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.     

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.     

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.     

Inhibition of cyclooxygenase-2 aggravates secretory phospholipase A2-mediated progression of acute liver injury

Our previous study [Bhave, V. S., Donthamsetty, S., Latendresse, J. R., Muskhelishvili, L., and Mehendale, H. M. 2008-this issue. Secretory phospholipase A(2) mediates progression of acute liver injury in the absence of sufficient COX-2. Toxicol Appl Pharmacol] showed that in the absence of sufficient induction and co-presence of cyclooxygenase-2 (COX-2), secretory phospholipase A(2) (sPLA(2)) appearing in the intercellular spaces for cleanup of post-necrotic debris seems to contribute to the progression of toxicant-initiated liver injury, possibly by hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas. To further test our hypothesis on the protective role of COX-2, male Fisher-344 rats were administered a selective COX-2 inhibitor, NS-398, and then challenged with a moderately toxic dose of CCl(4). This led to a 5-fold increase in the susceptibility of the COX-2 inhibited rats to CCl(4) hepatotoxicity and mortality. The CCl(4) bioactivating enzyme CYP2E1 protein, CYP2E1 enzyme activity, and the (14)CCl(4)-derived radiolabel covalently bound to the liver proteins were unaffected by the COX-2 inhibitor suggesting that the increased hepatotoxic sensitivity of the COX-2 inhibited rats was not due to higher bioactivation of CCl(4). Further investigation showed that this increased mortality was due to higher plasma and hepatic sPLA(2) activities, inhibited PGE(2) production, and progression of liver injury as compared to the non-intervened rats(.) In conclusion, inhibition of COX-2 mitigates the tissue protective mechanisms associated with COX-2 induction, which promotes sPLA(2)-mediated progression of liver injury in an acute liver toxicity model. Because increased sPLA(2) activity in the intercellular space is associated with increased progression of injury, and induced COX-2 is associated with hepatoprotection, ratios of hepatic COX-2 and sPLA(2) activities may turn out to be a useful tool in predicting the extent of hepatotoxicities.     

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.     

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.     

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.     

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.     

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.     

Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin

Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of exposure to (anti-)oestrogens and their mimics in fish [Sumpter, J.P., 1995. Feminized responses in fish to environmental estrogens. Toxicol. Lett. 82, 737-742; Arukwe, A., Goks?yr, A., 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp. Hepatol. 2, 4. ]. In some instances, links have been drawn between high level induction of Vtg and adverse health effects in fish [Herman, R.L., Kincaide, H.L., 1988. Pathological effects of orally administered estradiol to rainbow trout. Aquaculture 72, 165-172; Schwaiger, J., Spieser, O.H., Bauer, C., Ferling, H., Mallow, U., Kalbfus, W., Negele, R.D., 2000. Chronic toxicity of nonylphenol and ethinyloestraiol: haematological and histopathological effects in juvenile common carp (Cyprinus carpio). Aquat. Toxicol. 51, 69-78]. The widespread use of Vtg as a biomarker has led to the development of a variety of assays to quantitatively measure Vtg concentrations in tissue samples from fish, and hence a need for a standardization of the performance criteria and validation of such assays [Goks?yr, A., Eidem, J.K., Kristiansen, S.I., Nilsen, B.M., 2003. On the need for a standardized set-up for validation studies of fish vitellogenin assays as an endpoint in endocrine disruptor testing and screening-a proposal. ]. One of the most popular test fish species for assessing chemical effects is the fathead minnow (Pimephales promelas), which is now used widely for studies into endocrine disruption [Panter, G.H., Hutchinson, T.H., Lange, R., Lye, C.M., Sumpter, J.P., Zerulla, M., Tyler, C.R., 2002. Utility of a juvenile fathead minnow screening assay for detecting (anti)estrogenic substances. Environ. Toxicol. Chem. 21, 319-326; Hutchinson, T.H., Yokota, H., Hagino, S., Ozato, K., 2003. Development of fish tests for endocrine disruptors. Pure Appl. Chem. 75, 2343-2353]. This paper describes the development and validation of a new, homologous enzyme-linked immunosorbent assay (ELISA) for quantification of Vtg in this fish species.     

Streptozotocin-induced diabetic mice are resistant to lethal effects of thioacetamide hepatotoxicity

The effect of Type 1 diabetes on the toxicity of thioacetamide was investigated in a murine model. In streptozotocin-induced diabetic C57BL6 mice a LD90 dose of thioacetamide (1000 mg/kg, ip in saline) caused only 10% mortality. Alanine aminotransferase activity revealed approximately 2.7-fold less liver injury in the diabetic (DB) mice compared to the non-DB controls, at 36 h after thioacetamide (TA) administration, which was confirmed via histopathological analysis. HPLC analyses revealed lower plasma t(1/2) of TA in the DB mice. Covalent binding of [(14)C]TA to liver tissue was lower in the DB mice, suggesting lower bioactivation of TA. Compensatory hepatic S-phase stimulation as assessed by [(3)H]thymidine incorporation occurred much earlier and was substantially higher in the DB mice compared to the non-DB cohorts. Morphometric analysis of cells in various phases of cell division assessed via immunohistochemical staining for proliferating cell nuclear antigen revealed more cells in G(1), S, G(2), and M phases in the DB mice, indicating robust tissue repair in concordance with the findings of [(3)H]thymidine pulse labeling studies. The importance of tissue repair in the resistance of DB mice was further investigated by blocking cell division in the DB mice by colchicine (1 mg/kg, ip) at 40 h after TA administration, well after the bioactivation of TA. Antimitotic action of colchicine, confirmed by decreased S-phase stimulation, led to progression of liver injury and increased mortality in DB mice. These findings suggest that lower bioactivation of TA and early onset of liver tissue repair are the pivotal underpinnings for the resistance of DB mice.     

Type 2 diabetic rats are sensitive to thioacetamide hepatotoxicity

Previously, we reported high hepatotoxic sensitivity of type 2 diabetic (DB) rats to three dissimilar hepatotoxicants. Additional work revealed that a normally nonlethal dose of CCl4 was lethal in DB rats due to inhibited compensatory tissue repair. The present study was conducted to investigate the importance of compensatory tissue repair in determining the final outcome of hepatotoxicity in diabetes, using another structurally and mechanistically dissimilar hepatotoxicant, thioacetamide (TA), to initiate liver injury. A normally nonlethal dose of TA (300 mg/kg, ip), caused 100% mortality in DB rats. Time course studies (0 to 96 h) showed that in the non-DB rats, liver injury initiated by TA as assessed by plasma alanine or aspartate aminotransferase and hepatic necrosis progressed up to 48 h and regressed to normal at 96 h resulting in 100% survival. In the DB rats, liver injury rapidly progressed resulting in progressively deteriorating liver due to rapidly expanding injury, hepatic failure, and 100% mortality between 24 and 48 h post-TA treatment. Covalent binding of 14C-TA-derived radiolabel to liver tissue did not differ from that observed in the non-DB rats, indicating similar bioactivation-based initiation of hepatotoxicity. S-phase DNA synthesis measured by [3H]-thymidine incorporation, and advancement of cells through the cell division cycle measured by PCNA immunohistochemistry, were substantially inhibited in the DB rats compared to the non-DB rats challenged with TA. Thus, inhibited cell division and compromised tissue repair in the DB rats resulted in progressive expansion of liver injury culminating in mortality. In conclusion, it appears that similar to type 1 diabetes, type 2 diabetes also increases sensitivity to dissimilar hepatotoxicants due to inhibited compensatory tissue repair, suggesting that sensitivity to hepatotoxicity in diabetes occurs in the absence as well as presence of insulin.     

Relative potency based on hepatic enzyme induction predicts immunosuppressive effects of a mixture of PCDDS/PCDFS and PCBS

The toxic equivalency factor (TEF) approach was employed to compare immunotoxic potency of mixtures containing polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), using the antibody response to sheep erythrocytes (SRBC). Mixture-1 (MIX-1) contained TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,6,7,8,9-octachlorodibenzofuran (OCDF). Mixture-2 (MIX-2) contained MIX-1 and the following PCBs, 3,3',4,4'-tetrachlorobiphenyl (IUPAC No. 77), 3,3',4,4',5-pentachlorobiphenyl (126), 3,3',4,4',5,5N-hexachlorobiphenyl (169), 2,3,3',4,4'-pentachlorobiphenyl (105), 2,3',4,4',5-pentachlorobiphenyl (118), and 2,3,3',4,4',5-hexachlorobiphenyl (156). The mixture compositions were based on relative chemical concentrations in food and human tissues. TCDD equivalents (TEQ) of the mixture were estimated using relative potency factors from hepatic enzyme induction in mice [DeVito, M.J., Diliberto, J.J., Ross, D.G., Menache, M.G., Birnbaum, L.S., 1997. Dose-response relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I .CYP1A1 and CYP1A2 enzyme activity in liver, lung and skin. Toxicol. Appl. Pharmacol. 130, 197-208; DeVito, M.J., Menache, G., Diliberto, J.J., Ross, D.G., Birnbaum L.S., 2000. Dose-response relationships for induction of CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin in female mice following subchronic exposure to polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 167, 157-172] Female mice received 0, 1.5, 15, 150 or 450 ng TCDD/kg/day or approximately 0, 1.5, 15, 150 or 450 ng TEQ/kg/day of MIX-1 or MIX-2 by gavage 5 days per week for 13 weeks. Mice were immunized 3 days after the last exposure and 4 days later, body, spleen, thymus, and liver weights were measured, and antibody response to SRBCs was observed. Exposure to TCDD, MIX-1, and MIX-2 suppressed the antibody response in a dose-dependent manner. Two-way ANOVA indicated no differences in the response between TCDD and the mixtures for body weight, spleen/body weight and decreased antibody responses. The results support the use of the TEF methodology and suggest that immune suppression by dioxin-like chemicals may be of concern at or near background human exposures.     

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.     

Comparative safety testing of genetically modified foods in a 90-day rat feeding study design allowing the distinction between primary and secondary effects of the new genetic event

This article discusses the wider experiences regarding the usefulness of the 90-day rat feeding study for the testing of whole foods from genetically modified (GM) plant based on data from a recent EU-project [Poulsen, M., Schr?der, M., Wilcks, A., Kroghsbo, S., Lindecrona, R.H., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Shu, Q., Emami, K., Taylor, M., Gatehouse, A., Engel, K.-H., Knudsen, I., 2007a. Safety testing of GM-rice expressing PHA-E lectin using a new animal test design. Food Chem. Toxicol. 45, 364-377; Poulsen, M., Kroghsbo, S., Schr?der, M., Wilcks, A., Jacobsen, H., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Shu, Q., Emami, K., Sudhakar, D., Gatehouse, A., Engel, K.-H., Knudsen, I., 2007b. A 90-day safety in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA). Food Chem. Toxicol. 45, 350-363; Schr?der, M., Poulsen, M., Wilcks, A., Kroghsbo, S., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Emami, K., Gatehouse, A., Shu, Q., Engel, K.-H., Knudsen, I., 2007. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats. Food Chem. Toxicol. 45, 339-349]. The overall objective of the project has been to develop and validate the scientific methodology necessary for assessing the safety of foods from genetically modified plants in accordance with the present EU regulation. The safety assessment in the project is combining the results of the 90-day rat feeding study on the GM food with and without spiking with the pure novel gene product, with the knowledge about the identity of the genetic change, the compositional data of the GM food, the results from in-vitro/ex-vivo studies as well as the results from the preceding 28-day toxicity study with the novel gene product, before the hazard characterisation is concluded. The results demonstrated the ability of the 90-day rat feeding study to detect the biological/toxicological effects of the new gene product in the GM food. The authors consider on this basis that the 90-day, rodent feeding study with one high dose level and a dietary design based upon compositional data on the GM food and toxicity data on the gene product is sensitive and specific enough to verify the presence/absence of the biological/nutritional/toxicological effects of the novel gene insert and further by the use of spiking able to separate potentially unintended effects of the novel gene product from other unintended effects at the level of intake defined in the test and within the remit of the test. Recommendations for further work necessary in the field are given.     

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.     

Analyses of neurobehavioral screening data: benchmark dose estimation

Zhu et al. (Zhu, Y., Wessel, M., Liu, T., Moser, V.C., 2005. Analyses of neurobehavioral screening data: dose-time-response modeling of continuous outcomes. Regul. Toxicol. Pharmacol. 41, 240-255) have recently applied dose-time-response models to longitudinal or time-course neurotoxicity data, and have illustrated the modeling process using continuous data from a functional observational battery (FOB). Following the work of these authors, the purpose of this paper is to show that the benchmark dose (BMD) method for single time point dose-response data can be generalized and applied to longitudinal data such as those generated in neurotoxicity studies. We propose a statistical procedure called bootstrap method for computing the lower confidence limits for the BMD. We demonstrate the method using three previously published FOB datasets of triethyltin (Moser, V.C., Becking, G.C., Cuomo, V., Frantik, E., Kulig, B., MacPhail, R.C., Tilson, H.A., Winneke, G., Brightwell, W.S., DeSalvia, M.A., Gill, M.W., Haggerty, G.C., Hornychova, M., Lammers, J., Larsson, J., McDaniel, K.L., Nelson, B.K., Ostergaard, G., 1997a. The IPCS study on neurobehavioral screening methods: results of chemical testing. Neurotoxicology 18, 969-1056.) and the models of Zhu et al. (Zhu, Y., Wessel, M., Liu, T., Moser, V.C., 2005. Analyses of neurobehavioral screening data: dose-time-response modeling of continuous outcomes. Regul. Toxicol. Pharmacol. 41, 240-255).     

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.     

Chloroform Interaction with Chlordecone and Mirex: Correlation between Biochemical and Histological Indices of Toxicity and Quantitative Tissue Levels

Chloroform Interaction with Chlordecone and Mirex: Correlationbetween Biochemical and Histological Indices of Toxicity andQuantitative Tissue Levels. PLAA, G. L., CAILLÉ, G.,VÉZINA, M., IIJIMA, M., AND CÔTÉ M. G. (1987).Fundam Appl Toxicol 9, 198–207. The purpose of this studywas to investigate whether the tissue distnbution of chlordecone(CD) and mirex (M) might explain the difference in the potentiationof CHCl₃ liver injury. Male Sprague-Dawley rats received eithera single oral dose of CD or M (1, 2.5, 5, 10. 25, or 50 mg/kg)or three single daily doses of CD or M (0.5, 2, or 10 mg/kg).Eighteen hours following the last treatment, the animals weredivided into two groups. The first group was killed and residuesof CD or M in plasma, kidney, liver, and adipose tissue weremeasured by gas-liquid chromatography. The other group receivedCHCl₃ (0.5 ml/kg, po) and was killed 24 hr later. Biochemicaland histological indices of liver injury were evaluated. CDadministered either singly or repetitively is more effectivethan M in potentiating the CHCl₃-induced liver injury. Thiswas exhibited by a higher increase in the plasma activitiesof the enzymes alanine aminotransferase and ornithine carbamoyltransferase and a greater alteration of the morphological pattern.There was a very good correlation between biochemical and histologicalindices. However, the severity of the liver injury did not paralleltissue concentrations. With M, a relatively poor potentiationof the liver damage was observed although adequate hepatic concentrationswere present.