NNK Bioactivation Research Articles

Generation and characterization of a serum albumin‐humanized mouse model for biomarker discovery

The aim of this study is to develop a serum albumin‐humanized mouse model, for identifying human protein biomarkers of exposure to hazardous xenobiotics. The albumin‐humanized mouse was generated via cross‐breeding between a human albumin‐transgenic mouse and an albumin‐null mouse. The ability of human and mouse albumins to react with metabolites of the lung carcinogen 4‐(methylnitrosamino)‐l‐(3‐pyridyl)‐1‐butanone (NNK) was compared. Adult albumin‐humanized mice and wild‐type mice were treated with NNK (100 mg/kg body weight, IP). Blood, liver, and lung were analyzed 6 hours after NNK treatment. The NNK metabolite 4‐hydroxy‐l‐(3‐pyridyl)‐1‐butanone (HPB), free or as albumin‐HPB adduct, was detected in the plasma, whereas the O6‐methyl‐deoxyguanosine (O6‐mdG) DNA adduct, formed via NNK bioactivation, was detected in lung and liver. Results of quantitative analyses revealed lower levels of O6‐mdG in tissues of the humanized mice (10.4 and 85.5 pmol/μmol guanine, for lung and liver, respectively) than in wild‐type mice (18.4 and 240 pmol/μmol guanine, for lung and liver, respectively), a difference possibly explained by differing pharmacokinetic profiles of NNK. Interestingly, although a substantial genotype‐related difference was not observed in free HPB levels in the plasma (0.31 and 0.39 ng/ml, for wild‐type and humanized, respectively), human albumin‐HPB adduct levels in the humanized mouse were 6X higher than mouse albumin‐HPB adduct levels in the wild‐type mouse (per mg albumin). These data suggest that human and mouse albumins have different reactivities with reactive NNK metabolites and they illustrate the utility of the albumin‐humanized mouse model for biomarker discovery.Support or Funding InformationSupported in part by NIH grant CA092596

Contribution of Tobacco Use and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone to Three Methyl DNA Adducts in Urine

Human urinary DNA adducts may be useful surrogate biomarkers to estimate carcinogen exposure and activation, particularly if such adducts are of high selectivity from a specific carcinogen source. In this report, we provided evidence supporting tobacco use and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) being the dominant source for 3-methyladenine (3-mA), while nontobacco factors contribute significantly to 7-methylguanine and 1-methyladenine in the urine. Upon confirmation in human urine samples from larger populations in the future, urinary 3-mA may be used to estimate NNK bioactivation in smokers and to facilitate the development of a chemopreventive agent against NNK-induced carcinogenesis.

Dietary Dihydromethysticin Increases Glucuronidation of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol in A/J Mice, Potentially Enhancing Its Detoxification.

Effective chemopreventive agents are needed against lung cancer, the leading cause of cancer death. Results from our previous work showed that dietary dihydromethysticin (DHM) effectively blocked initiation of lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice, and it preferentially reduced 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-derived DNA adducts in lung. This study explored the mechanism(s) responsible for DHM's differential effects on NNK/NNAL-derived DNA damage by quantifying their metabolites in A/J mice. The results showed that dietary DHM had no effect on NNK or NNAL abundance in vivo, indicating that DHM does not affect NNAL formation from NNK. DHM had a minimal effect on cytochrome P450 2A5 (CYP2A5, which catalyzes NNK and NNAL bioactivation in A/J mouse lung), suggesting that it does not inhibit NNAL bioactivation. Dietary DHM significantly increased O-glucuronidated NNAL (NNAL-O-gluc) in A/J mice. Lung and liver microsomes from dietary DHM-treated mice showed enhanced activities for NNAL O-glucuronidation. These results overall support the notion that dietary DHM treatment increases NNAL detoxification, potentially accounting for its chemopreventive efficacy against NNK-induced lung tumorigenesis in A/J mice. The ratio of urinary NNAL-O-gluc and free NNAL may serve as a biomarker to facilitate the clinical evaluation of DHM-based lung cancer chemopreventive agents.

Effects of 8-methoxypsoralen on the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice

8-Methoxypsoralen (8-MOP) is a well established drug in the treatment of various skin diseases. Pretreatment of mice with 8-MOP before administration of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) significantly reduced the incidence of NNK-induced tumor. The present study was designed to evaluate the in vivo effects of 8-MOP on the bioactivation of NNK in mice. Decrease in the α-hydroxylation of NNK in mouse blood and tissues was observed as the most pronounced effect of 8-MOP. The catalytic property of cytochrome P450 2A5 (CYP2A5) enzyme in mice was determined by the coumarin 7-hydroxylation reaction, suggesting that 8-MOP produced remarkable inhibition on CYP2A5 in female C57BL/6 mice. These results implied that 8-MOP could prevent NNK-induced mutagenesis and tumorigenesis in mice through the inhibition of NNK α-hydroxylation, which may be achieved through the effect of 8-MOP on the bioactivities of CYP2A5.

Identification of cytochrome P450 enzymes critical for lung tumorigenesis by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): insights from a novel Cyp2abfgs-null mouse.

Cytochrome P450 (P450) enzymes encoded by the mouse Cyp2abfgs gene cluster are preferentially expressed in the respiratory tract. Previous studies have demonstrated that pulmonary P450-mediated bioactivation is necessary for lung tumorigenesis induced by the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and that CYP2A5 mediates a noteworthy fraction, but not all, of NNK bioactivation in the lung. The aim of this study was to determine whether other P450s encoded by the Cyp2abfgs gene cluster also play significant roles in NNK lung tumorigenesis. A novel Cyp2abfgs-null mouse was generated, in which all Cyp2a, 2b, 2g, 2f and 2s genes are deleted. The Cyp2abfgs-null mouse was viable, fertile and without discernible physiological abnormalities or compensatory increases in the expression of other P450s. NNK bioactivation in vitro and NNK-induced DNA adduction and lung tumorigenesis in vivo were determined for wild-type (WT) and Cyp2abfgs-null mice; the results were compared with previous findings from Cyp2a5-null mice. The Cyp2abfgs-null mice exhibited significantly lower rates of NNK bioactivation in lung and liver microsomes, compared with either WT or Cyp2a5-null mice. The levels of lung O(6)-methyl guanine DNA adduct were also substantially reduced in Cyp2abfgs-null mice, compared with either WT or Cyp2a5-null mice. Moreover, the Cyp2abfgs-null mice were largely resistant to NNK-induced lung tumorigenesis at both low (50mg/kg) and high (200mg/kg) NNK doses, in contrast to the WT or Cyp2a5-null mice. These results indicate for the first time that, collectively, the CYP2A, 2B, 2F, 2G, and 2S enzymes are indispensable for NNK-induced lung tumorigenesis.

Nicotine, cotinine, and β-nicotyrine inhibit NNK-induced DNA-strand break in the hepatic cell line HepaRG

Recent in vitro work using purified enzymes demonstrated that nicotine and/or a nicotine metabolite could inhibit CYPs (CYP2A6, 2A13, 2E1) involved in the metabolism of the genotoxic tobacco nitrosamine NNK. This observation raises the possibility of nicotine interaction with the mechanism of NNK bioactivation. Therefore, we hypothesized that nicotine or a nicotine metabolite such as cotinine might contribute to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes. The effect of nicotine and cotinine on DNA strand breaks was evaluated using the COMET assay in CYP competent HepaRG cells incubated with bioactive CYP-dependent NNK and CYP-independent NNKOAc (4-(acetoxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone). We report a dose-dependent reduction in DNA damage in hepatic-derived cell lines in the presence of nicotine and cotinine. Those results are discussed in the context of the in vitro model selected.

Simultaneous determination of NNK and its metabolites in mouse tissue for evaluating the effects of chronic alcohol consumption on the metabolism of NNK in mouse liver and lung.

A hydrophilic-interaction liquid chromatography-tandem mass spectrometry (HILIC-MS-MS) method was developed for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites in mouse liver and lung. The limits of detection of all analytes were in the range 0.017-0.057ngmL(-1), and recovery ranged from 88.4-119.8% with intra and inter-day precision in the range 0.89-6.03% and 1.01-6.97%, respectively. This simple and accurate method was used to evaluate the effect of chronic alcohol consumption on NNK bioactivation in mouse tissue. Time-course curves for NNK and its metabolites were generated, and the areas under the curves (AUCs) were compared. It was found that target tissues of NNK carcinogenesis in C57BL/6 mice contained high levels of α-hydroxylation metabolites of NNK and its carbonyl reduction metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The most pronounced effect of alcohol was to enhance α-hydroxylation of NNK in mouse lung and liver, which suggests that chronic alcohol consumption may increase the risk of carcinogenicity associated with NNK in mice.

Role of CYP2A13 in the bioactivation and lung tumorigenicity of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: in vivo studies using a CYP2A13-humanized mouse model.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is abundant in tobacco smoke, is a potent lung procarcinogen. The present study was aimed to prove that transgenic expression of human cytochrome P450 2A13 (CYP2A13), known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, will enhance NNK bioactivation and NNK-induced tumorigenesis in the mouse lung. Kinetic parameters of NNK bioactivation in vitro and incidence of NNK-induced lung tumors in vivo were determined for wild-type, Cyp2a5-null and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. As expected, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in Michaelis constant (K m) values for the formation of 4-oxo-4-(3-pyridyl)-butanal, representing the reactive intermediate that can lead to the formation of O(6)-methylguanine (O(6)-mG) DNA adducts; however, the gain of CYP2A13 at a fraction of the level of mouse lung CYP2A5 led to recovery of the activity in the lung, but not in the liver. The levels of O(6)-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of CYP2A13-humanized mice than in Cyp2a5-null mice. Moreover, incidences of lung tumorigenesis were significantly greater in CYP2A13-humanized mice than in Cyp2a5-null mice, and the magnitude of the differences in incidence was greater at low (30mg/kg) than at high (200mg/kg) NNK doses. These results indicate that CYP2A13 is a low K m enzyme in catalyzing NNK bioactivation in vivo and support the notion that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans.

Generation and Characterization of a CYP2A13‐Transgenic Mouse Model

CYP2A13, 2B6, and 2F1, neighboring P450 genes on human chromosome 19, are active toward many drugs, toxicants, and carcinogens. A CYP2A13/2B6/2F1‐transgenic mouse, in which CYP2A13 and 2F1 are both expressed in the respiratory tract, while CYP2B6 is expressed in the liver, was recently generated (Wei et al., Drug Metab. Dispos. 40:1144–1150, 2012). The aim of the present study is to generate a CYP2A13 (only) transgenic mouse, so that the specific activity of CYP2A13 can be determined. The CYP2B6 and 2F1 genes in the CYP2A13/2B6/2F1 genomic clone were inactivated via genetic manipulations, while CYP2A13 was kept intact. A CYP2A13 (only) transgenic (2A13‐TG) mouse was generated using the engineered construct, and then characterized, to confirm transgene integrity and determine copy numbers. The 2A13‐TG mice are normal in gross morphology, development, and fertility. As in the CYP2A13/2B6/2F1‐transgenic mouse, CYP2A13 expression in the 2A13‐TG mouse is limited to the respiratory tract; in contrast, CYP2B6 and 2F1 proteins are not detected. Additional studies using 2A13‐humanized (2A13‐TG/Cyp2abfgs‐null) mouse, produced by intercrossing between 2A13‐TG and newly generated Cyp2abfgs‐null mice, confirmed that the transgenic CYP2A13 is active in the bioactivation of NNK, a lung procarcinogen. The 2A13‐TG mouse should be valuable for assessing specific roles of human CYP2A13 in xenobiotic toxicity in the lung.

Abstract 5465: Role of CYP2A13 in NNK bioactivation and lung tumorigenesis: In vivo studies using a CYP2A13-humanized mouse model

Abstract The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung procarcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis depends on target tissue bioactivation by pulmonary P450 enzymes (Weng et al., 2007, Cancer Res 67:7825-7832). However, the specific P450 enzyme(s) responsible for NNK bioactivation in the lung have not been identified. The aim of the present study was to test the hypotheses that 1) mouse CYP2A5 plays an essential role in NNK bioactivation and tumorigenesis in mouse lung, and 2) transgenic expression of human CYP2A13, known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, can increase the rates of NNK bioactivation and incidence of tumorigenesis in the mouse lung. Kinetic parameters of microsomal NNK bioactivation in vitro, tissue levels of O6-methylguanine (O6-mG) DNA adducts formed in vivo, and incidence of NNK-induced lung tumors were determined for wild-type, Cyp2a5-null, and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. The results indicated that, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in the Km value for the formation of 4-oxo-4-(3-pyridyl)-butanal (OPB), which represents the reactive intermediate that can lead to the formation of O6-mG DNA adducts; but, the gain of CYP2A13 led to recovery of the activity in the lung, but not in the liver. The levels of O6-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of the wild-type mice and CYP2A13-humanized mice, than in the Cyp2a5-null mice. Furthermore, lung tumor bioassays, performed for the three mouse strains on A/J background, and using NNK at various doses (30 -200 mg/kg; single i.p. dose), revealed that lung tumor multiplicity determined at 4 months post-treatment was significantly greater in wild-type and CYP2A13-humanized mice than in Cyp2a5-null mice, for all NNK doses tested. The difference in lung tumor multiplicity between CYP2A13-humanized mice and Cyp2a5-null mice was greater at low (30 mg/kg) NNK dose (∼70%) than at high (200 mg/kg) NNK dose (∼30%). In control experiments, rates of systemic clearance of NNK and its major circulating metabolite, NNAL, were confirmed to be not different among the three mouse strains. These results indicated that CYP2A13 is a low-Km enzyme in catalyzing NNK bioactivation in vivo, and they provide strong support for the idea that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans. Moreover, the CYP2A13-humanized mouse model will be valuable for testing new, CYP2A13-targeted chemopreventive agents for lung carcinogenesis. (Supported in part by NIH grant CA092596) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5465. doi:1538-7445.AM2012-5465

Role of CYP2A5 in the Bioactivation of the Lung Carcinogen 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone in Mice

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis in mice depends on target-tissue bioactivation by pulmonary cytochrome P450 (P450) enzymes. The present study was designed to test the hypothesis that mouse CYP2A5 plays an essential role in NNK bioactivation in mouse lung. The role of CYP2A5 in NNK bioactivation was studied both in vitro and in vivo, by comparing the kinetic parameters of microsomal NNK metabolism and tissue levels of O(6)-methylguanine (O(6)-mG) (the DNA adduct highly correlated with lung tumorigenesis) between wild-type (WT) and Cyp2a5-null mice. In both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in the apparent K(m) values for the formation of 4-oxo-4-(3-pyridyl)butanone, which represents the reactive intermediate that produces O(6)-mG in vivo. The loss of CYP2A5 did not change circulating levels of NNK or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in mice treated intraperitoneally with NNK at either 20 or 100 mg/kg. However, the levels of lung O(6)-mG were significantly lower in Cyp2a5-null than in WT mice; the extent of the reduction was greater at the 20 mg/kg dose (∼40%) than at the 100 mg/kg dose (∼20%). These results indicate that CYP2A5 is the low-K(m) enzyme for NNK bioactivation in mouse lung. It is noteworthy that the remaining NNK bioactivation activities in the Cyp2a5-null mice could be inhibited by 8-methoxypsoralen, a P450 inhibitor used previously to demonstrate the role of CYP2A5 in NNK-induced lung tumorigenesis. Thus, P450 enzymes other than CYP2A5 probably also contribute to NNK-induced lung tumorigenesis in mice.

Abstract 3236: Role of mouse and human CYP2A enzymes in the bioactivation of the tobacco-specific lung procarcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Abstract NNK is a potent lung procarcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis depends on target-tissue metabolic activation by pulmonary P450 enzymes. The aim of this study is to test the hypotheses that 1) mouse CYP2A5 plays an essential role in NNK bioactivation in the lung, and 2) transgenic expression of human CYP2A13, known to be selectively expressed in the respiratory tract, and be the most efficient enzyme for NNK metabolic activation in vitro, can increase the rates of NNK metabolic activation in the mouse lung. We determined the rates of in vitro formation of 4-oxo-4-(3-pyridyl)-butanal (OPB), representing the reactive intermediate that can lead to the further formation of O6-mehtylguanine (O6-mG) DNA adduct, in liver and lung microsomes of wild-type, Cyp2a5-null, and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. We found that, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant decreases in the rates of formation of OPB; whereas, the gain of CYP2A13 led to recovery of the microsomal activity in the lung, but not in the liver. Furthermore, we observed that the levels of O6-mG, the DNA adduct known to be most highly correlated with incidences of lung tumor formation, were significantly higher in the lungs of the CYP2A13-humanized mice, than in the Cyp2a5-null mice. In control experiments, rates of systemic clearance of NNK and its major circulating metabolite, NNAL, were confirmed to be not different among the three mouse strains. These results indicate that CYP2A13 can catalyze NNK bioactivation in vivo, and they support the idea that CYP2A13 genetic polymorphisms can influence the risks of tobacco-induced lung tumorigenesis in humans. (Supported in part by NIH grant CA092596) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2011-3236

Abstract LB-432: Effect of chronic nicotine consumption on NNK-induced lung tumors in the A/J mouse

Abstract Nicotine replacement therapy (NRT) is often used to maintain smoking cessation. Some individuals continue to use NRT for years after smoking cessation, and some NRT users continue to smoke. It is important to understand the impact of chronic nicotine use on lung cancer risk. Therefore, we determined the effect of nicotine administration on NNK-induced lung tumors in the A/J mice. P450 2A enzymes are key catalysts in NNK bioactivation, and nicotine is both an inhibitor and a mechanism based inactivator of P450 2A-catalyzed nicotine metabolism. These data suggest nicotine might protect from NNK-induced tumorigenesis. However, several investigators have reported that nicotine inhibits apoptosis, enhances angiogenesis and enhances proliferation. We hypothesized that nicotine would prevent or delay initiation of NNK-induced tumors, but accelerate tumor growth by reducing apoptosis and promoting inflammation. To test this, female A/J mice were administered a single dose of NNK (100 mg/kg i.p) and 200 ug/ml nicotine in the drinking water. Nicotine was administered 2 weeks prior to NNK, 44 weeks after NNK, throughout the experiment, or without NNK treatment. There were several key outcomes of this study: Chronic nicotine consumption did not affect P450 2A5-catalyzed metabolism in the lung of liver. Nicotine administration alone for 44 weeks did not increase lung tumor multiplicity. Nicotine given before or after NNK administration did not increase, or decrease, tumor number or tumor size. There was also no difference in the number of carcinomas per mouse or the number of adenomas with dysplasia. In conclusion, nicotine consumption in the drinking water had no effect on the tumorigenicity of NNK in the A/J mouse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-432. doi:10.1158/1538-7445.AM2011-LB-432

Human cyt P450 mediated metabolic toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays

Electrochemiluminescent (ECL) arrays containing polymer ([Ru(bpy)(2)(PVP)(10)](2+), PVP = polyvinylpyridine), DNA, and selected enzymes were employed to elucidate cytochrome (cyt) P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Bioactivated NNK metabolites formed upon H(2)O(2)-enzymatic activation were captured as DNA adducts and detected simultaneously from 36 spot arrays by capturing and quantifying emitted ECL with an overhead CCD camera. Increased ECL emission was dependent on NNK exposure time. Of the enzymes tested, the activity toward NNK bioactivation was cyt P450 1A2 > 2E1 > 1B1 approximately chloroperoxidase (CPO) > myoglobin (Mb) in accordance with reported in vivo studies. Cyt P450/polyion films were also immobilized on 500 nm diameter silica nanospheres for product analysis by LC-MS. Analysis of the nanosphere film reaction media provided ECL array validation and quantitation of the bioactivated NNK hydrolysis product 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) confirming production of reactive metabolites in the films. Chemical screening in this fashion allows rapid clarification of enzymes responsible for genotoxic activation as well as offering insight into cyt P450-related toxicity and mechanisms.

Inhibition of rat hepatic CYP2E1 by quinacrine: molecular modeling investigation and effects on 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mutagenicity

Increased activity of CYP2E1 has been associated with increased risk of chemically-mediated cancers, through enhanced activation of a variety of procarcinogens. In this context, inhibition of CYP2E1 is potentially of significance in xenobiotic toxicity. The aim of the present study was to test the hypothesis that quinacrine inhibits hepatic CYP2E1. For this purpose, disulfiram (75 mg/kg i.p) as an inhibitor and isoniazid (100 mg/kg i.p) as an inducer of CYP2E1, as well as quinacrine (50 mg/kg i.p) were administered to Wistar rats and the hepatic activity of CYP2E1 was measured. The expression of CYP2E1 was further assessed by Western blot analysis. As expected, disulfiram inhibited, while isoniazid induced the activity and expression of the enzyme. Interestingly, treatment with quinacrine resulted in a significant decrease of CYP2E1 activity and expression. To investigate any similarities in the inhibition of CYP2E1 by quinacrine and disulfiram, molecular modeling techniques were adopted and revealed that quinacrine molecule anchors inside the same binding pocket of the protein where disulfiram is also attached. Finally, as assessed by the sister chromatid exchanges (SCE) assay, quinacrine was demonstrated to reduce the mutagenic effects of the tobacco-specific N-nitrosamine 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is known to be converted to active mutagen in the liver principally through CYP2E1. We suggest that these antimutagenic effects of quinacrine could be possibly attributed, at least in part, to its ability to block the bioactivation of NNK, mainly by the inhibition of CYP2E1. Our results, even preliminary, indicate that quinacrine as an inhibitor of CYP2E1 might be protective against chemically-induced toxicities such as NNK-induced mutagenicity.

Analysis of CYP2A Contributions to Metabolism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone in Human Peripheral Lung Microsomes

The objectives of this study were to determine the contributions of CYP2A13 and CYP2A6 to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism in human peripheral lung microsomes and to determine the influence of the genetic polymorphism, CYP2A13 Arg257Cys, on NNK metabolism. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the keto-reduced metabolite of NNK, was the major metabolite produced, ranging from 0.28 to 0.9%/mg protein/min. Based on total bioactivation of NNK and NNAL by alpha-carbon hydroxylation, subjects could be classified as either high (17 subjects) or low (12 subjects) bioactivators [(5.26 +/- 1.23) x 10(-2) and (6.49 +/- 5.90) x 10(-3)% total alpha-hydroxylation/mg protein/min, P < 0.05]. Similarly, for detoxification, subjects could be grouped into high (9 subjects) and low (20 subjects) categories [(2.03 +/- 1.65) x 10(-3) and (2.50 +/- 3.04) x 10(-4)% total N-oxidation/mg protein/min, P < 0.05]. When examining data from all individuals, no significant correlations were found between levels of CYP2A mRNA, CYP2A enzyme activity, or CYP2A immunoinhibition and the degree of total NNK bioactivation or detoxification (P > 0.05). However, subgroups of individuals were identified for whom CYP2A13 mRNA correlated with total NNK and NNAL alpha-hydroxylation and NNAL-N-oxide formation (P < 0.05). The degree of NNAL formation and CYP2A13 mRNA was also correlated (P < 0.05). Subjects (n = 84) were genotyped for the CYP2A13 Arg257Cys polymorphism, and NNK metabolism for the one variant (Arg/Cys) was similar to that for other subjects. Although results do not support CYP2A13 or CYP2A6 as predominant contributors to NNK bioactivation and detoxification in peripheral lung of all individuals, CYP2A13 may be important in some.

Determination of the Role of Target Tissue Metabolism in Lung Carcinogenesis Using Conditional Cytochrome P450 Reductase-Null Mice

Critical to mechanisms of chemical carcinogenesis and the design of chemopreventive strategies is whether procarcinogen bioactivation in an extrahepatic target tissue (e.g., the lung) is essential for tumor formation. This study aims to develop a mouse model capable of revealing the role of pulmonary microsomal cytochrome P450 (P450)-mediated metabolic activation in xenobiotic-induced lung cancer. A novel triple transgenic mouse model, with the NADPH-P450 reductase (Cpr) gene deleted in a lung-specific and doxycycline-inducible fashion (lung-Cpr-null), was generated. CPR, the obligate electron donor for microsomal P450 enzymes, is essential for the bioactivation of many procarcinogens. The lung-Cpr-null mouse was studied to resolve whether pulmonary P450 plays a major role in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer by producing carcinogenic metabolites in the target tissue. A liver-Cpr-null mouse was also studied to test whether hepatic P450 contributes predominantly to systemic clearance of NNK, thereby decreasing NNK-induced lung cancer. The numbers of NNK-induced lung tumors were reduced in the lung-Cpr-null mice but were increased in the liver-Cpr-null mice, relative to wild-type control mice. Decreased lung tumor multiplicity in the lung-Cpr-null mice correlated with reduced lung O6-methylguanine adduct levels, without decreases in NNK bioavailability, consistent with decreased NNK bioactivation in the lung. Moreover, lung tumors in lung-Cpr-null mice were positive for CPR expression, indicating that the tumors did not originate from Cpr-null cells. Thus, we have confirmed the essential role of pulmonary P450-mediated metabolic activation in NNK-induced lung cancer, and our mouse models should be applicable to studies on other procarcinogens that require P450-mediated metabolic activation.

Quantitation of Pyridyloxobutyl DNA Adducts of Tobacco-Specific Nitrosamines in Rat Tissue DNA by High-Performance Liquid Chromatography−Electrospray Ionization−Tandem Mass Spectrometry

The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce pyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN and NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitative analysis of four POB-DNA adducts with known structures. The corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis. The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis. The method was accurate and precise. Excellent sensitivity was achieved, especially for O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 12) was the most abundant adduct, followed by O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 8), O2-POB-dThd, and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O2-POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the in vitro data, 7-POB-Gua was the major POB-DNA adduct formed in vivo. However, levels of O6-POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in vivo. In contrast to the other three adducts, O6-POB-dGuo was more abundant in lung than in liver. O2-POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive quantitation of four POB-DNA adducts, support an important role of O6-POB-dGuo in NNK lung tumorigenicity in rats, and suggest that O2-POB-dThd may be a useful tobacco-specific DNA biomarker for future tobacco carcinogenesis studies.

Synergistic DNA damage and lipid peroxidation in cultured human white blood cells exposed to 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and ultraviolet A

4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen in cigarette smoke, while ultraviolet (UV) irradiation from sunlight is a major factor for causing skin aging and skin cancer. However, little is known about the effects of the interaction between NNK and UV light on the induction of DNA damage and oxidative stress. In this study, we incubated human white blood cells (WBCs) with NNK, followed by irradiating the cells with ultraviolet A (UVA) (320-380 nm), and we measured DNA strand breaks (by the Comet or single-cell gel electrophoresis assay), lipid peroxidation (as thiobarbituric acid-reactive substances, TABRS), and the levels of intracellular reactive oxygen species (ROS). We found that preincubation with 1.0 mM NNK, followed by UVA irradiation (7.6 kJ/m2) synergistically increased DNA damage, lipid peroxidation, and the level of intracellular ROS in WBCs, while NNK or UVA alone had little or no effect. Electron spin resonance spectroscopic analyses showed that NNK plus UVA enhanced the UVA-induced generation of singlet oxygen but not hydroxyl radicals. In addition to ROS, bioactivation of NNK by cytochromes P450 (CYP) to form reactive NNK intermediates may also be involved in the synergistic damage to WBCs by NNK plus UVA. This is evidenced by the synergistic increase in N7-methylguanine (7-mGua), a major DNA adduct produced by NNK. Overall, the present study demonstrates that exposure of WBCs to both NNK and UVA synergistically increases DNA damage and lipid peroxidation and that such effects involve enhanced generation of ROS, especially singlet oxygen, and activation of NNK to 7-mGua by CYP. The results imply that NNK is a phototoxic agent.

Molecular modelling of CYP2A enzymes: application to metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

1. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in a variety of animal models and a putative human lung carcinogen. Its tumorigenic potential is unmasked via cytochrome P450 (CYP)-mediated hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e. α-hydroxylation). Therefore, elucidation of enzyme–substrate interactions that facilitate α-hydroxylation is important to gain insight into the tumorigenic mechanism of NNK and to develop potent inhibitors of this detrimental reaction.2. Molecular models of CYP2A enzymes from mice, rats and humans that are catalysts of NNK bioactivation were constructed and used, in conjunction with docking experiments, to identify active-site residues that make important substrate contacts.3. Docking studies revealed that hydrophobic residues at positions 117, 209, 365 and 481, among others, play critical roles in orienting NNK in the active site to effect α-hydroxylation. These molecular models were then used to rationalize the stereo- and regioselectivity, as well as the efficiency, of CYP2A-mediated NNK metabolism.