Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol

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Abstract

Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (β-nicotyrine) and (−)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (-)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2 μM and 110 μM, respectively). β-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 μM). Neither menthol nor β-nicotyrine was an inactivator of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106 μM, kinact was 0.61 min−1). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that β-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.

Highlights

► β-Nicotyrine, menthol and menthofuran inhibit CYP2A6 and CYP2A13 activities in a time and dose dependent manner. ► β-Nicotyrine and menthofuran are much more potent inhibitors of both CYP2A6 and CYP2A13 activity than menthol. ► Like menthofuran, β-nicotyrine is an inactivator of CYP2A6 but not CYP2A13. ► Glutathione and semicarbazide do not protect CYP2A6 form either menthofuran or β-nicotyrine-mediated inactivation.

Introduction

Tobacco-use is driven by the addictive nature of nicotine [1]. However, the use of tobacco products results in the exposure to a diverse array of compounds. Some, like nicotine are naturally occurring. Others, for example menthol, are additives and some, such as the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) [2], form during the curing of the tobacco (Fig 1). Menthol is reported to modify nicotine metabolism and thus has been the focus of recent FDA regulation [3], [4], [5]. The primary route of nicotine elimination is CYP2A6-catalyzed metabolism [6], and a key pathway of NNK activation is CYP2A13 catalyzed hydroxylation [7]. Pharmacologically blocking nicotine metabolism has been reported to reduce the extent of smoking [8], [9]. Similarly, the inhibition of NNK metabolism could mitigate exposure to activated NNK. Therefore, the characterization of compounds that modify CYP2A6 and CYP2A13 activity is critical to understanding the influence of these enzymes on tobacco consumption and carcinogenesis.

CYP2A6 is primarily a hepatic enzyme [10] and CYP2A13 is expressed in the respiratory tract [11], [12]. Both enzymes efficiently catalyze nicotine 5′-oxidation, the first step in the conversion of nicotine to cotinine, the primary nicotine metabolism pathway. However, due to its presence in the liver, CYP2A6 plays the major role in nicotine elimination in smokers [6], [13]. NNK requires metabolic activation to exert its carcinogenic potential and CYP2A13 catalyzed activation likely plays a significant role in the induction of lung cancer in smokers [7], [12].

CYP2A6 and CYP2A13 share 94% sequence identity and catalyze the metabolism of many common substrates. However, they often have different catalytic efficiencies and generate unique metabolites. One common substrate, coumarin is frequently used to probe CYP2A activity. CYP2A6 selectively catalyzes the 7-hydroxylation of coumarin but, CYP2A13 catalyzes both 7-hydroxylation and 3,4-epoxidation [14]. Both CYP2A6 and CYP2A13 catalyze the metabolic activation of NNK however, CYP2A13 is a 200-fold more efficient catalyst (kcat/Km) than CYP2A6 [7]. CYP2A13 is also a somewhat superior catalyst of nicotine metabolism; catalyzing nicotine 5′-oxidation at a 5- to 20-fold higher catalytic efficiency than CYP2A6 [15], [16].

While mechanism-based inactivation of CYP2A6 and CYP2A13 has been reported for several compounds it is fully characterized for only a few. To be classified as a mechanism-based inactivator, a compound must be metabolically activated to an intermediate that covalently modifies the enzyme and renders it inactive [17]. Menthofuran has been identified as a potent mechanism-based inactivator of CYP2A6. Menthofuran-mediated loss of CYP2A6 activity is dependent on catalytic turnover and immunohistochemistry analysis has provided evidence that the apo-protein is modified [18]. Inactivation of both CYP2A6 and CYP2A13 occurs during nicotine metabolism [16]. Nicotine, however, is not the agent of inactivation; a secondary or even tertiary nicotine metabolite is likely the inactivator. The species responsible for enzyme inactivation has yet to be identified, however one possible contender is β-nicotyrine. It has been suggested that β-nicotyrine, which differs from nicotine in the presence of a pyrrole instead of a pyrrolidine ring (Fig 1), is a mechanism-based inactivator of CYP2A6 [19]. β-Nicotyrine is a urinary metabolite of nicotine in dogs and rats but has not been identified as a nicotine metabolite in humans [20]. Regardless, smokers are exposed to β-nicotyrine, since it is present in tobacco [21].

The importance of understanding the effects of tobacco constituents on CYP2A6 and CYP2A13 activity is underscored by their potential impact on smoking behavior and NNK activation. In addition, the characterization of potential mechanism-based inactivators may aid in the design of specific inactivators of these enzymes. The objectives of the current study were to determine the relative inhibition of CYP2A6 and CYP2A13 activity by β-nicotyrine, menthol and menthofuran and to assess inactivation of CYP2A6 and CYP2A13 by each of these compounds.

Section snippets

Chemicals and enzymes

Menthofuran, menthol, 7-hydroxycoumarin, coumarin, dilauroyl-l-α-phosphatidylcholine (DLPC), NADPH, bovine serum albumin, catalase, and all other biochemical reagents were obtained from Sigma–Aldrich (St. Louis, MO) and were of analytical grade. β-Nicotyrine (99% pure) was obtained from Toronto Research Chemicals. Trifluoroacetic acid (TFA) was obtained from Pierce Chemical (Rockford, IL). The enzymes used in this study were heterologously expressed in Escherichia coli and purified according to

Inhibition

The relative inhibition of CYP2A6 and CYP2A13-catalyzed coumarin 7-hydroxylation by menthol, β-nicotyrine and menthofuran was determined and the kinetic constants are presented in Table 1. As previously reported [28], the Km of CYP2A13-catalyzed coumarin 7-hydroxylation was lower than the Km for CYP2A6 (data not shown). Both menthofuran and β-nicotyrine were relatively potent inhibitors of CYP2A6 activity, with KI values of 0.29 μM and 1.07 μM, respectively. Inhibition of CYP2A6 by menthol was

Discussion

CYP2A6 and CYP2A13 are critical catalysts of nicotine and NNK metabolism in smokers [6], [12]. Therefore, inhibitors of these enzymes may influence both nicotine addiction and tobacco-induced carcinogenesis. In the study presented here, the inhibition and inactivation of CYP2A6 and CYP2A13 by the tobacco constituents, β-nicotyrine and menthol, were characterized and compared to the potent CYP2A6 inactivator, menthofuran [18]. Both compounds inhibited the coumarin 7-hydroxylation activity of

Conflict of interest statement

The authors declare no financial or commercial conflict of interest.

Acknowledgments

This work was supported by the National Institutes of Health [Grant CA-84529]; VMK was supported by a predoctoral traineeship from the National Institutes of Health [Grant GM 08700].

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