Elsevier

Environmental Research

Volume 151, November 2016, Pages 635-641
Environmental Research

Assessment of tobacco specific nitrosamines (TSNAs) in oral fluid as biomarkers of cancer risk: A population-based study

https://doi.org/10.1016/j.envres.2016.08.036Get rights and content

Highlights

  • TSNAs were quantified for the first time in oral fluid of smokers and non-smokers.

  • NNN has shown to be the most abundant TSNA present in human oral fluid.

  • NNK and NNAL could also be detected in fewer samples and lower concentrations.

  • NNN/cotinine ratio was significantly higher in non-smokers than in smokers.

  • Cotinine monitoring underestimates the exposure to NNN in non-smokers.

Abstract

Background

Smoke-free laws are expected to reduce smoking habits and exposure to secondhand smoke. The objective of this study was the measurement of tobacco specific carcinogens (TSNAs) in oral fluid to assess the most suitable biomarker of cancer risk associated with tobacco smoke.

Methods

TSNAs, N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), as well as nicotine and cotinine were measured in oral fluid samples from 166 smokers and 532 non-smokers of the adult population of Barcelona, Spain. A simple method with an alkaline single liquid-liquid extraction with dichloromethane/isopropanol was used and lower limits of quantification for cotinine, NNN, NNK and NNAL were set at 0.10 ng/mL, 1.0, 2.0 and 0.50 pg/mL respectively. The NNN/cotinine ratio was also calculated.

Results

NNN was the most abundant TSNA present in oral fluid with a significant difference between smokers and non-smokers (mean concentrations of 118 and 5.3 pg/mL, respectively, p<0.001). NNK and NNAL were detectable in fewer samples. NNN and cotinine concentrations had a moderate correlation within both groups (Spearman's rank correlation coefficient of 0.312, p<0.001 in smokers and 0.279, p=0.022 in non-smokers). NNN/cotinine ratio was significantly higher (p<0.001) in non-smokers than in smokers, in line with equivalent findings for the NNAL/cotinine ratio in urine.

Conclusions

TSNAs are detectable in oral fluid of smokers and non-smokers. NNN is the most abundant, in line with its association with esophageal and oral cavity cancers. The NNN/cotinine ratio confirms the relative NNN increase in second hand smoke. Findings provide a new oral fluid biomarker of cancer risk associated with exposure to tobacco smoke.

Introduction

Tobacco smoking is the world's leading preventable cause of cancer death (World Health Organization, 2003). The tobacco specific nitrosamines (TSNAs) are responsible of an important part of tobacco carcinogenesis (IARC, 2007). 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N′-nitrosonornicotine (NNN), the most prevalent of TSNAs (Fig. 1), are classified as group 1 carcinogen to humans by the International Agency for Research on Cancer (IARC) (International Agency for Research on Cancer, 1985). NNK and its main metabolite NNAL are closely associated with lung cancer and its ability to induce adenocarcinoma tumors is especially notable (Hoffmann et al., 1996, Hecht, 1999, Rivenson et al., 1988, Upadhyaya et al., 1999, Yuan et al., 2009, Church et al., 2009), while NNN plays an important role in the induction of esophagus and oral cavity cancers (Hecht, 2003, Stepanov and Hecht, 2005a, Yuan et al., 2011, Stepanov et al., 2014, Balbo et al., 2013). In the region of Catalonia (Spain), oral cavity and pharynx is the fifth more frequent cancer in men with a crude incidence rate (absolute risk) of 22.8 new cases/10,000 inhabitants-year in 2003–2007 (Clèries et al., 2014). NNK and NNN, present in virtually all cigarettes brands, are formed from tobacco alkaloids by reactions with nitrites during tobacco curing and processing (Hecht, 2014). Highs amounts of these nitrosamines pass to smoke during the cigarette smoking inhalation, are present in mainstream and secondhand smoke (Tricker et al., 1991, Sleiman et al., 2009) and also in thirdhand smoke (Hang et al., 2013, Ramírez et al., 2014). Nitrosamines are absorbed through the small airways and alveoli of the lung, and rapidly distributed by the blood (Benowitz et al., 2009a). NNN can be also endogenously formed from nicotine and nornicotine in human oral fluid (Knezevich et al., 2013) and in the stomach with the swallowed oral fluid, through the gastric acidic environment, optimal for nitrosation (Camels et al., 1991).

Biomarkers of tobacco smoke exposure are crucial to evaluate the metabolism of tobacco products and its potential health risk. Nicotine and its metabolites (mainly cotinine) have been extensively used as specific markers of exposure to cigarette smoke in smokers and non-smokers exposed to secondhand smoke (SHS) (Benowitz et al., 2009a, Martínez-Sánchez et al., 2014a, Al-Delaimy, 2002, Bhalala, 2003, Florescu et al., 2009, Benowitz et al., 2009b, Jacob et al., 2011, Pascual et al., 2003, Mahoney and Al-Delaimy, 2001). Human exposure to these carcinogens occurs specifically during contact with tobacco products. Therefore, monitoring TSNAs specifically implicates tobacco smoke as the source of this carcinogen and it is useful in understanding the tobacco-related cancer mechanisms (Hecht, 2002). Exposure to TSNAs has been mainly achieved by quantifying NNAL, the main NNK metabolite, either in serum or urine, in smokers and non-smokers (Hecht, 2002, Carmella et al., 1995, Carmella et al., 2005, Anderson et al., 2001, Kavvadias et al., 2009, Pan et al., 2004).

The average concentration of NNAL in urine of smokers is around 300 pg/mL (Xia et al., 2011), while NNN has been detected ranging 3.5–25 pg/mL in different studies (Yuan et al., 2011, Kavvadias et al., 2009, Stepanov et al., 2009, Stepanov and Hecht, 2005b, Urban et al., 2009, Xia et al., 2014). The lower values of total NNN compared to total NNAL, despite the higher content of NNN in most tobacco products, is due to the very relevant metabolic conversion of NNK to NNAL which is then rapidly excreted in urine (Hecht, 2014).

There is significant inter-individual variation of endogenous formation of NNN (Knezevich et al., 2013, Stepanov et al., 2009). Dietary and host factors, contributing to the wide range of urinary NNN concentrations found in smokers and to the remarkably strong association between the urinary NNN levels and risk of esophageal cancer in smokers (Yuan et al., 2011).

NNN concentrations give us an idea of the overall intake of this carcinogen, sum of the exogenous NNN coming from tobacco smoke and the endogenously produced NNN, synthesized with the help of bacteria in oral fluid and at the acidic pH of stomach. Therefore, NNN in oral fluid may be a suitable biomarker to understand tobacco-related cancer mechanisms (Hecht, 2002).

Oral fluid testing is a well-known alternative to plasma or urine for drug monitoring and tobacco exposure (Concheiro et al., 2010). Oral fluid has the primary advantage of being a relatively non-invasive matrix. Sample collection is very easy and well accepted by the participants. TSNAs have never been tested in oral fluid of individuals exposed to tobacco smoke. However studies performed on smokeless tobacco users have shown that NNN and NNK were significantly extracted into the oral fluid from tobacco snuff (Brunnemann et al., 1987, Idris et al., 1992, Brunnemann et al., 1996, Hecht et al., 2008).

The objective of this project was the analysis of TSNAs and other tobacco biomarkers in oral fluid in a sample of the adult population of the city of Barcelona (Spain), in an attempt to identify the most suitable tobacco specific carcinogen associated with tobacco smoke.

Section snippets

Safety hazards

NNK, NNN and NNAL are carcinogenic and mutagenic and should be handled with extreme care.

Chemicals

(−)-Nicotine and (−)-cotinine 1.0 mg/mL standard solutions in methanol as well as HPLC grade formic acid were purchased from Fluka-Sigma-Aldrich (Madrid, Spain). (±)-nicotine-d4 (2,4,5,6-tetradeutero-3-(1-methylpyrrolidin-2-yl)-pyridine) 100 μg/mL solution in acetonitrile and (±)-cotinine-d3 (5-(3-pyridinyl)-1-trideuterometyl-2-pyrrolidinone) 1.0 mg/mL in methanol, were purchased from Cerilliant Corp (Round

Results

Among the 698 individuals, 532 were non-smokers and 166 were smokers. 54% were females, with a median age of 58 years. From them, 43% had university studies. 46% were males, with a median age of 55 years and 45% had university studies. The prevalence of current e-cigarette users was 1.7% (12 individuals). From them, 9 were dual users of tobacco and e-cigarettes (classified as smokers) and 3 were exclusive e-cigarette users (excluded from calculations). None of the subjects was user of any other

Discussion

A single liquid-liquid extraction method was applied to the analysis of NNN, NNK and NNAL as well as cotinine in 698 oral fluid samples from an adult population of a major city in Spain. The sensitive UPLC/MS/MS instrumentation allowed limits of quantification in the lower pg/mL range for TSNAs, in line with other publications (Yuan et al., 2011, Xia et al., 2014, Benowitz et al., 2010) and compatible with the expected concentrations of those compounds. The ubiquitous presence of tobacco smoke

Acknowledgments

The authors wish to thank all the volunteers who kindly participated in this study.

This study was funded by Instituto de Salud Carlos III, Subdirección General de Evaluación y Fomento de la Investigación, PN I+D+I 2008–2011, co-funded by the European Regional Development Fund (FEDER), Government of Spain (Grant PI12/01119, recipients: J.A. Pascual, R. Pérez-Ortuño; Grant PI12/01114, recipients: J.M. Martínez-Sánchez, M. Ballvè, M. Fu) and Ministry of Universities and Research, Government of

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