UDP-glucuronosyltransferases (UGTs) play an important role in metabolism and detoxification of amine-containing chemicals. However, the disposition mechanisms for amines via UGT metabolism are not fully clear. Here we aim to investigate a potential role of UGT2B10 in N-glucosidation, and to determine the transporters for excretion of N-glucosides. We first established a HEK293 cell line stably overexpressing UGT2B10. Incubation of mianserin or cyclizine with the cells generated one N-glucuronide and one N-glucoside. Chemical inhibition (using specific chemical inhibitor Ko143) and biological inhibition (using specific shRNA) of BCRP resulted in a significant reduction in efflux of N-glucuronide. Similar results were observed when MRP4 was inhibited. Furthermore, inhibition of BCRP led to increased intracellular N-glucoside, whereas inhibition of MRP4 caused no changes in disposition of N-glucosides. The data overall indicated that BCRP not MRP4 was responsible for excretion of N-glucosides, whereas both BCRP and MRP4 contributed to excretion of N-glucuronides. Interestingly, down-regulation of N-glucuronidation led to increased N-glucosidation in the cells, supporting the glucosidation as a potential complementary pathway for conventional glucuronidation. In conclusion, UGT2B10 was for the first time identified as a N-glucosidation enzyme. Generated N-glucosides were excreted primarily by the BCRP transporter.
N -glucuronidation is an important pathway for metabolism and disposition of tertiary amines in humans. This reaction is mainly catalyzed by the enzymes UGT1A4 and UGT2B10. However, the metabolic patterns of UGT1A4- and UGT2B10-mediated N -glucuronidation are not fully clear. In this study, we first optimized in vitro reaction conditions for N -glucuronidation by using specific substrates (i.e., trifluoperazine for UGT1A4, cotinine and amitriptyline for UGT2B10). Furthermore, we found that hepatic N -glucuronidation showed significant species differences. In addition, UGT1A4 and UGT2B10 were primarily responsible for N -glucuronidation of many tertiary amines, including asenapine, loxapine, clozapine, chlorpromazine, dothiepin, doxepin, mirtazapine, mianserin, chlorcyclizine, cyclizine, promethazine, cyclobenzaprine, imatinib, retrorsine, strychnine and brucine. In conclusion, this study provides an in vitro assay system for evaluating N -glucuronidation of amines. Also, UGT1A4- and UGT2B10-mediated N -glucuronidation might play significant roles in metabolism and detoxification of tertiary amines in humans.
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