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...

Although there is a robust influence of APOE polymorphism on functional recovery after some types of brain injury in humans, it does not exert a major influence on injury severity or functional recovery following ischemic stroke. Arch Neurol. 2000;57:1480-1484

Cotinine formation is the major pathway of nicotine metabolism in smokers, and the primary pathway of cotinine metabolism is trans-3'-hydroxylation. trans-3'-Hydroxycotinine and its glucuronide conjugate account for up to 50% of the nicotine metabolites excreted by smokers. Minor metabolites of cotinine excreted by smokers include norcotinine and cotinine N-oxide, each of which account for <5% of the nicotine dose. It has been reported that P450 2A6 is the catalyst of cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown human metabolite of cotinine. N-(Hydroxymethyl)norcotinine was chemically synthesized, and its stability under the conditions of the enzyme reactions was confirmed. The products of P450 2A6-catalyzed [5-3H]cotinine metabolism were quantified by radioflow HPLC. The identification of N-(hydroxymethyl)norcotinine as the major metabolite was based on HPLC analysis on three unique systems and coelution with N-(hydroxymethyl)norcotinine standard. 5'-Hydroxycotinine and trans-3'-hydroxycotinine were minor products of P450 2A6-catalyzed cotinine metabolism, accounting for 14 and 8% of the total cotinine metabolites, respectively. N-(Hydroxymethyl)norcotinine was a product of cotinine metabolism by the extrahepatic P450, 2A13, but it was a minor one. The major product of P450 2A13-catalyzed cotinine metabolism was 5'-hydroxycotinine, which was formed at twice the rate of trans-3'-hydroxycotinine. The identification of all cotinine metabolites formed by both enzymes was confirmed by LC/MS/MS analysis. Kinetic parameters for cotinine metabolism were determined for P450 2A6 and P450 2A13. This work has confirmed that the major metabolite of cotinine in smokers, trans-3'-hydroxycotinine, is only a minor metabolite of P450 2A6-catalyzed cotinine metabolism.

Uromodulin, an 85-kDa glycoprotein isolated from pregnancy urine, has been shown to inhibit antigeninduced proliferation of human lymphocytes in vitro. The present investigation was undertaken to determine its mechanism of action. Uromodulin was found to be a potent inhibitor of interleukin 1 (IL-1)-induced thymocyte proliferation. Uromodulin was compared to a previously described 30-to 35-kDa IL-1 inhibitor isolated from urine of febrile patients (febrile inhibitor). Uromodulin and the febrile inhibitor blocked the effects of both human IL-1 and recombinant murine IL-1, but the activity of uromodulin was greater than that of the only partially purified febrile inhibitor preparation. However, in contrast to the febrile inhibitor, uromodulin markedly enhanced interleukin 2-induced thymocyte proliferation. Antigenic analysis of the two preparations by ELISA and immunoblot analysis demonstrated that the febrile inhibitor did not cross-react with uromodulin using monoclonal or polyclonal antisera. These findings indicate that uromodulin is a potent IL-1 inhibitor that is probably distinct from the IL-1 inhibitor derived from the urine offebrile individuals. Whether this IL-1 inhibitory activity underlies its immunosuppressive activity on human lymphocytes remains to be established.Interleukin 1 (IL-1) mediates a large number of diverse immunostimulatory and inflammatory events in mammals. IL-1 activates prostaglandin E2 synthesis and collagenase release, as well as acting on the central nervous system to control febrile responses and on the liver to control the synthesis of acute phase proteins (1). In addition to its maturational effects on thymocytes, IL-1 also regulates the activity of B cells (2, 3) and NK cells (4). With such a broad range of biologic activity, it would be extremely informative to characterize factors that may play a role in regulating the activity of this potent monokine.We have recently described an inhibitor of IL-i-induced thymocyte proliferation that is present in increased amounts in the urine offebrile individuals (5), which appears to consist of one or more 30-to 35-kDa glycoproteins (6). Workers in other laboratories have found several inhibitors of lymphocyte proliferation in human serum (7) and urine (8), and in the supernatants of cultured human macrophages (9), macrophage cell lines (10, 11), and B cell lines (12). Dayer and his coworkers (13) have also reported on a urine-derived 20-to 30-kDa protein that inhibits IL-i-induced fibroblast prostaglandin E2 synthesis.It has been known for many years that the urine ofpregnant women contains potent immunosuppressive factors that block mitogen- (14) and antigen-induced (15) lymphocyte proliferation of human blood lymphocytes in vitro. The immunosuppressive activity of pregnancy serum and urine has been ascribed to a number of high and low molecular weight materials, including pregnancy-associated a2-glycoprotein (16), a-fetoprotein (17), pregnancy-specific 81-glycoprotein (14, 18), and human placenta lactogen (14). These pregnan...

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