Tobacco-Specific Nitrosamines and Their Pyridine-N-glucuronides in the Urine of Smokers and Smokeless Tobacco Users

The Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street Southeast, Minneapolis, MN 55455, USA.
Cancer Epidemiology Biomarkers & Prevention (Impact Factor: 4.13). 05/2005; 14(4):885-91. DOI: 10.1158/1055-9965.EPI-04-0753
Source: PubMed


Tobacco-specific nitrosamines are believed to play a significant role as causes of cancer in people who use tobacco products. Whereas the uptake of one tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, has been shown by analysis of its metabolites in urine, there are no published studies on urinary levels of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), and N'-nitrosoanabasine (NAB) or their metabolites in human urine. We developed a method for quantitation of NNN, NAT, NAB, and their pyridine-N-glucuronides NNN-N-Gluc, NAT-N-Gluc, and NAB-N-Gluc in human urine. Total NNN (NNN plus NNN-N-Gluc) was assayed using 5-methyl-N'-nitrosonornicotine as internal standard. Urine was treated with beta-glucuronidase. Following solvent partitioning and solid-phase extraction, total NNN was determined using gas chromatography with nitrosamine-selective detection. Total NAT and total NAB were quantified in the same samples. Separate quantitation of NNN and NNN-N-Gluc was accomplished by extraction of the urine with ethyl acetate before beta-glucuronidase hydrolysis; NNN was analyzed in the ethyl acetate extract, and after enzyme treatment, NNN released from NNN-N-Gluc was quantified in the extracted urine. Separate analyses of NAT, NAT-N-Gluc, NAB, and NAB-N-Gluc proceeded similarly. Analyte identities were confirmed by gas chromatography-tandem mass spectrometry. Mean levels of total NNN, NAT, and NAB in the urine of 14 smokers were (pmol/mg creatinine) 0.18 +/- 0.22, 0.19 +/- 0.20, and 0.040 +/- 0.039, respectively, whereas the corresponding amounts in the urine of 11 smokeless tobacco users were 0.64 +/- 0.44, 1.43 +/- 1.10, and 0.23 +/- 0.19, respectively. Pyridine-N-glucuronides accounted for 59% to 90% of total NNN, NAT, and NAB. The results of this study show the presence of NNN, NAT, NAB, and their pyridine-N-glucuronides in human urine and provide a quantitative method for application in mechanistic and epidemiologic studies of the role of tobacco-specific nitrosamines in human cancer.

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    • "In contrast to the majority of BoE reductions, the urinary NNAL level in MSC was statistically significantly greater compared to SMK overall. These results are consistent with previous findings, which have indicated that NNK is metabolized to NNAL to a greater extent ($3-to 4-fold) in consumers of ST compared to SMK (Hecht et al., 2007, 2008; Stepanov & Hecht, 2005). Additionally, data from NHANES (2007–2008) indicated that NNAL concentrations were 4.7-to 6.0-fold higher in ST consumers than in SMK (Naufal et al., 2011). "
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    ABSTRACT: A study was conducted to evaluate biomarkers of biological effect and physiological assessments related to cardiovascular disease (CVD) among adult male cigarette smokers (SMK), moist snuff consumers (MSC) and non-consumers of tobacco (NTC). Additionally, biomarkers of tobacco and tobacco smoke exposure (BoE) were measured in spot urines and are reported here. Except for the BoE to nicotine and NNK, BoE were generally greater in SMK compared with MSC, and BoE were generally not different in comparisons of MSC and NTC. Results demonstrated that MSC had lower systemic exposures to many harmful and potentially harmful constituents than SMK, which is consistent with epidemiological data that indicate a differential in CVD risk between these groups.
    Inhalation Toxicology 03/2015; 27(3):1-8. DOI:10.3109/08958378.2015.1013228 · 2.26 Impact Factor
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    • "At basic pH the formation of tobacco specific amines occur, which makes the SLT products, potentially toxic to consumers. The production of nitrosamines are major contributors to the increased risk of chewing SLT products for cancer of the upper digestive tract and in the case of tobacco smoking created risk of respiratory tract cancer (Stepanov and Hecht, 2005). For total TEs the triplicate samples of each brand of mainpuri were analyzed after microwave assisted acid digestion method. "
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    ABSTRACT: It has been extensively investigated that smokeless tobacco chewing can lead mainly to inflammation of oral cavity. In present study, the total and artificial saliva extracted toxic elements, arsenic, cadmium, nickel and lead were estimated in smokeless tobacco product, mainpuri. Cloud point extraction has been used for the preconcentration of arsenic, cadmium, nickel and lead in artificial saliva extract, using complexing reagent, ammonium pyrrolidinedithiocarbamate. Total and extractable toxic elements were measured by electrothermal atomic absorption spectrometry. The chemical variables of cloud point extraction were optimized. The validity of methodology was tested by simultaneously analyzing certified reference material (Virginia tobacco leaves) and spike recovery test. The artificial saliva extractable levels of arsenic, cadmium, nickel and lead ranged from 15-22, 45-70, 35-58, and 18-32%, respectively, of total elemental contents in mainpuri samples. It was estimated that intake of 10g of different brands of mainpuri contributing the 5.88, 55.0, 45.0 and 40.3% of the provisional maximum tolerable daily intake for arsenic, cadmium, nickel and lead, respectively for adults of ~60kg.
    Ecotoxicology and Environmental Safety 04/2013; 92. DOI:10.1016/j.ecoenv.2013.03.001 · 2.76 Impact Factor
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    • "This nitrosamine is present in higher amounts than any other esophageal carcinogen in tobacco smoke [8]. It and/or its glucuronide conjugate have been detected in the urine and toenails of smokers and smokeless tobacco users [21–25]. Based on animal studies, NNK and NNN are listed as Group 1 human carcinogens by the International Agency for Cancer Research [6, 8]. "
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    ABSTRACT: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are tobacco-specific nitrosamines present in tobacco products and smoke. Both compounds are carcinogenic in laboratory animals, generating tumors at sites comparable to those observed in smokers. These Group 1 human carcinogens are metabolized to reactive intermediates that alkylate DNA. This paper focuses on the DNA pyridyloxobutylation pathway which is common to both compounds. This DNA route generates 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine, O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytosine, O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxythymidine, and O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine as well as unstable adducts which dealkylate to release 4-hydroxy-1-{3-pyridyl)-1-butanone or depyriminidate/depurinate to generate abasic sites. There are multiple repair pathways responsible for protecting against the genotoxic effects of these adducts, including adduct reversal as well as base and nucleotide excision repair pathways. Data indicate that several DNA adducts contribute to the overall mutagenic properties of pyridyloxobutylating agents. Which adducts contribute to the carcinogenic properties of this pathway are likely to depend on the biochemistry of the target tissue.
    Journal of nucleic acids 09/2010; 2010. DOI:10.4061/2010/284935
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