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Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

ABSTRACT: Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277-1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1-7, were tentatively identified in human liver microsomal incubations using liquid chromatography-tandem mass spectrometry (LC/ MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1-4, under the same conditions. Of particular interest was the formation of FLU-G5-7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5-7, respectively. The identities of FLU-G5-7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5-7 was completely blocked by the addition of a reversible CPR inhibitor, alpha-lipoic acid, to the incubations of FLU under aerobic conditions. In summary, these results clearly demonstrate that nitroreduction of FLU by CPR contributes to bioactivation and potentially to hepatotoxicity of FLU.

SUBMITTER: Wen B

PROVIDER: S-EPMC2802856 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

Wen Bo B Coe Kevin J KJ Rademacher Peter P Fitch William L WL Monshouwer Mario M Nelson Sidney D SD

Chemical research in toxicology 20081201 12

Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromati ...[more]

PMID: 19548358

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Project description:The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.

Project description:Cyclophosphamide is commonly used as an important component in conditioning prior to hematopoietic stem cell transplantation, a curative treatment for several hematological diseases. Cyclophosphamide is a prodrug activated mainly by cytochrome P450 2B6 (CYP2B6) in the liver. A high degree of inter- and intra-individual variation in cyclophosphamide kinetics has been reported in several studies.Hydroxylation of cyclophosphamide was investigated in vitro using three microsomal batches of CYP2B6*1 with different ratios of POR/CYP expression levels. Twenty patients undergoing hematopoietic stem cell transplantation were also included in the study. All patients received an i.v. infusion of cyclophosphamide (60 mg/kg/day, for two days) as a part of their conditioning. Blood samples were collected from each patient before cyclophosphamide infusion, 6 h after the first dose and before and 6 h after the second dose. POR gene expression was measured by mRNA analysis and the pharmacokinetics of cyclophosphamide and its active metabolite were determined.A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found. The apparent Km for CYP2B6.1 was almost constant (3-4 mM), while the CLint values were proportional to the POR/CYP ratio (3-34 ?L/min/nmol CYP). In patients, the average expression of the POR gene in blood was significantly (P <0.001) up-regulated after cyclophosphamide infusion, with high inter-individual variations and significant correlation with the concentration ratio of the active metabolite 4-hydroxy-cyclophosphamide/cyclophosphamide. Nine patients were carriers for POR*28; four patients had relatively high POR expression.This investigation shows for the first time that POR besides CYP2B6 can influence cyclophosphamide metabolism. Our results indicate that not only CYPs are important, but also POR expression and/or activity may influence cyclophosphamide bioactivation, affecting therapeutic efficacy and treatment related toxicity and hence on clinical outcome. Thus, both POR and CYP genotype and expression levels may have to be taken into account when personalizing treatment schedules to achieve optimal therapeutic drug plasma concentrations of cyclophosphamide.

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Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

Publications

Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

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