Kuehl Gwendolyn E, Murphy Sharon E
University of Minnesota Cancer Center, Department of Biochemistry, Molecular Biology & Biophysics, Minneapolis, Minnesota 55455, USA.
Drug Metab Dispos. 2003 Nov;31(11):1361-8. doi: 10.1124/dmd.31.11.1361.
Nicotine is considered the major addictive agent in tobacco. Tobacco users extensively metabolize nicotine to cotinine. Both nicotine and cotinine undergo N-glucuronidation. Human liver microsomes have been shown to catalyze the formation of these N-glucuronides. However, which UDP-glucuronosyltransferases contribute to this catalysis has not been identified. To identify these enzymes, we initially measured the rates of glucuronidation by 15 human liver microsome samples. Fourteen of the samples glucuronidated both nicotine and cotinine at rates ranging from 146 to 673 pmol/min/mg protein and 140 to 908 pmol/min/mg protein, respectively. The rates of nicotine glucuronidation and cotinine glucuronidation by these 14 samples were correlated, r = 0.97 (p < 0.0001). The glucuronidation of nicotine and cotinine by heterologously expressed UGT1A3, UGT1A4, and UGT1A9 was also determined. All three enzymes catalyzed the glucuronidation of nicotine. However, the rate of catalysis by UGT1A4 Supersomes was more than 30-fold greater than that by either UGT1A3 Supersomes or UGT1A9 Supersomes. Interestingly, when expressed per UGT1A protein, measured by a UGT1A specific antibody, cell lysate from V79-expressed UGT1A9 catalyzed nicotine glucuronidation at a rate 17-fold greater than did UGT1A9 Supersomes. UGT1A4 Supersomes also catalyzed cotinine N-glucuronidation, but at one-tenth the rate of nicotine glucuronidation. Cotinine glucuronidation by either UGT1A3 or UGT1A9 was not detected. Both propofol, a UGT1A9 substrate, and imipramine, a UGT1A4 substrate, inhibited the glucuronidation of nicotine and cotinine by human liver microsomes. Taken together, these data support a role for both UGT1A9 and UGT1A4 in the catalysis of nicotine and cotinine N-glucuronidation.
尼古丁被认为是烟草中的主要成瘾成分。烟草使用者会将尼古丁大量代谢为可替宁。尼古丁和可替宁都会发生N-葡萄糖醛酸化。已证明人肝微粒体能催化这些N-葡萄糖醛酸苷的形成。然而，尚未确定哪些尿苷二磷酸葡萄糖醛酸基转移酶（UDP- glucuronosyltransferases）参与了这种催化作用。为了鉴定这些酶，我们首先测量了15个人肝微粒体样品的葡萄糖醛酸化速率。其中14个样品对尼古丁和可替宁的葡萄糖醛酸化速率分别为146至673 pmol/分钟/毫克蛋白和140至908 pmol/分钟/毫克蛋白。这14个样品对尼古丁的葡萄糖醛酸化速率和对可替宁的葡萄糖醛酸化速率呈正相关，r = 0.97（p < 0.0001）。我们还测定了异源表达的UGT1A3、UGT1A4和UGT1A9对尼古丁和可替宁的葡萄糖醛酸化作用。这三种酶都能催化尼古丁的葡萄糖醛酸化。然而，UGT1A4微粒体的催化速率比UGT1A3微粒体或UGT1A9微粒体的催化速率高30倍以上。有趣的是，当用UGT1A特异性抗体测量，以每个UGT1A蛋白表达量来衡量时，V79细胞表达的UGT1A9的细胞裂解液催化尼古丁葡萄糖醛酸化的速率比UGT1A9微粒体高17倍。UGT1A4微粒体也能催化可替宁的N-葡萄糖醛酸化，但速率仅为尼古丁葡萄糖醛酸化速率的十分之一。未检测到UGT1A3或UGT1A9对可替宁的葡萄糖醛酸化作用。UGT1A9的底物丙泊酚和UGT1A4的底物丙咪嗪都能抑制人肝微粒体对尼古丁和可替宁的葡萄糖醛酸化。综上所述，这些数据支持UGT1A9和UGT1A4在催化尼古丁和可替宁的N-葡萄糖醛酸化过程中发挥作用。
