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.
Highlights
Since Mehendale and coworkers [26] postulated a role for phospholipase A2 (PLA2) in progression of liver injury induced by APAP toxicity we previously examined its role in APAP hepatotoxicity and the effect of TFP
We previously reported that two neuronal nitric oxide synthase (nNOS) inhibitors (7-nitroindazole and N-[(4S)-4-amino-5-[(2-aminoethyl) amino] pentyl]-N’-nitroguanidinetris (NANT)) blocked APAP toxicity in freshly isolated hepatocytes
In this manuscript we found that addition of TFP at time 0 to the APAP containing hepatocyte incubations inhibited APAP toxicity (Fig. 1), protein nitration (Fig. 2A), nitric oxide (NO) formation (Fig. 2B), reactive oxygen formation (Fig. 2C), and GSNO formation (Fig. 2D)
Summary
We recently reported that the specific nNOS inhibitor NANT inhibited APAP toxicity in freshly isolated hepatocytes and blocked production of reactive nitrogen and oxygen species [19]. These data led to the hypothesis that nNOS is the source of the NO leading to oxidant stress in APAP hepatotoxicity. TFP inhibits the calcium-calmodulin activation of nNOS activity by inhibiting electron flow [27,28,29] In this manuscript we have examined the effect of TFP on APAP toxicity and reactive nitrogen and oxygen formation in freshly isolated hepatocytes, and hepatic reactive nitrogen formation in APAP treated mice
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