Metabolism-dependent inhibition of CYP2E1 by isoniazid, a mediator of idiosyncratic liver injury.

Liver injury is a well-known adverse effect of the anti-tuberculosis drug isoniazid (INH); however, animal models that accurately replicate this effect as seen in humans have not been constructed, and the mechanism of its pathogenesis remains unclear. Recently, an immune-mediated mechanism have been proposed based on clinical studies, suggesting the involvement of cytochrome P450-mediated formation of reactive metabolites and covalent adducts in severe cases. In the present study, we investigated the role of CYP2E1 in this mechanism. Liver microsomes from humans, rats, and mice were preincubated with INH and NADPH; thereafter, residual CYP2E1 activity was measured. The inhibition of CYP2E1 by INH was potentiated by preincubation, indicating time-dependent inhibition. There were no major species-based differences in inhibition among humans, rats, and mice. Further to our findings on the inhibition kinetics, resistance of the inhibition to glutathione and catalase indicated that the reactive metabolites of INH covalently bonded to CYP2E1 in a suicidal manner. A similar time-dependent inhibition was also observed for the known metabolites acetylhydrazine and hydrazine; however, the conditions that inhibited the hydrolysis or activated the acetylation of INH did not affect inhibition by INH, suggesting that the reactive metabolites contributing to the inhibition were generated via alternative pathways. This indicates that CYP2E1 alone generates reactive INH metabolites and that haptenized CYP2E1 may be involved in immune-mediated liver injury.

Efficacy of oltipraz in preventing acetaminophen-induced liver injury in mice.

Oltipraz (OPZ) is a synthetic dithiolethione with potential as a cancer chemopreventive agent, which can work by inducing detoxification enzymes. OPZ is an activator of nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting its involvement in enzyme induction and possible protection against drug-induced liver injury. In this study, we present OPZ-mediated protection of mice against acetaminophen (APAP)-induced liver injury and discuss its possible contributing factors. Overnight-fasted male CD-1 mice were administered APAP intraperitoneally, and some mice were administered OPZ 16 h before APAP. Hepatotoxicity was assessed by measuring serum alanine aminotransferase leakage and histopathological evaluation. The hepatic mRNA expressions of CYP2E1, glutamate cysteine ligase (GCL), and NAD(P)H:quinone oxidoreductase (NQO1) were measured by real-time reverse-transcription polymerase chain reaction. OPZ protected mice from APAP-induced liver injury in a dose-dependent manner, but did not alter hepatic glutathione (GSH) content or GCL expression in control mice, indicating that its hepatoprotective effect is not due to changes in basal GSH levels. OPZ did not affect CYP2E1 expression or APAP-induced early GSH depletion, suggesting it does not inhibit the metabolic activation of APAP to produce N-acetyl-p-benzoquinone imine. In contrast, after GSH depletion, OPZ accelerated hepatic GSH recovery. APAP significantly increased GCL expression during liver injury, but OPZ treatment only led to additional NQO1 expression. This suggests that NQO1 is responsible for the enhanced GSH recovery and protection against APAP-induced liver injury seen in OPZ-treated mice. In summary, OPZ protects against APAP-induced liver injury by inducing NQO1 expression and resulting in improved GSH recovery.

Protection of mice against carbon tetrachloride-induced acute liver injury by endogenous and exogenous estrogens.

Gender is a crucial factor determining susceptibility to drug-induced liver injury (DILI) in humans and experimental animals. However, no general concept of sex differences in DILI has been established, as metabolic events specific to one DILI model are difficult to apply to other DILI models. Herein, we examined sex differences in carbon tetrachloride (CCl4)-induced hepatotoxicity, a widely employed DILI model. Male and female CD-1 mice were intraperitoneally administered CCl4. Additionally, some male mice were administered genistein or another isoflavone to evaluate the effects of exogenous estrogens. Dose-dependent alanine aminotransferase leakage was observed at a CCl4 range of 0.5-10 mmol/kg, with male-dominant sex differences mainly observed at lower doses. No sex differences in hepatic glutathione levels or thiobarbituric acid-reactive substance formation were detected. CCl4 induced hepatic inflammatory genes, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, predominantly in female mice, which might be involved in DILI resistance, observed in female mice. Treatment of male mice with phytoestrogens, especially genistein, attenuated CCl4-induced hepatotoxicity. Moreover, genistein inhibited IL-6 and TNF-α expression, suggesting possible hepatoprotection via immunosuppression. In conclusion, female mice are resistant to CCl4-induced hepatotoxicity, and male mice were afforded protection by genistein, probably via mechanisms based on anti-estrogenic, antioxidant and/or anti-inflammatory effects.

Sex difference in susceptibility to acetaminophen hepatotoxicity is reversed by buthionine sulfoximine.

Gender is a factor that influences susceptibility of individuals to drug-induced liver injury in experimental animals and humans. In this study, we investigated the mechanisms underlying resistance of female mice to acetaminophen (APAP)-induced hepatotoxicity. Overnight-fasted male and female CD-1 mice were administered APAP intraperitoneally. A minor increase in serum alanine aminotransferase levels was observed in female mice after APAP administration at a dose that causes severe hepatotoxicity in males. Hepatic glutathione (GSH) depleted rapidly in the both genders prior to development of hepatotoxicity, whereas its recovery was more rapid in female than in male mice. This was consistent with higher induction of hepatic glutamate-cysteine ligase (GCL) in females. Pretreatment of mice with L-buthionine sulfoximine (BSO), an inhibitor of GCL, exaggerated APAP hepatotoxicity only in female mice, resulting in much higher hepatotoxicity in female than in male mice. In addition, hepatic GSH was markedly depleted in BSO-pretreated female mice compared with male mice, which supports severe hepatotoxicity in BSO-pretreated females. APAP treatment highly induced multidrug resistance-associated protein 4 (Mrp4) only in female mice. The resulting high Mrp4 expression could thus contribute to decreased hepatic GSH levels via sinusoidal efflux when GCL is inhibited. In conclusion, resistance to APAP hepatotoxicity in female mice and its reversal by pretreatment with BSO could be attributed to sex differences in disposition of hepatic GSH, which may generally determine susceptibility to drug-induced liver injury.

Protective effects of exogenous glutathione and related thiol compounds against drug-induced liver injury.

An overdose of acetaminophen (APAP) causes liver injury both in experimental animals and humans. N-acetylcysteine (NAC) is clinically used as an antidote for APAP intoxication, and it is thought to act by providing cysteine as a precursor of glutathione, which traps a reactive metabolite of APAP. Other hepatoprotective mechanisms of NAC have also been suggested. Here, we examined the effects of thiol compounds with different abilities to restore hepatic glutathione, on hepatotoxicity of APAP and furosemide in mice. Overnight-fasted male CD-1 mice were given APAP or furosemide intraperitoneally. NAC, cysteine, glutathione, or glutathione-monoethyl ester was administered concomitantly with APAP or furosemide. All thiol compounds used in this study effectively protected mice against APAP-induced liver injury. Only glutathione-monoethyl ester completely prevented APAP-induced early hepatic glutathione depletion. Cysteine also significantly restored hepatic glutathione levels. NAC partially restored glutathione levels. Exogenous glutathione had no effect on hepatic glutathione loss. NAC and glutathione highly stimulated the hepatic expression of cytokines, particularly interleukin-6, which might be involved in the alleviation of APAP hepatotoxicity. Furosemide-induced liver injury, which does not accompany hepatic glutathione depletion, was also attenuated by NAC and exogenous glutathione, supporting their protective mechanisms other than replenishment of glutathione. In conclusion, exogenous thiols could alleviate drug-induced liver injury. NAC and glutathione might exert their effects, at least partially, via mechanisms that are independent of increasing hepatic glutathione, but probably act through cytokine-mediated and anti-inflammatory mechanisms.

Flexible structure of cytochrome P450: promiscuity of ligand binding in the CYP3A4 heme pocket.

BACKGROUND: CYP3A4 is the most abundant xenobiotic-metabolizing cytochrome P450 isoform. We examined the structural features of the CYP3A4 molecule with regard to ligand access. MATERIALS AND METHODS: The deleted amino acid sequences of X-ray data sets of CYP3A4s were complemented by molecular modeling techniques. Molecular features of the ligand accessible regions in CYP3A4 were analyzed and their molecular parameters (e.g. dipole moment, solvation free energy, electrostatic potential fields) were determined. RESULTS: Three ligand accessible regions (region 1-3) were present in erythromycin-bound CYP3A4, and these dipole moments indicated the same features as ketoconazole- or metyrapone-bound CYP3A4 molecules. In progesterone-bound CYP3A4, four candidate ligand accessible regions were observed and progesterone could be bound by two selected ligand accessible regions. CONCLUSION: The heme pocket of CYP3A4 is very flexible and is able to interact with various types of substrate.

Th1/Th2 cytokine balance as a determinant of acetaminophen-induced liver injury.

Inflammation is an important pathophysiological event in drug-induced liver injury, which is subsequent to metabolic activation and covalent binding of the reactive metabolites to target proteins. Cytokines are recognized as pro- and anti-inflammatory mediators involved in the progression and regression of the toxicity. We thus hypothesized that disturbed balance of Th1/Th2 cytokines exacerbated the drug-induced hepatotoxicity. Acetaminophen-induced liver injury was investigated in two mouse strains, C57BL/6 and BALB/c, which develop predominantly Th1 and Th2 responses, respectively. More severe liver injury after intraperitoneal administration of acetaminophen was observed in C57BL/6 mice than in BALB/c mice. There was no strain difference in metabolism of acetaminophen into its reactive metabolite, N-acetyl-p-benzoquinone imine, which was assessed by early glutathione consumption. Liver mRNA expression of tumor necrosis factor-alpha (TNF-alpha) and IL-6 were measured as pro- and anti-inflammatory cytokines, respectively. TNF-alpha was highly induced 24 h after administration of acetaminophen in C57BL/6 mice, whereas no change in BALB/c mice. On the other hand, liver IL-6 mRNA expression in BALB/c mice was higher than C57BL/6 mice 24 h after the administration. In addition, treatment of CD-1 mice, another susceptible strain, with an anti-inflammatory polyphenol, resveratrol, protected mice against the acetaminophen-induced liver injury, and the mice with attenuated toxicity revealed lower expression of TNF-alpha and higher expression of IL-6. It is therefore suggested that acetaminophen-induced liver injury is associated with Th1-dominant response in Th1/Th2 cytokine balance, and TNF-alpha may play a pathological role in the toxicity.

Down-regulation of hepatic transporters for BSP in rats with indomethacin-induced intestinal injury.

Previous reports have demonstrated that an intestinal injury causes hypofunctions of the liver associated with down-regulations of cytochrome P450, but an influence on hepatic transporters remains unclear. Here, we tested hepatic transporter functions in a rat model of bowel injury using indomethacin (IDM). After administration of IDM (8.5 mg/kg, i.p., 3 d), the rats suffered the intestinal impairment indicated by a reduction of alkaline phosphatase activity in mucosa. In vivo pharmacokinetic experiments of bromosulfophthalein (BSP) showed that there was a reduction in its plasma elimination rate and cumulative biliary excretion in IDM-treated rats and systemic and biliary clearances reduced to nearly 50% of the control group. Protein expressions in plasma membrane and mRNA levels of organic anion transporting polypeptide 1b2 (Oatp1b2) and multidrug resistance-associated protein 2 (Mrp2), which play hepatic BSP uptake and biliary excretion, respectively, in the liver were significantly reduced following the IDM treatment. In portal plasma, the levels of proinflammatory cytokines were unchanged, while the level of nitric oxide metabolites (NO2- + NO3-) increased to 6.5-fold that of the control. The time-course on IDM treatment indicated that, firstly, intestinal injury was induced, the NO level increased, and the hepatic Oatp1b2 and Mrp2 expression began to fall followed by an increase in plasma ALT. In conclusion, IDM-induced injury to the small intestine causes the hypofunction of hepatic Oatp1b2 and Mrp2 independently on the hepatic impairment, and NO arising from bowel injury may be one of key factors for it through the remote effect.

Metabolic and non-metabolic factors determining troglitazone hepatotoxicity: a review.

Troglitazone (TGZ), a thiazolidinedione class of antidiabetic agent, causes serious idiosyncratic hepatotoxicity. TGZ is metabolized into reactive metabolites that covalently bind to cellular macromolecules, one of which is oxidation at the chromane ring, a unique structure of TGZ, and another involves oxidative cleavage of the thiazolidinedione ring, a structure common to less hepatotoxic antidiabetics, rosiglitazone and pioglitazone. TGZ is cytotoxic to HepG2 cells and rat and human hepatocytes. However, the role of the reactive metabolite on the TGZ toxicity is controversial, because there was no correlation of the generation of the reactive metabolites with susceptibility to the TGZ cytotoxicity, and chemical inhibitors of drug metabolizing enzymes could not protect the cells against the toxicity. Mitochondrial dysfunction, especially mitochondrial permeability transition, may be a pathophysiological event, which is mediated by TGZ itself and is a major non-metabolic factor. Other events such as apoptosis and PPARgamma-dependent steatosis could be also mediated by TGZ, while inhibition of bile salt export pump, a cause of TGZ-induced cholestasis, may be caused by the TGZ sulfate. In conclusion, although the TGZ is biotransformed into chemically reactive metabolites, there is currently no potential evidence for involvement of the reactive metabolite in the TGZ-induced liver injury.

Down-regulation of hepatic cytochrome P450 enzymes associated with cisplatin-induced acute renal failure in male rats.

Hepatic drug metabolism is impaired in experimental animals and humans with renal diseases. An anticancer drug, cisplatin induces acute renal failure (ARF) in rats. Under the same experimental conditions, cisplatin causes down-regulation of hepatic cytochrome P450 (P450) enzymes in an isozyme selective manner. The present study examined the pathological role of ARF in the down-regulation of hepatic P450 enzymes in the cisplatin-treated rats. Male rats with single dose of intraperitoneally cisplatin (5 mg/kg) caused marked changes in renal parameters, BUN and serum creatinine but not hepatic parameters, serum alanine aminotransferase or aspartate aminotransferase. The rats also suffered from down-regulation of hepatic microsomal CYP2C11 and CYP3A2, male specific P450 isozymes, but not CYP1A2, CYP2E1, or CYP2D2. The decrease in serum testosterone level was also observed in injured rats, which was consistent with the selective effects on male specific P450 enzymes. Protection of rats against cisplatin-induced ARF by dimethylthiourea, a hydroxyl radical scavenger, also protected rats against the decrease in serum testosterone levels and the down-regulation of CYP2C11 and CYP3A2. Carboplatin, an analogue to cisplatin but no ARF inducer, did not cause decrease in serum testosterone levels and down-regulation of hepatic male specific P450 enzymes. These results suggest that down-regulation of hepatic P450 enzymes in male rats given cisplatin is closely related to the cisplatin-induced ARF and the resultant impairment of testis function.

Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice.

BACKGROUND/AIMS: Although mitochondria have been demonstrated as primary targets in acetaminophen hepatotoxicity, the mechanism for mitochondria-mediated toxicity has not been defined. We examined the role of mitochondrial permeability transition (MPT) in the acetaminophen-induced liver injury. METHODS: Male CD-1 mice were given intraperitoneally acetaminophen (350 mg/kg) without or with cyclosporin A (50 mg/kg), a specific inhibitor of MPT. Serum alanine aminotransferase (ALT), a marker of liver injury, and other biochemical parameters were determined. RESULTS: Acetaminophen-induced ALT leakage was attenuated by co-administration of cyclosporin A. Cyclosporin A did not affect acetaminophen-induced early decrease in hepatic reduced glutathione (GSH) contents, indicating lack of the effect on the metabolic activation. Acetaminophen-induced decrease in mitochondrial GSH and ATP contents, and cytosolic leakage of cytochrome c were attenuated by cyclosporin A, suggesting that mitochondrial oxidative stress and ATP depletion resulting from MPT are principle mechanisms involved in acetaminophen-induced liver injury. Mitochondrial swelling by calcium was exacerbated in the mitochondria isolated from the acetaminophen-treated mice. In vitro exposure of intact mitochondria to N-acetyl-p-benzoquinone imine (NAPQI) with calcium caused mitochondrial swelling. CONCLUSIONS: The present data indicate that the MPT is the principal mechanism in the acetaminophen-induced liver injury and NAPQI is a candidate to open the transition pore.

Endotoxin-mediated disturbance of hepatic cytochrome P450 function and development of endotoxin tolerance in the rat model of dextran sulfate sodium-induced experimental colitis.

Hepatobiliary abnormalities have been described in patients with chronic inflammatory bowel diseases. Hepatic cytochrome P450 (P450)-dependent drug-metabolizing enzyme activities and susceptibility to a hepatotoxin, d-galactosamine, were determined in rats with dextran sulfate sodium (DSS)-induced colitis to assess whether liver function is affected in the model of inflammatory bowel disease. Colitis was induced by treatment of rats with 3% DSS in drinking water for 7 days. Liver microsomes for enzyme activities and serum for biological analysis were prepared from the rats with colitis, along with untreated and lipopolysaccharide (LPS)-treated rats. Other rats received intraperitoneal injection of d-galactosamine to assess their susceptibility to the toxin-induced liver injury. Treatment of rats with DSS resulted in not only colitis but also decreases in hepatic P450-dependent drug-metabolizing enzyme activities. Elevated endotoxin was found in portal blood, which was not associated with liver injury. The potency and the isoform selectivity in the suppression of the P450 enzymes by DSS treatment were similar to those of LPS-treated rats. Coadministration of antibiotics, polymyxin B or metronidazole, with DSS protected rats from decreases in some but not all P450 enzyme activities, indicating partial involvement of bacterial endotoxin in the P450 decreases. The rats with colitis were less susceptible than untreated rats to d-galactosamine-induced liver injury and TNF-alpha production, suggesting development of endotoxin tolerance in DSS-colitis. In conclusion, these results suggest that the DSS-colitis leads to endotoxin-mediated down-regulation of hepatic P450 enzymes and protection against d-galactosamine-induced liver injury, probably due to endotoxin tolerance.

Role of interleukin-6 in hepatic heat shock protein expression and protection against acetaminophen-induced liver disease.

Recent experimental data suggest that the idiosyncratic nature of drug-induced liver disease (DILD) may be due in part to a deficiency of one or more hepatoprotective factors. In this study we have investigated whether interleukin (IL)-6 may also be one of these factors. Following the induction of liver injury with acetaminophen (APAP), a time-dependent increase in liver mRNA expression of IL-6 and its family members IL-11, leukemia inhibitory factor, and oncostatin M was observed in wild type (WT) mice, suggesting a possible hepatoprotective role played by this cytokine family. Indeed, mice lacking IL-6 (IL-6-/-) were more susceptible than were WT mice to APAP-induced liver injury. The increased susceptibility of the IL-6-/- mice was associated with a deficiency in the expression of hepatic heat shock protein (HSP)25, 32, and 40 as well as inducible HSP70 following APAP treatment. These results suggest that IL-6 and possibly other family members may protect the liver from injury, at least in part, by up-regulating the hepatic expression of several cytoprotective HSPs.

Protection against acetaminophen-induced liver injury and lethality by interleukin 10: role of inducible nitric oxide synthase.

Mechanistic study of idiosyncratic drug-induced hepatitis (DIH) continues to be a challenging problem because of the lack of animal models. The inability to produce this type of hepatotoxicity in animals, and its relative rarity in humans, may be linked to the production of anti-inflammatory factors that prevent drug-protein adducts from causing liver injury by immune and nonimmune mechanisms. We tested this hypothesis by using a model of acetaminophen (APAP)-induced liver injury in mice. After APAP treatment, a significant increase was observed in serum levels of interleukin (IL)-4, IL-10, and IL-13, cytokines that regulate inflammatory mediator production and cell-mediated autoimmunity. When IL-10 knockout (KO) mice were treated with APAP, most of these mice died within 24 to 48 hours from liver injury. This increased susceptibility to APAP-induced liver injury appeared to correlate with an elevated expression of liver proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, and IL-1, as well as inducible nitric oxide synthase (iNOS). In this regard, mice lacking both IL-10 and iNOS genes were protected from APAP-induced liver injury and lethality when compared with IL-10 KO mice. All strains, including wild-type animals, generated similar amounts of liver APAP-protein adducts, indicating that the increased susceptibility of IL-10 KO mice to APAP hepatotoxicity was not caused by an enhanced formation of APAP-protein adducts. In conclusion, these findings suggest that an important feature of the normal response to drug-induced liver injury may be the increased expression of anti-inflammatory factors such as IL-10. Certain polymorphisms of these factors may have a role in determining the susceptibility of individuals to idiosyncratic DIH.