Venkataraman Harini, den Braver Michiel W, Vermeulen Nico P E, Commandeur Jan N M
Division of Molecular Toxicology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Faculty of Sciences, Vrije Universiteit , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Chem Res Toxicol. 2014 Dec 15;27(12):2071-81. doi: 10.1021/tx500288b. Epub 2014 Nov 18.
Mefenamic acid (MFA) has been associated with rare but severe cases of hepatotoxicity, nephrotoxicity, gastrointestinal toxicity, and hypersensitivity reactions that are believed to result from the formation of reactive metabolites. Although formation of protein-reactive acylating metabolites by phase II metabolism has been well-studied and proposed to be the cause of these toxic side effects, the oxidative bioactivation of MFA has not yet been competely characterized. In the present study, the oxidative bioactivation of MFA was studied using human liver microsomes (HLM) and recombinant human P450 enzymes. In addition to the major metabolite 3'-OH-methyl-MFA, resulting from the benzylic hydroxylation by CYP2C9, 4'-hydroxy-MFA and 5-hydroxy-MFA were identified as metabolites resulting from oxidative metabolism of both aromatic rings of MFA. In the presence of GSH, three GSH conjugates were formed that appeared to result from GSH conjugation of the two quinoneimines formed by further oxidation of 4'-hydroxy-MFA and 5-hydroxy-MFA. The major GSH conjugate was identified as 4'-OH-5'-glutathionyl-MFA and was formed at the highest activity by CYP1A2 and to a lesser extent by CYP2C9 and CYP3A4. Two minor GSH conjugates resulted from secondary oxidation of 5-hydroxy-MFA and were formed at the highest activity by CYP1A2 and to a lesser extent by CYP3A4. Additionally, the ability of seven human glutathione S-transferases (hGSTs) to catalyze the GSH conjugation of the quinoneimines formed by P450s was also investigated. The highest increase of total GSH conjugation was observed with hGSTP1-1, followed by hepatic hGSTs hGSTA2-2 and hGSTM1-1. The results of this study show that, next to phase II metabolites, reactive quinoneimines formed by oxidative bioactivation might also contribute to the idiosyncratic toxicity of MFA.
甲芬那酸（MFA）与罕见但严重的肝毒性、肾毒性、胃肠道毒性及过敏反应有关，据信这些反应是由活性代谢物的形成所致。尽管II相代谢产生的蛋白质反应性酰化代谢物的形成已得到充分研究，并被认为是这些毒副作用的原因，但MFA的氧化生物活化尚未完全明确。在本研究中，利用人肝微粒体（HLM）和重组人P450酶研究了MFA的氧化生物活化。除了由CYP2C9进行苄基羟基化产生的主要代谢物3'-羟基甲基-MFA外，4'-羟基-MFA和5-羟基-MFA也被鉴定为MFA两个芳香环氧化代谢产生的代谢物。在谷胱甘肽（GSH）存在的情况下，形成了三种GSH缀合物，它们似乎是由4'-羟基-MFA和5-羟基-MFA进一步氧化形成的两种醌亚胺与GSH结合产生的。主要的GSH缀合物被鉴定为4'-OH-5'-谷胱甘肽基-MFA，由CYP1A2以最高活性形成，CYP2C9和CYP3A4形成的程度较小。两种次要的GSH缀合物是由5-羟基-MFA的二次氧化产生的，由CYP1A2以最高活性形成，CYP3A4形成的程度较小。此外，还研究了七种人谷胱甘肽S-转移酶（hGST）催化P450形成的醌亚胺与GSH结合的能力。观察到hGSTP1-1使总GSH结合增加最多，其次是肝脏hGST hGSTA2-2和hGSTM1-1。本研究结果表明，除了II相代谢物外，氧化生物活化形成的活性醌亚胺也可能导致MFA的特异质性毒性。
