Sprague-Dawley rats (200-250 g) received a single intraperitoneal injection of individual toxicants as well as mixtures of these toxicants. Liver injury was monitored by plasma enzyme (ALT and SDH) levels and histopathology. Tissue regeneration was measured by [3H]thymidine incorporation into hepatic nuclear DNA. Individually, TCE, TA, and AA administration, over a 10- to 12-fold dose range, revealed a dose-related increase in injury as well as tissue repair up to a threshold dose. Beyond this threshold, tissue repair was delayed and attenuated, and liver injury progressed. Mixtures of the four chemicals at the higher doses used in individual dose-response studies resulted in 100% mortality. Hence, mixtures at the lower two doses were selected for further study. Additional lower doses were also included to better understand the dose-response relationship of mixtures. Results of these studies support the observations of individual chemicals. Higher and sustained repair was observed at low dose levels. These studies show that the extent of injury at early time points correlates well with the maximal stimulation of the opposing response of tissue repair. It appears that the toxicity of the mixture employed in these studies is roughly additive and correlates well with tissue repair response. These initial studies suggest that a biologically based mathematical model can be constructed and tested to extrapolate the outcome of toxicity from a given dose of individual compounds as well as their mixtures, where the responses measured are injury on the one hand and compensatory tissue repair on the other.

Role of tissue repair in toxicologic interactions among hepatotoxic organics.

It is widely recognized that exposure to combinations or mixtures of chemicals may result in highly exaggerated toxicity even though individual chemicals might not be toxic at low doses. Chemical mixtures may also cause additive or less than additive toxicity. From the perspective of public health, highly exaggerated toxicity is of significant concern. Assessment of risk from exposure to chemical mixtures requires knowledge of the underlying mechanisms. Previous studies from this laboratory have shown that nontoxic doses of chlordecone (10 ppm, 15 days) and carbon tetrachloride (CCl4) (100 microliters/kg) interact at the biologic interface, resulting in potentiated liver injury and 67-fold amplification of CCl4 lethality. In contrast, although interaction between phenobarbital and CCl4 leads to even higher injury, animal survival is unaffected because of highly stimulated compensatory tissue repair. A wide variety of additional experimental evidence confirms the central role of stimulated tissue repair as a decisive determinant of the final outcome of liver injury inflicted by hepatotoxicants. These findings led us to propose a two-stage model of toxicity. In this model, tissue injury is inflicted in stage one by the well-described mechanisms of toxicity, whereas in stage two the ultimate toxic outcome is determined by whether timely and sufficient tissue repair response accompanies this injury. In an attempt to validate this model, dose-response relationships for injury and tissue repair as opposing responses have been developed for model hepatotoxicants. Results of these studies suggest that tissue repair increases in a dose-dependent manner, restraining injury up to a threshold dose, whereupon it is inhibited, allowing an unrestrained progression of injury. These findings indicate that tissue repair is a quantifiable response to toxic injury and that inclusion of this response in risk assessment may help in fine-tuning prediction of toxicity outcomes.

Temporal changes in tissue repair permit survival of diet-restricted rats from an acute lethal dose of thioacetamide.

Although, diet restriction (DR) has been shown to substantially increase longevity while reducing or delaying the onset of age-related diseases, little is known about the mechanisms underlying the beneficial effects of DR on acute toxic outcomes. An earlier study (S. K. Ramaiah et al., 1998, Toxicol. Appl. Pharmacol. 150, 12-21) revealed that a 35% DR compared to ad libitum (AL) feeding leads to a substantial increase in liver injury of thioacetamide (TA) at a low dose (50 mg/kg, i.p.). Higher liver injury was accompanied by enhanced survival. A prompt and enhanced tissue repair response in DR rats at the low dose (sixfold higher liver injury) occurred, whereas at equitoxic doses (50 mg/kg in DR and 600 mg/kg in AL rats) tissue repair in AL rats was substantially diminished and delayed. The extent of liver injury did not appear to be closely related to the extent of stimulated tissue repair response. The purpose of the present study was to investigate the time course (0-120 h) of liver injury and liver tissue repair at the high dose (600 mg TA/kg, i.p., lethal in AL rats) in AL and DR rats. Male Sprague-Dawley rats (225-275 g) were 35% diet restricted compared to their AL cohorts for 21 days and on day 22 they received a single dose of TA (600 mg/kg, i.p.). Liver injury was assessed by plasma ALT and by histopathological examination of liver sections. Tissue repair was assessed by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) immunohistochemistry during 0-120 h after TA injection. In AL-fed rats hepatic necrosis was evident at 12 h, peaked at 60 h, and persisted thereafter until mortality (3 to 6 days). Peak liver injury was approximately twofold higher in DR rats compared to that seen in AL rats. Hepatic necrosis was evident at 36 h, peaked at 48 h, persisted until 96 h, and returned to normal by 120 h. Light microscopy of liver sections revealed progression of hepatic injury in AL rats whereas injury regressed completely leading to recovery of DR rats by 120 h. Progression of injury led to 90% mortality in AL rats vs 30% mortality in DR group. In the surviving AL rats, S-phase DNA synthesis was evident at 60 h, peaked at 72 h, and declined to base level by 120 h, whereas in DR rats S-phase DNA synthesis was evident at 36 h and was consistently higher until 96 h reaching control levels by 120 h. PCNA studies showed a corresponding increase in cells in S and M phase in the AL and DR groups. DR resulted in abolition of the delay in tissue repair associated with the lethal dose of TA in ad libitum rats. Temporal changes and higher tissue repair response in DR rats (earlier and prolonged) are the conduits that allow a significant number of diet restricted rats to escape lethal consequence.

Colchicine antimitosis abolishes resiliency of postnatally developing rats to chlordecone-amplified carbon tetrachloride hepatotoxicity and lethality.

We have previously reported that rats are resilient to the hepatotoxic and lethal combination of chlordecone (CD) and carbon tetrachloride (CCl4) during early postnatal development. The overall findings pointed to stimulated cell division and tissue repair mechanisms as the underlying cause of resistance. The objective of the current study was to investigate if the antimitotic effect of colchicine (CLC) abolishes this resiliency to CD + CCl4 by inhibiting ongoing and stimulated cell division. We used 45-day-old rats in this study because this age group exhibited partial sensitivity to CD + CCl4 in our previous studies. Male Sprague-Dawley rats were treated with a single low intraperitoneal dose of CCl4 (100 microl/kg) or corn oil after exposure to either 10 ppm CD in the diet or a normal diet (ND) for 15 days. CLC (1 mg/kg) was administered 6 or 30 hr after CCl4 to ND or CD rats, respectively. Administration of CLC resulted in increased CCl4-induced mortality from 25% to 85% in rats pretreated with CD, in contrast to 100% survival in ND rats. Liver injury was assessed by plasma alanine transaminase (ALT) and sorbitol dehydrogenase (SDH) elevations as well as by histopathology. Hepatocellular regeneration was assessed by 3H-thymidine (3H-T) incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) studies during 0-96 hr after CCl4. Administration of CLC to ND + CCl4 rats resulted in a slight delay in cell division and tissue repair, as indicated by 3H-T incorporation and PCNA, thereby leading to prolonged liver injury as revealed by elevations in plasma ALT, SDH, and histopathological lesions. In contrast, CLC administration to CD + CCl4-treated rats further delayed and diminished cell division by 80%, which led to unrestrained progression of CCl4-induced liver injury, resulting in 85% mortality. These findings underscore the importance of ongoing and toxicant-stimulated cell division and tissue repair mechanisms in hepatotoxicity, and the need for the inclusion of age factors in risk assessment of exposure to environmental and other chemicals.

Diet restriction enhances compensatory liver tissue repair and survival following administration of lethal dose of thioacetamide.

Diet restriction is known to prevent a plethora of age-associated diseases including cancer. However, the effects of diet restriction on noncancer end points are not known. The objective of this study was to investigate whether diet restriction protects against hepatotoxicity of thioacetamide (TA), and if so, to investigate the underlying mechanism. Male Sprague-Dawley rats (250-275 g) were maintained on 65% of their ad libitum (AL) food consumption for a period of 3 weeks and then treated with a single low dose of 50 mg TA/kg i.p.. Plasma enzymes (ALT and SDH), hepatic glycogen levels, and 3H-thymidine incorporation into hepatocellular nuclear DNA were measured during a time course (0-120 h) after TA administration. Liver sections were examined for histopathology, and cell-cycle progression was assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry. In AL rats hepatic necrosis was evident at 12 h, peaked at 36 h, persisted up to 72 h, and was resolved by 96 h. In the diet-restricted (DR) group hepatic necrosis was observed at 12 h, peaked at 24 h, persisted till 72 h, and was resolved by 96 h. Maximal injury indicated by enzyme elevation occurred in DR rats and was approximately sixfold greater than that observed in the AL group. Histopathological examination of the liver sections revealed liver injury concordant with plasma enzyme elevations. There was a higher and sustained S-phase synthesis in the DR rats compared to AL group. S-phase stimulation was evident at 36 h, peaked at 48 h, and persisted until 96 h in the DR rats, whereas in the AL rats peak S-phase stimulation occurred at 36 h and subsided by 72 h. PCNA studies revealed a corresponding stimulation of cell-cycle progression indicating highly stimulated compensatory tissue repair. The 14-day lethality experiments (600 mg TA/kg i.p.) indicated 70% survival in the DR rats compared to 10% survival in the AL group. Although diet restriction increases hepatotoxic injury of TA, it protects from the lethal outcome by enhanced liver tissue repair. Comparison of liver injury and tissue repair employing an equitoxic dose (600 mg TA/kg in AL rats yields similar liver injury as observed with 50 mg TA/kg in DR rats) revealed that in spite of near equal injury up to 36 h, tissue repair response in DR rats is much higher. The compensatory tissue repair allows the DR rats to escape death in contrast to much lower compensation in AL rats leading to progression of liver injury culminating in death.

Tissue repair response as a function of dose during trichloroethylene hepatotoxicity.

Trichloroethylene (TCE), a widely used organic solvent and degreasing agent, is regarded as a hepatotoxicant. The objective of the present studies was to investigate whether the extent and timeliness of tissue repair has a determining influence on the ultimate outcome of hepatotoxicity. Male Sprague-Dawley rats (200-250 g) were injected with a 10-fold dose range of TCE and hepatotoxicity and tissue repair were studied during a time course of 0 to 96 h. Light microscopic changes as evaluated by H&E-stained liver sections revealed a dose-dependent necrosis of hepatic cells. Maximum liver cell necrosis was observed at 48 h after the TCE administration. However, liver injury as assessed by plasma sorbitol dehydrogenase (SDH) showed a dose response over a 10-fold dose range only at 6 h, whereas alanine aminotransferase (ALT) did not show a dose response at any of the time points studied. A low dose of TCE (250 mg/kg) showed an increase in SDH at all time points up to 96 h without peak levels, whereas higher doses showed peak only at 6 h. At later time points SDH declined but remained above normal. In vitro addition of trichloroacetic acid, a metabolite of TCE to plasma, decreased the activities of SDH and ALT indicating that metabolites formed during TCE toxicity may interfere with plasma enzyme activities in vivo. This indicates that the lack of dose-related increase in SDH and ALT activities may be because of interference by the TCE metabolite. Tissue regeneration response as measured by [3H]thymidine incorporation into hepatocellular nuclear DNA was stimulated maximally at 24 h after 500 mg/kg TCE administration. A higher dose of TCE led to a delay and diminishment in [3H]thymidine incorporation. At a low dose of TCE (250 mg/kg) [3H]thymidine incorporation peaked at 48 h and this could be attributed to very low or minimal injury caused by this dose. With higher doses tissue repair was delayed and attenuated allowing for unrestrained progression of liver injury. These results support the concept that the toxicity and repair are opposing responses and that a dose-related increase in tissue repair represents a dynamic, quantifiable compensatory mechanism.

Temporal changes in tissue repair upon repeated exposure to thioacetamide.

In an earlier study it was reported that a single low dose of thioacetamide (TA, 50 mg/kg) administered 36 h prior to challenge with a high dose of 400 mg/kg offers protection from lethality of high dose (Mangipudy et al., Pharmacol. Toxicol. 77, 1995). The mechanism underlying this protection was found to be preplaced hepatocellular division and tissue repair that peaked by 36 h following the low-dose treatment. In a separate study using the dose-response paradigm, it was established that the rate and the extent of the tissue repair response following infliction of injury after acute exposure has a critical bearing on the ultimate outcome of toxicity (Mangipudy et al., Environ. Health Perspect. 103, 1995). The objective of this study was to investigate the cell proliferation dynamics after repeated exposure to TA (50 mg/kg i.p.). Male Sprague-Dawley rats (200-225 g) were administered TA at intervals of 96 h. Liver injury and tissue repair were studied over a time course following each treatment. Tissue repair was estimated by S-phase DNA synthesis measuring 3H-thymidine incorporation into hepatonuclear DNA while liver injury was estimated by serum alanine aminotransferase activity. After the first dose of 50 mg/kg, peak S-phase DNA synthesis was observed at 36 h. This returned to control values by 96 h at which time the rats are known to overcome liver injury. A second dose of TA (repeated dose 1, RD1) resulted in peak S-phase DNA synthesis 12 h later at 48 h. Following the third dose (RD2) a dramatic increase in S-phase DNA synthesis was noted from as early as 12 h. Much higher peak was observed at 72 h. Interestingly, following the fourth dose (RD3) S-phase stimulation did not occur. Instead, a significant latency was observed for cells in the S-phase DNA synthesis even at time points as late as 144 h following the treatment. Liver injury on the other hand exhibited no significant differences between repetitions until RD2. However, injury was sustained in the rats treated with the fourth dose (RD3) while it returned to control levels in the earlier three instances. Sustained prolongation of liver injury after the fourth dose is presumably because tissue repair was not operational. Thus repeated exposure to TA causes a significant increase in tissue repair response although it is temporally delayed until a threshold is reached. Repetition beyond the threshold results in a marked attenuation of the repair response. These findings suggest that toxicodynamics of cell proliferation are altered after repeated exposure.

Effects of blockade of Kupffer cells by gadolinium chloride on hepatobiliary function in cold ischemia-reperfusion injury of rat liver.

The mechanisms of liver injury from cold storage and reperfusion are not completely understood. The aim of the present study was to investigate: 1) whether the inactivation of Kupffer cells (KCs) by gadolinium chloride (GadCl) modulates cold ischemia-reperfusion injury of rat liver; and 2) whether cold storage of rat liver involves injury to biliary epithelial cells (BECs). Hepatobiliary function was assessed using an isolated perfused rat liver model. Compared with control livers, in livers subjected to cold storage at 4 degrees C in Euro-Collins solution (EC) for 18 hours or in University of Wisconsin solution (UW) for 48 hours, portal flow was lower and resistance significantly higher, taurocholate (TC) and bromosulfophthalein (BSP) elimination were markedly impaired, bile flow was reduced, and lactate dehydrogenase (LDH) leakage into the perfusate was increased. Pretreatment of rats with GadCl, a selective KC toxicant, abrogated disturbances of the microcirculation in both models, but it did not influence viability and functional parameters of the liver. Most of the parameters studied in livers stored in UW solution for 18 hours were not significantly different from those found in control livers. As to biliary activity of gamma-glutamyl transferase (GGT), as an index of BEC integrity, it was increased with increasing time of cold storage. The reabsorption of glucose from the bile decreased with longer storage time. The results suggest the following: 1) that cold ischemia-reperfusion injury of rat liver is mediated by KC-dependent (hepatic microcirculation) and -independent (parenchymal cell function) mechanisms; and 2) that cold storage of rat liver induces functional impairment of BECs.

Antimitotic intervention with colchicine alters the outcome of o-DCB-induced hepatotoxicity in Fischer 344 rats.

Although, hepatotoxic injury of 1,2-dichlorobenzene (o-DCB) is greater in Fischer 344 (F344) as compared to Sprague-Dawley (S-D) rats, this interstrain difference does not transcend into any difference in lethal effects of o-DCB. Interstrain difference in compensatory tissue repair has been suggested as the underlying mechanism for the lack of strain differences in lethality (S.G. Kulkarni, H. Duong, R. Gomila, and H.M. Mehendale, Strain differences in tissue repair response to 1,2-dichlorobenzene. Archives of Toxicology 1996; 70: 714-723). If higher tissue repair in F344 rats compensates for more severe liver injury, then antimitotic intervention after infliction of o-DCB-induced liver injury should lead to lethality in F344 rats. Colchicine (CLC, 1 mg/kg) functions as an effective antimitotic agent and does not cause any side effects apart from suppressing cellular proliferation. Two groups of male F344 rats (160-190 g) received a single dose of 0.6 ml o-DCB/kg: 30 h later one group of rats received CLC (1 mg/kg; i.p.) and the other received distilled water (1 ml/kg; i.p.). Liver injury was assessed by measuring plasma ALT and SDH activity, liver histopathology, and liver regeneration was estimated by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) assay in both groups. Similar liver injury was noted in both the o-DCB + vehicle and o-DCB + CLC treated F344 rats at 36 h indicating that CLC does not interfere with the uptake, bioactivation and causation of injury by o-DCB. S-phase synthesis which occurred at 36 h in the o-DCB + vehicle group was blocked in the o-DCB + CLC group. CLC administration 6 h prior to S-phase stimulation selectively abolished S-phase stimulation at 36 h, and led to 50% lethality. Since the effect of CLC antimitosis was transient, S-phase synthesis occurring at 48 h was not blocked and was sustained up to 72 h thereby allowing the other 50% of rats to overcome liver injury induced by o-DCB and survive the lethal outcome. These findings suggest that a significantly higher rate of compensatory tissue repair in F344 rats enables them to overcome more severe liver injury inflicted by o-DCB.

Tissue injury and repair as parallel and opposing responses to CCl4 hepatotoxicity: a novel dose-response.

Recent studies indicate that the rate and extent of tissue repair, elicited as an endogenous response to toxic insult, are critical determinants in the ultimate outcome of hepatic injury. Therefore, the objective of this study was to develop a dose-response relationship for CCl4 measuring liver injury and tissue repair as two simultaneous but opposing responses. Male Sprague-Dawley rats were injected with a 40-fold dose range of CCl4 (0.1-4 ml/kg i.p.) in corn oil vehicle. Liver injury was assessed by serum enzyme elevations and histopathology, and tissue repair was measured by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen immunohistochemistry over a time course of 0 to 96 h. Stimulation of cell division, evident even after a subtoxic dose of CCl4, increased in a dose-dependent manner until a threshold (2 ml/kg) was reached. Doses above this threshold yielded no further increase in tissue repair. Instead, tissue repair response was significantly delayed and diminished. Injury was markedly accelerated above the threshold indicating an unrestrained progression of injury. Although 4 ml CCl4/kg consistently caused 80% lethality by 48 h, tissue repair response in the 20% surviving rats was increased by about 5-fold, aptly demonstrating the critical role of tissue repair in overcoming injury and enabling these animals to survive. This study suggests that, in addition to the extent of tissue repair, the time of onset of tissue repair also determines the extent of hepatic injury and inter-individual differences in the magnitude of tissue repair may contribute significantly to inter-individual differences in susceptibility to toxic chemicals. Thus, while dose-related and prompt stimulation of tissue regeneration leads to recovery, delayed and attenuated repair response, occurring at higher doses, leads to progression of injury and animal mortality. Such dose-response relationships may lead to a better understanding of the underlying cellular mechanisms of injury inflicted by chemical toxicants and aid in fine-tuning risk assessment.

Stimulated tissue repair prevents lethality in isopropanol-induced potentiation of carbon tetrachloride hepatotoxicity.

Published reports on the alcohol potentiation of CCl4 toxicity 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 CCl4 administration. 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 + CCl4 group was higher than that in the H2O + CCl4 group. Plasma enzyme activity returned to control levels by 60 hr, indicating recovery from injury in both groups. Maximum 3H-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 + CCl4 group by 60 hr but continued up to 72 hr in the ISOP + CCl4 group. These findings support the hypothesis that in response to increased infliction of CCl4 injury 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 CCl4 alone (H2O + CCl4). These findings suggest that greater stimulation of tissue repair restrains the progression of ISOP-enhanced infliction of CCl4 liver 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.