ABSTRACT:In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5-oxidation. The nicotine ⌬ Nicotine is the major addictive agent in tobacco (Benowitz, 1988), and 5Ј-oxidation of this alkaloid is the primary pathway of its metabolism in smokers (Gorrod and Schepers, 1999). The product of this reaction, 5Ј-hydroxynicotine, is in equilibrium with nicotine ⌬ 5Ј(1Ј) -iminium ion (Fig. 1). The formation of cotinine from the iminium ion is catalyzed by a cytosolic enzyme, aldehyde oxidase (Brandänge and Lindblom, 1979). In humans, P450 2A6 is the major catalyst of nicotine 5Ј-oxidation (Cashman et al., 1992;Nakajima et al., 1996;Messina et al., 1997;Yamazaki et al., 1999), and the extent of nicotine 5Ј-oxidation by individuals who do not express P450 2A6 is decreased Ͼ85% (Kitagawa et al., 1999;Yamanaka et al., 2004). A role for P450 2A6 in nicotine 5Ј-oxidation in human liver microsomes was first reported by Cashman et al. (1992) and subsequently by Nakajima et al. (1996) and Messina et al. (1997). The latter two groups also reported kinetic parameters for cotinine formation by lymphoblastoid expressing P450 2A6 when coincubated with aldehyde oxidase. A more recent publication reported kinetic parameters for cotinine formation by P450 2A6 heterologously expressed in baculovirus/Sf21 cells (Yamazaki et al., 1999). In all of these studies, cotinine was monitored, not the ⌬ 5Ј(1Ј) -iminium ion or any other nicotine metabolites.Several years ago, we characterized the formation of amino ketone (Fig. 1) by P450 2A6 and human liver microsomes (Hecht et al., 2000). Amino ketone is a product of nicotine 2Ј-oxidation that is in equilibrium with 2Ј-hydroxynicotine and the ⌬ 2Ј(1Ј) -iminium ion (Fig. 1). Nicotine may also be oxidized at the methyl carbon to form NЈ-(hydroxymethyl)nornicotine that exists in equilibrium with the methylene-iminium ion (Fig. 1). It is unknown whether NЈ-(hydroxymethyl)-nornicotine is a product of either P450 2A6 or human liver microsomal metabolism. However, nornicotine, a nonenzymatic decomposition product of NЈ-(hydroxymethyl)nornicotine is a metabolite of nicotine in smokers (Benowitz et al., 1994). In the current study, the relative rates of nicotine 5Ј-, 2Ј-, and methyl-oxidation by P450 2A6 are investigated and compared with the rates for P450 2A13, an extrahepatic P450 thought to be important in the metabolic activation of the tobacco-specific lung carcinogen NNK (Jalas et al., 2005). In addition, nicotine metabolism by rodent P450 2A enzymes is studied.Rats and mice are routinely used to characterize the addictive effects of nicotine; however, there is minimal data on what P450s catalyze nicotine 5Ј-oxidation in these animals. There are data supporting a role for P450 2B1 as the catalyst of nicotine metabolism in the rat liver (Hammond et al., 1991;Nakayama et al., 1993). Nicotine metabolism by mouse P450s has not been reported previously. However, in both the lung and the liver of mice, there are two enzymes, P450 2A4 and 2A5, that are closely related to the human P45...

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Nicotine is the major addictive agent in tobacco. The primary catalyst of nicotine metabolism in humans is CYP2A6. However, the closely related enzyme CYP2A13 is a somewhat better catalyst. CYP2A13 is an extrahepatic enzyme that is an excellent catalyst of the metabolic activation of the tobaccospecific carcinogen 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK). Here we report that both CYP2A6 and CYP2A13 were inactivated during nicotine metabolism. Inactivation of both enzymes was dependent on NADPH and increased with time and concentration. Alternate substrates for CYP2A6 and CYP2A13 protected these enzymes from inactivation. Inactivation of CYP2A13 was irreversible upon extensive dialysis and seems to be mechanism-based. The K I of CYP2A13 inactivation by nicotine was 17 M, the rate of inactivation, k inact , was 0.1 min Ϫ1 , and the t 1/2 was 7 min. However, the loss in enzyme activity occurred after nicotine metabolism was complete, suggesting that a secondary or possible tertiary metabolite of nicotine may be responsible. H]Nicotine metabolism by CYP2A13 was monitored by radioflow high-pressure liquid chromatography during the course of enzyme inactivation; the major product was the ⌬ 1Ј(5Ј) iminium ion. However, cotinine was a significant metabolite even at short reaction times. The metabolism of the nicotine ⌬ 1Ј(5Ј) iminium ion to cotinine did not require the addition of aldehyde oxidase. CYP2A13 catalyzed this reaction as well as further metabolism of cotinine to 5Ј-hydroxycotinine, trans-3Ј-hydroxycotinine, and N-(hydroxymethyl)-norcotinine as enzyme inactivation occurred. Studies are on-going to identify the metabolite responsible for nicotinemediated inactivation of CYP2A13.Nicotine is the major addictive agent in tobacco, and individuals continue to smoke and use tobacco products to maintain plasma nicotine levels (Benowitz, 1988;Hukkanen et al., 2005). The plasma concentration of nicotine varies among tobacco users, due in part to differences in nicotine metabolism. The major catalyst of nicotine metabolism in humans is CYP2A6 (Hukkanen et al., 2005). However, the closely related enzyme CYP2A13 is a better catalyst (Bao et al., 2005;Murphy et al., 2005). Whether or not CYP2A13, an extrahepatic P450 expressed in the respiratory tract (Su et al., 2000), plays a significant role in nicotine metabolism in smokers is unknown. However, CYP2A13 is an excellent catalyst of the metabolic activation of the tobacco carcinogen NNK (Jalas et al., 2005;Wong et al., 2005b). NNK is only present in tobacco and tobacco smoke; therefore, exposure to NNK is always concurrent with nicotine exposure. NNK requires metabolic activation to exert its carcinogenic potential, and CYP2A13 is likely a key catalyst of activation in smokers. Therefore, any inactivation of CYP2A13 by nicotine will influence the metabolic activation of NNK, a potent lung carcinogen.Nicotine is primarily metabolized by 5Ј-oxidation, but 2Ј-oxidation and methyl-oxidation also occur ( Fig. 1) (Hecht et al., 2000;Hukkanen et al., 2005;Mu...