Toxicological analysis of low-nicotine and nicotine-free cigarettes.

Low-nicotine and nicotine-free cigarettes are commercially available under the brand-name Quest. Some consumers may believe that these are safer cigarettes, and they may smoke more cigarettes or inhale more smoke to compensate for low nicotine yields. Thus, we have studied the toxicological effects of these two cigarettes and compared them with the Kentucky reference cigarette 2R4F. Also, the availability of nicotine-free cigarettes allows for the assessing the role of nicotine in cigarette smoke. In addition to nicotine, some tobacco-specific nitrosamines, aldehydes, and volatile organic compounds were also reduced in the Quest cigarettes compared to the 2R4F. However, aromatic amines were higher in the nicotine-free compared with low nicotine cigarettes. The Ames test revealed that cigarette smoke condensates from the nicotine-free (CSC-F), low nicotine (CSC-L) and 2R4F (CSC-R) cigarettes had a similar mutagenic potency. Exposure to any CSC caused a similar dose-dependent LDH leakage from normal human bronchial epithelial cells. However, CSC-F had more inhibitory effects on the cell growth than CSC-L and CSC-R. Adding nicotine to the CSC-F attenuated this inhibition. Both Quest CSCs decreased gap junction intercellular communication and caused cell cycle arrest. CSC exposure increased cytoplasmic nucleosomes, sub-G1/G0 population and apoptotic comet tails. Proapoptotic protein Bax increased independent of p53 induction after exposure to CSC-F. In conclusion, these studies are not consistent with a perception that low-nicotine or nicotine-free cigarettes may have less toxicity in human cells. Nicotine, as it exists in CSC, attenuates cytotoxicity possibly in part through inhibition of apoptotic pathways.

Phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates inhibit malignant progression of lung adenomas induced by tobacco carcinogens in A/J mice.

We have shown previously that naturally occurring isothiocyanates derived from cruciferous vegetables and their N-acetylcysteine conjugates inhibit lung adenoma formation induced by tobacco carcinogens in A/J mice at the post-initiation stage. The tumor-inhibitory activity by these compounds is linked with activation of activator protein and induction of apoptosis in lung tissues, suggesting that these compounds may also inhibit the development of adenomas to adenocarcinomas in lung. In this study, the chemopreventive activity of phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates during progression of lung adenomas to malignant tumors was investigated in A/J mice. Mice were divided into 14 groups and treated with a mixture of 3 micromol benzo(a)pyrene [B(a)P] and 3 micromol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) given by gavage once weekly for 8 weeks. Twenty weeks after the beginning of carcinogen administration, a total of 20 mice in the treatment groups were sacrificed with an average yield of 7.3 +/- 4.5 lung adenomas per mouse. The remaining mice in each group were fed diets containing phenethyl isothiocyanate (3 and 1.5 mmol/kg diet), sulforaphane (3 and 1.5 mmol/kg diet), phenethyl isothiocyanate-N-acetylcysteine (8 and 4 mmol/kg diet), sulforaphane-N-acetylcysteine (8 and 4 mmol/kg diet) during weeks 21 to 42. Four mice in each of the high-dose treatment groups were sacrificed during weeks 28 and 36 and the bioassay was terminated during week 42; lung tissues were harvested for histopathologic examination of tumors and for cell proliferation (proliferating cell nuclear antigen) and apoptosis (caspase-3) assays using immunohistochemical staining. At termination, the incidence of adenocarcinoma in the 3 mmol/kg diet phenethyl isothiocyanate group and 8 mmol/kg diet phenethyl isothiocyanate-N-acetylcysteine group was reduced to 19% and 13%, respectively, compared with 42% in the carcinogen-treated control group. At the lower doses, phenethyl isothiocyanate and its N-acetylcysteine conjugate also inhibited the incidences of lung adenocarcinoma, however, the decreases were not statistically significant. The lung tumor incidences in groups treated with sulforaphane-N-acetylcysteine in the diet were also significantly reduced to 11% or 16%. Furthermore, the malignant lung tumor multiplicity was significantly reduced from 1.0 tumor/mouse in the carcinogen-treated control group to 0.3 in the sulforaphane low-dose group, 0.3 and 0.4 in the two sulforaphane-N-acetylcysteine groups, and 0.4 in the phenethyl isothiocyanate high-dose group. The malignant tumor multiplicities in other treatment groups were also reduced (0.5-0.8 tumors/mouse), but not significantly. Unlike lung adenocarcinomas, both incidences and multiplicities of lung adenomas were not much affected by treatment with isothiocyanates or their conjugates. Immunohistochemical examination of the lung tumors from all time points indicated that significant reduction in proliferating cell nuclear antigen and induction of apoptosis (terminal nucleotidyl transferase-mediated nick end labeling and caspase-3) were observed in the isothiocyanate and isothiocyanate-N-acetylcysteine-treated groups that showed inhibition of the development of lung adenocarcinomas. The results of the study provide a basis for future evaluation of the potential of phenethyl isothiocyanate and sulforaphane and their conjugates as chemopreventive agents in smokers and ex-smokers with early lung lesions.

K-ras mutations in lung carcinomas from nonsmoking women exposed to unvented coal smoke in China.

Lung cancer mortality rate in nonsmoking women in Xuan Wei (XW) County is the highest in China. The XW lung cancer rate is associated with exposure to coal smoke, containing high concentrations of polycyclic aromatic hydrocarbons (PAHs), in unvented homes. Here we investigated codon 12 K-ras mutations in lung tumors or sputum samples from 102 XW lung cancer patients (41 nonsmoking women and 61 smoking men). In addition, we analyzed specimens from 50 lung cancer patients (14 nonsmoking women, 33 smoking men and three nonsmoking men), from Beijing and Henan (B&H), where natural gas is the main domestic fuel. K-ras mutations were found in nine women (21.9%) and 14 men (22.9%) from XW, with G to T transversions accounting for 66.7 and 85.7%, respectively. Among B&H patients, one woman (7.1%) and six men (16.7%) had K-ras mutations, with G to T transversions accounting for 66.7% of the mutations in the men. Therefore, the frequency and type of K-ras mutations in XW nonsmoking women are similar to those of K-ras mutations found in both XW and B&H smoking men. On the other hand, the mutation frequency in XW women is higher than, although not statistically significant from, that in the B&H nonsmoking women (P=0.28, two-sided Fisher's Exact Test). These results suggest an association between exposure to coal smoke and the increased K-ras mutation frequency in XW nonsmoking female lung cancer patients. They also suggest that the mutagens and/or mechanisms of mutations in these nonsmoking women are similar to those responsible for K-ras mutations in cigarette smoking lung cancer patients, which are probably induced largely by chemicals such as PAHs.