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.     

Dose-dependent liver tissue repair after chloroform plus trichloroethylene binary mixture

The aim of the present study was to investigate the hypothesis that liver tissue repair induced by exposure to chloroform (CHCl(3))+trichloroethylene binary mixture (BM) is dose-dependent similar to that elicited by exposure to these compounds individually. Male Sprague-Dawley rats (250-300 g) received three dose combinations of binary mixture (74+250, 185+500 and 370+1250 mg CHCl(3)+trichloroethylene/kg, intraperitoneally) in corn oil (maximum of 0.5 ml/kg). Liver injury was assessed by plasma alanine amino transaminase (ALT) activity and histopathology by haematoxylin & eosin. Liver tissue repair was measured by (3)H-thymidine incorporation into hepatonuclear DNA. Blood and liver levels of both the parent compounds and two major metabolites of trichloroethylene (trichloroacetic acid and trichloroethanol) were quantified by gas chromatography. The blood and liver CHCl(3) levels after the administration of binary mixture were similar compared to the administration of CHCl(3) alone. The blood and liver trichloroethylene levels after the binary mixture were significantly lower compared to trichloroethylene alone due to higher elimination in presence of CHCl(3), resulting in decreased production of metabolites. The antagonistic toxicokinetics 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 elicited by the binary mixture was dose-dependent. The interactive toxicity of this binary mixture of CHCl(3) and trichloroethylene led to subadditive initial liver injury because of a combined effect of higher elimination of TCE and mitigated progression of liver injury was prevented by timely dose-dependent stimulation of compensatory tissue repair. Even though the doses of the toxicants employed in this study are much higher than found in the environment, the results suggest that a mixture of these two compounds at environmental levels is unlikely to cause any exaggerated interactive acute liver toxicity of any biological significance.     

Tissue Repair Response as a Function of Dose during Trichloroethylene Hepatotoxicity

Trichloroethylene (TCE), a widely used organic solvent and degreasingagent, is regarded as a hepatotoxicant. The objective of thepresent studies was to investigate whether the extent and timelinessof tissue repair has a determining influence on the ultimateoutcome of hepatotoxicity. Male Sprague-Dawley rats (200–250g) were injected with a 10-fold dose range of TCE and hepatotoxicityand tissue repair were studied during a time course of 0 to96 h. Light microscopic changes as evaluated by H&E-stainedliver sections revealed a dose-dependent necrosis of hepaticcells. Maximum liver cell necrosis was observed at 48 h afterthe TCE administration. However, liver injury as assessed byplasma sorbitol dehydrogenase (SDH) showed a dose response overa 10-fold dose range only at 6 h, whereas alanine aminotrans-ferase(ALT) did not show a dose response at any of the time pointsstudied. A low dose of TCE (250 mg/kg) showed an increase inSDH at all time points up to 96 h without peak levels, whereashigher doses showed peak only at 6 h. At later time points SDHdeclined but remained above normal. In vitro addition of trichloroaceticacid, a metabolite of TCE to plasma, decreased the activitiesof SDH and ALT indicating that metabolites formed during TCEtoxicity may interfere with plasma enzyme activities in vivo.This indicates that the lack of dose-related increase in SDHand ALT activities may be because of interference by the TCEmetabolite. Tissue regeneration response as measured by [³H]thymidineincorporation into hepatocellular nuclear DNA was stimulatedmaximally at 24 h after 500 mg/kg TCE administration. A higherdose of TCE led to a delay and diminishment in [³H]thymidineincorporation. At a low dose of TCE (250 mg/kg) [³H]thymidineincorporation peaked at 48 h and this could be attributed tovery low or minimal injury caused by this dose. With higherdoses tissue repair was delayed and attenuated allowing forunrestrained progression of liver injury. These results supportthe concept that the toxicity and repair are opposing responsesand that a dose-related increase in tissue repair representsa dynamic, quantifiable compensatory mechanism.     

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.     

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.     

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.     

Prior administration of a low dose of thioacetamide protects type 1 diabetic rats from subsequent administration of lethal dose of thioacetamide

Previously, we reported that an ordinarily non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic rats due to inhibited liver tissue repair, whereas 30 mg TA/kg allows 100% survival due to stimulated although delayed tissue repair. Objective of this investigation was to test whether prior administration of a low dose of TA (30 mg/kg) would lead to sustainable stimulation of liver tissue repair in type 1 diabetic rats sufficient to protect from a subsequently administered lethal dose of TA. Therefore, in the present study, the hypothesis that preplacement of tissue repair by a low dose of TA (30 mg TA/kg, ip) can reverse the hepatotoxicant sensitivity (autoprotection) in type 1 diabetic rats was tested. Preliminary studies revealed that a single intraperitoneal (ip) administration of TA causes 90% mortality in diabetic rats with as low as 75 mg/kg. To establish an autoprotection model in diabetic condition, diabetic rats were treated with 30 mg TA/kg (priming dose). Administration of priming dose stimulated tissue repair that peaked at 72h, at which time these rats were treated with a single ip dose of 75 mg TA/kg. Our results show that tissue repair stimulated by the priming dose enabled diabetic rats to overexpress, calpastatin, endogenous inhibitor of calpain, to inhibit calpain-mediated progression of liver injury induced by the subsequent administration of lethal dose, resulting in 100% survival. Further investigation revealed that protection observed in these rats is not due to decreased bioactivation. These studies underscore the importance of stimulation of tissue repair in the final outcome of liver injury (survival/death) after hepatotoxicant challenge. Furthermore, these results also suggest that it is possible to stimulate tissue repair in diabetics to overcome the enhanced sensitivity of hepatotoxicants.     

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.     

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.     

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.     

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 substantiallyincrease longevity while reducing or delaying the onset of agerelateddiseases, little is known about the mechanisms underlying thebeneficial effects of DR on acute toxic outcomes. An earlierstudy (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 injuryof thioacetamide (TA) at a low dose (50 mg/kg, ip). Higher liverinjury was accompanied by enhanced survival. A prompt and enhancedtissue repair response in DR rats at the low dose (sixfold higherliver injury) occurred, whereas at equitoxic doses (50 mg/kgin DR and 600 mg/kg in AL rats) tissue repair in AL rats wassubstantially diminished and delayed. The extent of liver injurydid not appear to be closely related to the extent of stimulatedtissue repair response. The purpose of the present study wasto investigate the time course (0–120 h) of liver injuryand liver tissue repair at the high dose (600 mg TA/kg, ip,lethal in AL rats) in AL and DR rats. Male Sprague-Dawley rats(225–275 g) were 35% diet restricted compared to theirAL cohorts for 21 days and on day 22 they received a singledose of TA (600 mg/kg, ip). Liver injury was assessed by plasmaALT and by histopathological examination of liver sections.Tissue repair was assessed by [³H]thymidine incorporation intohepatonuclear 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, peakedat 60 h, and persisted thereafter until mortality (3 to 6 days).Peak liver injury was approximately twofold higher in DR ratscompared to that seen in AL rats. Hepatic necrosis was evidentat 36 h, peaked at 48 h, persisted until 96 h, and returnedto normal by 120 h. Light microscopy of liver sections revealedprogression of hepatic injury in AL rats whereas injury regressedcompletely leading to recovery of DR rats by 120 h. Progressionof injury led to 90% mortality in AL rats vs 30% mortality inDR group. In the surviving AL rats, S-phase DNA synthesis wasevident at 60 h, peaked at 72 h, and declined to base levelby 120 h, whereas in DR rats S-phase DNA synthesis was evidentat 36 h and was consistently higher until 96 h reaching controllevels by 120 h. PCNA studies showed a corresponding increasein cells in S and M phase in the AL and DR groups. DR resultedin abolition of the delay in tissue repair associated with thelethal dose of TA in ad libitum rats. Temporal changes and highertissue repair response in DR rats (earlier and prolonged) arethe conduits that allow a significant number of diet restrictedrats to escape lethal consequence.     

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.     

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.     

The influence of administered mass on the subcellular distribution and binding of mercury in rat liver and kidney

The influence of the administered mass on the tissue and sub-cellular distribution of mercury (Hg) was investigated in rats, using ²⁰³Hg. The fraction of the dose deposited in liver increased threefold over the dose range 0.17–1.65 mg Hg · kg^–1, while the retention in the kidney decreased by a factor of 2. The uptake in other organs, lung, spleen, brain, thymus, salivary glands showed no dose-dependent variation.Subcellular fractionation studies showed a dose-dependent increase in the Hg content of the liver cytosol, with corresponding decreases in the deposition in the lysosomal and nuclei-cell debris fractions. In contrast, no clear changes in the distribution of Hg amongst the subcellular organelles of the kidney were observed.The amount of Hg bound to metallothionein in the liver cytosol rose steeply with increasing dose. However, in the kidney cytosol the mass of Hg bound to metallothionein increased with dose up to 0.55 mg Hg · kg^–1, thereafter remaining approximately constant. These observations suggest that the Hg-binding metallothionein in the kidney was saturated by administered doses greater than 0.55 mg Hg · kg^–1, whereas in liver saturation levels of the metal were not reached even at the highest dose tested, 1.65 mg Hg · kg^–1.     

Applications of dosimetry modeling to assessment of neurotoxic risk

Risk assessment procedures can be improved through better understanding and use of tissue dose information and linking tissue dose level to adverse outcomes. For volatile organic compounds, such as toluene and trichloroethylene (TCE), blood and brain concentrations can be estimated with physiologically based pharmacokinetic (PBPK) models. Acute changes in the function of the nervous system can be linked to the concentration of test compounds in the blood or brain at the time of neurological assessment. This set of information enables application to a number of risk assessment situations. For example, we have used this approach to recommend duration adjustments for acute exposure guideline levels (AEGLs) for TCE such that the exposure limits for each exposure duration yield identical tissue concentrations at the end of the exposure period. We have also used information on tissue concentration at the time of assessment to compare sensitivity across species, adjusting for species-specific pharmacokinetic differences. Finally this approach has enabled us to compare the relative sensitivity of different compounds on a tissue dose basis, leading to expression of acute solvent effects as ethanol-dose equivalents for purposes of estimating cost–benefit relationships of various environmental control options.     

Moving from external exposure concentration to internal dose: duration extrapolation based on physiologically based pharmacokinetic derived estimates of internal dose

The potential human health risk(s) from chemical exposure must frequently be assessed under conditions for which adequate human or animal data are not available. The default method for exposure-duration adjustment, based on Haber's rule, C (external exposure concentration) or C(n) (the ten Berge modification) x t (exposure duration) = K (a constant toxic effect), has been criticized for prediction errors. A promising alternative approach to duration adjustment is based on equivalence of internal dose, that is, target-tissue dose levels, across different exposure durations. A proposed methodology for dose-duration adjustments for acute exposure guideline levels (AEGLs) based on physiologically based pharmacokinetic (PBPK) estimates of dose is illustrated with trichloroethylene (TCE). Steps in this methodology include: (1) selection and evaluation, or development and evaluation, of an appropriate PBPK model; (2) determination of an appropriate measure of internal dose; (3) estimation with the PBPK model of the tissue dose (the target tissue dose) resulting from the external exposure conditions (concentration, duration) of the critical effect; (4) estimation of the external exposure concentrations required to achieve tissue doses equivalent to the target tissue dose at exposure durations of interest; and (5) evaluation of sources of variability and uncertainty. For TCE, this PBPK modeling approach has allowed determination of dose metrics predictive of the acute neurotoxic effects of TCE and dose-duration adjustments based on estimates of internal dose.     

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.     

Contribution of direct solvent injury to the dose-dependent kinetics of trichloroethylene: portal vein administration to rats

Presystemic elimination of trichloroethylene (TCE), a common contaminant of drinking water, has been shown by Lee et al. (Toxicol. Appl. Pharmacol. 139, 262-271, 1996) to be inversely related to dose. When relatively high doses were administered to rats via the portal vein (PV), first-pass hepatic extraction became negligible. This phenomenon could result not only from metabolic saturation, but from suicidal destruction of cytochromes P450 and hepatocellular injury as well. The objectives of the current investigation were to: (a) clarify the relative roles of P450 depletion and hepatocellular toxicity in the apparent cessation of hepatic elimination of TCE in animals given relatively high doses of TCE via the PV; and (b) investigate mechanism(s) of hepatocellular injury under such exposure conditions. TCE (16 and 64 mg/kg body weight (bw) was incorporated into a 5% aqueous Alkamuls emulsion and injected via an indwelling jugular vein (JV) or PV cannula into male Sprague-Dawley rats. Some animals received 73.5 micromol/kg of p-nitrophenol (PNP), a competitive metabolic inhibitor of TCE, through the PV cannula 3 min before TCE. Administration of TCE via the PV resulted in deposition of relatively high levels of TCE in the liver. PV dosing resulted in lower total hepatic P450 levels than did JV dosing. PV dosing produced marked elevations of cytoplasmic enzymes in serum, but JV dosing did not. Decreases in hepatic P450 were not selective for cytochrome P4502E1. Histological examination of the liver of PV-dosed rats revealed periportal rather than centrilobular necrosis. PNP pretreatment failed to prevent the increase in serum enzymes, decrease in hepatic P450 content, and hepatic necrosis following PV TCE. It is concluded that PV injection of bolus doses of TCE >/= 16 mg/kg causes liver injury within minutes in rats, primarily through direct solvent action on hepatocellular membranes rather than by P450-mediated effects. This liver damage likely plays a modest role in reducing the liver's capacity to metabolize high PV doses of TCE.     

2-Methylfuran toxicity in rats--role of metabolic activation in vivo

Administration of a single ip dose of 2-methylfuran (2-MF) to male Sprague-Dawley rats at a dose of 100 mg/kg produced centrilobular necrosis of the liver and bronchial injury of the lung, the severity of the lesions increasing with increasing doses up to 400 mg/kg. Kidneys, however, showed no visible evidence of tissue damage even at the highest dose. Liver injury was also evidenced by an increase in serum glutamic pyruvic transaminase (SGPT) levels. Tissue distribution and covalent binding studies conducted over a dose of 50-200 mg/kg of [14C]2-MF indicated that the total radioactivity present per gram of wet tissue was in the order of liver greater than kidney greater than lung greater than blood. Covalent binding of the label to protein was greatest in the liver followed by kidney and the lung. Radioactivity bound covalently per milligram of DNA was also highest in the liver followed by kidney. Tissue distribution and covalent binding studies were conducted over a period of 0.5 to 24 hr after an ip dose of 100 mg/kg of [14C]2-MF. Maximal covalent binding was observed in the liver at 4 hr. At all time points binding of the label was greatest in liver, followed by kidney. Liver glutathione levels were depressed following 2-MF administration. Pretreatment of rats with phenobarbital markedly increased the covalent binding to protein and DNA and caused a twofold increase in SGPT compared to rats treated with 2-MF alone. Pretreatment with 3-methylcholanthrene had no effect on either parameter. Administration of N-octylimidazole, an inhibitor of cytochrome P-450, prior to administration of the radiolabeled 2-MF decreased the covalent binding of the label to protein and DNA. Moreover, the SGPT levels remained the same in the pretreated rats compared to the rats treated with vehicle alone. Thus, pretreatment with phenobarbital, an inducer of cytochrome P-450, enhanced both covalent binding and toxicity while prior treatment with N-octylimidazole, an inhibitor of cytochrome P-450 decreased covalent binding and prevented hepatotoxicity of 2-MF. These results support the view that at least some of the toxic effects of 2-MF are mediated by reactive metabolite(s) formed in vivo.