ABSTRACT:Thioacetamide (TA), a potent centrilobular hepatotoxicant, undergoes a two-step bioactivation mediated by microsomal CYP2E1 to TA sulfoxide (TASO), and further to TA-S,S-dioxide (TASO 2 ), a reactive metabolite that initiates cellular necrosis. Our earlier studies showed that bioactivation-mediated liver injury of TA is not doseproportional. The objective of this study was to examine whether increasing doses of TA lead to enzyme saturation, thereby resulting in lack of dose-response for injury: bioactivation of TA 3 TASO 3 TASO 2 may follow zero-order kinetics. A 12-fold dose range of TA (50, 300, and 600 mg/kg i.p.) was injected into male SpragueDawley rats. TA and TASO were quantified in plasma, liver, and urine by high-performance liquid chromatography. With increasing doses, the apparent elimination half-lives of TA and TASO increased linearly, indicating that TA bioactivation exhibits saturation kinetics. Increasing TA dose resulted in greater-than-proportional increases in plasma TA and TASO levels. The TASO/TA ratio was inversely proportional to the dose of TA. Covalent binding of 14 C-TA-derived radiolabel to liver macromolecules showed a lessthan-dose-proportionate increase with a 12-fold higher dose. Less than dose-proportional covalent binding was confirmed in liver microsomal incubations with 14 C-TA. Three-fold higher excretion of TASO was seen in urine at the highest dose (600 mg/kg) compared with the lowest dose (50 mg TA/kg). Incubation of TA with rat liver microsomes and purified baculovirus-expressed rat and human CYP2E1 Supersomes, over a concentration range of 0.01 to 10 mM, revealed saturation of TA conversion to TASO at and above 0.05 mM TA concentration, comparable to in vivo plasma and liver levels achieved upon administration of higher doses. Calculated K m values for TA (0.1 mM) and TASO (0.6 mM) suggest that the second step of TA bioactivation is 6-fold less efficient. Collectively, the findings indicate saturation of CYP2E1 at the first (TA to TASO) and second (TASO to TASO 2 ) steps of TA bioactivation.

Acute kidney injury (AKI) is a common and potentially lifethreatening complication after ischemia/reperfusion and exposure to nephrotoxic agents. In this study, we examined the efficacy and mechanism(s) of suramin in promoting recovery from glycerol-induced AKI, a model of rhabdomyolysis-induced AKI. After intramuscular glycerol injection (10 ml of 50% glycerol per kilogram) into male Sprague-Dawley rats, serum creatinine maximally increased at 24 to 72 h and then decreased at 120 h. Creatinine clearance (CrCl) decreased 75% at 24 to 72 h and increased at 120 h. Suramin (1 mg/kg i.v.) administered 24 h after glycerol accelerated recovery of renal function as demonstrated by increased CrCl, decreased renal kidney injury molecule-1, and improved histopathology 72 h after glycerol injection. Suramin treatment decreased interleukin-1␤ (IL-1␤) mRNA, transforming growth factor-␤ 1 (TGF-␤ 1 ), phospho-p65 of nuclear factor-B (NF-B), and cleaved caspase-3 at 48 h compared with glycerol alone. Suramin treatment also decreased glycerol-induced activation of intracellular adhesion molecule-1 (ICAM-1) and leukocyte infiltration at 72 h. Urinary/ renal neutrophil gelatinase-associated lipocalin 2 (NGAL) levels, hemeoxygenase-1 expression, and renal cell proliferation were increased by suramin compared with glycerol alone at 72 h. Mechanistically, suramin decreases early glycerol-induced proinflammatory (IL-1␤ and NF-B) and growth inhibitory (TGF-␤ 1 ) mediators, resulting in the prevention of late downstream inflammatory effects (ICAM-1 and leukocyte infiltration) and increasing compensatory nephrogenic repair. These results support the hypothesis that delayed administration of suramin is effective in abrogating apoptosis, attenuating inflammation, and enhancing nephrogenic repair after glycerol-induced AKI.

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Recent studies in mice suggest that stress-activated c-Jun N-terminal protein kinase 2 (JNK2) plays a pathologic role in acetaminophen (APAP)-induced liver injury (AILI), a major cause of acute liver failure (ALF). In contrast, we present evidence that JNK2 can have a protective role against AILI. When male C57BL/6J wild type (WT) and JNK2 −/− mice were treated with 300mg APAP/kg, 90% of JNK2 −/− mice died of ALF compared to 20% of WT mice within 48 h. The high susceptibility of JNK2 −/− mice to AILI appears to be due in part to deficiencies in hepatocyte proliferation and repair. Therefore, our findings are consistent with JNK2 signaling playing a protective role in AILI and further suggest that the use of JNK inhibitors as a potential treatment for AILI, as has been recommended by other investigators, should be reconsidered. KeywordsAcetaminophen; Cyclin D1; Hepatoprotection; c-Jun N-terminal kinase 2; JNK2; Liver injury; Proliferating cell nuclear antigen; Repair Overdose of APAP, a popular antipyretic and analgesic, is a leading cause of ALF resulting in approximately 1,800 deaths per year in the United States [1]. Although the initiating events in AILI have been attributed to reactive metabolite and protein adduct formation as well as glutathione depletion [2][3][4], the downstream signaling pathways controlling or promoting the severity of AILI have become of a great interest to many researchers [5][6][7][8][9][10][11][12][13][14]. One of these pathways involves JNK, which when activated is known to influence important cellular events, including alterations in gene expression [15], cell death [16], cellular proliferation and survival [17][18][19][20].Recent studies involving AILI in JNK2 −/− mice have led to conflicting results where JNK2 −/− mice were found to be either less susceptible [21] Animals and APAP treatmentSeven to 9 week-old male WT, JNK1 −/− and JNK2 −/− mice on a C57BL/6J background were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were acclimated for at least 1 week to a 12-h light/dark cycle in a humidity and temperature-controlled, specific-pathogenfree environment in microisolator autoclaved cages. Mice were allowed autoclaved food and water ad libitum until experimental use. Before each study, mice were fasted overnight (14-16h; free access to water) to uniformly deplete hepatic GSH stores [23]. Food supplies were restored after intraperitoneal administration of APAP (300mg/kg in warm saline; 20ml/kg) or saline vehicle (20ml/kg). All maintenance of animals conformed to the guidelines for humane treatment set by the Association for Assessment and Accreditation for Laboratory Animal Care International's Guide for the Care and Use of Laboratory Animals and by the National Institutes of Health. Sera and tissue collectionBlood samples were collected and allowed to clot in microtainer serum separator tubes (Becton Dickinson and Co., Franklin Lakes, NJ) for approximately 2h at room temperature and then centrifuged. Serum was separated and used for ALT measurements. A por...

In a recent study, we reported that interleukin (IL)-4 had a protective role against acetaminophen (APAP)-induced liver injury (AILI), although the mechanism of protection was unclear. Here, we carried out more detailed investigations and have shown that one way IL-4 may control the severity of AILI is by regulating glutathione (GSH) synthesis. In the present studies, the protective role of IL-4 in AILI was established definitively by showing that C57BL/6J mice made deficient in IL-4 genetically (IL-4−/−) or by depletion with an antibody, were more susceptible to AILI than mice not depleted of IL-4. The increased susceptibility of IL-4−/− mice was not due to elevated levels of hepatic APAP-protein adducts, but was associated with a prolonged reduction in hepatic GSH that was attributed to decreased gene expression of γ-glutamylcysteine ligase (γ-GCL). Moreover, administration of recombinant IL-4 to IL-4−/− mice post-acetaminophen treatment diminished the severity of liver injury and increased γ-GCL and GSH levels. We also report that the prolonged reduction of GSH in APAP-treated IL-4−/− mice appeared to contribute towards increased liver injury by causing a sustained activation of c-Jun-N-terminal kinase (JNK), since levels of phosphorylated JNK remained significantly higher in the IL-4−/− mice up to 24 hours after APAP treatment Conclusion Overall these results show for the first time that IL-4 has a role in regulating the synthesis of GSH in the liver under conditions of cellular stress. This mechanism appears to be responsible at least in part for the protective role of IL-4 against AILI in mice and may have a similar role not only in AILI in humans, but also in pathologies of the liver caused by other drugs and etiologies.