Human Arylacetamide Deacetylase Is a Principal Enzyme in Flutamide Hydrolysis

Abstract

ABSTRACT:Flutamide, an antiandrogen drug, is widely used for the treatment of prostate cancer. The initial metabolic pathways of flutamide are hydroxylation and hydrolysis. It was recently reported that the hydrolyzed product, 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1), is further metabolized to N-hydroxy FLU-1, an assumed hepatotoxicant. However, the esterase responsible for the flutamide hydrolysis has not been characterized. In the present study, we found that human arylacetamide deacetylase (AADAC) efficiently hydrolyzed flutamide using recombinant AADAC expressed in COS7 cells. In contrast, carboxylesterase1 (CES1) and CES2, which are responsible for the hydrolysis of many drugs, could not hydrolyze flutamide. AADAC is specifically expressed in the endoplasmic reticulum. Flutamide hydrolase activity was highly detected in human liver microsomes (K m , 794 ؎ 83 M; V max , 1.1 ؎ 0.0 nmol/min/mg protein), whereas the activity was extremely low in human liver cytosol. The flutamide hydrolase activity in human liver microsomes was strongly inhibited by bis-(nonylphenyl)-phenylphosphate, diisopropylphosphorofluoride, and physostigmine sulfate (eserine) but moderately inhibited by sodium fluoride, phenylmethylsulfonyl fluoride, and disulfiram. The same inhibition pattern was obtained with the recombinant AADAC. Moreover, human liver and jejunum microsomes showing AADAC expression could hydrolyze flutamide, but human pulmonary and renal microsomes, which do not express AADAC, showed slight activity. In human liver microsomal samples (n ‫؍‬ 50), the flutamide hydrolase activities were significantly correlated with the expression levels of AADAC protein (r ‫؍‬ 0.66, p < 0.001). In conclusion, these results clearly showed that flutamide is exclusively hydrolyzed by AADAC. AADAC would be an important enzyme responsible for flutamideinduced hepatotoxicity.Flutamide (3Ј-trifluoro-2-methyl-4Ј-nitro-2-methyl-propinoylanilide) is a nonsteroidal antiandrogen drug used for the treatment of prostate cancer. The combination of luteinizing hormone-releasing hormone agonist results in prolonged survival in prostate cancer patients (Crawford et al., 1989). However, flutamide occasionally causes severe hepatotoxicity (Thole et al., 2004). Flutamide itself is not toxic when used at the appropriate clinical dose, but bioactivation of flutamide has been considered to be the cause of flutamide-induced hepatotoxicity (Fau et al., 1994).Flutamide is mainly metabolized to 2-hydroxyflutamide by human CYP1A2. It has been suggested that 2-hydroxyflutamide is associated with the therapeutic effect of flutamide (Katchen and Buxbaum, 1975). Flutamide is also hydrolyzed to 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1) by esterase (Katchen and Buxbaum, 1975;Schulz et al., 1988). FLU-1 is considered to have no therapeutic effect (Aizawa et al., 2003). Goda et al. (2006) recently reported that FLU-1 is further metabolized to N-hydroxyl FLU-1 by human CYP3A4. Many researchers have reported on the relationship between the toxicity and metabolism...

Arylacetamide Deacetylase Is a Determinant Enzyme for the Difference in Hydrolase Activities of Phenacetin and Acetaminophen

“…The enzyme preparations were stored at Ϫ80°C until use. The AADAC expression was confirmed with Western blotting according to the previous report (Watanabe et al, 2009). The protein concentrations were determined according to Bradford (1976).…”

“…PNPA, flutamide, imidapril, and CPT-11 hydrolase activities were determined as described previously (Takahashi et al, 2009;Watanabe et al, 2009;Maruichi et al, 2010). The substrate concentrations of PNPA, flutamide, imidapril, and CPT-11 were 200, 500, 100, and 2 M, respectively.…”

“…It was reported that flutamide and imidapril are substrates specific for AADAC and CES1A1, respectively (Takai et al, 1997;Takahashi et al, 2009;Watanabe et al, 2009). On the other hand, it was reported that CPT-11 could be hydrolyzed by both CES1A and CES2, but the catalytic efficiency of CES2 is 60-fold higher than that of CES1A (Humerickhouse et al, 2000).…”

“…The hydrolase activities of PNPA, a general esterase substrate, at a concentration of 200 M by AADAC, CES1A1, and CES2 were 453.6 Ϯ 32.2, 867.6 Ϯ 21.1, and 364.7 Ϯ 17.7 nmol/(min ⅐ mg) protein, respectively. Moreover, flutamide, imidapril, and CPT-11, which are typical substrates for AADAC, CES1A1, and CES2, respectively, were also measured (Takai et al, 1997;Humerickhouse et al, 2000;Watanabe et al, 2009). The flutamide hydrolase activity at a concentration of 500 M was detected by AADAC [0.68 Ϯ 0.03 nmol/(min ⅐ mg) protein] but not by CES1A1 and CES2.…”

“…Thus, AADAC, as well as CES enzymes, is a major serine hydrolase in HLM. AADAC was identified as the enzyme responsible for deacetylation of the carcinogen, 2-acetylaminofluorene (Probst et al, 1991), and we first demonstrated that AADAC is a principal enzyme in the hydrolysis of flutamide, an antiandrogen drug (Watanabe et al, 2009). In this study, we hypothesized that phenacetin is hydrolyzed by AADAC and investigated the difference in the catalytic efficiencies between phenacetin and APAP hydrolysis in HLM and AADAC.…”