TSNA levels in machine-generated mainstream cigarette smoke: 35 years of data

This paper characterizes historical and current tobacco specific nitrosamine (TSNA) levels in mainstream (MS) cigarette smoke of US commercial cigarettes. To conduct this analysis, we gathered 35 years of published data of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) levels in MS cigarette smoke. We also assessed internal data of MS smoke NNK and NNN levels generated from various market monitoring initiatives and from control cigarettes used in a multi-year program for testing cigarette ingredients. In all, we analyzed machine smoking data from 401 cigarette samples representing a wide range of products and design characteristics from multiple manufacturers and market leaders. There was no indication that TSNA levels systematically increased in cigarette MS smoke over the 35-year analysis period. In particular, TSNA levels expressed as either per cigarette or normalized for tar suggest a downward trend in MS smoke over the past 10 years. The apparent downward trend in TSNA levels in MS smoke may reflect industry and agricultural community efforts to reduce levels of TSNAs in tobacco and cigarette smoke.     

Mass spectrometric analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung

An improved analytical method was developed for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung samples of patients undergoing surgery for lung cancer. HPB-releasing adducts can be formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, and have been reported to play an important role in tobacco carcinogenesis. [2,2,3,3-D(4)]HPB (D(4)-HPB) was used as an internal standard, and HPB released by acid hydrolysis of DNA was determined by gas chromatography/mass spectrometry in the negative ion chemical ionisation mode. The method is sensitive with a limit of detection of 5.9 fmol HPB and a limit of quantification of 15.2 fmol HBP/mg DNA. The recovery of HPB was 82+/-17% and the background response was 10.1+/-1.8 fmol HPB/sample. The concentration of HPB-releasing lung DNA adducts was significantly higher (p<0.0001) in 21 self-reported smokers compared to in 11 self-reported nonsmokers (404+/-258 fmol versus 59+/-56 fmol HPB/mg DNA, respectively). HPB-releasing hemoglobin adduct concentrations were only marginally higher in a subset of 12 smokers compared to in 7 nonsmokers (63+/-53 fmol versus 42+/-34 fmol HPB/g hemoglobin; p=0.36). No correlation was found between HPB-releasing adducts in DNA and hemoglobin (p=0.074).     

Toxicity and Carcinogenicity of Ozone in Combination with 4-（N-methyl-N-nitrosamino）-1-（3-pyridyl）-1-butanone and Dibutyl Phthalate in B6C3F1 Mice for 16 and 32 Weeks

Objective To evaluate the toxic and carcinogenic potential of ozone alone or in combination with 4-（N-methyl-N-nitrosamino）-1-（3-pyridyl）-1-butanone （NNK） and/or dibutyl phthalate （DBP）. Methods Male and female B6C3F1 mice were exposed, through inhalation, intravenous administration and diet, to 0.5 ppm of ozone, 1.0 mg/kg of NNK and 5000 ppm of DBP, individually and in combination for 16 and 32 weeks. Results No treatment-related death was seen, but significant differences in body and organ weights between control and treated mice were observed during the study. No incidence of lung tumor incidence was recorded in mice exposed to either ozone alone or combined treatment. Oviductal carcinomas were observed in female mice exposed to ozone or DBP alone for 16 weeks and ozone in combination with NNK and DBP for 32 weeks. Conclusion Although ozone alone and in conjunction with NNK and/or DBP does not induce lung cancer under our experimental conditions, they induce oviductal carcinomas in B6C3F1 mice.     

The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: Dual role in nicotine addiction and lung cancer

More than 1 billion people around the world smoke, with 10 million cigarettes sold every minute. Cigarettes contain thousands of harmful chemicals including the psychoactive compound, nicotine. Nicotine addiction is initiated by the binding of nicotine to nicotinic acetylcholine receptors, ligand-gated cation channels activated by the endogenous neurotransmitter, acetylcholine. These receptors serve as prototypes for all ligand-gated ion channels and have been extensively studied in an attempt to elucidate their role in nicotine addiction. Many of these studies have focused on heteromeric nicotinic acetylcholine receptors containing α4 and β2 subunits and homomeric nicotinic acetylcholine receptors containing the α7 subunit, two of the most abundant subtypes expressed in the brain. Recently however, a series of linkage analyses, candidate-gene analyses and genome-wide association studies have brought attention to three other members of the nicotinic acetylcholine receptor family: the α5, α3 and β4 subunits. The genes encoding these subunits lie in a genomic cluster that contains variants associated with increased risk for several diseases including nicotine dependence and lung cancer. The underlying mechanisms for these associations have not yet been elucidated but decades of research on the nicotinic receptor gene family as well as emerging data provide insight on how these receptors may function in pathological states. Here, we review this body of work, focusing on the clustered nicotinic acetylcholine receptor genes and evaluating their role in nicotine addiction and lung cancer.     

Identification and Assessment of Tumor-Promoting and Cocarcinogenic Agents: State-of-the-Art in Vitro Methods

Abstract

Chemical-analytical studies during the past 4 years led to several new observations on the formation of tobacco-specificN-nitrosamines (TSNA) and their occurrence in smokeless tobacco, mainstream smoke (MS), and sidestream smoke (SS) of American and foreign cigarettes. When snuff was extracted by means of supercritical fluid extraction with carbon dioxide containing 10% methanol, analysis of this material confirmed that the extraction with organic solvents had been partially incomplete.

Epidemiological studies in the northern Sudan showed a high risk for oral cancer for users of toombak, a home-made oral snuff. Toombak contains 100-fold higher levels of TSNA than commercial snuff in the U.S. and Sweden. The TSNA content in the saliva of toombak dippers is at least ten times higher than that reported in the saliva of dippers of commercial snuff. Biomarker studies have shown corresponding high levels of hemoglobin adducts with metabolites of NNN and NNK as well as for urinary metabolites of NNK. These data supported the epidemiological findings.

The analyses of MS of U.S. and foreign cigarettes smoked under FTC conditions revealed comparable data for the smoke of nonfilter cigarettes and filter cigarettes except in the case of low- and ultralow-yield cigarettes, which showed reduced TSNA yields. The MS of cigarettes made from Burley or dark tobacco is exceptionally high in TSNA, primarily because of the high nitrate content of those tobacco types. Taking puffs of larger volume and drawing puffs more frequently, practices observed among most smokers of cigarettes with low nicotine yield, results in high TSNA values in the MS. The formation of the lung carcinogen NNK is favored during the smoldering of cigarettes, between puffs, when SS is generated. Consequently, in most samples from indoor air polluted with environmental tobacco smoke (ETS), the highest concentration of an individual TSNA is that of NNK. When nonsmokers had remained for up to 2 h in a test laboratory with high ETS pollution, they excreted measurable amounts of NNK metabolites in the urine, indicative of the uptake of TSNA.     

Effects of K-ras Gene Mutations in the Development of Lung Lesions Induced by 4-(N- Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone in A/J Mice

The relationship between the development of peripheral lung lesions induced by tobacco-specific 4-( N -methyl- N -nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and K- ras gene mutation in A/J mice, and the correlations between histological alterations and the course of lung lesion development after NNK treatment and K- ras gene mutation were investigated. The acquisition of a selective growth advantage by the lung lesions with mutations was also examined using immunohistochemical labeling with bromodeoxyuridine. Thirty female 5 weeks old A/J mice were each injected intraperitoneally with a single dose of NNK (100 mg/kg body weight) and subdivided into 6 groups according to the time after NNK treatment. The lung lesions were characterized histologically as alveolar/bronchiolar hyperplasia, adenoma and adenocarcinoma, and point mutations in codons 12 and 61 of the K- ras gene were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and dideoxy sequencing methods. K- ras gene mutations were identified in 7 (58.3%) of 12 hyperplasias, 42 (75.0%) of 56 adenomas and 3 (75.0%) of 4 adenocarcinomas. The most frequent K- ras gene mutation was a G-to-A transition at the second base of codon 12 and this accounted for 86.5% of all the mutations detected. Neither the frequency of activation of this gene nor the specific mutation was affected by the time after NNK treatment and there was no positive correlation between the proliferative activity of lung lesions and the presence of K- ras gene mutations. Thus, K- ras gene mutation is closely associated with the development of NNK-induced peripheral lung lesions in A/J mice, but it plays no role in the selective growth advantage of these lesions.     

Effects of compounds of plant origin on the mutagenicity and metabolism of the tobacco-specific nitrosamine NNK

We have investigated the effects of five phytochemicals on the microsomal-dependent mutagenicity and metabolism of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Two compounds, d-limonene and silymarin, had no effect on NNK-induced mutagenesis in Salmonella typhimurium TA1535 over the concentration range of 0.1–0.4 μmol/plate. Diallyl sulphide was weakly antimutagenic at a concentration of 0.4 μmol/plate. Both capsaicin and tannic acid showed a dose-dependent inhibition of mutagenesis in TA1535. Metabolism studies using [³H]NNK indicated that the effects of the phytochemicals on NNK-induced mutagenesis did not always correlate with the effects on NNK metabolism. α-Carbon hydroxylation reactions are considered the most significant pathways involved in the metabolic activation of NNK to mutagenic and carcinogenic species. D-Limonene and silymarin (0,4 μmol) had the least inhibitory effect on the total α-carbon hydroxylation reactions, 19% and 28%. Capsaicin and diallyl sulphide inhibited these pathways by 74% and 70%. Tannic acid, the most potent phytochemical tested in this study, inhibited total α-carbon hydroxylation pathways by 99%.     

Effects of tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the activation of ERK1/2 MAP kinases and the proliferation of human mammary epithelial cells

Cigarette smoking is a risk factor in the developing of various cancers including breast tumors. There are more than 60 chemical carcinogens in the cigarette smoke; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) being one of the strongest tobacco-specific carcinogens. In this study, we demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases and stimulated proliferation in human normal mammary epithelial cells. MEK1/2 specific inhibitor UO126 completely blocked NNK-induced ERK1/2 activation and cell proliferation, whereas nicotinic receptor nAchR antagonist mecamylamine partially and the selective ₇-nAchR antagonist -bungarotoxin essentially inhibited the NNK-induced ERK1/2 activation and cell proliferation. Surprisingly, receptor tyrosine kinase inhibitor genistein, the selective β₁-adrenergic antagonist atenolol, and the selective β₂-adrenergic antagonist ICI118.551 had a strong inhibitory effect on ERK1/2 activation and cell proliferation induced by NNK. These results suggest that there are at least two different routes in activating ERK1/2 by NNK. One is through nicotinic receptor ₇-nAchR to MEK1/2; the other is from β₁/β₂-adrenergic transactivation of tyrosine kinase containing receptor(s) to MEK1/2. In human cancer mammary epithelial cell lines, we found that ERK MAPK signaling pathway was deregulated: (1) ERK1/2 was constitutively activated at various levels; (2) ERK1/2 was further significantly activated in response to NNK induction; (3) UO126 partially or totally failed to inhibit ERK1/2 activation induced by NNK; (4) The expression levels of ERK1/2 in the cancer cell lines were much higher than those in the normal mammary epithelial cells. The tobacco-specific carcinogen NNK showed a strong proliferative effect on human normal and cancer mammary epithelial cells; the proliferation multitudes of these cells are well correlated with the activation levels of ERK1/2 MAP kinases.     

Determinants of a genotoxic effect of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human diploid fibroblasts

Summary The induction of micronuclei was studied in human diploid fibroblasts incubated in the presence of the tobacco-specific nitrosamine NNK. We used four fibroblast strains having a high capacity of 0⁶-alkylguanine DNA alkyltransferase (13.0–23.3 pmol 0⁶-methylguanine repaired per 8 × 10⁶ cells) and four strains that showed no detectable repair capacity. Incubation with NNK doubled the frequency of micronuclei in repair-deficient cells but failed to evoke any effect in the proficient cell strains. Control experiments were performed with the direct methylating agent MNNG and in the presence of inhibitors of either metabolic activation or alkyltransferase. The results showed that the genotoxicity of NNK is dependent on the relationship between its metabolic activation and the constitutive DNA repair. This supports earlier findings that low constitutive levels of 0⁶-alkylguanine DNA alkyltransferase may increase susceptibility to lung cancer after exposure to DNA methylating agents.Abbreviations NNK 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone - MNNG N-methyl-N-nitro-N-nitrosoguanidin - MN micronuclei - O⁶-AT O⁶-alkylguanine-DNA-alkyl-transferase This work is part of a thesis for a medical doctorate of CP     

人支气管上皮细胞恶性转化过程中FHIT蛋白表达的研究

背景与目的目前大多数研究者分别研究了正常支气管上皮、癌前病变及肺癌组织（吸烟或不吸烟者）标本FHIT蛋白表达，而没有在肺癌发生发展过程中研究其表达及意义。本研究的目的是在烟草特异性亚硝胺（NNK）诱发人支气管上皮细胞（BEAS-2B）恶性转化过程中，探讨FHIT蛋白表达的变化及其意义。方法用500mg／LNNK诱发BEAS-2B细胞（对照组）恶性转化为BEAS-2BNNK细胞（实验组），并在此过程中用免疫细胞化学方法动态观察FHIT蛋白表达的情况。结果㈠NNK诱发BEAS-2B细胞恶性转化模型的建立：①第5代BEAS-2BNNK细胞血清抗性显著增强；②第15代BEAS-2BNNK细胞具有锚着独立性生长特性（软琼脂克隆形成）；③第20代BEAS-2BNNK细胞超微结构出现明显异型性；④第25代BEAS-2BNNK细胞在裸鼠体内成瘤，病理类型为高分化鳞癌。㈡FHIT蛋白表达：BEAS-2B细胞FHIT蛋白表达稳定，在各代之间的差异无统计学意义（P〉0．05）；第5代BEAS-2BNNK细胞FHIT蛋白表达即有所降低，并随传代次数增加而进行性下降，但第25代细胞FHIT蛋白却呈高表达。结论①500mg／LNNK能成功诱发BEAS-2B细胞恶性转化，为进一步探讨肺癌尤其是吸烟致肺癌的发生机制提供了理想模型。②FHIT蛋白表达减弱在肺癌发生过程中可能属早期事件，但FHIT蛋白在细胞恶性转化晚期上调表达值得进一步研究。