* Graduate Program in Toxicology, Department of Veterinary Sciences, Utah State University, 4620 Old Main Hill, Logan, Utah, 84322; and
R. J. Reynolds Tobacco Co., EMT Division, Winston-Salem, North Carolina 27102
Received May 22, 2001; accepted September 4, 2001
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ABSTRACT |
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Key Words: nicotine; cotinine; ACTE; human CYP 2E1; inhibition.
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INTRODUCTION |
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CYP 2E1 probably plays a minor role, if any, in nicotine metabolism. Rat liver CYP 2E1 did not oxidize nicotine (Nakayama et al., 1993), while another report suggests that human liver 2E1 oxidizes nicotine, but only at relatively high (500 µM) concentrations (Yamazaki et al., 1999
). 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).
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MATERIALS AND METHODS |
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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, 1986; Reinke and Moyer, 1985
). Reaction mixtures (total volume: 25 µl) containing 5 pmoles CYP 2E1, 5 mM NADPH, 5 mM MgCl2, 0.05 M Tris, inhibitor, and pNP (01.25 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
= 515 nm. A range of supersome concentrations was used to insure that CYP 2E1 was the limiting factor in reactions at all pNP concentrations (01.25 mM). Preliminary assays also showed that the 30-min incubation time was well within initial velocity (< 10% of substrate converted). Absorbance spectra (200750 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, 1989; Jenski and Kleyle, 1989
; Melnick et al., 1998
):
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RESULTS |
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DISCUSSION |
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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., 1996). Cotinine or cigarette smoke were also shown to reduce the formation of NNK-induced O6-methylguanine DNA adducts in vivo (Brown et al., 1999
). 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., 1996
). 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., 1999
). 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., 1987; Caldwell and Conner, 1990
; Tricker et al., 1991
; Tricker and Preussmann, 1991
; Trushin and Hecht, 1999
), results of this study show that ACTE was approximately 5-fold more potent at 2E1 inhibition than nicotinethe 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. 7
), while 0.32 mg/ml pure nicotine would inhibit 2E1 activity by 11% (calculated from linear regression analysis; Fig. 5A
). Because ACTE contains 5 mM nicotine (Lee et al., 1996
), 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. 5A
), 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., 1996
).
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., 1994). Of the tea components studied, (-)-epigallocatechin-3-gallate was the most effective antimutagen and inhibitor of CYP 2E1 (Shi et al., 1994
). Ellagic acid (EA) also inhibits CYP 2E1 and reportedly reduces nitrosamine-induced tumors in rats (Ahn et al., 1996
). Red ginseng saponins also inhibit CYP 2E1 (Kim et al., 1997
). 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., 1997
; Wang et al., 1996
). DAS, a flavor component of garlic, inhibits the formation of NNK-induced lung tumors in mice (Hong et al., 1992
). 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., 1996; Kazakoff et al., 1994
; Lin et al., 1998
; Shu and Hollenberg, 1997
), 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, 1986
). 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, 1999
).
While nonpolar condensates from tobacco smoke also contain potent inhibitors of CYPs (Shimada and Guengerich, 1991), 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., 1996
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (435) 797-1601. E-mail: rogerc{at}cc.usu.edu.
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