Nicotine Glucuronidation and the Human UDP-Glucuronosyltransferase UGT2B10

Abstract

Nicotine biotransformation affects the smoking habits of addicted individuals and therefore their health risk. Using an improved analytical method, we have discovered that the human UDP-glucuronosyltransferase (UGT) 2B10, a liver enzyme previously unknown to conjugate nicotine or exhibit considerable activity toward any compound, plays a major role in nicotine inactivation by direct conjugation with glucuronic acid at the aromatic nitrogen atom. The K m value of recombinant UGT2B10 for nicotine (0.29 mM) was similar to that determined for human liver microsomes (0.33 mM), whereas the K m value of UGT1A4 for nicotine was almost 10-fold greater (2.4 mM). UGT2B10 was also more active than UGT1A4 in N-glucuronidation of cotinine (oxidative nicotine metabolite), whereas UGT2B7 exhibited only low nicotine glucuronidation activity and was essentially inactive toward cotinine. UGT1A9 did not glucuronidate nicotine or cotinine. Quantitative reverse transcription-polymerase chain reaction showed that UGT2B10 mRNA was exclusively expressed in human liver, whereas UGTs 1A4 and 2B7 were expressed at comparable, although somewhat lower, levels in liver and several other extrahepatic tissues, including kidney and intestine. These findings for UGT2B10 (but not for UGT1A4 and UGT2B7) were mirrored by human tissue activities because nicotine and cotinine glucuronidation rates in intestine microsomes were less than 0.1% that of human liver microsomes. These novel findings solve two seemingly separate questions: which UGT is primarily responsible for nicotine glucuronidation in human liver, and what conjugation reactions are catalyzed by UGT2B10.Nicotine is not carcinogenic by itself and might even have some beneficial therapeutic effects in some neurological diseases. Nonetheless, it is the major perpetrator of tobaccorelated diseases because nicotine addiction drives smokers to pursue the habit despite the known health hazards. Nicotine concentration in the blood increases sharply during cigarette smoking and then decreases rapidly because of metabolism and clearance, driving the addicted individual to reach for another cigarette. Hence, better understanding of nicotine metabolism can assist the development of treatments to reduce the health risks associated with nicotine addiction.Cytochrome P450 monooxygenase 2A6 (CYP2A6) catalyzes nicotine oxygenation and plays an important role in nicotine metabolism (Hukkanen et al., 2005;Nakajima and Yokoi, 2005). However, there is more to nicotine metabolism than CYP2A6 because both nicotine and its primary oxidation metabolite, cotinine, undergo direct N-glucuronidation at the aromatic nitrogen (Fig.

Human UDP-Glucuronosyltransferase (UGT) 2B10: Validation of Cotinine as a Selective Probe Substrate, Inhibition by UGT Enzyme-Selective Inhibitors and Antidepressant and Antipsychotic Drugs, and Structural Determinants of Enzyme Inhibition

“…Although initially considered an orphan enzyme, more recent studies have shown that UGT2B10, like UGT1A4, catalyzes the N-glucuronidation of a number of xenobiotics that incorporate an aliphatic tertiary amine or aromatic N-heterocyclic group (Kaivosaari et al, 2011). Known substrates are nicotine and its oxidation product cotinine (Chen et al, 2007;Kaivosaari et al, 2007); desloratadine (Kazmi et al, 2015a); medetomidine (Kaivosaari et al, 2008); the tricyclic antidepressants (TCAs) amitriptyline, clomipramine, imipramine, and trimipramine (Chen et al, 2007;Zhou et al, 2010;Kato et al, 2013); several tobacco-specific nitrosamines (Chen et al, 2008); RO5263397 (Fowler et al, 2015); and miscellaneous drugs that include diphenhydramine, ketoconazole, ketotifen, midazolam, olanzapine, pizotifen, and tamoxifen (Erickson-Ridout et al, 2011;Kato et al, 2013). Consistent with the known selectivity of UGT1A4 for N-glucuronidation (Kubota et al, 2007), most, if not all, UGT2B10 substrates are additionally glucuronidated by UGT1A4 and biphasic kinetics are frequently observed when human liver microsomes (HLM) are used as the enzyme source (Kaivosaari et al, 2011;Kato et al, 2013).…”

Bucolome N-Glucuronide Formation: Species Differences and Identification of Human UDP-Glucuronosyltransferase Isoforms

“…However, BCP-NG formation by hUGT1A4 was <10 pmol equivalents/min/mg protein (Figure 3). The level of hUGT1A4 mRNA in tissues other than the liver is either very low [18,19] or below the detection limit [25]. Therefore, it seems that the contribution of UGT1A4 to BCP-NG formation is negligible.…”

“…In recent years, it has been reported that hepatic UGT1A4 and 2B10 catalyze the N-glucuronidation of aromatic N-heterocycles in humans [18,19]. However, BCP-NG formation by hUGT1A4 was <10 pmol equivalents/min/mg protein (Figure 3).…”

“…Therefore, it seems that the contribution of UGT1A4 to BCP-NG formation is negligible. hUGT2B10 has only been detected in the liver and small intestine [18,19], and the level of hUGT2B10 mRNA in the small intestine is only 0.05% of that in the liver. The ortholog of human 2B10 has not been detected in animals [20], while the UGT1A1 family has been detected in various species [20,[26][27] and a number of different tissues [28][29][30].…”

“…A number of aromatic N-heterocycles with five-and six-member rings, such as imidazoles, pyrazoles, triazines, tetrazoles, and pyridines are subject to N-glucuronidation. However, N-glucuronidation of the pyrimidine skelton of barbiturates has not been mentioned in recent review articles [18][19][20].…”