Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes

摘要

Abbreviations: APAP, Acetaminophen; NAPQI, N-Acetyl-p-benzoquinone imine; GSH, reduced glutathione; APAP-cys, 3-(cystein-S-yl)-acetaminophen; NO, Nitric Oxide; iNOS, Inducible nitric oxide synthase (NOS2); nNOS, Neuronal nitric oxide synthase (NOS1); GSNO, S-Nitrosoglutathione; GSSG, glutathione disulfide; NANT, N-[(4S)-4-amino-5-[(2-aminoethyl) amino] pentyl]-N’-nitroguanidinetris (trifluoroacetate); MPT, Mitochondrial Permeability Transition; LDH, Lactate Dehydrogenase; OCR, Oxygen Consumption Rate; 3-NT, 3-Nitrotyrosine; TFP, TrifluoperazineKeywords: Acetaminophen, Neuronal nitric oxide, Oxidative stress, Mitochondria

Abstract

The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.