Metabolism and hepatic toxicity of flutamide in cytochrome P450 1A2 knockout SV129 mice

ABSTRACT:Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH-and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.

Section: Discussionsupporting

confidence: 91%

“…Ichimura et al (1999) have also illustrated the necessity of enhanced flutamide metabolism for development of severe hepatotoxicity. More recently, Matsuzaki et al (2006) have demonstrated flutamide-induced toxicity after administering flutamide to CYP1A2 knockout SV129 mice.…”

Section: Flutamidementioning

confidence: 99%

Identification of a Novel Glutathione Conjugate of Flutamide in Incubations with Human Liver Microsomes

Kang¹,

Dalvie²,

Smith³

et al. 2007

“…Ichimura and coworkers have also illustrated the necessity of enhanced flutamide metabolism for development of severe hepatotoxicity (Ichimura et al, 1999). More recently, Matsuzaki and coworkers have demonstrated flutamide-induced toxicity in CYP1A2 knockout SV129 mice administered with 400 mg/kg dose after the mice were fed with an amino acid-deficient diet for 2 weeks, which reduced the glutathione (GSH) content to 27% of the initial content (Matsuzaki et al, 2006). In vitro conjugation with GSH is widely used in the characterization of reactive metabolites in probing the mechanism of bioactivation (Samuel et al, 2003).…”

mentioning

confidence: 99%

Bioactivation of Flutamide Metabolites by Human Liver Microsomes

Kang¹,

Dalvie²,

Smith³

et al. 2008

ABSTRACT:Flutamide, a widely used nonsteroidal antiandrogen drug for the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. A novel N-S glutathione adduct has been identified in a previous bioactivation study of flutamide (Kang et al., 2007). Due to the extensive first pass metabolism, flutamide metabolites such as 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)phenylamine (Flu-1) have achieved plasma concentrations higher than the parent in prostate cancer patients. In vitro studies in human liver microsomes were conducted to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide metabolites and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. Several GSH adducts (G1, Flu-1-G1, Flu-1-G2, Flu-6-Gs) derived from the metabolites of flutamide were identified and characterized. A comprehensive bioactivation mechanism was proposed to account for the formation of the observed GSH adducts. Of interest were the formation of a reactive intermediate by the desaturation of the isopropyl group of M5 and the unusual bioactivation of Flu-1. Studies using recombinant P450s suggested that the major P450 isozymes involved in the bioactivation of flutamide and its metabolites were CYP1A2, CYP3A4, and CYP2C19. These findings suggested that, in addition to the direct bioactivation of flutamide, the metabolites of flutamide could also be bioactivated and contribute to flutamideinduced hepatotoxicity.2-Methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide (flutamide), a widely prescribed nonsteroidal antiandrogen drug, has been shown to increase survival time of prostate cancer patients in combination therapy with luteinizing hormone-releasing agonists or orchiectomy (Brogden and Clissold, 1989;McLeod, 1993;Schmitt et al., 2001). However, a number of hepatotoxicity cases were reported to be associated with the clinical use of this drug, such as temporary increases in transaminase markers and rare incidences of severe liver dysfunction (Gomez et al

“…Plasma concentration of FLU-1 was considerably higher in patients who had exhibited liver dysfunction, and lower CYP1A2 activity in patients was found to be associated with onset of liver injury (Ozono et al, 2002;Aizawa et al, 2003). In addition, Cyp1a2-null mice that mainly produced FLU-1 displayed hepatotoxicity by continuous administration of flutamide in contrast to the metabolites formed by wild-type mice (Matsuzaki et al, 2006). These findings suggest that FLU-1 may possibly mediate flutamide-induced liver injury.…”

mentioning

confidence: 85%

“…This flutamide-induced liver injury is not acute but delayed (Thole et al, 2004;Manso et al, 2006). Although reactive metabolites of flutamide have been considered to be involved in hepatotoxicity induction (Berson et al, 1993;Fau et al, 1994;Matsuzaki et al, 2006;Tevell et al, 2006;Kang et al, 2007), the mechanism of flutamideinduced hepatotoxicity in humans remains obscure.…”

mentioning

confidence: 99%

Role of EnzymaticN-Hydroxylation and Reduction in Flutamide Metabolite-Induced Liver Toxicity

Ohbuchi¹,

Miyata²,

Nagai³

et al. 2008

Self Cite

ABSTRACT:Flutamide is used for prostate cancer therapy but occasionally induces severe liver injury. Flutamide is hydrolyzed in the body into 5-amino-2-nitrobenzotrifluoride (FLU-1) and then further oxidized. In our previous study, N-hydroxy FLU-1 (FLU-1 N-OH) was detected in the urine of patients and exhibited cytotoxicity in rat primary hepatocytes. In the present study, we have assessed the roles of FLU-1 N-oxidation and hepatic glutathione (GSH) depletion in liver injury. FLU-1 (200 mg/kg p.o.) was administered to C57BL/6 mice for 5 days together with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) (3 mg/kg i.p.) for the first 3 days. Mice were fasted for the last 2 days to deplete hepatic GSH. Administration of FLU-1 alone did not affect serum alanine aminotransferase activities (ALT), whereas coadministration of FLU-1 and TCPOBOP significantly increased ALT in fasted mice but not in nonfasted mice.Microsomal FLU-1 N-hydroxylation was enhanced approximately 5 times by TCPOBOP treatment. Flutamide metabolite-protein adducts were detected in liver microsomes incubated with FLU-1 N-OH, but not with FLU-1 and flutamide, by immunoblotting using antiflutamide antiserum. In the presence of mouse liver cytosol, FLU-1 N-OH was reduced back into FLU-1. This enzymatic reduction required NAD(P)H as a cofactor. The reduction was enhanced by the coexistence of NAD(P)H and GSH, whereas it was markedly inhibited by allopurinol (20 M). By using purified bovine xanthine oxidase, the reduction was observed in the presence of NAD(P)H. These results suggest that FLU-1 N-OH is involved in flutamideinduced hepatotoxicity and that cytosolic reduction of FLU-1 N-OH plays a major role in protection against flutamide-induced hepatotoxicity.