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Unlabelled: Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6h after APAP and a partial protection when given at 15 h., Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic., (Copyright © 2014 Elsevier Inc. All rights reserved.)

HIF-1α is a nuclear factor important in the transcription of genes controlling angiogenesis including vascular endothelial growth factor (VEGF). Both hypoxia and oxidative stress are known mechanisms for the induction of HIF-1α. Oxidative stress and mitochondrial permeability transition (MPT) are mechanistically important in acetaminophen (APAP) toxicity in the mouse. MPT may occur as a result of oxidative stress and leads to a large increase in oxidative stress. We previously reported the induction of HIF-1α in mice with APAP toxicity and have shown that VEGF is important in hepatocyte regeneration following APAP toxicity. The following study was performed to examine the relative contribution of hypoxia versus oxidative stress to the induction of HIF-1α in APAP toxicity in the mouse. Time course studies using the hypoxia marker pimonidazole showed no staining for pimonidazole at 1 or 2h in B6C3F1 mice treated with APAP. Staining for pimonidazole was present in the midzonal to periportal regions at 4, 8, 24 and 48h and no staining was observed in centrilobular hepatocytes, the sites of the toxicity. Subsequent studies with the MPT inhibitor cyclosporine A showed that cyclosporine A (CYC; 10mg/kg) reduced HIF-1α induction in APAP treated mice at 1 and 4h and did not inhibit the metabolism of APAP (depletion of hepatic non-protein sulfhydryls and hepatic protein adduct levels). The data suggest that HIF-1α induction in the early stages of APAP toxicity is secondary to oxidative stress via a mechanism involving MPT. In addition, APAP toxicity is not mediated by a hypoxia mechanism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Our recent investigations have focused on the possible involvement of glutathione-derived APAP metabolites in APAP nephrotoxicity and have demonstrated that administration of acetaminophen-cysteine (APAP-CYS) potentiated APAP-induced renal injury with no effects on APAP-induced liver injury. Additionally, APAP-CYS treatment alone resulted in a dose-responsive renal GSH depletion. This APAP-CYS-induced renal GSH depletion could interfere with intrarenal detoxification of APAP or its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and may be the mechanism responsible for the potentiation of APAP nephrotoxicity. Renal-specific GSH depletion has been demonstrated in mice and rats following administration of amino acid gamma-glutamyl acceptor substrates for gamma-glutamyl transpeptidase (gamma-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the gamma-glutamyl cycle through interaction with gamma-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the gamma-glutamyl transpeptidase (gamma-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a gamma-glutamyl acceptor for both murine and bovine renal gamma-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a gamma-glutamyl-cysteinyl-acetaminophen metabolite. These findings are consistent with the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by depletion of renal GSH stores through interaction with the gamma-glutamyl cycle.

Repeated treatment with clofibrate (CFB) significantly increased hepatic glutathione (GSH) content and also diminished acetaminophen's (APAP) selective protein arylation, GSH depletion, and severity of hepatocellular necrosis. The present work was conducted to evaluate the role of elevated GSH and APAP detoxifying pathways in the amelioration of APAP's toxicity by CFB. Male CD-1 mice received 500 mg CFB/kg, i.p., daily for 10 days. Controls were given corn oil vehicle. They were challenged with 700 mg APAP/kg in 50% propylene glycol/water after an overnight fast. Results indicate that CFB pretreatment had no effect on 24-hr urinary excretion of APAP-glucuronide, sulfate, or GSH-derived conjugates; however, there was 50% less unchanged APAP excreted in urine of CFB-pretreated mice. CFB also did not alter microsomal UDP-glucuronyl transferase activity toward APAP in vitro. However, elimination of APAP from plasma and liver was much greater in CFB-pretreated mice. This was accompanied by elevated biliary APAP-GSH content in CFB-pretreated mice at 2 hr after APAP dosing with diminished levels in bile at 12 hr. The CFB-induced increase in biliary excretion of APAP-GSH may mediate the protection against APAP-induced hepatotoxicity.

Peroxisome proliferators have been shown to diminish acetaminophen (APAP) hepatotoxicity (Biochem. Pharmacol. 43, 1395, 1992). To investigate the mechanistic basis for this protection CD-1 male mice were given corn oil or 500 mg clofibrate (CFB)/kg, ip, daily for 10 days. They were then fasted overnight and either killed without challenge or at 4 or 12 hr after challenge with 800 mg APAP/kg (in 50% propylene glycol). At 12 hr, hepatotoxicity was evidenced by elevated plasma sorbitol dehydrogenase and histopathology in corn oil but not in CFB-pretreated mice. At 4 hr after APAP treatment, hepatic glutathione (GSH) depletion and selective arylation of the major APAP target proteins were both greatly diminished by CFB pretreatment. Western blot analysis with the anti-58 antibody of liver cytosol from unchallenged mice showed no apparent changes in the levels of the 58-kDa major APAP target protein with CFB treatment. These findings suggest that protection could be the result of diminished net availability of generated electrophile. In vitro, measurements indicated that the specific activity in microsomes for APAP oxidation by cytochrome P450 was not changed by CFB treatment; whereas GSH S-transferase activity in cytosol was decreased by 25%. Pretreatment with CFB also produced a significant elevation in hepatic GSH. These studies indicate that protection by CFB might result from increased availability of hepatic GSH which could trap APAP electrophile nonenzymatically, thereby decreasing covalent binding and preventing toxicity.

Previously, we demonstrated that oltipraz [OTP: 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] prevented the hepatotoxicity of acetaminophen (AAP) in hamsters and that the observed protection was not related to increases in hepatic reduced glutathione (GSH) levels. These experiments were designed to elucidate the mechanism of OTP-induced protection with respect to an apparent non-GSH-dependent system. Marked differences in the relative amounts of hepatic GSH content depleted by AAP in control vs OTP-treated hamsters occurred. Urinary recoveries of AAP and metabolites indicated that more AAP-glucuronide was formed at the expense of other major metabolites (AAP-GSH, -N-acetylcysteine, and -sulfate) in OTP-treated hamsters, while plasma toxicokinetic modeling suggested a greater rate of AAP systemic clearance. An increased apparent formation rate constant for AAP glucuronidation (135%), in concert with significantly lower apparent formation rate constants for those metabolites which reflect the production of the reactive intermediate from AAP (glutathione and N-acetylcysteine), provide the rationale for this shift of metabolism. The biochemical basis for metabolic shunting is significantly elevated hepatic UDP-glucuronic acid content, an increased calculated UDP-glucuronic acid synthetic rate, and an increased liver microsomal UDP-glucuronyl transferase activity in OTP-treated animals. These changes in AAP conjugation were concomitant with decreased fractional clearance of AAP via bioactivation and less in vivo AAP covalent binding. These data support the hypothesis that OTP provides a protecting effect from AAP hepatotoxicity due to an augmented and predisposing glucuronidation capacity.

These studies were designed to test the hypothesis that oltipraz (OTP) provided protection against AAP intoxication in a sensitive species, the hamster; and further, to show that the sparing effect was related to the marked increase in hepatic reduced glutathione (GSH) levels. Dose-response and time-course experiments demonstrated that maximal increases in liver GSH occurred at 48 hr after an oltipraz dose of approximately 2.0 mmol/kg (po). Accompanying greater GSH levels were increased glutathione disulfide (GSSG) levels. Decreased indices of the oxidation state of glutathione and of hepatic pyridine nucleotides indicated a greater share of glutathione existed as GSH and that increased reducing equivalents were present, respectively. Additionally, glutathione disulfide reductase activity was greater in OTP-treated groups. Glutathione S-transferase activities were only marginally increased. OTP treatment did not elicit observable hepatotoxicity, whereas AAP (2.6 mmol/kg, ip) resulted in a reproducible model of liver damage. OTP-treated groups were protected from AAP-induced toxicity, as shown by decreased plasma appearance of liver enzymes and unremarkable histopathology. However, the degree of liver GSH depletion by AAP was fourfold greater in non-OTP treated groups compared to those which had received the dithiolthione. To test the importance of increased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH content to 50% of control in hamsters which either had or had not received OTP. The groups receiving BSO and AAP incurred 83% lethality, while no lethality, unremarkable liver histopathology, and plasma enzyme levels consistent with control were found in the group receiving OTP, BSO, and AAP. Treatment with BSO only had no influence on hepatotoxicity parameters. These results indicate that the increased GSH levels in the OTP-treated hamster are coincidental to the sparing effect of OTP and are not central to the protection scheme in AAP-induced hepatotoxicity.

Inhibition of the cytochrome P-450-dependent formation of the acetaminophen-reactive metabolite was investigated as a possible mechanism for cysteamine protection against acetaminophen hepatotoxicity. Studies in isolated hamster hepatocytes indicated that cysteamine competitively inhibited the cytochrome P-450 enzyme system as represented by formation of the acetaminophen-glutathione conjugate. However, cysteamine was not a potent inhibitor of glutathione conjugate formation (Ki = 1.17 mM). Cysteamine also weakly inhibited the glucuronidation of acetaminophen (Ki = 2.44 mM). In vivo studies were in agreement with the results obtained in isolated hepatocytes; cysteamine moderately inhibited both glucuronidation and the cytochrome P-450-dependent formation of acetaminophen mercapturate. The overall elimination rate constant (beta) for acetaminophen was correspondingly decreased. Since cysteamine decreased both beta and the apparent rate constant for mercapturate formation (K'MA), the proportion of the dose of acetaminophen which is converted to the toxic metabolite (K'MA/beta) was not significantly decreased in the presence of cysteamine. Apparently, cysteamine does inhibit the cytochrome P-450-dependent formation of the acetaminophen-reactive metabolite, but this effect is not sufficient to explain antidotal protection.

Acetaminophen (ACM) administration to mice of the (C57BL/6 X DBA/2)F1 strain produced a typical hepatic centrilobular necrosis similar to that observed in rodents and humans. To determine the effects of this drug-induced necrosis on cadmium (Cd) metabolism, mice were given a sublethal dose of CdCl2 . 2.5 H2O containing 109CdCl2 and maintained for a period of time sufficient for Cd-metallothionein (Cd-MT) to be synthesized and distributed. Subsequent administration of ACM ip or po evoked a marked redistribution of Cd from livers to kidneys of mice, and increased the amount of Cd excreted in urine and feces. There were only minimal or no effects on Cd concentrations in other organs assessed. The effect of ACM on Cd redistribution was antagonized by administration of cysteine, a glutathione precursor, and was enhanced by pretreatment with phenobarbital, a potent inducer of the cytochrome P-450 mixed-function oxidase system. Pretreatment of mice with ACM 6 or 24 hr prior to Cd administration caused aberrations of the normal Cd distribution pattern, but no effect was noted when Cd administration was delayed for 48 hr after ACM injection, indicating recovery of the mechanisms of Cd-MT synthesis and sequestration. Sephadex G-75 gel filtration chromatography of serum from ACM-treated mice showed that most of the Cd was associated with high-molecular-weight proteins, and only a minor portion was present as Cd-MT. Cd excreted in urine was predominantly in a low-molecular-weight form, but there was evidence of two minor components of higher molecular weight, neither of which eluted as Cd-MT. Cd excreted in feces was insoluble following homogenization in 0.25 M sucrose solution. Cd in livers and kidneys of ACM-treated mice eluted as Cd-MT. It was concluded that persons who have a moderately high Cd burden may be at risk of Cd nephrotoxicity if they incur hepatic necrosis subsequent to ACM abuse.

The protective effect of a single dose of ethanol with regard to hepatotoxicity caused by acetaminophen (APAP) can be a consequence of either direct or indirect inhibition of APAP oxidation to its hepatotoxic intermediate (N-acetyl-p-benzoquinoneimine, NAPQI), or augmentation of repair mechanisms following the hepatotoxic insult. The mechanism of hepatoprotection appears to be species dependent. By varying the time of ethanol administration relative to APAP in mice (30 min before to 240 min after APAP), it was shown that ethanol must be administered early relative to APAP for hepatoprotection to be maximized. The role of direct inhibition of cytochrome P450 in the hepatoprotective effect of ethanol was evaluated by comparing the hepatoprotection afforded by ethanol and 4-methylpyrazole (4-MP) in vivo to the inhibition of APAP oxidation to NAPQI caused by each in mouse liver microsomes. At their respective peak in vivo concentrations attained following hepatoprotective doses, both ethanol and 4-MP inhibited the oxidation of APAP in microsomes by 25-30%. This result suggests that direct inhibition of cytochrome P450 by ethanol plays a role in the protection against acetaminophen-induced hepatotoxicity in mice. In human liver microsomes the inhibition of APAP oxidation to the hepatotoxic intermediate by 48 mM ethanol is less than half of the apparent inhibition of APAP oxidation reported in clinical studies in which the maximum ethanol concentration would have been 15-20 mM. Thus, in contrast to the mouse, inhibition of APAP oxidation to NAPQI in humans appears to be largely indirect, as has been reported previously in the rat.

The purpose of this investigation was to assess protection by zinc against acetaminophen induced hepatotoxicity and to evaluate possible mechanisms of protection. Mice were treated with zinc (3 mg/kg, ip) or saline (ip) 48 and 24 hr before and sacrificed 12 hr after acetaminophen administration (375, 500, or 750 mg/kg, po). Liver toxicity was then assessed by histological examination. The incidence of hepatotoxicity was significantly less at 375 and 500 mg/kg of acetaminophen in zinc treated animals. The same dosage of zinc was not hepatoprotective when given 1 hr after acetaminophen. Mice were also treated with 1 to 10 mg/kg of zinc (ip) 48 and 24 hr prior to sacrifice, and metallothionein, cytochrome P-450, glutathione, and UDP-glucuronosyl transferase (GT) were determined in the liver. Metallothionein and UDP-GT were increased and P-450 and glutathione decreased at the higher dosages of zinc; however, only metallothionein was significantly changed at the dosage of zinc (3 mg/kg) used in the hepatoprotection experiments. Further, mice were similarly treated with 3 mg/kg of zinc before administration of 375 mg/kg of [3H]acetaminophen (po) and the amount of acetaminophen and acetaminophen bound to metallothionein were determined in the liver for 0.5 to 24 hr. In addition, after 6 hr the subcellular distribution and covalent binding to protein of acetaminophen were also determined. Zinc treatment had no significant effect in any of the above determinations. These results indicate that zinc protects against acetaminophen induced hepatotoxicity and that the observed protection is probably due to an induced biochemical change, but it is apparently not the result of any of the commonly invoked mechanisms.

Experiments were undertaken to examine the ability of selenium to protect against acetaminophen-induced hepatotoxicity and to examine possible mechanisms for this protective effect. Pretreatment of male, Sprague-Dawley rats with sodium selenite (12.5 mumol Se/kg, ip) 24 hr prior to acetaminophen administration produced a significant protection against the hepatotoxic effects of acetaminophen as assessed by a decrease in the plasma appearance of alanine aminotransferase and aspartate aminotransferase activities following acetaminophen. This was accompanied by an increase in the hepatic glutathione levels in selenium-treated animals and an inhibition in the decrease in hepatic glutathione content observed in animals receiving hepatotoxic doses of acetaminophen. Selenium pretreatment decreased the in vivo covalent binding of acetaminophen metabolites to hepatic protein, but did not alter hepatic microsomal cytochrome P-450 content or NADPH cytochrome c reductase activity, suggesting that selenium does not significantly alter the metabolism of acetaminophen to reactive electrophilic metabolites by the cytochrome P-450-dependent mixed-function oxidase enzyme system. Selenium produced an increase in the activity of gamma-glutamylcysteine synthetase which may account for the increased glutathione availability in selenium-treated animals and increased the activities of glutathione S-transferase and glucose-6-phosphate dehydrogenase. Examination of the urinary metabolite profile in selenium-treated animals revealed that the urinary excretion of acetaminophen and its metabolites was significantly increased over a 72-hr period. The increase occurred in the AAP-glucuronide metabolite while parent AAP and AAP-sulfate were actually decreased in selenium-treated rats. No change in recovery was observed in the AAP-glutathione or AAP-mercapturate urinary metabolites. While the glutathione conjugating system is enhanced by selenium treatment, amelioration of acetaminophen toxicity is most likely the result of enhanced glucuronidation which effectively diverts the amount of acetaminophen to be converted by the cytochrome P-450 system to the toxic metabolite.