Identification of the UDP-glucuronosyltransferase isozyme involved in senecionine glucuronidation in human liver microsomes.
Senecionine (SEN) is a representative of the hepatotoxic pyrrolizidine alkaloids. Although phase I metabolism for cytochrome P450-mediated metabolic activation of SEN was investigated extensively, phase II metabolism for glucuronidation of this compound has not been investigated until now. In our present study, one unique glucuronidation product of SEN in human liver microsomes (HLMs) was identified as SEN N-glucuronide using an authentically synthesized product for which the structure was identified via (1)H and (13)C NMR analysis. Subsequently, kinetics indicated that SEN N-glucuronidation followed the typical Michaelis-Menten model and only one major isozyme participated in it. Finally, this isozyme was demonstrated to be UDP-glucuronosyltransferase (UGT) 1A4, with the direct evidence that recombinant UGT1A4 exhibited predominant and exclusive activity on SEN N-glucuronidation. This result was confirmed by other experiments including chemical inhibition by selective inhibitors and a correlation study between activities of SEN N-glucuronidation and various UGT isozymes. The exclusive role of UGT1A4 on SEN N-glucuronidation was strengthened additionally by its inhibitory kinetic study in which the selective inhibitor of UGT1A4 showed a similar inhibition pattern and K(i) values in both HLM and recombinant UGT1A4 systems. Because UGT2B10 activity failed to correlate with SEN N-glucuronidation in HLMs from 10 individuals, it was impossible for UGT2B10 to play an important role in this metabolism.
He
Yu-Qi
YQ
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, China.
Liu
Yong
Y
Zhang
Bin-Feng
BF
Liu
Hui-Xin
HX
Lu
Yan-Liu
YL
Yang
Li
L
Xiong
Ai-zhen
AZ
Xu
Ling-Ling
LL
Wang
Chang-Hong
CH
Yang
Ling
L
Wang
Zheng-Tao
ZT
D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000378:metabolism*; D011763:Pyrrolizidine Alkaloids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012502:Sapogenins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D000972:Antineoplastic Agents, Phytogenic; D020719:Glucuronides; D007202:Indicators and Reagents; D007527:Isoenzymes; D011763:Pyrrolizidine Alkaloids; D011994:Recombinant Proteins; D012502:Sapogenins; C019787:bilirubin glucuronoside glucuronosyltransferase; C007908:hecogenin; C009237:senecionine; D014453:Glucuronosyltransferase