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Stephen S. Hecht - One of the best experts on this subject based on the ideXlab platform.
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metabolism and dna adduct formation of carcinogenic tobacco specific nitrosamines found in smokeless tobacco products
2020Co-Authors: Stephen S. HechtAbstract:Abstract Tobacco-specific nitrosamines are the most abundant strong carcinogens in smokeless tobacco products. This chapter focuses on three of the most important of these compounds: N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The carcinogenic activities of NNN, NNK, and NNAL are discussed, and the metabolic pathways of these carcinogens are presented. Specific metabolite biomarkers have been developed to assess exposure to tobacco-specific nitrosamines in smokeless tobacco users; these are presented and discussed. DNA and protein adduct formation by tobacco-specific nitrosamines are also summarized, based mainly on studies in laboratory animals.
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Mass Spectrometric Quantitation of Pyridyloxobutyl DNA Phosphate Adducts in Rats Chronically Treated with N'-Nitrosonornicotine.
Chemical Research in Toxicology, 2019Co-Authors: Qing Cao, Silvia Balbo, Pramod Upadhyaya, Lijiao Zhao, Stephen S. HechtAbstract:The tobacco-specific carcinogens N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) require metabolic activation to exert their carcinogenicity. NNN and NNK are metabolized to the same reactive diazonium ions, which alkylate DNA forming pyridyloxobutyl (POB) DNA base and phosphate adducts. We have characterized the formation of both POB DNA base and phosphate adducts in NNK-treated rats and the formation of POB DNA base adducts in NNN-treated rats. However, POB DNA phosphate adducts in NNN-treated rats are still uncharacterized. In this study, we quantified the levels of POB DNA phosphate adducts in tissues of rats chronically treated with (S)-NNN or (R)-NNN for 10, 30, 50, and 70 weeks during a carcinogenicity study. The highest amounts of POB DNA phosphate adducts were observed in the esophagus of the (S)-NNN-treated rats, with a maximum level of 5400 ± 317 fmol/mg DNA at 50 weeks. The abundance of POB DNA phosphate adducts in the esophagus was consistent with the resul...
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DNA Adduct Formation from Metabolic 5′-Hydroxylation of the Tobacco-Specific Carcinogen N′-Nitrosonornicotine in Human Enzyme Systems and in Rats
Chemical Research in Toxicology, 2016Co-Authors: Adam T. Zarth, Jing Yang, Pramod Upadhyaya, Stephen S. HechtAbstract:N′-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2′-hydroxylation and 5′-hydroxylation. While most previous studies have focused on 2′-hydroxylation in target tissues of rats, available evidence suggests that 5′-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5′-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2′-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7–500 ppm), py-py-dI was the major DNA adduct resulting from 5′-hydroxylation of NNN in vivo. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (S)-NNN or (R)-NNN, the ratios of formation of (R...
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Transcriptome profiling in oral cavity and esophagus tissues from (S)-N'-nitrosonornicotine-treated rats reveals candidate genes involved in human oral cavity and esophageal carcinogenesis.
Molecular Carcinogenesis, 2016Co-Authors: Ali Khammanivong, Stephen S. Hecht, Pramod Upadhyaya, Arunkumar Anandharaj, Xuemin Qian, Jung Min Song, Silvia Balbo, Dipankar Bandyopadhyay, Erin B. Dickerson, Fekadu KassieAbstract:Recently, we have shown that (S)-N'-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 wk and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate P-value
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exposure and metabolic activation biomarkers of carcinogenic tobacco specific nitrosamines
Accounts of Chemical Research, 2016Co-Authors: Stephen S. Hecht, Irina Stepanov, Steven G. CarmellaAbstract:ConspectusLung cancer is the leading cause of cancer death in the world, and cigarette smoking is its main cause. Oral cavity cancer is another debilitating and often fatal cancer closely linked to tobacco product use. While great strides have been made in decreasing tobacco use in the United States and some other countries, there are still an estimated 1 billion men and 250 million women in the world who are cigarette smokers and there are hundreds of millions of smokeless tobacco users, all at risk for cancer. Worldwide, lung cancer kills about three people per minute.This Account focuses on metabolites and biomarkers of two powerful tobacco-specific nitrosamine carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN), considered to be among the main causes of lung cancer and oral cavity cancer in people who use tobacco products. Three properties of NNK and NNN are critical for successful biomarker studies: they are present in all tobacco products, they are tobac...
Irina Stepanov - One of the best experts on this subject based on the ideXlab platform.
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Presence of the Carcinogen N′-Nitrosonornicotine in Saliva of E-cigarette Users
Chemical Research in Toxicology, 2018Co-Authors: Gabriela Bustamante, Katrina Yershova, Galina Yakovlev, Joni Jensen, Dorothy K. Hatsukami, Irina StepanovAbstract:Many harmful constituents are present in e-cigarettes at much lower levels than in cigarette smoke, and the results of analysis of urinary biomarkers in e-cigarette users are consistent with these findings. However, understanding the health effects of chronic exposures to e-cigarette aerosols may require thinking beyond these comparisons. In this study, we investigated the endogenous formation of the tobacco-specific oral and esophageal carcinogen N′-nitrosonornicotine (NNN) in e-cigarette users. Salivary NNN, nornicotine, and nicotine as well as urinary tobacco biomarkers, including total NNN, were analyzed in 20 e-cigarette users, 20 smokers, and 19 nonsmokers. Nornicotine and NNN levels in e-cigarettes used by the study participants were also analyzed. The mean of NNN in saliva of e-cigarette users was 14.6 (±23.1) pg/mL, ranging from nonquantifiable (below the limit of quantitation, LOQ) to 76.0 pg/mL. In smokers, salivary NNN ranged from below LOQ to 739 pg/mL, with 80% of smokers having salivary NNN...
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exposure and metabolic activation biomarkers of carcinogenic tobacco specific nitrosamines
Accounts of Chemical Research, 2016Co-Authors: Stephen S. Hecht, Irina Stepanov, Steven G. CarmellaAbstract:ConspectusLung cancer is the leading cause of cancer death in the world, and cigarette smoking is its main cause. Oral cavity cancer is another debilitating and often fatal cancer closely linked to tobacco product use. While great strides have been made in decreasing tobacco use in the United States and some other countries, there are still an estimated 1 billion men and 250 million women in the world who are cigarette smokers and there are hundreds of millions of smokeless tobacco users, all at risk for cancer. Worldwide, lung cancer kills about three people per minute.This Account focuses on metabolites and biomarkers of two powerful tobacco-specific nitrosamine carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN), considered to be among the main causes of lung cancer and oral cavity cancer in people who use tobacco products. Three properties of NNK and NNN are critical for successful biomarker studies: they are present in all tobacco products, they are tobac...
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(S)-N'-Nitrosonornicotine, a Constituent of Smokeless Tobacco, is a Powerful Oral Cavity Carcinogen in Rats
Carcinogenesis, 2013Co-Authors: Silvia Balbo, Irina Stepanov, Steven G. Carmella, Dipankar Bandyopadhyay, Fekadu Kassie, Sandra James-yi, Charles S. Johnson, Michael G. O’sullivan, Mingyao Wang, Pramod UpadhyayaAbstract:Currently, smokeless tobacco products are being proposed as an alternative mode of tobacco use associated with less harm. All of these products contain the tobacco-specific carcinogen N′-nitrosonornicotine (NNN). The major form of NNN in tobacco products is (S)-NNN, shown in this study to induce a total of 89 benign and malignant oral cavity tumors in a group of 20 male F-344 rats treated chronically with 14 p.p.m. in the drinking water. The opposite enantiomer (R)-NNN was weakly active, but synergistically enhanced the carcinogenicity of (S)-NNN. Thus, (S)-NNN is identified for the first time as a strong oral cavity carcinogen in smokeless tobacco products and should be significantly reduced or removed from these products without delay in order to prevent debilitating and deadly oral cavity cancer in people who use them.
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nornicotine nitrosation in saliva and its relation to endogenous synthesis of n nitrosonornicotine in humans
Nicotine & Tobacco Research, 2013Co-Authors: Aleksandar Knezevich, Dorothy K. Hatsukami, Stephen S. Hecht, John Muzic, Irina StepanovAbstract:INTRODUCTION We recently reported that certain amounts of the carcinogen N'-nitrosonornicotine (NNN) can be formed endogenously from nicotine and/or nornicotine in some users of oral nicotine replacement therapy products. Although the acidic environment of the stomach creates the most favorable conditions for nitrosation, this reaction could also occur in the oral cavity in the presence of bacteria that catalyze nitrosation at neutral pH. METHODS To test the hypothesis that endogenous formation of NNN could occur in the oral cavity, we investigated nitrosation of nicotine and nornicotine in human saliva. To specifically identify NNN as derived from precursors added to saliva, we incubated saliva samples with [pyridine-D(4)]nicotine and [pyridine-D(4)]nornicotine, with and without the addition of nitrite, and subsequently analyzed [pyridine-D(4)]NNN by liquid chromatography-tandem mass spectrometry. RESULTS Consistent with kinetic studies on nicotine and nornicotine nitrosation, incubation of saliva with [pyridine-D(4)]nornicotine alone produced detectable amounts of [pyridine-D(4)]NNN, whereas only traces of [pyridine-D(4)]NNN were found in samples incubated with [pyridine-D(4)]nicotine and sodium nitrite. Incubation of saliva samples from 10 nonsmoking volunteers with [pyridine-D(4)]nornicotine resulted in the formation of [pyridine-D(4)]NNN in 8 samples, with yields ranging from 0.003% to 0.051% of the added alkaloid. CONCLUSION Our results demonstrate that NNN can be formed from nornicotine in human saliva without deliberate addition of any other substance. Therefore, nornicotine, as present in tobacco or in nicotine replacement products, is a carcinogen precursor.
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Levels of (S)-N'-nitrosonornicotine in U.S. tobacco products.
Nicotine & Tobacco Research, 2012Co-Authors: Irina Stepanov, Pramod Upadhyaya, Steven G. Carmella, Katrina Yershova, Stephen S. HechtAbstract:Introduction: N′-nitrosonornicotine (NNN), an esophageal and oral carcinogen present in tobacco products, has a chiral center in its structure. Of its two enantiomers, (S)-NNN exhibits higher tumorigenic potency than (R)-NNN. There is no information available on the levels of (S)-NNN in various tobacco products currently marketed in the United States.
Steven G. Carmella - One of the best experts on this subject based on the ideXlab platform.
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exposure and metabolic activation biomarkers of carcinogenic tobacco specific nitrosamines
Accounts of Chemical Research, 2016Co-Authors: Stephen S. Hecht, Irina Stepanov, Steven G. CarmellaAbstract:ConspectusLung cancer is the leading cause of cancer death in the world, and cigarette smoking is its main cause. Oral cavity cancer is another debilitating and often fatal cancer closely linked to tobacco product use. While great strides have been made in decreasing tobacco use in the United States and some other countries, there are still an estimated 1 billion men and 250 million women in the world who are cigarette smokers and there are hundreds of millions of smokeless tobacco users, all at risk for cancer. Worldwide, lung cancer kills about three people per minute.This Account focuses on metabolites and biomarkers of two powerful tobacco-specific nitrosamine carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN), considered to be among the main causes of lung cancer and oral cavity cancer in people who use tobacco products. Three properties of NNK and NNN are critical for successful biomarker studies: they are present in all tobacco products, they are tobac...
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Combined analysis of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the urine of cigarette smokers and e-cigarette users.
Journal of Chromatography B, 2015Co-Authors: Delshanee Kotandeniya, Steven G. Carmella, Makenzie E. Pillsbury, Stephen S. HechtAbstract:Abstract A liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI+–MS/MS) method for the analysis of the tobacco-specific carcinogens N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and their glucuronides (total NNN and total NNAL) in human urine was developed. The method has excellent accuracy and intra-day and inter-day precision, and limits of quantitation of 0.015 and 0.075 pmol/mL urine, respectively, for total NNN and total NNAL. A unique aspect of this method is internal assessment of possible artifactual formation of NNN by inclusion of the monitor amine [pyridine-D4]nornicotine. We found that artifactual formation of NNN comprised only 2.5% of the measured amounts of total NNN in urine of cigarette smokers, under our conditions using ammonium sulfamate as an inhibitor of nitrosation. The method was applied to urine samples from cigarette smokers and e-cigarette users. Levels of total NNN and total NNAL in the urine of cigarette smokers averaged 0.060 ± 0.035 pmol/mL and 2.41 ± 1.41 pmol/mL urine, (N = 38), respectively, which were both significantly greater than in the urine of 27 e-cigarette users.
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(S)-N'-Nitrosonornicotine, a Constituent of Smokeless Tobacco, is a Powerful Oral Cavity Carcinogen in Rats
Carcinogenesis, 2013Co-Authors: Silvia Balbo, Irina Stepanov, Steven G. Carmella, Dipankar Bandyopadhyay, Fekadu Kassie, Sandra James-yi, Charles S. Johnson, Michael G. O’sullivan, Mingyao Wang, Pramod UpadhyayaAbstract:Currently, smokeless tobacco products are being proposed as an alternative mode of tobacco use associated with less harm. All of these products contain the tobacco-specific carcinogen N′-nitrosonornicotine (NNN). The major form of NNN in tobacco products is (S)-NNN, shown in this study to induce a total of 89 benign and malignant oral cavity tumors in a group of 20 male F-344 rats treated chronically with 14 p.p.m. in the drinking water. The opposite enantiomer (R)-NNN was weakly active, but synergistically enhanced the carcinogenicity of (S)-NNN. Thus, (S)-NNN is identified for the first time as a strong oral cavity carcinogen in smokeless tobacco products and should be significantly reduced or removed from these products without delay in order to prevent debilitating and deadly oral cavity cancer in people who use them.
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Levels of (S)-N'-nitrosonornicotine in U.S. tobacco products.
Nicotine & Tobacco Research, 2012Co-Authors: Irina Stepanov, Pramod Upadhyaya, Steven G. Carmella, Katrina Yershova, Stephen S. HechtAbstract:Introduction: N′-nitrosonornicotine (NNN), an esophageal and oral carcinogen present in tobacco products, has a chiral center in its structure. Of its two enantiomers, (S)-NNN exhibits higher tumorigenic potency than (R)-NNN. There is no information available on the levels of (S)-NNN in various tobacco products currently marketed in the United States.
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Presence of the carcinogen N'-nitrosonornicotine in the urine of some users of oral nicotine replacement therapy products
Cancer Research, 2009Co-Authors: Irina Stepanov, Steven G. Carmella, Dorothy K. Hatsukami, Anna Briggs, Louise A. Hertsgaard, Bruce R. Lindgren, Stephen S. HechtAbstract:N′-nitrosonornicotine (NNN) is a strong carcinogen present in unburned tobacco and cigarette smoke. We here analyze data obtained in two studies, in which a biomarker of exposure to NNN – the sum of NNN and its pyridine-N-glucuronide, referred to as total NNN – was quantified in the urine of people who had stopped smoking and used various nicotine replacement therapy (NRT) products. In 13 out of 34 nicotine gum or lozenge users from both studies, total NNN at one or more time points after biochemically confirmed smoking cessation was comparable to, or considerably higher than, the baseline levels. For most of the subjects who used the nicotine patch as a smoking cessation aid, urinary total NNN at all post-quit time points was less than 37% of their mean baseline levels. These results indicate that endogenous formation of significant amounts of NNN may occur sporadically in some users of oral NRT. Given the carcinogenicity of NNN and the frequent use of nicotine gum as a smoking cessation aid, further studies are needed so that preventive measures can be developed.
Erik Dybing - One of the best experts on this subject based on the ideXlab platform.
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Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N′-Nitrosonornicotine (NNN) by isolated rabbit lung cells
Cell Biology and Toxicology, 1990Co-Authors: Jon E. Dahl, Rune Becher, Hildegard M. Schuller, Erik DybingAbstract:The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [^3H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.
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Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) by isolated rabbit lung cells.
Cell Biology and Toxicology, 1990Co-Authors: Jon E. Dahl, Rune Becher, Hildegard M. Schuller, Marit Låg, Erik DybingAbstract:The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [3H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.
Elmar Richter - One of the best experts on this subject based on the ideXlab platform.
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Ultrasensitive method for the determination of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts by gas chromatography–high resolution mass spectrometry in mucosal biopsies of the lower esophagus
Analytical and Bioanalytical Chemistry, 2009Co-Authors: Christopher W. Heppel, Anne-kathrin Heling, Elmar RichterAbstract:4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N ′-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3–2.0 g of human target tissues such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of 14.9 fmol HBP and a recovery of 45 ± 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of
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Metabolism of N’-nitrosonornicotine by precision-cut rodent lung slices.
Cancer Research, 2004Co-Authors: Bettina Lassnack, Elmar Richter, Anthony R. TrickerAbstract:3198 Precision-cut tissue slices in dynamic organ culture retain their cellular architecture and metabolic activity for several hours, suggesting that organ slices are a suitable in vitro model for studying tissue metabolism of xenobiotics. N’-Nitrosonornicotine (NNN) is reported to induce tumors of the lung in A/J mice, and esophagus and nasal mucosa in the F344 rat. The major routes of NNN metabolism are α-hydroxylation to yield either 2’-hydroxyNNN or 5’-hydroxyNNN, pyridine-N-oxidation, and concerted denitrosation and oxidation to norcotinine. Minor pathways of metabolism include β-hydroxylation to yield either 3’-hydroxyNNN or 4’-hydroxyNNN. Of all the potential routes of NNN metabolism, 2’-hydroxylation is thought to be the major activation pathway yielding target tissue DNA adducts, and the ratio of 2’-hydroxylation to 5’-hydroxylation is often considered to indicate the extent of NNN activation. 2’-Hydroxylation of 0.004 to 1.2 μM [5-3H]NNN (27 Ci/mmol) in precision-cut A/J mouse lung tissue slices (6 h at 37 °C) proceeded on average 8.6±1.4 fold faster (Km: 3.09±5.19 μmol, Vmax: 3178±4104 fmol/min/mg protein) than 2’-hydroxylation of [5-3H]NNN in precision-cut F344 rat lung tissue slices under identical experimental conditions (Km: 0.08±0.07 μmol, Vmax: 60±12 fmol/min/mg protein). In the same concentration range the difference for 5’-hydroxylation was less pronounced, 4.5±0.8 fold higher turnover in mouse lung slices (Km: 1.42±1.63 μmol, Vmax: 792±567 fmol/min/mg protein) compared to rat lung slices (Km: 0.14±0.11 μmol, Vmax: 61±13 fmol/min/mg protein). The ratio of 2’-hydroxylation to 5’-hydroxylation was significantly higher in A/J mouse lung (2.3±0.1) compared to in F344 rat lung (1.2±0.1) over the whole concentration range. These results, which support increased metabolic activation of NNN in the A/J mouse lung compared to in the F344 rat lung, are also consistent with the reported target organ specificity of NNN in both rodent species.
