Human multidrug resistance protein 4 (MRP4) is a cellular efflux transporter for paracetamol glutathione and cysteine conjugates.

Paracetamol (acetaminophen, APAP) overdose is a leading cause of acute drug-induced liver failure. APAP hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is inactivated by conjugation with glutathione (GSH) to APAP-GSH, which is further converted into its cysteine derivative APAP-CYS. Before necrosis of hepatocytes occurs, APAP-CYS is measurable in plasma of the affected patient and it has been proposed as an early biomarker of acetaminophen toxicity. APAP-GSH and APAP-CYS can be extruded by hepatocytes, but the transporters involved are unknown. In this study we examined whether ATP-binding cassette (ABC) transporters play a role in the cellular efflux of APAP, APAP-GSH, and APAP-CYS. The ABC transport proteins P-gp/ABCB1, BSEP/ABCB11, BCRP/ABCG2, and MRP/ABCC1-5 were overexpressed in HEK293 cells and membrane vesicles were produced. Whereas P-gp, BSEP, MRP3, MRP5, and BCRP did not transport any of the compounds, uptake of APAP-GSH was found for MRP1, MRP2 and MRP4. APAP-CYS appeared to be a substrate of MRP4 and none of the ABC proteins transported APAP. The results suggest that the NAPQI metabolite APAP-CYS can be excreted into plasma by MRP4, where it could be a useful biomarker for APAP exposure and toxicity. Characterization of the cellular efflux of APAP-CYS is important for its development as a biomarker, because plasma concentrations might be influenced by drug-transporter interactions and upregulation of MRP4.

Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants.

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated inactivation of reactive quinoneimine metabolites of diclofenac and mefenamic acid.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4'- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites.

Reconstitution of the interplay between cytochrome P450 and human glutathione S-transferases in clozapine metabolism in yeast.

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.

Metabolism related toxicity of diclofenac in yeast as model system.

Diclofenac is a widely used drug that can cause serious hepatotoxicity, which has been linked to metabolism by cytochrome P450s (P450). To investigate the role of oxidative metabolites in diclofenac toxicity, a model for P450-related toxicity was set up in Saccharomyces cerevisiae. We expressed a drug-metabolizing mutant of cytochrome P450 BM3 (BM3 M11) in yeast. Importantly, BM3 M11 yielded similar oxidative metabolite profiles of diclofenac as human P450s. It was found that yeast strains expressing BM3 M11 grew significantly slower when exposed to diclofenac than strains without BM3 M11. Furthermore, the amount of reactive oxygen species (ROS) after incubation with diclofenac was higher in strains expressing BM3 M11 than in strains without this enzyme, confirming that P450 activity increases diclofenac toxicity. Interestingly, 4'- and 5-hydroxydiclofenac had no effect on cell growth or ROS formation in cells expressing BM3 M11, although hydroxydiclofenac-derived quinone imines were identified in these strains by detection of their glutathione conjugates. This suggests that 4'- and 5-hydroxydiclofenac, as well as their quinone imines, are not involved in toxicity in yeast. Rather, the P450-related toxicity of diclofenac is caused by primary metabolites such as arene oxides resulting in hydroxydiclofenac or radical species formed during decarboxylation.