Variability in addictive and carcinogenic potential of smokeless tobacco products marketed in Mumbai, India: a surveillance study.

Background: India has the highest incidence worldwide of smokeless tobacco (SLT)-associated oral cancer, accounting for nearly 70% of all SLT users globally. Nicotine and tobacco-specific N-nitrosamines (TSNA) play critical roles in the addictive and carcinogenic potential, respectively, of SLT products. Our group has previously reported substantial variability in nicotine and TSNA levels across a small SLT product sample in India, calling for systematic surveillance. However, there is no information available on the current levels of these constituents in Indian SLT. Methods: We analysed 321 samples representing 57 brands of eight popular types of manufactured SLT products purchased from five local markets in Mumbai, India between August, and September 2019. The sampling locations were Mumbai Central, Kurla, Thane, Vashi, and Airoli. Product pH, moisture content, total and unprotonated (biologically available) nicotine, and TSNA levels were measured at the Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC) in Mumbai. Findings: Total nicotine content ranged from 0.45 to 35.1 mg/g across products. The unprotonated nicotine fraction contributed 0.1-100% of the total nicotine content. The carcinogenic TSNA levels ranged 0.06-76 ug/g for N'-nitrosonornicotine (NNN), 0.02-19.2 ug/g for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 0.01-6.51 ug/g for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Consistent with our previous study, we observed substantial variations across different brands of the same product type. Interpretation: This is the most extensive and the first within-country study to report brand-specific nicotine and TSNA levels in SLT products marketed in Mumbai, India. Our results show that levels of these constituents remain extremely variable across Indian SLT and are strikingly high in many products. Enhanced public education and continued efforts to reduce SLT use prevalence in India are critical for reducing the global burden of SLT-associated morbidity and mortality. Regulation of nicotine and TSNA levels in SLT products should be considered. Funding: This work was supported by the National Institutes of Health (USA) grant R01-TW010651 and, in part, by grants R01-CA180880 and R50-CA211256. The LC-MS/MS analysis was supported in part by XII Plan project funding from the Department of Atomic Energy, Government of India.

From cultivation to cancer: formation of N-nitrosamines and other carcinogens in smokeless tobacco and their mutagenic implications.

Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific N-nitrosamines (TSNAs), such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.

Investigation of exposure biomarkers in human plasma following differing levels of tobacco-specific N-nitrosamines and nicotine in cigarette smoke.

Tobacco-specific N-nitrosamines (TSNAs) are strong carcinogens widely found in tobacco products, environmental tobacco smoke, lake, and wastewater. The main objective of this study was to investigate the effects of cigarette smoke with different yields of TSNAs (NNK, NNN, NAT, NAB) and nicotine on the levels of biomarkers of exposure in smokers' plasma. Three hundred healthy volunteers were recruited comprising 60 smokers of each of 3 mg, 8 mg and 10 mg ISO tar yield cigarettes and 60 smokers who smoked 10 mg, 8 mg, and 3 mg for 14 days sequentially and 60 non-smokers. All study participants were male, aged from 21 to 45 years old, and were recruited from a same unit in Hebei, China. We measured the levels of NNAL, NAT, NNN, NAB and cotinine in plasma from 240 smokers and 60 non-smokers using a novel method established by online two-dimensional solid phase extraction-liquid chromatography-tandem mass spectrometry. The results showed that NNAL, NAT, NNN, NAB and cotinine in the plasma of smokers smoking cigarette with low TSNAs and nicotine were lower than that with high TSNAs and nicotine. When smokers switched from higher to lower TSNA yields of cigarettes, their plasma NNAL, NAT, NNN, NAB levels significantly decreased. The plasma concentrations of NNAL were significantly correlated with those of cotinine, NNN, NAT and NAB for smokers (p < 0.001). Similarly, the plasma concentrations of cotinine were significantly correlated with those of NNN, NAT and NAB for smokers (p < 0.001). The plasma NNAL, NAT, NNN, NAB and cotinine levels for smokers were significantly higher than those for non-smokers. These findings suggested that the total NNAL, NNN, NAT, NAB and cotinine in plasma were valid and reliable biomarkers for human exposure to TSNAs and nicotine.

Metabolism and DNA Adduct Formation of Tobacco-Specific N-Nitrosamines.

The tobacco-specific N-nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) always occur together and exclusively in tobacco products or in environments contaminated by tobacco smoke. They have been classified as "carcinogenic to humans" by the International Agency for Research on Cancer. In 1998, we published a review of the biochemistry, biology and carcinogenicity of tobacco-specific nitrosamines. Over the past 20 years, considerable progress has been made in our understanding of the mechanisms of metabolism and DNA adduct formation by these two important carcinogens, along with progress on their carcinogenicity and mutagenicity. In this review, we aim to provide an update on the carcinogenicity and mechanisms of the metabolism and DNA interactions of NNK and NNN.

Magnetic solid-phase extraction of tobacco-specific N-nitrosamines using magnetic graphene composite as sorbent.

Tobacco-specific N-nitrosamines are carcinogenic components in mainstream cigarette smoke. To explore tobacco-specific N-nitrosamine release levels in cigarettes, a magnetic solid-phase extraction procedure using magnetic graphene composite as sorbent for fast enrichment of tobacco-specific N-nitrosamine was developed. Under optimal conditions, a tobacco-specific N-nitrosamine determination method was successfully proposed by combining magnetic solid-phase extraction procedure and high-performance liquid chromatography coupled with tandem mass spectrometry. The method's limit of detection for tobacco-specific N-nitrosamines in mainstream cigarette smoke ranged from 0.018 to 0.057 ng/cigarette. Good linearities were obtained with correlation coefficients above 0.9992. The accuracies of tobacco-specific N-nitrosamines in a spiked mainstream cigarette smoke sample were from 89.3 to 109.4%, with a relative standard deviation of less than 11.2%. The proposed method has the merits of rapidity and high sensitivity. Finally, the method was successfully applied to tobacco-specific N-nitrosamine analysis in real samples.

Quantitative method for analysis of tobacco-specific N-nitrosamines in mainstream cigarette smoke by using heart-cutting two-dimensional liquid chromatography with tandem mass spectrometry.

A heart-cutting two dimensional liquid chromatography coupled with tandem mass spectrometry method was developed for the analysis of tobacco-specific N-nitrosamines (TSNAs) at low concentration level in Virginia-type cigarette smoke. A strong cation exchange column was utilized for the first dimensional separation, which effectively removed acidic and neutral components in the smoke, followed by a reversed phase liquid chromatography coupled with tandem mass spectrometric analysis. To capture components of the TSNAs in the effluent on the trapping column, a compensating pump was applied for online dilution and pH adjustment during the period of the TSNAs fraction transferring and enrichment. Highly sensitive determination of the TSNAs in mainstream cigarette smoke was achieved by isotope deuterated internal standards under the multiple reaction monitoring mode. Compared with traditional methodologies, the method was almost no matrix interference. Limits of quantity for the TSNAs were within 0.027-0.094 ng/mL, and the results showed good reproducibility and accuracy. Finally, the new method was applied for analysis of the Kentucky reference cigarettes and the results agreed well with joint experiments of Cooperation Centre for Scientific Research Relative to Tobacco.

Effect of smokeless tobacco products on human oral bacteria growth and viability.

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log10 fold, 18 strains showed enhanced growth of 0.3-0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log10 fold, 8 strains showed enhanced growth of 0.3-1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 µg/ml; while the growth of P. micros was enhanced 0.31-0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria.

Simultaneous determination of polycyclic aromatic hydrocarbons and tobacco-specific N-nitrosamines in mainstream cigarette smoke using in-pipette-tip solid-phase extraction and on-line gel permeation chromatography-gas chromatography-tandem mass spectrometry.

In this study, a silica/primary secondary amine (SiO2/PSA) was used as an in-pipette-tip solid phase extraction (SPE) sorbent for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs) and tobacco-specific N-nitrosamines (TSNAs) in mainstream cigarette smoke (MSS). We investigated several parameters including an extraction procedure of total particulate matter, type and amount of sorbent and on-line gel permeation chromatography parameters to obtain optimum conditions for a new strategy to target analytes. Under the optimized conditions, we developed a method for the simultaneous determination of PAHs and TSNAs in MSS by coupling in-pipette-tip SPE procedures to an on-line gel permeation chromatography-gas chromatography-tandem mass spectrometry (on-line GPC-GC-MS(2)). Our method had limits of detection for target analytes ranging from 0.01 to 0.23ng/cig. Good linearities were obtained with coefficients of determination (R(2)) greater than 0.9984 for all target analytes. Good reproducibility was obtained as intra- and inter-day precisions, and the relative standard deviations were less than 11.4 and 13.3%, respectively. The recoveries were in the range of 77.1-108.6% at different concentrations for real samples. Compared to previous standard methods for the determination of PAHs and TSNAs in MSS, our method was highly effective, fast, and had low consumption of organic solvent and a high degree of automation. Finally, our method successfully analyzed PAHs and TSNAs in real samples, and no significant deviations were observed when compared to similar analysis using standard methods.

Fully automated analysis of four tobacco-specific N-nitrosamines in mainstream cigarette smoke using two-dimensional online solid phase extraction combined with liquid chromatography-tandem mass spectrometry.

A fully automated method for the detection of four tobacco-specific nitrosamines (TSNAs) in mainstream cigarette smoke (MSS) has been developed. The new developed method is based on two-dimensional online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE/LC-MS/MS). The two dimensional SPE was performed in the method utilizing two cartridges with different extraction mechanisms to cleanup disturbances of different polarity to minimize sample matrix effects on each analyte. Chromatographic separation was achieved using a UPLC C18 reversed phase analytical column. Under the optimum online SPE/LC-MS/MS conditions, N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were baseline separated with good peak shapes. This method appears to be the most sensitive method yet reported for determination of TSNAs in mainstream cigarette smoke. The limits of quantification for NNN, NNK, NAT and NAB reached the levels of 6.0, 1.0, 3.0 and 0.6 pg/cig, respectively, which were well below the lowest levels of TSNAs in MSS of current commercial cigarettes. The accuracy of the measurement of four TSNAs was from 92.8 to 107.3%. The relative standard deviations of intra-and inter-day analysis were less than 5.4% and 7.5%, respectively. The main advantages of the method developed are fairly high sensitivity, selectivity and accuracy of results, minimum sample pre-treatment, full automation, and high throughput. As a part of the validation procedure, the developed method was applied to evaluate TSNAs yields for 27 top-selling commercial cigarettes in China.