RT Journal Article
SR Electronic
T1 Evaluation of Carbazeran 4-Oxidation and O6-Benzylguanine 8-Oxidation as Catalytic Markers of Human Aldehyde Oxidase: Impact of Cytosolic Contamination of Liver Microsomes
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP dmd.118.082099
DO 10.1124/dmd.118.082099
A1 Jiarong Xie
A1 Nur Fazilah Saburulla
A1 Shiyan Chen
A1 Siew Ying Wong
A1 Ze Ping Yap
A1 Linghua Harris Zhang
A1 Aik Jiang Lau
YR 2018
UL http://dmd.aspetjournals.org/content/early/2018/10/18/dmd.118.082099.abstract
AB The current study investigated the contribution of microsomal cytochrome P450 and cytosolic aldehyde oxidase-1 (AOX-1) to carbazeran 4-oxidation and O6-benzylguanine 8-oxidation in human liver microsomal, cytosolic, and S9 fractions. Incubations containing carbazeran and human liver microsomes with or without exogenously added NADPH yielded comparable levels of 4-oxo-carbazeran. O6-Benzylguanine 8-oxidation occurred in microsomal incubations, and the extent was increased by NADPH. Human recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 did not catalyze carbazeran 4-oxidation, whereas CYP1A2 was highly active in O6-benzylguanine 8-oxidation. 1-Aminobenzotriazole, a pan-cytochrome P450 inhibitor, decreased O6-benzylguanine 8-oxidation, but not carbazeran 4-oxidation, in microsomal incubations, whereas 1-aminobenzotriazole and furafylline (a CYP1A2-selective inhibitor) did not inhibit carbazeran 4-oxidation or O6-benzylguanine 8-oxidation in human liver S9 fraction. Carbazeran 4-oxidation in incubations containing human liver microsomes (from multiple donors and commercial suppliers) was attributed to microsomal preparations contaminated with AOX-1, as suggested by liver microsomal experiments indicating a decrease in carbazeran 4-oxidation by an AOX-1 inhibitor (hydralazine), and detection of AOX-1 protein (at one-third the level of that in liver cytosol). Cytosolic contamination of liver microsomes was further demonstrated by the formation of dehydroepiandrosterone sulfate (catalyzed by cytosolic sulfotransferases) in liver microsomal incubations containing dehydroepiandrosterone. In conclusion, carbazeran 4-oxidation and O6-benzylguanine 8-oxidation are enzyme-selective catalytic markers of human AOX-1, as shown in human liver S9 fraction expressing cytochrome P450 and AOX-1. This study highlights the negative impact of cytosolic contamination of liver microsomes on the interpretation of reaction phenotyping data collected in an in vitro study performed in microsomal fractions.