Peroxisome proliferator-activated receptor alpha-null mice lack resistance to acetaminophen hepatotoxicity following clofibrate exposure.

The purpose of this study was to investigate whether activation of the nuclear receptor PPARalpha is needed for protection from acetaminophen (APAP) hepatotoxicity produced by repeated administration of the peroxisome proliferator clofibrate (CFB). Female wild-type and PPARalpha-null mice received corn oil vehicle or 500 mg CFB/kg, ip, daily for 10 days. They were then fasted overnight (18 h) and either killed at 4 or 24 h after challenge with 400 mg APAP/kg. Controls received 50% propylene glycol vehicle only. In this model of CFB hepatoprotection, liver injury was assessed by measuring plasma sorbitol dehydrogenase activity and by histopathology at 24 h after APAP challenge. Significant hepatocellular necrosis was evident in both corn oil-pretreated PPARalpha-null and wild-type mice at 24 h after APAP challenge. In agreement with previous studies, CFB-pretreated wild-type mice showed marked protection against APAP toxicity. In contrast, CFB did not provide protection against APAP hepatotoxicity in the PPARalpha-null mice. Similarly, at 4 h after APAP challenge, hepatic glutathione depletion and selective arylation of cytosolic proteins were reduced significantly in CFB-pretreated wild-type mice, but not in PPARalpha-null mice. The lack of changes in APAP binding and NPSH depletion in CFB-pretreated, PPARalpha-null mice is consistent with the presence of significant liver injury at 24 h in this treatment group. These findings demonstrate that the protection against APAP hepatotoxicity by peroxisome proliferator treatment is mediated by the activation of PPARalpha.     

Type 1 diabetic mice are protected from acetaminophen hepatotoxicity.

Streptozotocin (STZ)-induced diabetic (DB) mice challenged with single ordinarily lethal doses of acetaminophen (APAP), carbon tetrachloride (CCl4), or bromobenzene (BB) were resistant to all three hepatotoxicants. Mechanisms of protection against APAP hepatotoxicity were investigated. Plasma alanine aminotransferase, aspartate aminotransferase, and liver histopathology revealed significantly lower hepatic injury in DB mice after APAP administration. HPLC analysis of plasma and urine revealed lower plasma t1/2, increased volume of distribution (Vd), and increased plasma clearance (CLp) of APAP in the DB mice and no difference in APAP-glucuronide, a major metabolite in mice. Interestingly, covalent binding of 14C-labeled APAP to liver target proteins; arylation of APAP to 58, 56, and 44 kDa acetaminophen binding proteins (ABPs); and glutathione (GSH) depletion in the liver did not differ between nondiabetic (non-DB) and DB mice in spite of downregulated hepatic microsomal CYP2E1 and 1A2 proteins in the DB mice, known to be involved in bioactivation of APAP. Compensatory cell division measured via 3H-thymidine pulse labeling and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) indicated earlier onset of S-phase in the DB mice after exposure to APAP. Antimitotic intervention of liver cell division by colchicine (CLC) after administration of APAP led to significantly higher mortality in the DB mice suggesting a pivotal role of liver cell division and tissue repair in the protection afforded by diabetes. In conclusion, the resistance of DB mice against hepatotoxic and lethal effects of APAP appears to be mediated by a combination of enhanced APAP clearance and robust compensatory tissue repair.     

THE PPAR ACTIVATOR DOCOSAHEXAENOIC ACID PREVENTS ACETAMINOPHEN HEPATOTOXICITY IN MALE CD-1 MICE

Acetaminophen (APAP)-induced hepatocellular necrosis can be prevented by treatment with peroxisome proliferators. This protection is associated with lowered protein arylation and glutathione depletion in mice. Peroxisome proliferators have been shown to activate nuclear receptors. These receptors, termed peroxisome proliferator activated receptors (PPARs), can also be activated by free fatty acids. This study was designed to determine if treatment with the PPAR activator docosahexaenoic acid (DHA) would also lower APAP toxicity. Male CD-1 mice received 250 mg DHA/kg or 500 mg clofibrate (CFB)/kg, ip, for 5 d. Controls received corn oil vehicle, ip. After overnight fasting, mice received 800 mg APAP/kg, po. At 24 h after APAP, hepatotoxicity was evident in control mice by elevated plasma sorbitol dehydrogenase activity (SDH) and histologic evidence of hepatic degeneration and necrosis. As expected, CFB pretreatment significantly decreased this. Similarly, DHA protected against APAP-induced hepatotoxicity at 24 h after challenge. However, treatment with DHA did not increase hepatic glutathione prior to APAP, as previously shown with CFB. Interestingly, DHA did not increase palmitoyl coenzyme A (CoA) oxidase activity or other biochemical parameters associated with peroxisome proliferation after 5 d of treatment at 250 mg/kg. No significant alterations in microsomal APAP glucuronidation or cytochrome P-450-mediated bioactivation were detected either. Collectively, these results show that DHA also prevents APAP-induced hepatotoxicity at 24 h after challenge. However, the association between resistance against APAP-induced liver injury, PPAR activation, and peroxisome proliferation is not clearly understood.     

Acetaminophen metabolism does not contribute to gender difference in its hepatotoxicity in mouse.

Gender is an important factor in pharmacokinetics and pharmacodynamics. In the current study, gender difference in acetaminophen (APAP)-induced hepatotoxicity has been examined. Male and female mice were injected with a toxic dose of APAP (500 mg/kg, ip). Female mice were resistant to the hepatotoxic effects of APAP, depicted by serum alanine aminotransferase and sorbital dehydrogenase activities and histological analysis. Basal hepatic reduced glutathione (GSH) levels were lower in females than in males, suggesting that basal GSH level may not be a factor in determining the gender difference of APAP hepatotoxicity. APAP metabolism was slower in females than males, revealed by lower levels of glucuronidation and sulfation and higher amounts of free APAP in the livers of female mice. Lower basal Cyp1a2 mRNA levels and lower expression of Cyp1a2 and Cyp3a11 mRNAs after APAP dosing were also observed in females compared with males. However, there was no gender difference in N-acetyl-p-benzoquinone imine covalent binding 2 h after APAP administration, suggesting similar APAP bioactivation between genders. Moreover, liver Gst pi mRNA levels were significantly lower in females than males. This finding is consistent with a previous report, which showed that Gst pi knockout mice are protected from APAP-induced liver toxicity. In conclusion, gender difference of APAP-induced hepatotoxicity is not likely due to APAP metabolism. Perhaps, it is in part due to gender-dependent Gst pi expression. However, the mechanism underlying the association between reduction in Gst pi expression and hepatoprotective effect against APAP toxicity remains to be further explored.     

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.     

Increased hepatotoxicity of acetaminophen in Hsp70i knockout mice

The effect of the inducible forms of 70 kDa heat shock protein (Hsp70i) on acetaminophen (APAP) hepatotoxicity was assessed in an Hsp70i knockout mouse model. Absence of the Hsp70i protein in liver was verified by monitoring Hsp levels in knockout and control mice after heat stress (41.5 degrees C water bath immersion for 30 min). Hsp70i knockout mice were more susceptible to APAP-induced hepatotoxicity than controls, as indicated by elevated serum alanine aminotransferase activities 24 and 48 h after the APAP dose. Increased APAP hepatotoxicity in knockout mice was verified by morphological evaluation of liver sections. The difference in toxic response to APAP between knockout and control strain mice could not be attributed to differences in APAP bioactivation, assessed by measurement of CYP2E1 and glutathione S-transferase activities, hepatic nonprotein sulfhydryl content, or covalent binding of reactive APAP metabolites to proteins. Pretreatment with transient hyperthermia to produce a general upregulation of Hsps resulted in decreased APAP hepatotoxicity in both the knockout and control strains. Among thermally-pretreated mice, hepatotoxicity of APAP was greater in the knockouts compared with the control strain. These observations suggest that increased Hsp70i expression in response to APAP acts to limit the extent of tissue injury. Results further suggest that other factors related to heat stress can also contribute to protection against APAP toxicity.     

The PPAR activator docosahexaenoic acid prevents acetaminophen hepatotoxicity in male CD-1 mice.

Acetaminophen (APAP)-induced hepatocellular necrosis can be prevented by treatment with peroxisome proliferators. This protection is associated with lowered protein arylation and glutathione depletion in mice. Peroxisome proliferators have been shown to activate nuclear receptors. These receptors, termed peroxisome proliferator activated receptors (PPARs), can also be activated by free fatty acids. This study was designed to determine if treatment with the PPAR activator docosahexaenoic acid (DHA) would also lower APAP toxicity. Male CD-1 mice received 250 mg DHA/kg or 500 mg clofibrate (CFB)/kg, i.p., for 5 d. Controls received corn oil vehicle, i.p. After overnight fasting, mice received 800 mg APAP/kg, p.o. At 24 h after APAP, hepatotoxicity was evident in control mice by elevated plasma sorbitol dehydrogenase activity (SDH) and histologic evidence of hepatic degeneration and necrosis. As expected, CFB pretreatment significantly decreased this. Similarly, DHA protected against APAP-induced hepatotoxicity at 24 h after challenge. However, treatment with DHA did not increase hepatic glutathione prior to APAP, as previously shown with CFB. Interestingly, DHA did not increase palmitoyl coenzyme A (CoA) oxidase activity or other biochemical parameters associated with peroxisome proliferation after 5 d of treatment at 250 mg/kg. No significant alterations in microsomal APAP glucuronidation or cytochrome P-450-mediated bioactivation were detected either. Collectively, these results show that DHA also prevents APAP-induced hepatotoxicity at 24 h after challenge. However, the association between resistance against APAP-induced liver injury, PPAR activation, and peroxisome proliferation is not clearly understood.     

Secretory phospholipase A₂-mediated progression of hepatotoxicity initiated by acetaminophen is exacerbated in the absence of hepatic COX-2

We have previously reported that among the other death proteins, hepatic secretory phospholipase A₂ (sPLA₂) is a leading mediator of progression of liver injury initiated by CCl₄ in rats. The aim of our present study was to test the hypothesis that increased hepatic sPLA₂ released after acetaminophen (APAP) challenge mediates progression of liver injury in wild type (WT) and COX-2 knockout (KO) mice. COX-2 WT and KO mice were administered a normally non lethal dose (400 mg/kg) of acetaminophen. The COX-2 KO mice suffered 60% mortality compared to 100% survival of the WT mice, suggesting higher susceptibility of COX-2 KO mice to sPLA₂-mediated progression of acetaminophen hepatotoxicity. Liver injury was significantly higher at later time points in the KO mice compared to the WT mice indicating that the abatement of progression of injury requires the presence of COX-2. This difference in hepatotoxicity was not due to increased bioactivation of acetaminophen as indicated by unchanged cyp2E1 protein and covalently bound ¹⁴C-APAP in the livers of KO mice. Hepatic sPLA₂ activity and plasma TNF-α were significantly higher after APAP administration in the KO mice. This was accompanied by a corresponding fall in hepatic PGE₂ and lower compensatory liver regeneration and repair (³H-thymidine incorporation) in the KO mice. These results suggest that hindered compensatory tissue repair and poor resolution of inflammation for want of beneficial prostaglandins render the liver very vulnerable to sPLA₂-mediated progression of liver injury. These findings are consistent with the destructive role of sPLA₂ in the progression and expansion of tissue injury as a result of continued hydrolytic breakdown of plasma membrane phospholipids of perinecrotic hepatocytes unless mitigated by sufficient co-induction of COX-2.     

Differential gene expression in mouse liver associated with the hepatoprotective effect of clofibrate

Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPARalpha) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPARalpha-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPARalpha-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.     

Acute acetaminophen toxicity in transgenic mice with elevated hepatic glutathione

Previous studies demonstrated that elevation of hepatic glutathione (GSH) concentrations protect against acetaminophen (APAP) hepatotoxicity in mice. Employing transgenic mice overexpressing glutathione synthetase, this study was conducted to determine if sustained elevation of hepatic GSH concentrations could ameliorate or prevent APAP toxicity. International Cancer Research transgenic mouse males and matched (ie same strain, sex, and age) control nontransgenic mice were pretreated ip with GSH synthetase substrate gamma-glutamylcysteinyl ethyl ester (gamma-GCE) or with saline. After a 16-h fast, mice received a single dose of 500 mg APAP/kg bw in saline ip and were sacrificed 4 h later. Other mice similarly pretreated were killed without APAP challenge. The elevated GSH concentrations in transgenic mice livers did not lessen APAP hepatotoxicity. Instead higher degrees of hepatotoxicity and nephrotoxicity were observed in transgenic mice than in controls as indicated by higher serum alanine aminotransferase activity and more severe histopathological lesions in transgenic mice livers and kidneys. Pretreatment with gamma-GCE did not affect either initial or post-APAP treatment tissue GSH concentrations or observed degrees of toxicity. Detection of a higher level of serum APAP in transgenic mice and the histopathological lesions found in transgenic mice kidneys together with no observable nephrotoxicity in control mice indicated early kidney damage in transgenic mice. Our findings suggest that high levels of GSH-APAP conjugates resulting from increased GSH concentrations in the livers of transgenic mice caused rapid kidney damage. Compromised excretory ability may have caused retention of APAP, which, in effect, elicited higher hepatotoxicity than that observed in nontransgenic mice.     

Sex- and Age-Dependent Acetaminophen Hepato- and Nephrotoxicity in Sprague-Dawley Rats: Role of Tissue Accumulation, Nonprotein Sulfhydryl Depletion, and Covalent Binding

Acetaminophen (APAP) produces sex-dependent nephrotoxicity andhepatotoxicity in young adult Sprague-Dawley (SD) rats and age-dependenttoxicity in male rats. There is no information re garding thesusceptibility of aging female SD rats to APAP toxicity. Therefore,the present studies were designed to determine if sex-dependentdifferences in APAP toxicity persist in aging rats and to elucidatefactors contributing to sex- and age-dependent APAP hepatotoxicityand nephrotoxicity. Young adult (3 months old) and aging (18months old) male and female rats were killed from 2 through24 hr after receiving APAP (0–1250 mg/kg, ip) containing[ring-¹⁴C]APAP. Trunk blood was collected for determinationof blood urea nitrogen (BUN) concentration, serum alanine aminotransferase(ALT) activity, and plasma APAP concentration; urine was collectedfor determination of glucose and protein excretion; and liverand kidneys were removed for determination of tissue glutathione(GSH) concentration, APAP concentration, and covalent binding.APAP at 1250 mg/kg induced nephrotoxicity (as indicated by elevationsin BUN concentration) in 3-month-old females but not males,whereas APAP induced hepatotoxicity (as indicated by elevationsin serum ALT activity) in 3-month-old males but not females.Sex differences in APAP toxicity were no longer apparent in18-month-old rats. APAP at 750 mg/kg ip produced liver and kidneydamage in 18-month-old but not 3-month-old male and female rats.No consistent sex- or age-dependent differences in serum, hepatic,and renal APAP concentrations were observed that would accountfor differences in APAI toxicity. No sex- or age-dependent differencesin tissue GSH depletion or covalent binding of radiolabel fromAPAP in livers or kidneys were observed following APAP administration.Utilizing an affinity-purified polyclonal antibody raised againstAPAP, arylated proteins with electrophoretic mobility similarto those observed in mice were prominent in rat livers followingAPAP administration to 3- and 18-month-old rats of both sexes.In contrast, no arylated proteins were detected in any rat kidneysfollowing APAP administration. Absence of immunochemically detectableproteins in rat kidney following APAP administration is in directcontrast to observations in mice and supports the hypothesisthat mechanisms of APAP hepatotoxicity and nephrotoxicity inrats and mice are distinctly different. In conclusion, sex differencesin APAP toxicity are observed only in young adult (3-month-old)rats and sex differences are organ-specific with males moresusceptible to hepatotoxicity and females more susceptible tonephrotoxicity. Aging rats are more susceptible to APAP-induceddamage to both the liver and the kidney than are 3-month-oldrats but sex differences are no longer apparent in 18-month-oldrats. The mechanisms contributing to sex- and age-dependentdifferences in APAP toxicity cannot be attributed to differencesin tissue APAP concentrations, GSH depletion, or covalent binding.     

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.     

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.     

Hepatoprotective role of endogenous interleukin-13 in a murine model of acetaminophen-induced liver disease

Recent evidence suggests that a deficiency in one or more hepatoprotective regulatory mechanisms may contribute to determining susceptibility in drug-induced liver disease. In the present study, we investigated the role of interleukin (IL)-13 in acetaminophen (APAP)-induced liver disease (AILD). Following APAP (200 mg/kg) administration to male C57BL/6 wild-type (WT) mice, hepatotoxicity developed up to 24 h post-APAP, with a concomitant increase in serum IL-13 concentration. Pretreatment of these mice with an IL-13-neutralizing antibody exacerbated liver injury, as did APAP administration to IL-13 knockout (KO) mice in comparison to WT mice. No difference was observed in either overall APAP-protein adduct formation or liver glutathione levels between KO and WT mice following APAP administration, suggesting that the increased susceptibility of IL-13 KO mice to AILD was not due to enhanced APAP bioactivation but rather injurious downstream events. In this regard, multiplex antibody arrays were used to identify potential IL-13-regulated biomarkers, including various cytokines and chemokines, as well as nitric oxide (NO), associated with AILD that were present at higher concentrations in the sera of APAP-treated IL-13 KO mice than in WT mice. Subsequent inhibition studies determined interferon-gamma, NO, neutrophils, natural killer cells, and natural killer cells with T-cell receptors had pathologic roles in AILD in IL-13 KO mice. Taken together, these results suggest that IL-13 is a critical hepatoprotective factor modulating the susceptibility to AILD and may provide hepatoprotection, in part, by down-regulating protoxicant factors and cells associated with the innate immune system.     

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.     

Prevention of acetaminophen-induced hepatotoxicity by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) has previously been shown to protect against acetaminophen (APAP)-induced hepatotoxicity, but the mechanism of this effect was not clear. Treatment of mice with 1 mg/kg DMSO 4 h before 250 mg/kg APAP resulted in significantly less hepatotoxicity than with APAP alone, as measured by serum glutamic pyruvic transaminase (SGPT) content 24 h after APAP. Protection was also evident when 1 ml/kg DMSO was given 4, but not 8 h after 250 mg/kg APAP. The APAP-induced depletion of liver glutathione was prevented in mice pretreated with DMSO, although DMSO alone had no effect on liver glutathione levels. The hepatic concentration of cytochrome P-450 (P450) 4 h after treatment of mice with 1 ml/kg DMSO, was significantly decreased compared to saline-treated animals. However, while this DMSO pretreatment significantly decreased the activity of cytochrome P-450-linked aminopyrine-N-demethylase, it increased the activity of aniline hydroxylase. Covalent binding of [14C]APAP to hepatic protein in vivo was significantly decreased in mice pretreated with DMSO. Covalent binding of [14C]APAP to hepatic microsomal protein in vitro was not significantly altered after in vivo treatment with DMSO. However, the presence of DMSO in the in vitro incubation mixture significantly decreased covalent binding of [14C]APAP in a dose-dependent manner compared to microsomal fractions from untreated, saline-treated or DMSO pretreated animals. These data suggest that the DMSO-induced alterations in cytochrome P-450 content and activity may not be the cause of the observed protective action of this chemical. The ability to competitively inhibit APAP bioactivation or to directly scavenge free radicals produced during APAP metabolism, including the activated species which covalently binds to protein, may account for the hepatoprotection afforded by DMSO.     

Protection against Acetaminophen Hepatotoxicity by a Single Dose of Clofibrate: Effects on Selective Protein Arylation and Glutathione Depletion

Previous reports demonstrated that repeated administration ofperoxisome proliferators protects against acetaminophen (APAP)hepatotoxicity in mice. This protection was associated witha decrease in APAP's selective protein arylation and glutathionedepletion. This study was conducted to determine if a singledose of clofibrate (CFB), rather than repeated doses, wouldsimilarly prevent APAP toxicity. CD-1 male mice received a singledose of 500 mg CFB/kg and controls were given corn oil 24 hrprior to APAP challenge. After an 18-hr fast, mice were challengedwith 800 mg APAP/kg (in 50% propylene glycol) and killed at4 or 12 hr. Other mice similarly pretreated were killed withoutAPAP challenge. The results showed that pretreatment with asingle CFB dose significantly decreased APAP-induced hepatotoxicity.At 12 hr after APAP plasma sorbitol dehydrogenase activity andthe severity of hepatocellular necrosis were decreased in CFBpretreated mice. Surprisingly, no differences in hepatic nonproteinsulfhydryl (NPSH) depletion or selective arylation of targetproteins in cytosol were observed at 4 hr after APAP challenge.Neither did a single dose of CFB significantly alter hepaticNPSH content prior to APAP challenge. These results indicatethat protection against APAP hepatotoxicity by CFB does notrequire repeated administration, and the absence of significantalterations in APAP's selective protein arylation or glutathionedepletion suggests that the protection against APAP hepatotoxicityafter a single treatment with CFB may differ mechanisticallyfrom the protection observed after repeted CFB dosing.     

Role of galectin-3 in acetaminophen-induced hepatotoxicity and inflammatory mediator production

Galectin-3 (Gal-3) is a β-galactoside-binding lectin implicated in the regulation of macrophage activation and inflammatory mediator production. In the present studies, we analyzed the role of Gal-3 in liver inflammation and injury induced by acetaminophen (APAP). Treatment of wild-type (WT) mice with APAP (300 mg/kg, ip) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was associated with increased hepatic expression of Gal-3 messenger RNA and protein. Immunohistochemical analysis showed that Gal-3 was predominantly expressed by mononuclear cells infiltrating into necrotic areas. APAP-induced hepatotoxicity was reduced in Gal-3-deficient mice. This was most pronounced at 48-72 h post-APAP and correlated with decreases in APAP-induced expression of 24p3, a marker of inflammation and oxidative stress. These effects were not due to alterations in APAP metabolism or hepatic glutathione levels. The proinflammatory proteins, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, and MIP-3α, as well as the Gal-3 receptor (CD98), were upregulated in livers of WT mice after APAP intoxication. Loss of Gal-3 resulted in a significant reduction in expression of iNOS, MMP-9, MIP-3α, and CD98, with no effects on IL-1β. Whereas APAP-induced increases in MIP-2 were augmented at 6 h in Gal-3(-/-) mice when compared with WT mice, at 48 and 72 h, they were suppressed. Tumor necrosis factor receptor-1 (TNFR1) was also upregulated after APAP, a response dependent on Gal-3. Moreover, exaggerated APAP hepatotoxicity in mice lacking TNFR1 was associated with increased Gal-3 expression. These data demonstrate that Gal-3 is important in promoting inflammation and injury in the liver following APAP intoxication.     

Prevention of acetaminophen (APAP)-induced hepatotoxicity by leflunomide via inhibition of APAP biotransformation to N-acetyl-p-benzoquinone imine

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl-p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH(2)-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77-1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.