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. 2016 Apr 28;5(4):1115–1121. doi: 10.1039/c6tx00016a

The inhibition of cytochrome P450 2A13-catalyzed NNK metabolism by NAT, NAB and nicotine†

Xingyu Liu a, Jie Zhang a, Chen Zhang a, Bicheng Yang b, Limeng Wang c,d, Jun Zhou a,✉
PMCID: PMC6062012  PMID: 30090417

graphic file with name c6tx00016a-ga.jpg4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic of the four tobacco-specific nitrosamines (TSNAs) and it needs to be metabolically activated to exert its carcinogenic effect on humans.

Abstract

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic of the four tobacco-specific nitrosamines (TSNAs) and it needs to be metabolically activated to exert its carcinogenic effect on humans. For the simultaneous intake of NNK and other compounds with similar molecular structures in the context of tobacco smoke, whether (R,S)-N-nitrosoanatabine (NAT), (R,S)-N-nitrosoanabasine (NAB) and nicotine contribute to the inhibitory potency of the cytochrome P450 (CYP) enzyme-catalyzed NNK metabolism or not needs to be investigated. In the in vitro study, 4-oxo-4-(3-pyridyl) butanal (OPB), 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-oxo-4-(3-pyridyl) butanoic acid (OPBA) were established as the products of the CYP2A13-catalyzed NNK metabolism and the kinetic parameters were calculated from the Michaelis-Menten equation. Addition of NAT, NAB or nicotine resulted in a competitive inhibition for the NNK metabolism catalyzed by CYP2A13. The inhibition constant Ki values were calculated to be 0.21 μM (NAT), 0.23 μM (NAB) and 8.51 μM (nicotine) for OPB formation; 0.71 μM (NAT), 0.87 μM (NAB) and 25.01 μM (nicotine) for HPB formation and 0.36 μM (NAT), 0.50 μM (NAB) and 6.57 μM (nicotine) for OPBA formation, respectively. In addition, the study of the transformation of the three metabolites revealed OPB was not only an end product but also an intermediate product of the CYP2A13-catalyzed NNK metabolism. These results suggest that structurally similar tobacco constituents with weak or no carcinogenicity influence the metabolic activation of NNK, which interferes with its carcinogenicity to some extent.

1. Introduction

Tobacco-specific nitrosamines (TSNAs) are widely considered to be among the most harmful carcinogens in tobacco products and cigarette smoke.1–3 One tobacco alkaloid derived compound, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is carcinogenic to humans and was classified as “Group 1” by the International Agency for Research on Cancer (IARC) in 2012.4 The other two compounds studied, classified as “Group 3”,4 are (R,S)-N-nitrosoanatabine (NAT) and (R,S)-N-nitrosoanabasine (NAB), which have weak carcinogenicity. NAT was even reported to be apparently lacking in carcinogenicity.1 Nicotine is the major alkaloid present in tobacco, playing a critical role in establishing and maintaining tobacco addiction. Nicotine and its related alkaloids are reported to be precursors of tobacco TSNAs. Nicotine mostly contributes to the formation of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNN).5,6 Nornicotine was found to be one of the major precursors of NNN.6,7 The other two TSNAs, NAT and NAB, are derived from anatabine and anabasine, respectively.6 In spite of similar molecule structure and mutual transformation, the interaction of these compounds with the cytochrome P450 (CYP) enzymes-catalyzed metabolism has been unclear until now.

Various CYP enzymes, including CYP2A13, CYP2A6, CYP2B6, CYP1A1, CYP1A2, CYP2D6, CYP2E1 and CYP3A4, have been shown to take part in the metabolism of TSNAs and nicotine.1,8 Due to the structural similarities between TSNAs and nicotine, some of the phase I metabolism was catalyzed by the same cytochrome enzyme.8–10 For example, NNK and nicotine were simultaneously metabolized by CYP2A13.11 Although the kinetic parameters for the metabolism of the single chemical of TSNAs were established, the biological interactions, especially the metabolism of TSNAs and nicotine by CYP enzymes, were ambiguous in vitro and in vivo. Tobacco smoke containing TSNAs and nicotine, as a whole, is inhaled by smokers. Considering the biological interactions (e.g., addition, synergism and antagonism) of different chemicals in tobacco smoke, the adverse effects of a single chemical is not equivalent to that in the smoke matrix.3 A number of publications using in vitro systems have reported that nicotine or nicotine metabolites were the inhibitors of diverse CYPs involved in NNK bioactivation including CYP2A13, CYP2A6 and CYP2E1.12 Moreover, in vitro work using purified enzymes has demonstrated that nicotine or nicotine metabolites could protect against CYP2A13- and 2A6-dependent NNK bioactivation.9,11 Further investigation identified the nicotine Delta5′(1′) iminium ion as a mechanism based inhibitor of CYP2A6 and CYP2A13.13 β-nicotyrine, a nicotine related alkaloid, has also been shown to inhibit CYP2A13 and CYP2A6 in vitro.10,14 CYP2E1, which has been shown to activate nitrosamines in human and rat livers,15 is also inhibited by nicotine and cotinine, albeit modestly.16 Interestingly, cotinine is an uncompetitive inhibitor of CYP2E1, which indicates that nicotine or nicotine metabolites do not necessarily have to be an enzyme substrate to exert an inhibitory activity.16 Many tobacco smoke constituents have been shown to inhibit the activation of CYP enzymes and the mutagenicity of N-nitrosamines in vivo and in vitro.17–19 Compounds that inhibit NNK or N-nitrosodimethyamine (NDMA) mutagenicity, such as ellagic acid, (–)-epigallocatechin-3-gallate and alkyl sulfides, have also been shown to be CYP2E1 inhibitors.20–23 Because of the structural similarity between pyridine alkaloids and N-nitrosamines, it is possible that this protection is due to competitive inhibition of CYP2E1, although to our knowledge this has never been studied.16

NNK metabolism has been comprehensively investigated in vitro and in vivo for many years. A simplified metabolism pathway of NNK has been drawn from past reviews and is shown in Fig. 1.1,8 Studies on NNK metabolism have been carried out in a number of human tissues and laboratory animals and have been reported to produce various metabolites. Human liver microsomes have been demonstrated to metabolize NNK to generate 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), HPB, OPB, NNK-N-oxide etc.1,24,25 While purified CYP enzymes-mediated reactions of NNK were shown to produce metabolites of OPB, HPB etc.,26,27 Fig. 1 reveals the distinct metabolism pathways of NNK namely carbonyl reduction (NNAL formation), pyridine oxidation (NNK-N-oxide formation), α-methyl hydroxylation (HPB formation) and α-methylene hydroxylation (OPB and OPBA formation), occurring simultaneously in the study of in vitro and in vivo NNK metabolism.1,28,29 In fact, the reactive intermediates (e.g., methyldiazonium ions and diazonium ions) in the metabolic pathway, not NNK itself, react with DNA bases to form methyl or pyridyloxobutyl DNA adducts, which were demonstrated to be crucial to exert TSNAs’ carcinogenesis. Thus, NNK metabolism is the prerequisite for NNK to exert a carcinogenic effect.

Fig. 1. A simplified representation of the NNK metabolism pathway.

Fig. 1

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To obtain strong evidence of carcinogenesis in humans, it is necessary to investigate the catalysis of NNK by phase I CYP enzymes. In the context of tobacco smoke chemical interactions, nicotine and its deviates-dependent inhibition of CYP enzymes raise an interesting and potentially controversial question regarding the possibility that weak or non-carcinogenic chemicals, including NAT, NAB or nicotine, could inactivate or inhibit CYP enzymes for the metabolism of NNK. From previous studies it has been concluded that several members of the CYP2A subfamily were efficient catalysts for NNK or nicotine metabolism. Since CYP2A13 and CYP2A6 share a similar sequence identity and catalyze the metabolism of many common substrates, their efficiency for catalyzing NNK metabolism was determined to choose the enzyme to carry out the inhibition kinetics study. Herein, we comprehensively investigated the inhibition of NAT, NAB and nicotine on NNK metabolism catalyzed by human CYP enzymes.

2. Materials and methods

2.1. Chemicals and reagents

NNK, d4-NNK, NAT, NAB, nicotine, OPB, HPB, d4-HPB, OPBA and d4-OPBA were purchased from Toronto Research Chemicals (Ontario, Canada). d4-HPB was used as the internal standard for both OPB and HPB quantitation and d4-OPBA was used for OPBA quantitation. Human CYP2A13 and CYP2A6 enzymes were purchased from Cypex Ltd (Scotland, UK). Triphosphopyridine nucleotide disodium salt trihydrate (NADP+) was purchased from Amresco LLC. (OH, USA). d-Glucose 6-phosphate disodium salt (G6P) and glucose-6-phosphate dehydrogenase (G6PDH) were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Methanol, acetic acid, ammonium acetate and other reagents of HPLC grade were purchased from Sigma-Aldrich Inc. (MO, USA).

2.2. In vitro NNK metabolism catalyzed by CYP2A13 and CYP2A6

The NNK metabolism was determined using a modified technique adapted from Jalas et al.'s study.30 Human CYP2A13 or CYP2A6 enzymes, derived from Escherchia coli, were combined with CYP-reductase co-expressed in one common bacterial host. The reaction mixtures contained enzyme solution (25 pM CYP2A13 or CYP2A6), substrates (10 μM NNK), a NADPH-generating system (1 mM NADP+, 5 mM G6P and 0.5 units per mL G6PDH) and 3 mM MgCl2 in 200 μL 0.1 mM Tris buffer, pH 7.4. The reaction was allowed to proceed for 10 min at 37 °C prior to termination by addition of 200 μL of pre-cooled acetonitrile at 4 °C. Analysis of NNK and its metabolites was carried out using high-performance liquid chromatography (HPLC, Spark Holland, Emmen, Netherlands) coupled with an API 5500 triple quadruple mass spectrometer (Applied Biosystems, Foster City, CA, USA).

2.3. Inhibition of in vitro NNK metabolism by NAT, NAB and nicotine

NNK metabolism catalyzed by CYP2A13 was measured either alone or in the presence of NAT (1, 5 μM), NAB (1, 5 μM) or nicotine (10, 50 μM), respectively. Experiments were carried out within the linear range of each product formation at 12.5 pM of the concentration of CYP2A13 in 5 min of reaction time, which was confirmed by previous studies in our lab. Other relative reaction conditions were modified from Jalas et al.'s study.30Km, Vmax and Ki values were calculated using the Sigma Plot kinetics program from Systat Software Inc. (San Jose, California). Ki estimates were determined using nonlinear regression analysis. All data were fit to a competitive inhibition model (V0 = Vmax[S]/(Km(1 + ([I]/Ki)) + [S])) and tested using the Runs test of residuals to determine statistically whether experimental data are randomly distributed around the curve with 95% confidence. Additionally, global R2 values to assess the goodness of fit confirmed that the competitive model fit the data sets well (R2 ≥ 0.98).

2.4. Transformation of NNK metabolites mediated by CYP2A13

In order to determine the transformation of NNK metabolites, a CYP2A13 enzyme system was used to incubate with OPB, HPB and OPBA, respectively. The reaction mixture contained 25 pM CYP2A13, an NNK metabolite (<10 μM), an NADPH-generating system (1 mM NADP+, 5 mM G6P and 0.5 units per mL G6PDH at 37 °C) prior to termination by the addition of 200 μL of pre-cooled acetonitrile at 4 °C.

2.5. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of NNK and its metabolites

NNK analysis was carried out by the previously reported method in our lab.31 The analytical method for metabolites was described in detail in the Appendix, which employed a symbiosis liquid chromatography system (Spark Holland, Emmen, Netherlands) coupled with an API 5500 triple quadruple mass spectrometer (Applied Biosystems, Foster City, CA, USA).

3. Results

3.1. Quantitative analysis of NNK reduction and its metabolites

Table 1 shows the CYP2A13-catalyzed enzymatic reaction after 30 min, resulting in 58.4% NNK reduction, while no change in the NNK level was obtained from the CYP2A6-catalyzed enzymatic reaction even after a longer incubation time. Accordingly, the three products of NNK metabolism catalyzed by CYP2A13 were observed to be as follows: OPB, HPB and OPBA. Other reported products, such as NNAL and NNK-N-oxide, were not detected in the study.

Table 1. NNK reduction and its metabolites by a CYP2A13- and 2A6-catalyzed reaction a .

  Control (μM) 2A13 (μM) 2A6 (μM)
NNK 10.41 ± 0.20 4.33 ± 0.29 10.04 ± 0.20
OPB 0 0.96 ± 0.032 — b
HPB 0 1.76 ± 0.012 — b
OPBA 0 0.46 ± 0.015 — b
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aIndicates that the enzymatic reaction was performed at 37 °C for 10 min with 25 pM CYP2A13 (or 50 pM CYP2A6), 10 μM NNK, NADPH-generating system and 3 mM MgCl2 in 0.1 mM Tris buffer at pH 7.4. Values are means ± SD (n = 3).

bRepresents the metabolites level was under the limit of quantitation in the recent analytical method.

Under the same reaction conditions, as shown in Table 1, it was found that HPB was the predominant product of the CYP2A13-catalyzed NNK metabolism and OPBA was the minor product. Corresponding with the lack of change in the NNK level, there was no product detected in the CYP2A6-catalyzed enzymatic reaction. It was concluded that one common substrate catalyzed by two CYPs generates various products, although the two CYPs belong to one subfamily with a highly similar sequence identity. In the following study, the CYP2A13 enzyme system was chosen as the research object for the kinetics of enzyme-catalyzed reactions and an inhibition study mediated by NAT, NAB and nicotine.

3.2. Kinetic parameters of CYP2A13 for the NNK metabolism

The kinetic parameters for NNK metabolism by CYP2A13 were determined in the study, which were fit with the Michaelis–Menten equation. It is shown in Table 2 that the Km and Vmax values for OPB formation were 3.5 μM and 6.3 pmol min–1 pmol–1 2A13, respectively, for HPB formation were 9.3 μM and 10.7 pmol min–1 pmol–1 2A13, respectively and for OPBA formation were 4.1 μM and 1.3 pmol min–1 pmol–1 2A13, respectively. Unequal values of Km and Vmax of different metabolites indicate the different generating velocity of the CYP2A13-catalyzed enzymatic reaction. The ratio of Vmax to Km of OPB formation, the maximum value, indicated OPB was more readily to be generated than other metabolites. The Vmax of HPB formation, the largest value among that of three metabolites, meant CYP2A13-catalyzed NNK metabolism produced larger amount of HPB than other metabolites.

Table 2. The effects of NAT, NAB and nicotine on the kinetic parameters of CYP2A13-catalyzed NNK metabolism a .

  NNK NNK + NAT (1 μM) NNK + NAB (1 μM) NNK + nicotine (10 μM)
K m (μM)
OPB 3.5 22.9 23.7 7.4
HPB 9.3 17.8 20.1 13.1
OPBA 4.1 15.5 14.5 11.0
V max (pmol min–1 pmol–1 2A13)
OPB 6.3 6.6 7.0 6.3
HPB 10.6 9.7 10.9 10.8
OPBA 1.3 1.3 1.4 1.2
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aMeans the reaction was performed at 37 °C for 5 min with NNK (1–100 μM), CYP2A13 (12.5 pM), a NADPH-generating system and MgCl2 (3 mM), in the absence or presence of NAT, NAB or nicotine.

3.3. Inhibition of the NNK metabolism by NAT, NAB and nicotine

To explore the inhibitory potency of NAT, NAB and nicotine on NNK metabolism, various concentrations of inhibitors were imposed on the CYP2A13-mediated reaction. As shown in Fig. 2(A–I), NAT, NAB and nicotine inhibited CYP2A13 activity over a range of substrate concentrations (1–100 μM NNK) in a dose-dependent manner. When NAT, NAB or nicotine were added to the incubation mixture, the CYP2A13-catalyzed formation of OPB, HPB and OPBA were significantly inhibited, respectively, with increasing NNK concentration. Table 2 shows the effects of NAT, NAB and nicotine on the kinetics of NNK metabolism catalyzed by CYP2A13. Km values for OPB, HPB and OPBA were increased to 22.9, 17.8 and 15.5 μM, respectively, by addition of NAT at 1 μM of final concentration, while Vmax values remained unchanged. This was also the case with NAB addition. For nicotine added at 10 μM of the final concentration, Km values for OPB, HPB and OPBA were increased to 7.4, 13.1 and 11.0 μM, respectively, while Vmax values remained unchanged. It is concluded from these data that the inhibition is competitive in nature since the addition of NAT, NAB or nicotine caused a 2- to 6-fold increase in the Km value but did not affect the Vmax (Table 2). Lineweaver–Burke analysis further suggested a competitive mechanism for NAT, NAB and nicotine-mediated inhibition, which is not shown in the article.

Fig. 2. Kinetics of OPB, HPB and OPBA formation from CYP2A13-catalyzed NNK metabolism and the effects of NAT, NAB and nicotine. NNK (1–100 μM) was incubated at 37 °C for 5 min with CYP2A13 (12.5 pM), a NADPH-generating system and MgCl2 (3 mM) either alone or in the presence of NAT (1, 5 μM), NAB (1, 5 μM) or nicotine (10, 50 μM). Data points are the means ± SD (error bars) (n = 3).

Fig. 2

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Estimated Ki values for NAT, NAB and nicotine were calculated from Michaelis–Menten equation parameters (Table 3). Data presented analogous Ki values as NAT- and NAB-inhibited the three metabolites’ formation. On the other hand, nicotine exhibited an 18–40 fold higher Ki value than that of NAT and NAB. Ki values for nicotine-inhibited HPB formation was the maximum (25.01 μM) and that for NAT-inhibited OPB formation was the minimum (0.21 μM). It is concluded that NAT and NAB exerted much more of an inhibitory effect than nicotine in the CYP2A13-catalyzed NNK metabolism.

Table 3. Calculated inhibition constants for the inhibition of CYP2A13 by NAT, NAB and nicotine.

  K i (μM)
NNK + NAT NNK + NAB NNK + nicotine
OPB 0.21 0.23 8.51
HPB 0.71 0.87 25.01
OPBA 0.36 0.50 6.57
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3.4. Mutual transformation of metabolites

Each of the three metabolites was incubated with a CYP2A13 enzyme system to determine mutual transformation between OPB, HPB and OPBA. It was found only in the OPB reaction mixture that OPB was decreased by 42.3% and HPB appeared from zero to 3.09 μM in 10 min of reaction time (Fig. 3). The CYP2A13 enzyme system did not induce any transformation of HPB or OPBA. Herein, this is the first report of an investigation into whether OPB can be hydrogenated to HPB by an in vitro CYP2A13 enzymatic reaction. As shown in Fig. 4, as NNK increased, the OPB/HPB ratio declined and the HPB/OPBA ratio rose, which means the HPB formation is dependent on not only the OPB transformation, but also another independent pathway directly mediated by the CYP2A13 enzyme system.

Fig. 3. CYP2A13-catalyzed transformation of OPB to HPB. Data bars are the means ± SD (error bars) (n = 3). a,b,c Bars with different letters were significantly different compared to control (p < 0.05).

Fig. 3

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Fig. 4. Ratio values of different metabolites produced from the CYP2A13-catalyzed NNK metabolism. The enzymatic reaction was performed as described in Table 2, but without competitor addition.

Fig. 4

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4. Discussion

This is the first report on the use of CYP2A enzymes to investigate the mutual effects of four TSNAs and nicotine with similar molecular structures. Because of the explicit carcinogenesis, NNK was used as the substrate for CYP enzymes-mediated metabolism to study the inhibitory potency of CYP2A enzymes induced by the weak- or non-carcinogenic compounds NAT, NAB and nicotine. Moreover, the results showed that CYP2A13, not 2A6, possessed catalytic activity toward NNK. Our study firstly concluded that NAT, NAB and nicotine exert competitively inhibitory effects on the CYP2A13-mediated NNK metabolism. This is in accordance with the following studies. Weymarn et al. demonstrated that nicotine or its metabolites is a mechanism-based inhibitor of CYP2A13 and CYP2A6.11 In addition, many studies have investigated the inhibitory potency of nicotine or its related alkaloids on the CYP2A13- or CYP2A6-mediated NNK metabolism. Denton et al. showed that nicotine and beta-nicotyrine were mechanism-based inhibitors of human CYP2A6,10 which was consistent with Kramlinger et al.'s further study, which showed that beta-nicotyrine was a mechanism based inhibitor and produced time-dependent inhibition and confirmed that beta-nicotyrine is an irreversible inactivator of CYP2A6.14 As for other CYP enzymes, Vleet et al. determined that nicotine and its major metabolite, cotinine, inhibit the catalytic activity of human CYP2E1, which confirmed that pyridine alkaloid-mediated CYP enzymes inhibition is a possible mechanism for the observed inhibition of NNK and NDMA mutagenicity by nicotine and cotinine in vitro.16 Bao et al. found that nicotine or NNN competitively inhibit NNK metabolism and considered nicotine as substrates of CYP2A13.9 Ordonez et al. evaluated the effect of nicotine and cotinine on DNA strand breaks using the COMET assay and found that nicotine contributed to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes.12 Although there have been many reports on inhibition studies of NNK metabolism induced by nicotine or its metabolites, few studies have focused on the inhibitory potency of CYP2A13 resulting from NAT and NAB. This article firstly used the two weak carcinogens NAT and NAB to investigate the inhibitory potency of CYP2A13-mediated NNK metabolism.

The structures of CYP2A13 and CYP2A6 share a high degree of sequence identity and catalyze the metabolism of many common substrates.32 CYP2A6 was detected in human lung and liver microsomes, but it predominantly exists in the liver. Although CYP2A13 is predominantly expressed in the respiratory tract33 and presents at lower levels than CYP2A6 in human liver microsomes, it is widely considered to contribute more to NNK metabolic activation.34 NNK requires metabolic activation to exert its carcinogenesis and CYP2A13-catalyzed activation likely plays a significant role in the induction of lung cancer in smokers.8,33 However, the two enzymes often exhibit discrepant catalytic efficiencies and produce unique metabolites.14 The present study compared the two CYP2A subfamily enzymes in their catalysis of one common compound. CYP2A13-mediated NNK metabolism generates OPB, HPB and OPBA, while CYP2A6-mediated NNK metabolism generates small quantities of products under the limit of detection in the present analytical method.

Although HPB and OPB were reported to be most easily formed by NNK metabolism, some discrepancies exist in those investigations. Peterson et al. found that HPB was preferentially formed in the absence of b5 but OPB preferentially formed in the presence of b5. The effect of adding b5 on the kinetics of P450-mediated NNK metabolism has not been further investigated. The result of our experiment without b5 addition showed that HPB was the most metabolite generated from, at least, two pathways: 1) CYP2A13-catalyzed reaction of NNK and 2) CYP2A13-catalyzed reaction of OPB, although the intermediate product between OPB and HPB was not clear in the article. Both our study and past reports exhibited that OPB was more likely to be formed than OPBA from CYP-mediated NNK metabolism, although OPBA was easily generated from oxidation of OPB in ambient air. In addition, OPBA was obviously detected in our study in relatively low levels than both OPB and HPB in CYP2A13-catalyzed NNK metabolism, which has been seldom reported in previous studies. The inconsistent results might be ascribed to various hydrogen donators and CYPs used in those studies.

Our study showed a lower value of Km and Km/Vmax for OPB formation, indicating an incline for this product generation in CYP2A13-catalyzed NNK metabolism. It was demonstrated that although OPB was a significant metabolite of NNK, the ratio of OPB : HPB formation was different in liver and lung microsomes.35 In our in vitro study, the ratio of OPB/HPB and OPB/OPBA exhibited their respective downward and steady trend with the increasing concentration of NNK. It can be concluded that the end product HPB was not only reduced from OPB, but also directly derived from CYP2A13-catalyzed NNK metabolism in spite of unknown intermediates. Peterson et al. also calculated apparent Km values for OPB and HPB formation in the liver and lung microsomes and concluded that more than one CYP enzyme participated in the NNK metabolism.35 Through the distinct incubation mixture, the results of our in vitro study were consistent with Peterson's in vivo study, in which the Km and Km/Vmax for OPB formation were lower than HPB. However, the kinetic parameters of CYP-mediated metabolism in our study were somewhat inconsistent with the past reports, which might be as a result of the purified CYP enzymes expressed in different host cells (e.g., Saccharomyces cerevisiae, E. coli) and different CYP-reductases used in those studies.9

In summary, our study firstly concluded the competitively inhibitory potency on CYP2A13-catalyzed NNK metabolism induced by weak carcinogenic compounds including NAT and NAB and confirmed the competitively inhibitory role on CYP2A13 activity played by the non-carcinogen nicotine. Metabolites of OPB, HPB and OPBA with respective kinetic parameters were obtained in our in vitro study on CYP2A13-catalyzed NNK metabolism. The metabolite of OPB was found to be easily reduced to HPB by the CYP2A13-mediated reaction. The present study suggests that the biological interaction of tobacco constituents may occur through competitive inhibition of CYP activity in the bioactivation stage and further interfere in the carcinogenic effects of carcinogens, such as NNK and NNN, which will need future studies to elucidate the inhibition mechanism.

Abbreviations

CYP

Cytochrome P450

NNK

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

NNN

N′-Nitrosonornicotine

NAT

(R,S)-N-Nitrosoanatabine

NAB

(R,S)-N-Nitrosoanabasine

TSNAs

Tobacco-specific nitrosamines

NNAL

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

NNK-N-oxide

4-(Methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone

OPB

4-Oxo-4-(3-pyridyl)butanal

HPB

4-Hydroxy-1-(3-pyridyl)-1-butanone

OPBA

4-Oxo-4-(3-pyridyl) butanoic acid

Supplementary Material

Click here for additional data file. (215.5KB, pdf)

Footnotes

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tx00016a

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