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Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Lefluno-mide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP)-catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH 2-terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-X L inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepato-toxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabi-lization. (HEPATOLOGY 2007;45:412-421.) A cetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses. However, when taken at high doses or, rarely in particularly susceptible people at therapeutic doses, APAP can precipitate severe liver injury that can develop into fulminant liver failure. 1 The clinical significance of this adverse effect is underscored by APAP being, among all drugs, the single major cause of drug-induced hepatotoxicity in the United States and the United Kingdom. 2 The mechanisms underlying APAP-induced liver injury have been studied for several decades, and excellent recent reviews have summarized the cellular and molecular pathways of this toxic response. 3-5 Although the initial steps in the sequence of events leading to hepatocyte ne-crosis (bioactivation of APAP and glutathione depletion) have been well known for many years, the more distal events (signaling pathways that lead to the precipitation of cell death) are less clear. However, recently, the mito-chondrial permeability transition (mPT) has been identified as a pivotal mechanism mediating APAP-induced cell death. 6,7 According to this concept, a combination of mi-tochondrial oxidant stress, increased Ca 2 levels, and other factors may favor ...

Troglitazone, a first-generation thiazolidinedione antidiabetic drug, was withdrawn from the market due to an unacceptable risk of idiosyncratic hepatotoxicity. Troglitazone does not cause hepatotoxicity in normal healthy rodents, but it produces mitochondrial injury in vitro at high concentrations. The aim of this study was to explore whether genetic mitochondrial abnormalities might sensitize mice to hepatic adverse effects of troglitazone. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model of clinically silent mitochondrial stress. Troglitazone was daily administered for 4 weeks (0, 10 or 30 mg/kg/day, ip). We found that troglitazone caused overt liver injury in the high-dose group, manifested by increased serum alanine aminotransferase activity (> twofold) and midzonal areas of hepatic necrosis, in Sod2(+/-) but not in wild-type mice. No signs of hepatotoxicity were apparent at 2 weeks of treatment. Hepatic mitochondria isolated from troglitazone-treated mice exhibited decreased activities of aconitase (by 45%) and complex I (by 46%) and increased (by 58%) protein carbonyls, indicative of enhanced mitochondrial oxidant stress. This was paralleled by compensatory increases in mitochondrial glutathione levels. Finally, in hepatocytes isolated from untreated Sod2(+/-), but not wild-type mice, troglitazone caused a concentration-dependent increase in superoxide anion levels as demonstrated with a selective mitochondria-targeting fluorescent probe. In conclusion, prolonged administration of troglitazone can superimpose oxidant stress, potentiate mitochondrial damage, and induce delayed hepatic necrosis in mice with genetically compromised mitochondrial function. These data are consistent with our hypothesis that inherited or acquired mitochondrial abnormalities may be one of the contributing determinants of susceptibility to troglitazone-induced idiosyncratic liver injury.

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Exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). The organochlorine pesticide dieldrin is one of the environmental chemicals potentially linked to PD. Because recent evidence indicates that abnormal accumulation and aggregation of ␣-synuclein and ubiquitin-proteasome system dysfunction can contribute to the degenerative processes of PD, in the present study we examined whether the environmental pesticide dieldrin impairs proteasomal function and subsequently promotes apoptotic cell death in rat mesencephalic dopaminergic neuronal cells overexpressing human ␣-synuclein. Overexpression of wild-type ␣-synuclein significantly reduced the proteasomal activity. Dieldrin exposure dose-dependently (0 -70 M) decreased proteasomal activity, and 30 M dieldrin inhibited activity by more than 60% in ␣-synuclein cells. Confocal microscopic analysis of dieldrintreated ␣-synuclein cells revealed that ␣-synuclein-positive protein aggregates colocalized with ubiquitin protein. Further characterization of the aggregates with the autophagosomal marker mondansyl cadaverine and the lysosomal marker and dot-blot analysis revealed that these protein oligomeric aggregates were distinct from autophagosomes and lysosomes. The dieldrin-induced proteasomal dysfunction in ␣-synuclein cells was also confirmed by significant accumulation of ubiquitin protein conjugates in the detergent-insoluble fraction. We found that proteasomal inhibition preceded cell death after dieldrin treatment and that ␣-synuclein cells were more sensitive than vector cells to the toxicity. Furthermore, measurement of caspase-3 and DNA fragmentation confirmed the enhanced sensitivity of ␣-synuclein cells to dieldrin-induced apoptosis. Together, our results suggest that increased expression of ␣-synuclein predisposes dopaminergic cells to proteasomal dysfunction, which can be further exacerbated by environmental exposure to certain neurotoxic compounds, such as dieldrin.

Chronic ethanol ingestion mildly damages liver through oxidative stress and lipid oxidation, which is ameliorated by dietary supplementation with the anti-inflammatory β-amino acid taurine. Kidney, like liver, expresses cytochrome P450 2E1 that catabolizes ethanol with free radical formation, and so also may be damaged by ethanol catabolism. Sudden loss of kidney function, and not liver disease itself, foreshadows mortality in patients with alcoholic hepatitis [J. Altamirano, Clin Gastroenterol Hepatol. 2012, 10:65]. We found ethanol ingestion in the Lieber-deCarli rat model increased kidney lipid oxidation, 4-hydroxynonenal protein adduction, and oxidatively truncated phospholipids that attract and activate leukocytes. Chronic ethanol ingestion increased myeloperoxidase-expressing cells in kidney and induced an inflammatory cell infiltrate. Apoptotic Terminal deoxynucleotidyl transferase Nick End Labeling (TUNEL)-positive cells and active caspase-3 increased in kidney after ethanol ingestion, with reduced filtration with increased circulating Blood Urea Nitrogen and creatinine. These events were accompanied by release of albumin, myeloperoxidase, and the Acute Kidney Injury biomarkers Kidney Injury Molecule-1 (KIM-1), Neutrophil Gelatinase-associated Lipocalin (NGAL), and Cystatin c to urine. Taurine sequesters HOCl from myeloperoxidase of activated leukocytes, and taurine supplementation reduced renal lipid oxidation, reduced leukocyte infiltration, and reduced the increase in myeloperoxidase-positive cells during ethanol feeding. Taurine supplementation also normalized circulating BUN and creatinine levels, and suppressed enhanced myeloperoxidase, albumin, KIM-1 and cystatin c in urine. Thus, chronic ethanol ingestion oxidatively damages kidney lipids and proteins, damages renal function, and induces Acute Kidney Injury through an inflammatory cell infiltrate. The anti-inflammatory nutraceutical taurine effectively interrupts this ethanol-induced inflammatory cycle in kidney.

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is one of the most potent environmental contaminants, which has been shown to induce oxidative stress in testis and epididymal sperm of rats. However, the nature and mechanism of action of TCDD on the epididymis is not clear. The aim of the present study was to investigate whether induction of oxidative stress in epididymal sperm was direct effect of TCDD on epididymis. In the present studies, TCDD (0.1, 1.0 and 10 micro g/kg body weight per day) was administered orally to rats for 4 days. Twenty-four hours after the last treatment the animals were killed using anesthetic ether. Both epididymides were dissected out and epididymal sperm were collected by cutting the epididymides into small pieces in Ham's F-12 medium at 35 degrees C. The epididymal sperm and caput, corpus and cauda epididymides were homogenized and used for biochemical studies. Epididymal sperm counts did not decrease in the rats treated with TCDD. Administration of TCDD increased the production of reactive oxygen species such as hydrogen peroxide while the activities of antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase were found to be decreased in the epididymal sperm as well as in cauda epididymides. Lipid peroxidation also increased in the epididymal sperm and in the various regions of the epididymides after exposure to TCDD. The results indicated that TCDD induces oxidative stress in the epididymis and epididymal sperm by decreasing the antioxidant enzymes through induction of reactive oxygen species. Thus, the adverse effects of TCDD on the epididymal sperm were due to direct effect of TCDD on epididymis.