Article

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

Department of Drug Metabolism and Pharmacokinetics, Roche Palo Alto, Palo Alto, California 94304, USA.
Chemical Research in Toxicology (impact factor: 3.78). 01/2009; 21(12):2393-406. DOI:10.1021/tx800281h pp.2393-406
Source: PubMed

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.

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  • Article: Distribution of nitroreductive activity toward nilutamide in rat.
    Kjetil Ask, Nathalie Décologne, Nana Asare, Jørn A Holme, Yves Artur, Hélène Pelczar, Philippe Camus
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    ABSTRACT: Nilutamide is a pneumotoxic and hepatotoxic nitroaromatic (R-NO2) antiandrogen used in the treatment of prostate carcinoma in man. Previously, we established that in the rat lung, the drug is metabolized into the corresponding hydroxylamine (R-NHOH) and amine (R-NH2) derivatives. These results evidenced a cytosolic oxygen-sensitive (type II) nitroreductase activity in lung. In the present studies, we extended the characterization of nilutamide metabolism in liver, brain, kidney, heart, blood, intestine (small, cecum, and large, and their respective luminal contents) of male Sprague-Dawley rats. Subcellular fractions for all tissues (except blood) examined (postmitochondrial, cytosolic, and microsomal) were prepared by differential ultracentrifugation. Blood and intestinal contents were sonicated before investigation. Incubations were run in the presence or absence of O2 to assess type I and II nitroreductase activities. Organic extracts were analyzed by HPLC methods and results were expressed as pmoles of R-NH2 formed per milligram protein per minute. Four distinct nitroreductive activities were evidenced. Cytosolic and microsomal type II nitroreductase activities were detected in all tissue samples studied. Type I NR activity was not observed in any of the cytosols, but was detected in the small intestine, lung, kidney, and liver microsomes. Nilutamide was also reduced in the intestinal lumen, possibly by a bacterial type I nitroreductase. Highest activities were observed in cytosols and were oxygen sensitive. These results evidence and characterize previously unknown nitroreductive activities toward nilutamide in rat tissues that might provide some explanation to the side effects of nilutamide and other nitroaromatic compounds observed in human therapeutics.
    Toxicology and Applied Pharmacology 12/2004; 201(1):1-9. · 4.45 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

aerobic conditions
 
alpha-lipoic acid
 
amino group
 
anaerobic conditions
 
CPR contributes
 
cryopreserved human hepatocytes
 
cytochrome P450
 
FLU-induced hepatotoxicity [Coe
 
form corresponding para-diimine intermediates
 
form GSH adducts FLU-G5-7
 
four corresponding GSH adducts
 
human liver microsomal incubations
 
liver microsomal incubations
 
microsomal incubations
 
nitroaromatic group
 
nitroreduction metabolite FLU-6
 
nonsteroidal antiandrogen drug
 
reactive metabolite(s)
 
synthesized standard FLU-6
 
tentative pathway