AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences (CPS), VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Toxicol Lett. 2013 Oct 9;222(3):247-56. doi: 10.1016/j.toxlet.2013.07.023. Epub 2013 Aug 2.
Clozapine, an often-prescribed antipsychotic drug, is implicated in severe adverse drug reactions (ADRs). Formation of reactive intermediates by cytochrome P450s (CYPs) has been proposed as a possible explanation for these ADRs. Moreover, a protective role for human glutathione S-transferases (hGSTs) was recently shown using purified enzymes. We investigated the interplay between CYP bioactivation and GST detoxification in a reconstituted cellular context using recombinant yeast expressing a bacterial CYP BM3 mutant (M11), mimicking the drug-metabolizing potential of human CYPs, combined with hGSTA1-1, M1-1 or P1-1. Clozapine and the N-desmethylclozapine metabolite caused comparable growth inhibition and reactive oxygen species (ROS) formation, whereas the clozapine-N-oxide metabolite was clearly less toxic. Clozapine metabolism by BM3 M11 and the hGSTs in yeast was confirmed by identification of stable clozapine metabolites and hGST isoform-specific glutathione-conjugates. Oxidative metabolism of clozapine by BM3 M11 increased ROS formation and growth inhibition. Co-expression of hGSTP1-1 protected yeast from BM3 M11 induced growth inhibition in presence of clozapine, whereas similar expression levels of hGSTA1-1 and hGSTM1-1 did not. ROS formation was not lowered by hGSTP1-1 co-expression and was unrelated to mitochondrial electron transport chain (mETC) activity. We present a novel cellular model to study the effect of CYP and GST interplay in drug toxicity.
氯氮平是一种常用的抗精神病药物，与严重的药物不良反应（ADR）有关。细胞色素 P450s（CYPs）形成的反应中间体被认为是这些 ADR 的可能解释。此外，最近使用纯化酶证明了人类谷胱甘肽 S-转移酶（hGSTs）的保护作用。我们使用表达细菌 CYP BM3 突变体（M11）的重组酵母在重组细胞环境中研究了 CYP 生物活化和 GST 解毒之间的相互作用，该突变体模拟了人类 CYP 的药物代谢能力，并与 hGSTA1-1、M1-1 或 P1-1 结合。氯氮平和 N-去甲基氯氮平代谢物引起相当的生长抑制和活性氧（ROS）形成，而氯氮平-N-氧化物代谢物明显毒性较低。通过鉴定稳定的氯氮平代谢物和 hGST 同工型特异性谷胱甘肽缀合物，证实了酵母中 BM3 M11 和 hGSTs 对氯氮平的代谢。BM3 M11 对氯氮平的氧化代谢增加了 ROS 的形成和生长抑制。在存在氯氮平的情况下，hGSTP1-1 的共表达保护酵母免受 BM3 M11 诱导的生长抑制，而 hGSTA1-1 和 hGSTM1-1 的相似表达水平则没有。hGSTP1-1 的共表达并没有降低 ROS 的形成，与线粒体电子传递链（mETC）活性无关。我们提出了一种新的细胞模型，用于研究 CYP 和 GST 相互作用在药物毒性中的作用。
