In vivo and in vitro hepatotoxicity and metabolism of acetaminophen in Syrian hamsters

The purpose of this investigation was to correlate the in vitro and in vivo toxicity of the hepatotoxicant, acetaminophen. Hamsters were pretreated with either phenobarbital (70 mg/kg) or 3-methylcholanthrene (20 mg/kg) or an appropriate vehicle for 3 days. In non-pretreated hamsters, single doses of acetaminophen (200-400 mg/kg i.p.) caused elevations in serum alanine aminotransferase and sorbitol dehydrogenase activities in a dose-related manner. 3-Methylcholanthrene significantly potentiated, while phenobarbital significantly reduced acetaminophen-induced elevations in serum liver enzyme activities. Both phenobarbital and 3-methylcholanthrene significantly reduced acetaminophen plasma T1/2 while only 3-methylcholanthrene increased APAP clearance. Phenobarbital pretreatment increased the urinary excretion of APAP-glucuronide. Exposure of isolated hepatocytes to acetaminophen (0.01-2.0 mM) resulted in concentration-related decreases in hepatocyte viability. Cells from 3-methylcholanthrene-pretreated hamsters were more markedly susceptible to acetaminophen toxicity than cells isolated from non-induced animals. Hepatocytes isolated from phenobarbitol pretreated animals were slightly but significantly more susceptible to acetaminophen toxicity than cells from control animals. Hepatocytes isolated from 3-methylcholanthrene pretreated animals had increased formation of an acetaminophen-glutathione conjugate compared to control. Pre-treatment with either phenobarbital or 3-methylcholanthrene enhanced glucuronidation of acetaminophen in vitro. These data demonstrate a lack of correlation between in vivo hepatotoxicity and in vitro cytotoxicity in that phenobarbital pre-treatment protected hamsters from acetaminophen-induced liver toxicity, but failed to protect hepatocytes exposed to acetaminophen in vitro.     

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.     

Potential role of caveolin-1 in acetaminophen-induced hepatotoxicity

Caveolin-1 (Cav-1) is a membrane scaffolding protein, which functions to regulate intracellular compartmentalization of various signaling molecules. In the present studies, transgenic mice with a targeted disruption of the Cav-1 gene (Cav-1^−/−) were used to assess the role of Cav-1 in acetaminophen-induced hepatotoxicity. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was correlated with decreased expression of Cav-1 in the liver. Acetaminophen-induced hepatotoxicity was significantly attenuated in Cav-1^−/− mice, an effect that was independent of acetaminophen metabolism. Acetaminophen administration resulted in increased hepatic expression of the oxidative stress marker, lipocalin 24p3, as well as hemeoxygenase-1, but decreased glutathione and superoxide dismutase-1; no differences were noted between the genotypes suggesting that reduced toxicity in Cav-1^−/− mice is not due to alterations in antioxidant defense. In wild-type mice, acetaminophen increased mRNA expression of the pro-inflammatory cytokines, interleukin-1β, and monocyte chemoattractant protein-1 (MCP-1), as well as cyclooxygenase-2, while 15-lipoxygenase (15-LOX), which generates anti-inflammatory lipoxins, decreased. Acetaminophen-induced changes in MCP-1 and 15-LOX expression were greater in Cav-1^−/− mice. Although expression of tumor necrosis factor-α, a potent hepatocyte mitogen, was up-regulated in the liver of Cav-1^−/− mice after acetaminophen, expression of proliferating cell nuclear antigen and survivin, markers of cellular proliferation, were delayed, which may reflect the reduced need for tissue repair. Taken together, these data demonstrate that Cav-1 plays a role in promoting inflammation and toxicity during the pathogenesis of acetaminophen-induced injury.     

Protective effect of arabic gum against acetaminophen-induced hepatotoxicity in mice

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This study was undertaken to examine the effects of arabic gum (AG), which is commonly used in processed foods, on acetaminophen-induced hepatotoxicity in mice. Mice were given arabic gum orally (100 g l(-1)) 5 days before a hepatotoxic dose of acetaminophen (500 mg kg(-1)) intraperitoneally. Arabic gum administration dramatically reduced acetaminophen-induced hepatotoxicity as evidenced by reduced serum alanine (ALT) and aspartate aminotransferase (AST) activities. Acetaminophen-induced hepatic lipid peroxidation was reduced significantly by arabic gum pretreatment. The protection offered by arabic gum does not appear to be caused by a decrease in the formation of toxic acetaminophen metabolites, which consumes glutathione, because arabic gum did not alter acetaminophen-induced hepatic glutathione depletion. Acetaminophen increased nitric oxide synthesis as measured by serum nitrate plus nitrite at 4 and 6 h after administration and arabic gum pretreatment significantly reduced their formation. In conclusion, arabic gum is effective in protecting mice against acetaminophen-induced hepatotoxicity. This protection may involve the reduction of oxidative stress.     

Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.     

Differential induction of heme oxygenase-1 in macrophages and hepatocytes during acetaminophen-induced hepatotoxicity in the rat: effects of hemin and biliverdin

Heme oxygenase-1 (HO-1), also known as heat shock protein 32, has been shown to protect against oxidant-induced tissue injury. In the present studies, we analyzed expression of this enzyme in macrophages and hepatocytes following acetaminophen administration and its potential role in hepatotoxicity. Treatment of rats with a hepatotoxic dose of acetaminophen (1 g/kg, ip) resulted in a time-dependent induction of HO-1 in the liver. This was observed within 6 h of acetaminophen administration in both hepatocytes and macrophages. Hepatocytes were found to be more sensitive than macrophages to the effects of acetaminophen on HO-1. Up regulation of HO-1 in the liver following acetaminophen administration correlated with induction of ferritin and manganese superoxide dismutase (MnSOD). To determine if HO-1 was hepatoprotective, rats were pretreated with hemin (30 micromol/kg, ip), a potent inducer of the enzyme. Following hemin treatment, we observed a time-dependent increase in HO-1 protein in the liver and in serum bilirubin levels. Pretreatment of rats with hemin was found to prevent acetaminophen-induced hepatotoxicity, as measured histologically and biochemically by decreased serum transaminase levels. This was correlated with more rapid increases in expression of hepatic ferritin and MnSOD. Heme metabolism via HO-1 generates biliverdin, which is rapidly converted to bilirubin by biliverdin reductase. Pretreatment of rats with biliverdin (40 micromol/kg, ip) was also found to block acetaminophen-induced injury. These data suggest that HO-1 is an important component of antioxidant defense during acetaminophen-induced hepatotoxicity.     

The Concentration and Temporal Relationships of Acetaminophen-Induced Changes in Intracellular and Extracellular Total Glutathione in Freshly Isolated Hepatocytes from Untreated and 3-Methylcholanthrene Pretreated Sprague-Dawley and Fischer Rats*

Abstract: Fischer rats are more sensitive to acetaminophen-induced hepatotoxicity than Sprague-Dawley rats, however, the mechanisms for this enhanced sensitivity remain unclear. The susceptability to hepatotoxicity is determined largely by the balance between acetaminophen toxification and detoxification. Since glutathione plays a critical role in the detoxification process, it would be of interest to compare the effects of acetaminophen on hepatic glutathione homeostasis in the Sprague-Dawley and Fischer rat, and relate these effects to cytotoxicity. To this end, we measured the sequential changes of intracellular and extracellular total glutathione in freshly isolated hepatocytes from untreated and 3-methylchol-anthrene pretreated Fischer and Sprague-Dawley rats, both in the absence (basal) and presence of acetaminophen. In the basal state, the intracellular total glutathione content was significantly (P < 0.01) increased in hepatocytes from untreated Fischer rats. Nevertheless, the sequential release of total glutathione into the medium and the sequential depletion of intracellular total glutathione were quantitatively similar in hepatocytes from untreated Fischer and Sprague-Dawley rats. Following exposure to acetaminophen, there was a striking dose and time associated depletion of intracellular total glutathione in untreated hepatocytes from both rat strains, and quantitatively the depletion was similar in untreated hepatocytes from both rat strains. This degree of depletion of intracellular total glutathione was not associated with acetaminophen-induced cytotoxicity in Sprague-Dawley hepatocytes, whereas significant (P < 0.05) cytotoxicity was demonstrated in Fischer hepatocytes. In hepatocytes from 3-methylcholanthrene pretreated rats, there was an even more marked dose and time associated depletion of intracellular total glutathione as compared with untreated hepatocytes from both rat strains. Although the magnitude of this depletion was also similar in hepatocytes from both rat strains, the acetaminophen-induced cytotoxicity was significantly (P < 0.01) more prominent in hepatocytes from the Fischer rat. Our observations establish that basal hepatocellular glutathione concentrations are higher in Fischer than Sprague-Dawley rats, however sequential changes in basal intracellular and extracellular glutathione are similar in hepatocytes of both strains. Moreover, the effect of acetaminophen on these glutathione measures was also similar in hepatocytes from both rat strains. Finally, our observations reveal a discordance between acetaminophen-induced cytotoxicity and cellular glutathione content in the two rat strains, implying that the Fischer rats'enhanced susceptibility to hepatocellular injury may be related to a defective inherent cellular defense mechanism against attack by the reactive metabolite of acetaminophen.     

Effects of butylated hydroxyanisole on acetaminophen hepatotoxicity and glucuronidation in vivo

The present study examined the effects of butylated hydroxyanisole (BHA) on acetaminophen-induced hepatotoxicity and metabolism in vivo with emphasis on possible changes in the glucuronidation pathway. Female Swiss-Webster mice received BHA in the diet (1% w/w) for 12 days (600 to 800 mg/kg/day). BHA prevented acetaminophen hepatotoxicity (600 mg/kg, ip), based on serum alanine and aspartate aminotransferase activities and histopathological examination. The rate of elimination of acetaminophen from blood was 10-fold higher in BHA-fed mice (clearance, 49 ml/min/kg) than in controls (4.4 ml/min/kg). In general, the urinary metabolite excretion patterns in control and BHA-treated mice were the same. However, the rates of acetaminophen conjugation via the sulfation, glucuronidation, and mercapturic acid pathways were enhanced with the rate of glucuronide formation, the major biotransformation pathway of acetaminophen, increased sevenfold in BHA-treated mice (0.041 min-1) compared to controls (0.006 min-1). BHA increased hepatic UDP-glucuronosyltransferase activity twofold, as well as hepatic UDP-glucuronic acid concentrations. In addition, after acetaminophen administration, UDP-glucuronic acid in BHA-treated mice was depleted to a lesser extent and returned to control values more rapidly than in untreated animals. BHA had a similar but less pronounced effect on hepatic glutathione levels. The findings indicate that the rate of acetaminophen glucuronidation is increased in vivo during BHA feeding to mice. This effect appears to play a role in the enhanced excretion of acetaminophen as well as protection against acetaminophen-induced hepatotoxicity.     

The concentration and temporal relationships of acetaminophen-induced changes in intracellular and extracellular total glutathione in freshly isolated hepatocytes from untreated and 3-methylcholanthrene pretreated Sprague-Dawley and Fischer rats.

Fischer rats are more sensitive to acetaminophen-induced hepatotoxicity than Sprague-Dawley rats, however, the mechanisms for this enhanced sensitivity remain unclear. The susceptibility to hepatotoxicity is determined largely by the balance between acetaminophen toxification and detoxification. Since glutathione plays a critical role in the detoxification process, it would be of interest to compare the effects of acetaminophen on hepatic glutathione homeostasis in the Sprague-Dawley and Fischer rat, and relate these effects to cytotoxicity. To this end, we measured the sequential changes of intracellular and extracellular total glutathione in freshly isolated hepatocytes from untreated and 3-methylcholanthrene pretreated Fischer and Sprague-Dawley rats, both in the absence (basal) and presence of acetaminophen. In the basal state, the intracellular total glutathione content was significantly (P less than 0.01) increased in hepatocytes from untreated Fischer rats. Nevertheless, the sequential release of total glutathione into the medium and the sequential depletion of intracellular total glutathione were quantitatively similar in hepatocytes from untreated Fischer and Sprague-Dawley rats. Following exposure to acetaminophen, there was a striking dose and time associated depletion of intracellular total glutathione in untreated hepatocytes from both rat strains, and quantitatively the depletion was similar in untreated hepatocytes from both rat strains. This degree of depletion of intracellular total glutathione was not associated with acetaminophen-induced cytotoxicity in Sprague-Dawley hepatocytes, whereas significant (P less than 0.05) cytotoxicity was demonstrated in Fischer hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetaminophen-induced hepatic glycogen depletion and hyperglycemia in mice

Two hours following administration of a hepatotoxic dose of acetaminophen (500 mg/kg, i.p.) to mice, liver sections stained with periodic acid Schiff reagent showed centrilobular hepatic glycogen depletion. A chemical assay revealed that following acetaminophen administration (500 mg/kg) hepatic glycogen was depleted by 65% at 1 hr and 80% at 2 hr, whereas glutathione was depleted by 65% at 0.5 hr and 80% at 1.5 hr. Maximal glycogen depletion (85% at 2.5 hr correlated with maximal hyperglycemia (267 mg/100 ml at 2.5hr). At 4.0 hr following acetaminophen administration, blood glucose levels were not significantly different from saline-treated animals; however, glycogen levels were still maximally depleted. A comparison of the dose-response curves for hepatic glycogen depletion and glutathione depletion showed that acetaminophen (50–500 mg/kg at 2.5 hr) depleted both glycogen and glutathione by similar percentages at each dose. Since acetaminophen (100 mg/kg at 2.5 hr) depleted glutathione and glycogen by approximately 30%, evidence for hepatotoxicity was examined at this dose to determine the potential importance of hepatic necrosis in glycogen depletion. Twenty-four hours following administration of acetaminophen (100 mg/kg) to mice, histological evidence of hepatic necrosis was not detected and serum glutamate pyruvate transaminase (SGPT) levels were not significantly different from saline-treated mice. The potential role of glycogen depletion in altering the acetaminophen-induced hepatotoxicity was examined subsequently. When mice were fasted overnight, hepatic glutathione and glycogen were decreased by 40 and 75%, respectively, and fasted animals showed a dramatic increase in susceptibility to acetaminophen-induced hepatotoxicity as measured by increased SGPT levels. Availability of glucose in the drinking water (5%) overnight resulted in glycogen levels similar to those in fed animals, whereas hepatic glutathione levels were not significantly different from those of fasted animals. Fasted animals and animals given glucose water overnight were equally susceptible to acetaminophen-induced hepatotoxicity, as quantitated by increases in SGPT levels 24 hr after drug administration. The potential role of a reactive metabolite in glycogen depletion was investigated by treating mice with N-acetylcysteine to increase detoxification of the reactive metabolite. N-Acetylcysteine treatment of mice prevented acetaminophen-induced glycogen depletion.     

Sensitivities of gel entrapped hepatocytes in hollow fibers to hepatotoxic drug

The aim of this study was to determine the feasibility of detecting hepatotoxicity using gel entrapped hepatocytes in simple hollow fibers. Four typical hepatotoxic drugs were tested for hepatotoxicity in gel entrapped hepatocyte as opposed to hepatocyte monolayer, a hepatocyte system extensively used for hepatotoxicity studies in vitro. Hepatotoxicity or cell damage was assessed by the methyl tetrazolium (MTT) assay, liver-specific functions and the intracellular glutathione (GSH) content. After exposure to acetaminophen, significant cell damage of gel entrapped hepatocytes was detected at 48 h while hepatocyte monolayer was not so sensitive except for albumin synthesis and this difference between two hepatocyte systems was similar on hepatotoxic response to antituberculosis drugs including rifampicin and isoniazid. At low concentrations of either rifampicin or isoniazid, time-dependent hepatotoxicity was only evidenced in gel entrapped hepatocytes after treatment and no cell damage occurred in hepatocyte monolayer at an incubation time as long as 96 h. Interestingly, hepatotoxicities of acetaminophen, isoniazid and rifampicin are all reportedly relevant to drug metabolisms of cytochrome P450. For sodium salicylate whose hepatotoxicity is unassociated to P450 activities, more significant reductions on cell viability and albumin synthesis at 5 mM than those at 1 mM apparently illustrated the concentration-dependent hepatotoxicities of gel entrapped hepatocytes as well as hepatocyte monolayer. It is highly suggested that gel entrapped hepatocyte are more sensitive in evaluation of hepatotoxicities than hepatocyte monolayer if this hepatotoxicity is related to drug metabolism. Thus, gel entrapment culture of hepatocytes with simple hollow fibers could be recommended for hepatotoxicity studies in vitro.     

Protective effect of s-allyl cysteine and s-propyl cysteine on acetaminophen-induced hepatotoxicity in mice.

In vivo protective effects of s-allyl cysteine (SAC) and s-propyl cysteine (SPC) against acetaminophen-induced hepatotoxicity in Balb/cA mice were studied. SAC and SPC at 1g/L were added into drinking water for four weeks and followed by acetaminophen treatment. Acetaminophen treatment significantly depleted glutathione content, increased oxidation stress and elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities (P < 0.05); however, the intake of SAC or SPC significantly alleviated glutathione depletion and the elevation of ALT and AST, enhanced glutathione peroxidase activity, and lowered malondialdehyde formation (P < 0.05). Plasma levels of C-reactive protein (CRP), von Willebrand factor (vWF), IL-6, IL-10 and TNF-alpha were significantly increased by acetaminophen treatment (P < 0.05); and SAC or SPC intake significantly suppressed acetaminophen-induced elevation of CRP, vWF and the three cytokines (P < 0.05). Acetaminophen treatment also significantly increased plasminogen activator inhibitor-1 (PAI-1) activity and plasma fibrinogen level, and decreased antithrombin III (AT-III) and protein C activities (P < 0.05). SAC or SPC intake alleviated AT-III and protein C reduction (P < 0.05); but did not affect PAI-1 activity and plasma fibrinogen level (P > 0.05). These data suggest that SAC and SPC are potential multiple-protective agents against acetaminophen-induced hepatotoxicity.     

Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: potential role of tumor necrosis factor-alpha and interleukin-10

Macrophage-derived inflammatory mediators have been implicated in tissue injury induced by a number of hepatotoxicants. In the present studies, we used transgenic mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOS II) to analyze the role of nitric oxide in inflammatory mediator production in the liver and in tissue injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis, which was evident within 3 h and reached a maximum at 18 h. This was correlated with NOS II expression and nitrotyrosine staining of the liver, which was most prominent after 6 h. Expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), matrix metalloproteinase-9, and connective tissue growth factor (CTGF) also increased in the liver following acetaminophen treatment of wild-type mice. NOS II knockout mice were found to be less sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This did not appear to be due to differences in acetaminophen-induced glutathione depletion or adduct formation. In NOS II knockout mice treated with acetaminophen, hepatic expression of TNF-alpha, as well as CTGF, was significantly increased compared to wild-type mice. In contrast, IL-10 expression was reduced. These data demonstrate that nitric oxide is important in hepatotoxicity induced by acetaminophen. Moreover, some of its effects may be mediated by altering production of pro- and antiinflammatory cytokines and proteins important in tissue repair.     

The protective potential of metformin against acetaminophen-induced hepatotoxicity in BALB/C mice

Context: Acetaminophen overdose is regarded to a common cause of acute liver failure. The hepatotoxicity leads to mitochondrial oxidative stress and subsequent necrotic hepatocellular death.

Objective: This study examines the protective effect of metformin on acetaminophen-induced oxidative stress, inflammation and subsequent hepatotoxicity in mice.

Materials and methods: Male BALB/c mice were orally administered to acetaminophen (250?mg/kg/d) for a 7-day period. The mice received metformin (100 and 200?mg/kg/d, p.o.) for 21 days. To evaluate acetaminophen-induced oxidative stress, liver tissue level of malodialdehyde (MDA), end product of membrane lipid peroxidation, and activities of superoxide dismutase (SOD) and glutathione (GSH) were measured. Histological analysis and measurement of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were performed. Moreover, tissue concentrations of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), along with, C-reactive protein (CRP) were assessed.

Results: Acetaminophen caused focal hepatocyte necrosis, inflammation and fatty degeneration, as well as increased tissue levels of AST, ALT, ALP and MDA, and also decreased GSH and SOD activities. Moreover, IL-6, TNF-α and CRP levels were increased following acetaminophen hepatotoxicity. Metformin (200?mg/kg/d) significantly normalized MDA, SOD and GSH levels (p?<?0.001), and exerted a hepatoprotective effect by significant decreasing ALT, AST and ALP concentrations (p?<?0.001). The tissue levels of IL-6, TNF-α and CRP were markedly decreased by 21-day treatment with metformin (200?mg/kg/d) (p?<?0.001).

Discussion: The results suggest metformin protects hepatocytes against acute acetaminophen toxicity. Metformin is indicated to diminish oxidative stress, proinflammatory cytokines, and hepatocyte necrosis.     

Acetaminophen-induced inhibition of Fas receptor-mediated liver cell apoptosis: mitochondrial dysfunction versus glutathione depletion

We reported previously that acetaminophen overdose interrupts the signaling pathway of Fas receptor-mediated apoptosis. The aim of our study was to investigate the mechanism of this effect. Male C3Heb/FeJ mice received a single dose of acetaminophen (300 mg/kg ip) and/or anti-Fas antibody Jo-2 (0.6 mg/kg iv). Some animals were treated with allopurinol (100 mg/kg po) 18 and 1 h before acetaminophen injection. After 90 min of Jo treatment, there was processing of procaspase-3 and a significant increase in liver caspase-3 activity, which is consistent with apoptotic cell death. Treatment with acetaminophen 2.5 h before Jo inhibited the increase in hepatic caspase-3 activity by preventing the processing of the proenzyme. When administered alone, acetaminophen did not induce caspase-3 activation but caused significant liver injury. Acetaminophen treatment alone caused mitochondrial cytochrome c release, depletion of the hepatic ATP content by 55%, and a 10-fold increase in mitochondrial glutathione disulfide levels. Pretreatment with allopurinol prevented the mitochondrial oxidant stress and liver injury due to acetaminophen toxicity but had no effect on Jo-mediated apoptosis. Allopurinol did not affect the initial glutathione depletion after acetaminophen. However, allopurinol restored the sensitivity of hepatocytes to Fas receptor signaling in acetaminophen-treated animals. Histochemical evaluation of DNA fragmentation with the TUNEL assay showed that acetaminophen eliminated Fas receptor-mediated apoptosis in all hepatocytes not just in the damaged cells of the centrilobular area. Our data suggest that acetaminophen-induced mitochondrial dysfunction and not the initial glutathione depletion is responsible for the interruption of Fas receptor-mediated apoptotic signaling in hepatocytes.     

Evidence that genetic deletion of the TNF receptor p60 or p80 inhibits Fas mediated apoptosis in macrophages

Almost 19 members of the tumor necrosis factor (TNF) superfamily have been identified that interact with 29 different receptors. Whether these receptors communicate with each other is not understood. Recently, we have shown that receptor activator of NF-kappaB ligand signaling is modulated by genetic deletion of the TNF receptor. In the current report, we investigated the possibility of a cross-talk between Fas and TNF-alpha signaling pathway in macrophage cell lines derived from wild-type (WT) mice and from mice with genetic deletion of the type 1 TNF receptor (p60(-/-)), the type 2 TNF receptor (p80(-/-)), or both receptors (p60(-/-)p80(-/-)). We found that the macrophages expressing TNF receptors were highly sensitive to apoptosis induced by anti-Fas. The genetic deletion of TNF receptors, however, made the cells resistance to anti-Fas-induced apoptosis. Anti-Fas induced activation of caspase-3 and PARP cleavage in WT cells but not in TNF receptor-deleted cells. This difference was found to be independent of the expression of Fas, Fas-associated protein with death domain (FADD) or TNF receptor-associated death domain (TRADD). We found that anti-Fas induced recruitment of TNFR1 into Fas-complex. We also found that TRADD, which mediates TNF signaling, was constitutively bound to Fas receptor in TNF receptor-deleted cells but not in wild-type cells. Transient transfection of TNFR1 in TNFR1-deleted cells sensitized them to anti-Fas-induced apoptosis. Overall our results demonstrate that Fas signaling is modulated by the TNF receptors and thus provide the evidence of cross-talk between the receptors of two cytokines.     

Thymoquinone supplementation reverses acetaminophen-induced oxidative stress,nitric oxide production and energy decline in mice liver

This study was undertaken to evaluate the protective effect of thymoquinone (TQ) against acetaminophen-induced hepatotoxicity. Mice were given TQ orally at three different doses (0.5, 1 and 2 mg/kg/day) for 5 days before a single hepatotoxic dose of acetaminophen (500 mg/kg i.p.). TQ supplementation dramatically reduced acetaminophen-induced hepatotoxicity, in a dose-dependent manner, as evidenced by decreased serum alanine aminotransferase (ALT) activities.Acetaminophen (500 mg/kg i.p.) resulted in a significant increase in serum ALT and total nitrate/nitrite, hepatic lipid peroxides and a significant decrease in hepatic reduced glutathione (GSH) and ATP in a time-dependent manner. Interestingly, supplementation of TQ (2 mg/kg/day) for 5 days before acetaminophen administration resulted in reversal of acetaminophen-induced increase in ALT, total nitrate/nitrite, lipid peroxide and a decrease in GSH and ATP. Moreover, TQ did not affect acetaminophen-induced early decrease in hepatic GSH indicating lack of the effect on the metabolic activation of acetaminophen.In conclusion, TQ is effective in protecting mice against acetaminophen-induced hepatotoxicity possibly via increased resistance to oxidative and nitrosative stress as well as its ability to improve the mitochondrial energy production.     

Effects and mechanisms of rifampin on hepatotoxicity of acetaminophen in mice

This study examined the effects and possible mechanisms of rifampin against acetaminophen-induced hepatotoxicity in mice. Rifampin significantly enhanced the biotransformation of acetaminophen, evidenced by the increase in p-aminophenol formation in rifampin-treated microsomes and the increase in plasma clearance rate of acetaminophen. Pretreatment with rifampin significantly decreased serum alanine transaminase (ALT) activities, aspartate transaminase (AST) activities and prevented severe liver necrosis following acetaminophen overdose. The contents and activities of microsomal drug-metabolizing enzyme were less affected in rifampin-pretreated mice in comparison to the animals treated with acetaminophen alone. Rifampin was capable of increasing glutathione (GSH) level and GSH reductase activity and reducing GSH depletion and the decrease in GSH reductase activity by acetaminophen in mice. In addition, it was found that the microsomal Ca²⁺-ATPase activity was not directly related to acetaminophen toxic species generated in the P450 enzyme system in vitro. These findings suggest that rifampin has species-specific effects on the liver against acetaminophen-induced hepatotoxicity in mice, which increase the level of GSH by promoting GSH regeneration.     

Effects of tumor necrosis factor type 1 and 2 receptor deficiencies on anorexia, growth and IgA dysregulation in mice exposed to the trichothecene vomitoxin.

Dietary exposure of mice to vomitoxin (VT), a trichothecene mycotoxin, causes anorexia and impaired growth as well as inducing elevated serum IgA and kidney mesangial IgA deposition in a manner analogous to human IgA nephropathy. Based on the observations that TNF-alpha is induced by in vitro and in vivo VT exposure, it was hypothesized that this cytokine plays a role in the nutritional and immunological effects of this toxin. To test this hypothesis, the effects of dietary VT on feed intake, weight gain, serum IgA levels and kidney mesangial IgA deposition in mice homozygous for targeted disruption of the two known TNF-alpha cell surface receptors, TNFR1(p55) or TNFR2(p75), were compared to effects in corresponding C57BL/6J wild-type (WT) mice with normal receptor function. The capacity of VT to cause feed refusal or impair weight gain over a 12-week feeding period was not impaired in TNFR1 knockout (KO) or TNFR2-KO as compared to WT mice. Both WT and TNFR-KO mice fed VT exhibited reduced (P<0.05) feed conversion efficiency, but surprisingly, feed conversion efficiency was significantly higher (P<0.05) in TNFR1-KO and TNFR2-KO fed either control or VT diets than in corresponding WT mice. By week 12, serum IgA concentrations in all three mouse groups fed VT were significantly higher than those for corresponding mice fed control diets (P<0.05). Serum IgA levels in the VT-fed TNFR1-KO group were significantly less (P<0.05) than those for the VT-fed WT mice at 4, 8 and 12 weeks, whereas no differences in this parameter were found between the TNFR2-KO and WT groups. Serum IgA immune complex concentrations were measured at wk 12 and found to follow an identical pattern to IgA. Kidneys taken from VT-fed TNFR2-KO and WT mice after 12 weeks had significantly increased mesangial IgA deposition as compared to controls. While slight increases in mesangial IgA were also observed in VT-fed TNFR1-KO mice, these levels were significantly less (P<0.05) than that found in VT-fed TNFR2-KO and WT mice. Taken together, the data suggest that while VT-mediated anorexic and growth effects were largely independent of TNF-alpha, VT-induced dysregulation of IgA production was dependent, in part, on the interaction of TNF-alpha with TNFR1.