title: Reconstitution of the interplay between cytochrome P450 and human glutathione S-transferases in clozapine metabolism in yeast.
abstract: 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.
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences (CPS), VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Galvin
authors.initials: G
authors.lastname: Vredenburg
authors.forename: Kadene P T
authors.initials: KP
authors.lastname: Vassell
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.forename: J Chris
authors.initials: JC
authors.lastname: Vos
mesh_terms: D003024:Clozapine / Q000009:adverse effects / Q000378:metabolism* / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism* / Q000502:physiology; D005982:Glutathione Transferase / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D017382:Reactive Oxygen Species; D003577:Cytochrome P-450 Enzyme System; D005982:Glutathione Transferase; D003024:Clozapine
keywords: 1-chloro-2,4-dinitrobenzene; ADR; Adverse drug reactions; BM3 M11; CDNB; CYP; Clozapine; Cytochrome P450; GSH; Glutathione S-transferase; ROS; Reactive oxygen species; Yeast; adverse drug reaction; cytochrome P450; cytochrome P450 BM3 M11 mutant; glutathione; hGST; human glutathione S-transferase; mETC; mitochondrial electron transport chain; reactive oxygen species