Inhibition of Human Cytochrome P450 2E1 by Nicotine, Cotinine, and Aqueous Cigarette Tar Extract in Vitro

Terry R. Van Vleet*, David W. Bombick{dagger} and Roger A. Coulombe, Jr.*,1

* Graduate Program in Toxicology, Department of Veterinary Sciences, Utah State University, 4620 Old Main Hill, Logan, Utah, 84322; and {dagger} R. J. Reynolds Tobacco Co., EMT Division, Winston-Salem, North Carolina 27102

Received May 22, 2001; accepted September 4, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cigarette smoke is a complex mixture containing, among other chemicals, pyridine alkaloids and N-nitrosamines. Carcinogenic tobacco-specific N-nitrosamines, N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are both activated by cytochrome P450 (CYP) 2E1 in rats. Previous reports indicate that nicotine and the main nicotine metabolite, cotinine, reduce the mutagenicity of both NNK and NDMA in Salmonella typhimurium. To study the mechanism of this effect, we examined inhibition of CYP 2E1 activity, as assessed by p-nitrophenol (pNP) hydroxylation, by nicotine, cotinine, and an aqueous cigarette tar extract (ACTE) in human 2E1-expressing microsomes. At all substrate concentrations (0–1.25 mM) nicotine was a significantly more potent inhibitor of CYP 2E1 activity compared to cotinine. Estimated Ki values for nicotine and cotinine (both at 10 mM) were 13 mM (2 mg/ml) and 308 mM (54 mg/ml) respectively. The Ki for ACTE was 0.2 mg/ml at a concentration of 0.32 mg/ml. This rank order for inhibition was also seen when the data was expressed as IC50. When compared on a mass/vol basis, ACTE was a significantly more potent CYP 2E1 inhibitor relative to nicotine and cotinine. Double-reciprocal plots indicated that nicotine and ACTE inhibited by a competitive, while cotinine inhibited CYP 2E1 by an uncompetitive mechanism. Although the contribution of nicotine to ACTE-mediated 2E1 inhibition is probably modest, pyridine alkaloid-mediated CYP 2E1 inhibition is a possible mechanism for the observed inhibition of NNK and NDMA mutagenicity by nicotine and cotinine in vitro.

Key Words: nicotine; cotinine; ACTE; human CYP 2E1; inhibition.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyridine alkaloids and N-nitrosamines are 2 components of the complex chemical mixture that comprises tobacco smoke. The concentration of pyridine alkaloids, including nicotine, is several orders of magnitude greater than that of N-nitrosamines (Adams et al., 1987Go; Caldwell and Conner, 1990Go; Tricker et al., 1991Go; Tricker and Preussmann, 1991Go). Tobacco specific N-nitrosamines include N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Hoffmann et al., 1994Go). As shown in Figure 1Go, these compounds require activation by cytochromes P450 (CYP) to produce mutagenic intermediates such as methyldithiazohydroxide and 4-(3-pyridyl)-4-oxobutyl diazohydroxide (Guo et al., 1992Go; Patten et al., 1996Go; Smith et al., 1998Go). While other CYP isozymes appear to be involved, activation of tobacco-specific nitrosamines has been strongly linked to CYP 2E1 activity in several mammalian species (Guengerich et al., 1994Go; Hecht, 1996Go; Smith et al., 1996Go; Yamazaki et al., 1992Go). For example, nitrosamine activation by human liver microsomes is mediated by several CYPs, including 2E1 (Bellec et al., 1996Go). Demethylation of NDMA to the reactive intermediate methyldiazohydroxide appears to be catalyzed by a high-affinity CYP 2E1 in human liver (Guengerich et al., 1994Go; Patten et al., 1992Go). Cytochrome P450 2E1 is also linked to activation of NNK and NDMA in human esophagus (Smith et al., 1998Go), and to NDMA activation in human fibroblasts (Lin et al., 1998Go). CYP 2E1 mRNA and protein are expressed in peripheral human lung and bronchial mucosa (Mace et al., 1998Go), human A549 lung cells (Hukkanen et al., 2000Go), and in human liver (Patten et al., 1997Go). In fact, expression of CYP 2E1 in human liver is second only to CYP 3A4 (Rodriguez-Antona et al., 2000Go).



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FIG. 1. Proposed metabolism and activation of NDMA and NNK (adapted from Lee et al., 1996Go; Pool-Zobel et al., 1992Go). Brackets designate intermediates.

 
Tobacco smoke constituents have been shown to inhibit the activation and mutagenicity of N-nitrosamines in vivo and in vitro (Brown et al., 1999Go; Lee et al., 1996Go; Schneider et al., 1999Go). Compounds that inhibit NNK or NDMA mutagenicity such as ellagic acid, (-)-epigallocatechin-3-gallate, and alkyl sulfides, have also been shown to be CYP 2E1 inhibitors (Ahn et al., 1996Go; Shi et al., 1994Go; Siess et al., 1997Go; Wang et al., 1996Go). Because of the structural similarity between pyridine alkaloids and N-nitrosamines, it is possible that this protection is due to competitive inhibition of CYP 2E1, although to our knowledge this has never been studied.

CYP 2E1 probably plays a minor role, if any, in nicotine metabolism. Rat liver CYP 2E1 did not oxidize nicotine (Nakayama et al., 1993Go), while another report suggests that human liver 2E1 oxidizes nicotine, but only at relatively high (500 µM) concentrations (Yamazaki et al., 1999Go). The purpose of the present study was to determine whether nicotine, and its major metabolite, cotinine, inhibit the catalytic activity of human CYP 2E1. The inhibitory effect of these compounds was compared to that of aqueous cigarette tar extract (ACTE) from a reference cigarette (K1R4F).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
CYP 2E1 supersomes had an activity of 2 pmole human CYP 2E1/ml spectrally ({lambda} = 450 nm) determined by the manufacturer (Gentest, Woburn, MA). Nicotine, cotinine, and p-nitrophenol (pNP) were purchased from Aldrich Chemical (Milwaukee, WI). 4-Nitrocatechol standard was from Acros Chemical (Pittsburgh, PA). NADPH was obtained from ICN Biochemicals (Costa Mesa, CA). Hanks buffered salt solution (HBSS) was from JRH Biosciences (Lenexa, KS). All other chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA).

Preparation of ACTE.
Cigarette smoke condensate was prepared from 1R4F Kentucky reference cigarettes on a smoking machine under standard Federal Trade Commission (FTC) conditions (35 ml puff volume, 2-s duration, once per min). The 1R4F cigarettes represent the average light cigarette with respect to tar, nicotine, and CO yields. The cigarette smoke condensate was collected on Cambridge filter pads, which were subsequently extracted with HBSS to yield an ACTE solution with a concentration of 10 mg total particulate matter (TPM)/ml. The ACTE solution was divided into smaller volumes and stored at –70°C until analysis. Aqueous rather than nonpolar extracts were used in this study, to avoid residues that might inhibit CYP 2E1 activity. For example, preliminary studies showed that DMSO significantly inhibited CYP 2E1 activity (data not shown).

CYP 2E1 assay.
The ability of nicotine, cotinine, and ACTE to inhibit CYP 2E1 activity was assayed spectrophotometrically by measuring the CYP 2E1-mediated conversion of p-nitrophenol to 4-nitrocatechol (Koop, 1986Go; Reinke and Moyer, 1985Go). Reaction mixtures (total volume: 25 µl) containing 5 pmoles CYP 2E1, 5 mM NADPH, 5 mM MgCl2, 0.05 M Tris, inhibitor, and pNP (0–1.25 mM), were incubated at 37°C for 30 min before the reaction was stopped by adding 1 volume of ice cold MeOH. Samples were stored at –20°C overnight before centrifuging at 13,000 x g. Supernatants were removed and 10 µl of 10 N NaOH was added. The spectrophotometer (Model DU 64, Beckman Instruments, Fullerton, CA) was zeroed with the negative control (no supersomes), and the production of 4-nitrocatechol from p-nitrophenol was measured at {lambda} = 515 nm. A range of supersome concentrations was used to insure that CYP 2E1 was the limiting factor in reactions at all pNP concentrations (0–1.25 mM). Preliminary assays also showed that the 30-min incubation time was well within initial velocity (< 10% of substrate converted). Absorbance spectra (200–750 nm) were determined for all reagents, cofactors, inhibitors, and substrate under assay conditions to insure that no component of the reaction mixture absorbed at wavelengths that would interfere with detection of the product (data not shown).

Concentration-response modeling and statistical analysis.
Enzyme activity plots (activity vs. [substrate]) were fit using an empirical 3-parameter Hill equation model (Jenski, 1989Go; Jenski and Kleyle, 1989Go; Melnick et al., 1998Go):

where V is the measured enzyme activity (nmoles 4-NC/min/nmole CYP 2E1), Km is the p-nitrophenol concentration yielding half of the maximal response (Vmax), and h is the Hill exponent, which is a measure of cooperativity. The parameters for this equation were calculated using Sigma Plot (Jandel Scientific, San Rafael, CA). Groups were compared for differences using 2-way ANOVA. Differences in means were judged significant when p < 0.05. Linear regression analysis was used to estimate IC50 values.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibition of CYP 2E1 by Nicotine and Cotinine
Preliminary experiments showed that pNP concentrations > 1.25 mM resulted in a decline in CYP 2E1 activity (Fig. 2Go). We therefore ran all subsequent assays at pNP concentrations <= 1.25 mM. Data from the resulting control curve (with no inhibitor) was fit with the Hill model equation yielding an estimated Km for p-nitrophenol of 156 µM, under these assay conditions (Fig. 3Go). Nicotine and cotinine inhibited CYP 2E1 activity over a range of substrate (p-Nitrophenol) concentrations (0–1.25 mM) in a dose-dependent manner (Figs. 3A and 3BGo). From Hill equation parameters, estimated Ki values for nicotine and cotinine were 13 mM and 308 mM, respectively (Figs. 3A and 3BGo). Lineweaver-Burke analyses suggested a probable competitive mechanism for nicotine-mediated inhibition, and an uncompetitive mechanism for cotinine-mediated inhibition (Figs. 4A and 4BGo).



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FIG. 2. Effect of pNP concentration on CYP-mediated production of 4-nitrocatechol from pNP by human CYP 2E1-expressing microsomes. Enzyme activity was determined spectrophotometrically as outlined in Materials and Methods. Data points are the means ± SD (n = 3).

 


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FIG. 3. The inhibitory effect of (A) nicotine (control, open circle; 5 mM, filled triangle; 10 mM, filled circle) and (B) cotinine (control, open circle; 10 mM, filled circle; 20 mM, filled triangle) on human CYP 2E1 activity over a range of substrate (pNP) concentrations. 2E1 activity is reduced by pyridine alkyloids in a concentration dependent manner. Data points are the means ± SD (error bars) (n = 3). a,b,cPoints with different letters are significantly different (p < 0.05).

 


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FIG. 4. Lineweaver-Burke plots of (A) nicotine and (B) cotinine inhibition of human CYP 2E1 activity. The resulting Ki values were 13 mM (2 mg/ml) for nicotine and 308 mM (54 mg/ml) cotinine, at 10 mM inhibitor concentrations. These plots indicate a possible competitive type of nicotine-mediated inhibition of CYP 2E1 (A), and an uncompetitive cotinine-mediated inhibition (B). Data points are the means ± SD (n = 3). Symbols same as in Figure 3Go.

 
The inhibitory action of nicotine and cotinine on CYP 2E1 activity was also apparent when a larger range of inhibitor concentrations was used at a fixed (i.e., 0.24 mM) pNP concentration (Figs. 5A and 5BGo). Because substrate saturation was not achievable under assay conditions, CYP 2E1 inhibition by nicotine, cotinine, and ACTE was also quantified by a log conversion of the data to obtain IC50 values (Figs. 5A and 5BGo). Results of this analysis showed that the concentration of nicotine resulting in a 50% inhibition of CYP 2E1 was 3.87 mM (Fig. 5AGo; r2 = 0.995). As is the case with nicotine, cotinine inhibited CYP 2E1 in a concentration-dependent manner (Fig. 5BGo), with an IC50 value of 37.71 mM (r2 =0 .960). These trends were similar at a lower pNP concentration (0.12 mM; data not shown).



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FIG. 5. Concentration dependency of (A) nicotine (0–10 mM) and (B) (0–100 mM) cotinine-mediated inhibition of human CYP 2E1 activity. A substrate concentration 0.24 mM was used for these data, but similar results were detected at 0.12 mM pNP (data not shown). Linear regression analysis was used to determine the concentration at which control enzyme activity was reduced by half. (A) Nicotine (b[0] = –1.278, b[1] = –0.071, r2 = 0.995, and IC50 = 3.87). (B) Cotinine (b[0] = –1.312, b[1] = –0.006, r2 = 0.960, and IC50 = 37.71 mM). Values are means ± SD (n = 3).

 
Inhibition of CYP 2E1 by ACTE
Like nicotine and cotinine, ACTE inhibited human CYP 2E1 activity over a range of pNP concentrations (Fig. 6Go). Hill equation parameters of ACTE-mediated inhibition yielded a Ki of 0.2 mg/ml, with a concentration of ACTE of 0.32 mg/ml (Fig. 6Go).



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FIG. 6. ACTE-mediated inhibition of human CYP 2E1 over a range of substrate concentrations (0–1.25 mM). (A) Two ACTE concentrations (0.08 and 0.32 mg/ml) were used (control, filled triangle; 0.08 mg/ml, open circle; 0.32 mg/ml, filled circle). a,b,cPoints with different letters are significantly different (p < 0.05). (B) Lineweaver-Burke plots reflect a probable competitive type inhibition of human CYP 2E1 by ACTE. The calculated Ki value is 0.2 mg/ml. Data points are means ± SD (n = 3).

 
The concentration-dependency of human CYP 2E1 inhibition could be clearly seen when a range of ACTE concentrations was used. Concentrations of ACTE between 0.08–0.32 mg/ml exerted an inhibitory effect on CYP 2E1 activity (Fig. 7Go). Using a pNP concentration of 0.24 mM, an IC50 value was calculated to be 0.12 mg/ml (r2 = 0.980; Fig. 7Go). The results were similar at a pNP concentration of 0.12 mM (data not shown).



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FIG. 7. Concentration dependent inhibition of human CYP 2E1 by ACTE (0–0.32 mg/ml). A p-Nitrophenol concentration of 0.24 mM was used. Linear regression of data was used to estimate IC50 (b[0] = 1.406, b[1] = -2.133, r2=0.980, and IC50 = 0.12 mg/ml). Values on graph are means ± SD (n = 3).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although other CYPs have a role, the {alpha}-hydroxylation of NDMA and NNK is catalyzed principally by CYP 2E1 in human liver, which produces a variety of genotoxic metabolites (Fig. 1Go). Methyldiazohydroxide, the active intermediate that can methylate DNA bases to form O6 and 7 Me-Gua, is formed by hydroxylation of either NDMA or NNK (Hecht and Hoffmann, 1989Go; Pool-Zobel et al., 1992Go). Of particular importance among these is O6-methylguanine, which has been shown to induce G -> A transitions in codon 12 of the K-ras oncogene in mouse lung (Ronai et al., 1993Go). Another metabolic route is the hydroxylation of the methyl carbon on NNK to produce 4-(3-pyridyl)-4-oxobutyl diazohydroxide, which also alkylates DNA (Lee et al., 1996Go). Thus, an inhibition of CYP-mediated conversion of NDMA and NNK would possibly reduce the production of one or more of these genotoxic metabolites.

Certain pyridine alkaloids present in tobacco have been shown to inhibit the biological action of certain tobacco-specific nitrosamines in vitro and in vivo. For example, N-nitrosamine mutagenesis in vitro was reduced by nicotine, cotinine, or ACTE (Lee et al., 1996Go). Cotinine or cigarette smoke were also shown to reduce the formation of NNK-induced O6-methylguanine DNA adducts in vivo (Brown et al., 1999Go). The present study demonstrates, for the first time, that nicotine, cotinine, and ACTE inhibit human CYP 2E1 activity. Therefore, it is plausible that the observed reduction in N-nitrosamine mutagenicity in vitro may be due, at least in part, to CYP 2E1 inhibition. The rank order of human CYP 2E1 inhibition in the present study (ACTE > nicotine > cotinine) was precisely the order in which reductions in mutagenicity from NNK and NDMA have been detected by these compounds (Lee et al., 1996Go). The competitive nature of CYP 2E1 inhibition by nicotine is supported by a previous report showing that nicotine is a CYP 2E1 substrate at high concentrations (Yamazaki et al., 1999Go). ACTE-mediated inhibition was also probably due to a competitive mechanism.

In any event, our data indicate that nicotine probably plays a minor role in ACTE-mediated CYP 2E1 inhibition. Although the amount of nicotine in cigarette smoke is as much as 30,000-fold greater than that of the N-nitrosamines (Adams et al., 1987Go; Caldwell and Conner, 1990Go; Tricker et al., 1991Go; Tricker and Preussmann, 1991Go; Trushin and Hecht, 1999Go), results of this study show that ACTE was approximately 5-fold more potent at 2E1 inhibition than nicotine—the IC50 value for nicotine was 0.63 mg/ml, compared to 0.12 mg/ml for ACTE. Thus, it is plausible that because ACTE is a complex mixture of chemicals, it is likely that chemicals other than nicotine in ACTE are responsible for the majority of CYP 2E1 inhibition. It is also possible that CYP 2E1 inhibition by ACTE may be the result of a synergistic effect of a combination of chemicals. At 0.32 mg/ml, ACTE inhibited 2E1 activity by 82% (0.24 mM pNP) (Fig. 7Go), while 0.32 mg/ml pure nicotine would inhibit 2E1 activity by 11% (calculated from linear regression analysis; Fig. 5AGo). Because ACTE contains 5 mM nicotine (Lee et al., 1996Go), the 0.32 mg/ml ACTE incubations contained 0.16 mM nicotine. A nicotine concentration of 0.16 mM would therefore theoretically represent an estimated 8% inhibition (calculated from linear regression analysis; Fig. 5AGo), which is very similar to the 11% estimate. Therefore, the contribution of nicotine to ACTE-mediated inhibition of 2E1 is probably modest. This observation is in agreement with a previous conclusion that nicotine was a minor contributor to ACTE-mediated inhibition of NNK and NDMA mutagenicity in S. typhimurium (Lee et al., 1996Go).

There is precedence for CYP 2E1 inhibition by naturally occurring compounds. Many naturally occurring phytochemicals have been shown to inhibit mutagenicity of nitrosamines either in vitro or in vivo. Green and black tea extracts inhibit the activation of NNK in vitro (Shi et al., 1994Go). Of the tea components studied, (-)-epigallocatechin-3-gallate was the most effective antimutagen and inhibitor of CYP 2E1 (Shi et al., 1994Go). Ellagic acid (EA) also inhibits CYP 2E1 and reportedly reduces nitrosamine-induced tumors in rats (Ahn et al., 1996Go). Red ginseng saponins also inhibit CYP 2E1 (Kim et al., 1997Go). The alkyl sulfides dipropyl sulfide (DPS), dipropyl disulfide (DPDS), diallyl disulfide (DADS), and diallyl sulfide (DAS) reduce nitrosodimethylamine N-demethylase and inhibit 2E1 expression (Siess et al., 1997Go; Wang et al., 1996Go). DAS, a flavor component of garlic, inhibits the formation of NNK-induced lung tumors in mice (Hong et al., 1992Go). Because 2E1 inhibitors are present in many plants, it is possible that tobacco also produces compound(s) that inhibit CYP 2E1.

While p-nitrophenol has been widely used as a substrate for measuring CYP 2E1 activity (Bellec et al., 1996Go; Kazakoff et al., 1994Go; Lin et al., 1998Go; Shu and Hollenberg, 1997Go), there are limitations to this assay. Our finding that the highest pNP concentrations (> 1.25 mM) inhibited 2E1 activity has also been noted by other researchers (Koop, 1986Go). The high (i.e., mM) concentrations of inhibitors used in this study (which are unlikely to be achieved in vivo), were necessary in this model system because of the spectrophotometric detection limits of 4-nitrocatechol and also because of attempts to approach enzyme saturation. Furthermore, because the concentrations of substrate used in this study were in the mM range, similar inhibitor concentrations were required. These high inhibitor concentrations may not be necessary if another assay for CYP 2E1 were used. In addition, in vitro measurements of CYP 2E1 metabolism of many substrates may be complicated by the existence of a rate-determining step between product formation and release (Bell-Parikh and Guengerich, 1999Go).

While nonpolar condensates from tobacco smoke also contain potent inhibitors of CYPs (Shimada and Guengerich, 1991Go), the water-extracted ACTE was more appropriate for use in the present study. An important objective of this work was to determine if CYP 2E1 inhibition by ACTE (in addition to nicotine and cotinine) was a potential mechanism underlying the reported inhibition of N-nitrosamine mutagenesis in Salmonella (Lee et al., 1996Go). Secondly, we found that several common solvents used to prepare nonpolar cigarette smoke condensates (DMSO, methanol, ethanol, and dimethylformamide, all at 0.5%) significantly inhibited CYP 2E1 activity (data not shown).

Our data provide evidence for the inhibitory effect of tobacco smoke constituents on the major CYP involved in nitrosamine activation. It is therefore plausible that the demonstrated protective effect of nicotine, cotinine, and other tobacco smoke constituents on the biological action of N-nitrosamines, is due, at least in part, to inhibition of this specific enzyme in lung, liver and other organs.


    ACKNOWLEDGMENTS
 
The authors wish to acknowledge support from the Utah State Agricultural Experiment Station, where this paper is designated number 7385.


    NOTES
 
Portions of this data were presented at the 39th annual meeting of the Society of Toxicology, March 2000, Philadelphia, PA.

1 To whom correspondence should be addressed. Fax: (435) 797-1601. E-mail: rogerc{at}cc.usu.edu. Back


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 DISCUSSION
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