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.     

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.     

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.     

Protective effects of salidroside against acetaminophen-induced toxicity in mice

The protective effect of salidroside (SDS) isolated from Rhodiola sachalinensis A. BOR. (Crassulaceae), was investigated in acetaminophen (APAP)-induced hepatic toxicity mouse model in comparison to N-acetylcysteine (NAC). Drug-induced hepatotoxicity was induced by an intraperitoneal (i.p.) injection of 300 mg/kg (sub-lethal dose) of APAP. SDS was given orally to mice at a dose of 50 or 100 mg/kg 2 h before the APAP administration in parallel with NAC. Mice were sacrificed 12 h after the APAP injection to determine aspartate aminotransferase (AST), alanine aminotransferase (ALT), and tumor necrosis factor-alpha (TNF-alpha) levels in serum and glutathione (GSH) depletion, malondialdehyde (MDA) accumulation, and caspase-3 expression in liver tissues. SDS significantly protected APAP-induced hepatotoxicity for SDS improved mouse survival rates better than NAC against a lethal dose of APAP and significantly blocked not only APAP-induced increases of AST, ALT, and TNF-alpha but also APAP-induced GSH depletion and MDA accumulation. Histopathological and immunohistochemical analyses also demonstrated that SDS could reduce the appearance of necrosis regions as well as caspase-3 and hypoxia inducible factor-1alpha (HIF-1alpha) expression in liver tissue. Our results indicated that SDS protected liver tissue from the APAP-induced oxidative damage via preventing or alleviating intracellular GSH depletion and oxidation damage, which suggested that SDS would be a potential antidote against APAP-induced hepatotoxicity.     

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.     

Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2

Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.     

Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice.

The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP-protein adducts and centrilobular necrosis in the liver. N-acetylcysteine (NAC), a cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)-mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.     

Acetaminophen hepatotoxicity: Is there a role for prostaglandin synthesis?

The hepatotoxicity of acetaminophen (APAP) overdose depends on metabolic activation to a toxic reactive metabolite via hepatic mixed function oxidase. In vitro studies have indicated that APAP may also be cooxidized by prostaglandin H synthetase. The present experiments were designed to assess the possible contribution of hepatic prostaglandin synthesis to APAP toxicity. Adult fed male mice were overdosed with 400 mg APAP/kg. Liver toxicity was estimated by measurement of serum transaminases. Hypertonic xylitol or sodium chloride (2250 mOsm/l), administered intragastrically to stimulate prostaglandin synthesis, increased APAP toxicity. By contrast, the cyclooxygenase inhibiting drugs aspirin (at 25 mg/kg) and indomethacin (at 10 mg/kg) protected against APAP-induced toxicity. APAP kinetics were not affected by hypertonic xylitol or indomethacin, nor were hepatic glutathione levels in overdosed mice. Imidazole, a nonspecific thromboxane synthetase inhibitor, also protected overdosed mice. This drug prolonged hexobarbital sleeping time and prevented the depletion of hepatic glutathione that followed APAP intoxication. Thus, the data support the conclusion that APAP-induced hepatotoxicity may be modulated not only by inhibition of cytochrome P450 mediated oxidation, but also by controlling hepatic cyclooxygenase activity.The Eugene Hecht Chair in Clinical Pharmacology     

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.     

Green tea extract can potentiate acetaminophen-induced hepatotoxicity in mice

Green tea extract (GTE) has been advocated as a hepatoprotective compound and a possible therapeutic agent for acetaminophen (APAP) overdose. This study was conducted to determine if GTE can provide protection against APAP-induced hepatotoxicity. Three different exposure scenarios were tested. The first involved administering APAP (150 mg/kg, orally) to mice followed 6 h later by GTE (500 or 1000 mg/kg). The other two involved administering GTE prior to the APAP dose. GTE (500 or 1000 mg/kg, orally) was administered 3 h prior to APAP (200 mg/kg, orally) or for three consecutive days (once-daily) followed by APAP (300 mg/kg) on the fourth day. Indices of hepatotoxicity were assessed 24 h after the APAP dose. GTE potentiated APAP-induced hepatotoxicity when administered after the APAP dose. GTE caused significant glutathione depletion and this effect likely contributed to the observed potentiation. In contrast, GTE provided protection against APAP-induced hepatotoxicity when administered prior to the APAP dose. GTE dramatically decreased APAP covalent binding to protein indicating that less reactive metabolite was available to cause hepatocellular injury. These results highlight the potential for drug-dietary supplement interactions and the importance of testing multiple exposure scenarios to adequately model different types of potential interactions.     

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.     

S-Adenosylmethionine (SAMe) attenuates acetaminophen hepatotoxicity in C57BL/6 mice

Hepatic toxicity is associated with excessive dosages of the over the counter analgesic, acetaminophen (APAP). The aim of this study was to explore protection by the nutritional agent S-adenosylmethionine (SAMe) on APAP hepatotoxicity. Male C57BL/6 mice were injected intraperitoneal (i.p.) with 500 mg/kg (15 ml/kg) APAP or water vehicle (VEH). SAMe was injected i.p. at a dose of either 1000 mg/kg (5 ml/kg) just prior or 500 mg/kg SAMe 15 min prior to administration of VEH or APAP. Comparison of groups showed that SAMe reduced APAP toxicity. Plasma alanine aminotransferase (ALT) levels were increased 2 and 4 h after APAP administration when compared to vehicle (VEH) controls. Liver weight was increased relative to the VEH group within 4 h after APAP treatment. Histological examination by light microscopy confirmed small changes in morphology within 2 h after APAP injection and marked centrilobular necrosis within 4 h in the APAP group. In contrast, when APAP was administered to SAMe pretreated mice, ALT and liver weights were comparable to the VEH and SAMe groups. Histological examination also showed that SAMe produced a marked protection in APAP mediated centrilobular necrosis at 4 h after APAP injection. APAP administration depressed hepatic glutathione levels when monitored at 2 and 4 h. Lipid peroxidation was induced above VEH values 2 and 4 h after APAP injection. Consistent with the SAMe protection of APAP hepatic toxicity, the expected depletion of hepatic glutathione (GSH) levels by APAP was prevented by SAMe pretreatment. SAMe pretreatment also prevented the induction of lipid peroxidation at 2 and 4 h post-APAP administration. In conclusion, SAMe provides protection from APAP hepatic toxicity at 2 and 4 h post-APAP injection. SAMe pretreatment prevented APAP associated depletion in hepatic glutathione and induction of lipid peroxidation as part of its mechanism of protection.     

The potential protective role of alpha-lipoic acid against acetaminophen-induced hepatic and renal damage

The potential protective role of alpha-lipoic acid (alpha-LA) in acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity was investigated in rats. Pretreatment of rats with alpha-LA (100mg/kg) orally protected markedly against hepatotoxicity and nephrotoxicity induced by an acute oral toxic dose of APAP (2.5 g/kg) as assessed by biochemical measurements and by histopathological examination. None of alpha-LA pretreated animals died by the acute toxic dose of APAP. Concomitantly, APAP-induced profound elevation of nitric oxide (NO) production and oxidative stress, as evidenced by increasing of lipid peroxidation level, reducing of glutathione peroxidase (GSH-Px) activity and depleting of intracellular reduced glutathione (GSH) level in liver and kidney, were suppressed by pretreatment with alpha-LA. Similarly, daily treatment of rats with a smaller dose of alpha-LA (25mg/kg) concurrently with a smaller toxic dose of APAP (750 mg/kg) for 1 week protected against APAP-induced hepatotoxicity and nephrotoxicity. This treatment also completely prevented APAP-induced mortality and markedly inhibited APAP-induced NO overproduction and oxidative stress in hepatic and renal tissues. These results provide evidence that inhibition of NO overproduction and maintenance of intracellular antioxidant status may play a pivotal role in the protective effects of alpha-LA against APAP-induced hepatic and renal damage.     

Resistance to acetaminophen-induced hepatotoxicity in glutathione S-transferase Mu 1-null mice

We investigated the role of glutathione S-transferases Mu 1 (GSTM1) in acetaminophen (APAP)-induced hepatotoxicity using Gstm1-null mice. A single oral administration of APAP resulted in a marked increase in plasma alanine aminotransferase accompanied by hepatocyte necrosis 24 hr after administration in wild-type mice, but its magnitude was unexpectedly attenuated in Gstm1-null mice. Therefore, it is suggested that Gstm1-null mice are resistant to APAP-induced hepatotoxicity. To examine the mechanism of this resistance in Gstm1-null mice, we measured phosphorylation of c-jun N-terminal kinase (JNK), which mediates the signal of APAP-induced hepatocyte necrosis, by Western blot analysis 2 and 6 hr after APAP administration. A marked increase in phosphorylated JNK was observed in wild-type mice, but the increase was markedly suppressed in Gstm1-null mice. Therefore, it is suggested that suppressed phosphorylation of JNK may be a main mechanism of the resistance to APAP-induced hepatotoxicity in Gstm1-null mice, although other possibilities of the mechanism cannot be eliminated. Additionally, phosphorylation of glycogen synthase kinase-3β and mitogen-activated protein kinase kinase 4, which are upstream kinases of JNK in APAP-induced hepatotoxicity, were also suppressed in Gstm1-null mice. A decrease in liver total glutathione 2 hr after APAP administration, which is an indicator for exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of APAP, were similar in wild-type and Gstm1-null mice. In conclusion, Gstm1-null mice are considered to be resistant to APAP-induced hepatotoxicity perhaps by the suppression of JNK phosphorylation. This study indicates the novel role of GSTM1 as a factor mediating the cellular signal for APAP-induced hepatotoxicity.     

Therapeutic effect of liposomal-N-acetylcysteine against acetaminophen-induced hepatotoxicity

Abstract

Background: Acetaminophen (APAP) is an antipyretic analgesic drug that when taken in overdose causes depletion of glutathione (GSH) and hepatotoxicity. N-acetylcysteine (NAC) is the antidote of choice for the treatment of APAP toxicity; however, due to its short-half-life repeated dosing of NAC is required.

Purpose: To determine whether a NAC-loaded liposomal formulation (Lipo-NAC) is more effective than the conventional NAC in protecting against acute APAP-induced hepatotoxicity.

Methods: Male Sprague–Dawley rats were challenged with an intragastric dose of APAP (850?mg/kg b.wt.); 4?h later, animals were administered saline, NAC, Lipo-NAC or empty liposomes and sacrificed 24?h post-APAP treatment.

Results: APAP administration resulted in hepatic injury as evidenced by increases in plasma bilirubin, alanine (AST) and aspartate (ALT) aminotransferase levels and tissue levels of lipid peroxidation and myeloperoxidase as well as decreases in hepatic levels of reduced GSH, GSH peroxidase and GSH reductase. Treatment of animals with Lipo-NAC was significantly more effective than free NAC in reducing APAP-induced hepatotoxicity. Histological evaluation showed that APAP caused periacinar hepatocellular apoptosis and/or necrosis of hepatocytes around the terminal hepatic venules which was reduced by NAC treatment, the degree of reduction being greater for Lipo-NAC.

Conclusion: These data suggest that administration of Lipo-NAC ameliorated the APAP-induced hepatotoxicity.     

Role of nitric oxide and reduced glutathione in the protective effects of aminoguanidine, gadolinium chloride and oleanolic acid against acetaminophen-induced hepatic and renal damage

The potential protective role of aminoguanidine (AG), gadolinium chloride (GdCl(3)) and oleanolic acid (OA) in acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity was investigated in rats. Pretreatment of rats with AG (50mg/kg) orally, GdCl(3) (10mg/kg) intramuscularly or OA (25mg/kg) intramuscularly protected markedly against hepatotoxicity and nephrotoxicity induced by an acute oral toxic dose of APAP (2.5g/kg) as assessed by biochemical measurements and by histopathological examination. None of AG-, GdCl(3)- or OA-pretreated animals died by the acute toxic dose of APAP. Concomitantly, pretreatment of rats with these agents suppressed the profound elevation of nitric oxide (NO) production and obvious reduction of intracellular reduced glutathione (GSH) levels in liver and kidney induced by the acute toxic dose of APAP. Similarly, daily treatment of rats with a smaller dose of AG (10mg/kg), GdCl(3) (3mg/kg) or OA (5mg/kg) concurrently with a smaller toxic dose of APAP (750mg/kg) for 1 week protected against APAP-induced hepatotoxicity and nephrotoxicity. This treatment also completely prevented APAP-induced mortality and markedly inhibited APAP-induced NO overproduction as well as hepatic and renal intracellular GSH levels reduction. These results provide evidence that inhibition of NO overproduction and consequently maintenance of intracellular GSH levels may play a pivotal role in the protective effects of AG, GdCl(3) and OA against APAP-induced hepatic and renal damages.     

Prevention of acetaminophen-induced liver toxicity by 2(R,S)-n-propylthiazolidine-4(R)-carboxylic acid in mice

The cysteine (Cys) precursor 2(R,S)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA) was shown previously to maintain near normal levels of hepatic GSH and GSSG at 24 hr and to protect against hepatic necrosis and mortality at 48 hr after toxic doses of acetaminophen (APAP) in mice. Studies were performed in C57BL/6 mice to determine: (a) the time course of APAP-induced hepatic sulfhydryl depletion, and (b) the effectiveness of PTCA in preventing APAP-induced decreases in sulfhydryl concentrations at the time of maximal depletion. APAP (400-800 mg/kg in 50% propylene glycol; 2.65-5.29 mmol/kg) and PTCA (1-5 mmol/kg 30 min after APAP) were administered i.p. Hepatic GSH, GSSG, and Cys concentrations were determined by HPLC. Hepatocellular damage was assessed by elevations in serum glutamate-pyruvate transaminase (SGPT) activity and histopathologic examination. APAP and PTCA produced dose-dependent effects. At 4 hr after the highest dose of APAP, hepatic GSH and Cys concentrations were reduced to 5 and 14%, respectively, of values in vehicle-treated controls, and the GSSG concentration was below the sensitivity of the analytical method. At 24 hr, recovery of hepatic sulfhydryls was incomplete, and there was hepatic necrosis with an approximately 100-fold increase in SGPT activity. At the highest dose of PTCA, the concentrations of GSH, Cys, and GSSG at 4 hr after APAP (800 mg/kg) were 66, 116, and 111%, respectively, of vehicle controls. PTCA in doses of 1.75 to 5 mmol/kg attenuated the APAP-induced increases in SGPT activity. It was concluded that the protective effect of PTCA is most likely related to prevention of hepatic sulfhydryl depletion.     

A nutrient mixture prevents acetaminophen hepatic and renal toxicity in ICR mice

Acetaminophen (APAP) overdose is often fatal, leading to fulminant hepatic and renal tubular necrosis in humans and animals. We studied the effect of a nutrient mixture (NM) containing, among other nutrients, lysine, proline, ascorbic acid, N-acetyl cysteine, and green tea extract, which has previously been demonstrated to exhibit a broad spectrum of therapeutic properties on APAP-induced hepatic and renal damage in ICR (Imprinting Control Region) mice. Seven-week-old male ICR mice were divided into four groups (A-D) of five animals each. Groups A and C mice were fed a regular diet for 2 weeks, while groups B and D mice were supplemented with 0.5% NM (w/w) during that period. Groups A and B received saline i.p., while groups C and D received APAP (600 mg/kg) i.p. All animals were killed 24 h after APAP administration, serum was collected to assess the liver and kidney functions, and the livers and kidneys were excised for histology. Mean serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, BUN (Blood Urea Nitrogen), creatinine, and BUN/creatinine ratios were comparable in groups A and B, increased markedly in group C and significantly lower in group D compared with group C. APAP caused significant centrilobular necrosis and glomerular damage in unsupplemented animals, while NM prevented these alterations. The results indicate that NM has potential to protect against APAP-induced liver and kidney damage.     

Inhibition of matrix metalloproteinases minimizes hepatic microvascular injury in response to acetaminophen in mice.

The acetaminophen (APAP)-induced hepatic centrilobular necrosis is preceded by hepatic microcirculatory dysfunction including the infiltration of erythrocytes into the space of Disse. The purpose of this study was to examine the involvement of matrix metalloproteinases (MMPs) in the hepatic microvascular injury elicied by APAP. Male C57Bl/6 mice were pretreated with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor (5 mg/kg, ip) 30 min before oral gavage with 600 mg/kg of APAP. The hepatic microvasculature in anesthetized mice was observed using established in vivo microscopic methods 2 and 6 h after APAP. The levels of mRNAs and activities of MMP-2 and MMP-9 in the liver were increased from 1 h through 6 h after APAP gavage. APAP increased alanine transferase (ALT) levels (41.1-fold) and resulted in centrilobular hemorrhagic necrosis at 6 h. Pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid attenuated ALT values by 71% as well as the necrosis. APAP decreased the numbers of perfused sinusoids in centrilobular regions by 30% and increased the area occupied by infiltrated erythrocytes into Disse space. 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid restored the sinusoidal perfusion to 90% of control levels and minimized extrasinusoidal area occupied by erythrocytes. The present study showed that increased MMPs during APAP intoxication are associated with hepatocellular damage and with hepatic microcirculatory dysfunction including impaired sinusoidal perfusion and infiltration of erythrocytes in Disse space. 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid attenuated APAP-induced parenchymal and microvascular injury. These results suggest that MMPs participate in APAP hepatotoxicity mediated by sinusoidal endothelial cell injury, which results in impairment of microcirculation.