Chemical and Enzymatic Transformations of Nimesulide to GSH Conjugates through Reductive and Oxidative Mechanisms

Nimesulide (NIM) is a nonsteroidal anti-inflammatory drug, and clinical treatment with NIM has been associated with severe hepatotoxicity. The bioactivation of nitro-reduced NIM (NIM-NH2), a major NIM metabolite, has been thought to be responsible for the hepatotoxicity of NIM. However, we found that NIM-NH2 did not induce toxic effects in primary rat hepatocytes. This study aimed to investigate other bioactivation pathways of NIM and evaluate their association with hepatotoxicity. After incubating NIM with NADPH- and GSH-supplemented human or rat liver microsomes, we identified two types of GSH conjugates: one was derived from the attachment of GSH to NIM-NH2 (NIM-NH2-GSH) and the other one was derived from a quinone-imine intermediate (NIM-OH-GSH). NIM-NH2-GSH was generated not only by the oxidative activation of NIM-NH2 but also from the reductive activation of NIM. Both NADPH and GSH could act as reducing agents. Moreover, aldehyde oxidase also participated in the reductive activation of NIM. NIM-OH-GSH was generated mainly from NIM via epoxidation with CYP1A2 as the main catalyzing enzyme. NIM was toxic to both primary human and rat hepatocytes, with IC50 values of 213 and 40 μM, respectively. Inhibition of the oxidative and reductive activation of NIM by the nonspecific CYP inhibitor 1-aminobenzotriazole and selective aldehyde oxidase inhibitor estradiol did not protect the cells from NIM-mediated toxicity. Moreover, pretreating cells with l-buthionine-sulfoximine (a GSH depletor) did not affect the cytotoxicity of NIM. These results suggested that oxidative and reductive activation of NIM did not cause the hepatotoxicity and that the parent drug concentration was associated with the cytotoxicity.