Inhibition of mutagenicity of N-nitrosamines by tobacco smoke and its constituents

Tobacco smoke is a complex chemical mixture including pyridine alkaloids and N-nitrosamines, with the concentration of the former several orders of magnitude higher than that of the N-nitrosamines. The major biologically important N-nitrosamines present in tobacco smoke are N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosonornicotine (NNN). These nitrosamines require metabolic activation by cytochrome P-450s for the expression of mutagenicity. Although nicotine, the major pyridine alkaloid in tobacco, has been shown to inhibit the metabolic activation of NNK, its effect on the mutagenicity of NNK and other N-nitrosamines has not been reported. In the present study, the ability of three pyridine alkaloids (nicotine, cotinine, nornicotine) and aqueous cigarette smoke condensate extract (ACE) to inhibit the mutagenicity of tobacco-related N-nitrosamines was tested on Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9). All three of the pyridine alkaloids tested, as well as ACE, inhibited the mutagenicity of NDMA and NNK, but not NNN, in a concentration-dependent manner. The induction of SCEs in mammalian cells (CHO) by NNK in the presence of metabolic activation was also significantly reduced by nicotine and cotinine. None of the observed reductions in mutagenicity could be explained by cytotoxicity. These results demonstrate that tobacco smoke contains chemicals, pyridine alkaloids and other unidentified constituent(s), which inhibit the mutagenicity of N-nitrosamines.     

Genotoxicity of tobacco smoke and tobacco smoke condensate: a review

This report reviews the literature on the genotoxicity of mainstream tobacco smoke and cigarette smoke condensate (CSC) published since 1985. CSC is genotoxic in nearly all systems in which it has been tested, with the base/neutral fractions being the most mutagenic. In rodents, cigarette smoke induces sister chromatid exchanges (SCEs) and micronuclei in bone marrow and lung cells. In humans, newborns of smoking mothers have elevated frequencies of HPRT mutants, translocations, and DNA strand breaks. Sperm of smokers have elevated frequencies of aneuploidy, DNA adducts, strand breaks, and oxidative damage. Smoking also produces mutagenic cervical mucus, micronuclei in cervical epithelial cells, and genotoxic amniotic fluid. These data suggest that tobacco smoke may be a human germ-cell mutagen. Tobacco smoke produces mutagenic urine, and it is a human somatic-cell mutagen, producing HPRT mutations, SCEs, microsatellite instability, and DNA damage in a variety of tissues. Of the 11 organ sites at which smoking causes cancer in humans, smoking-associated genotoxic effects have been found in all eight that have been examined thus far: oral/nasal, esophagus, pharynx/larynx, lung, pancreas, myeoloid organs, bladder/ureter, uterine cervix. Lung tumors of smokers contain a high frequency and unique spectrum of TP53 and KRAS mutations, reflective of the PAH (and possibly other) compounds in the smoke. Further studies are needed to clarify the modulation of the genotoxicity of tobacco smoke by various genetic polymorphisms. These data support a model of tobacco smoke carcinogenesis in which the components of tobacco smoke induce mutations that accumulate in a field of tissue that, through selection, drive the carcinogenic process. Most of the data reviewed here are from studies of human smokers. Thus, their relevance to humans cannot be denied, and their explanatory powers not easily dismissed. Tobacco smoke is now the most extreme example of a systemic human mutagen.     

Investigation of the mutagenic activity of tobacco smoke

The genotoxic effect of whole tobacco smoke was studied employing the Salmonella/microsome mutagenicity assay, the micronucleus test in mouse bone marrow and UDS in peripheral human lymphocytes. It was established that tobacco smoke (120-480 cm3 in a 16-1 glass chamber, at 1-10 min exposure time) induced a 3-9-fold increase of spontaneous his+ reversion mutation rate in S. typhimurium TA98, but not in strains TA97a, TA100 and TA102. Addition of S9 mix obtained from the liver of Aroclor 1254-treated rats was necessary to reveal the mutagenic activity of tobacco smoke. Treatment of BDF1 mice placed in a 14-1 glass chamber with tobacco smoke (600 cm3 smoke, 2 exposures of 30 min each, with a 1-min interval between them) caused a 2-fold dose-dependent elevation of the number of micronucleated PCE in bone marrow. No cumulative effect was detected when mice were treated with tobacco smoke during 2-28 consecutive days. The effect observed 24 h after tobacco-smoke exposure was abolished 48 h later. Tobacco smoke (180 or 360 cm3) passed through the culture medium (with or without S9 mix) of human peripheral lymphocytes (the cells were then incubated for 60 min at 37 degrees C) did not increase the spontaneous rate of UDS. Both the Salmonella/microsome mutagenicity assay employing S. typhimurium TA98 strain and the micronucleus test in mouse bone marrow might be useful in studying tobacco smoke-induced mutagenesis.     

Effect of tobacco smoke components on organogenesis in plant tissues

Callus tissue derived from haploid plants of Nicotiana tabacumdifferentiated when inoculated on media fortified with differentconcentrations of tobacco smoke components: benzo(a)pyrene (BaP),benzo(e)pyrene (BeP), dibenz(a, h)anthracene (DBA), pyrene andchrysene. Neither any auxins nor cytokinins were present inthe medium. BaP was found to be the most potent morphogeneticagent. However, these treatments did not induce any vegetativebuds on the callus obtained from diploid plants of tobacco. ¹ This study was carried out under Contract No. 12-14-100-10341(73)with the Agricultural Research Service, U. S. Department ofAgriculture, administered by the Eastern Marketing and NutritionResearch Division, 600 East Mermaid Lane, Philadelphia, Pennsylvania19118, U.S.A. (Received February 4, 1971; )     

Effect of tobacco smoke on the level of estrogens and DNA in the rat uterus

Tobacco smoke induced no changes in the rat uterus weight or in oestrus cycle but decreased estradiol (E2) concentration in the uterus tissue and increased and later decreased the proliferation index and percentage of the cells in the S-phase. The data obtained suggest a phasic character of changes in the reproductive system under the effect of tobacco smoke and corroborate the concept of the role of smoking in the shifting the type of hormonal carcinogenesis from promotional to genotoxic one.     

Oxidation of carotenoids by heat and tobacco smoke

The stability to autoxidation of the polar carotenoids, lutein and zeaxanthin, was compared to that of the less polar carotenoids, beta-carotene and lycopene at physiologically or pathophysiologically relevant concentrations of 2 and 6 microM, after exposure to heat or cigarette smoke. Three methodological approaches were used: 1) Carotenoids dissolved in solvents with different polarities were incubated at 37 and 80 degrees C for different times. 2) Human plasma samples were subjected to the same temperature conditions. 3) Methanolic carotenoid solutions and plasma were also exposed to whole tobacco smoke from 1-5 unfiltered cigarettes. The concentrations of individual carotenoids in different solvents were determined spectrophotometrically. Carotenoids from plasma were extracted and analyzed using high performance liquid chromatography. Carotenoids were generally more stable at 37 than at 80 degrees C. In methanol and dichloromethane the thermal degradation of beta-carotene and lycopene was faster than that of lutein and zeaxanthin. However, in tetrahydrofuran beta-carotene and zeaxanthin degraded faster than lycopene and lutein. Plasma carotenoid levels at 37 degrees C did not change, but decreased at 80 degrees C. The decrease of beta-carotene and lycopene levels was higher than those for lutein and zeaxanthin. Also in the tobacco smoke experiments the highest autoxidation rates were found for beta-carotene and lycopene at 2 microM, but at 6 microM lutein and zeaxanthin depleted to the same extent as beta-carotene. These data support our previous studies suggesting that oxidative stress degrade beta-carotene and lycopene faster than lutein and zeaxanthin. The only exception was the thermal degradation of carotenoids solubilized in tetrahydrofuran, which favors faster breakdown of beta-carotene and zeaxanthin.     

The mutagenic and clastogenic activity of tobacco smoke

Employing the Salmonella/microsome mutagenicity assay it was established that the mutagenic effect of tobacco smoke (TS) (240 cm3 in a 16-l glass chamber, at 1 min or 5 min exposure time) in S. typhimurium TA98 depended on the type of S9 mix used. Addition of S9 mix obtained from the liver of 3-methylcholanthrene- or Aroclor-1254-pretreated rats but not from the liver of phenobarbital-pretreated or untreated rats was required to demonstrate the mutagenic activity of TS. One might suggest that polycyclic aromatic hydrocarbons were involved in TS-induced mutagenesis in S. typhimurium TA98. In addition, treatment of BDF1 mice with TS (600 cm3 TS in a 14-l glass chamber, 2-6 exposures of 30 min each with a 1-min interval between them during which a total change of the air was made) caused an up to 3.5-fold increase of the number of micronucleated polychromatic erythrocytes (PCE) in mouse bone marrow detected 24 h after the TS exposure. Furthermore, a stable 2-5-fold elevation of the number of micronucleated normochromatic erythrocytes (NCE) was detected in the peripheral blood of mice treated daily (2 x 30 min) with TS, starting 48 h after the first TS exposure. The application of the micronucleus test in mouse peripheral blood, a more convenient and useful approach for detecting the chronic clastogenic activity of TS, allowed us to establish the cumulative genotoxic effect of TS in mice.     

Effect of alcohol and tobacco smoke on mtDNA damage and atherogenesis

Environmental tobacco smoke (ETS) exposure and alcohol (EtOH) consumption often occur together, yet their combined effects on cardiovascular disease development are currently unclear. A shared feature between ETS and EtOH exposure is that both increase oxidative stress and dysfunction within mitochondria. The hypothesis of this study was that simultaneous EtOH and ETS exposure will significantly increase atherogenesis and mitochondrial damage compared to the individual effects of either factor (ETS or EtOH). To test this hypothesis, apoE(-/-) mice were exposed to EtOH and/or ETS singly or in combination for 4 weeks and compared to filtered air, nonalcohol controls. Atherosclerotic lesion formation (oil red O staining of whole aortas), mitochondrial DNA (mtDNA) damage, and oxidant stress were assessed in vascular tissues. Combined exposure to ETS and EtOH had the greatest impact on atherogenesis, mtDNA damage, and oxidant stress compared to filtered air controls, alcohol, or ETS-exposed animals alone. Because moderate EtOH consumption is commonly thought to be cardioprotective, these studies suggest that the potential influence of common cardiovascular disease risk factors, such as tobacco smoke exposure or hypercholesterolemia, on the cardiovascular effects of alcohol should be considered.     

Early effects of tobacco smoke exposure on vascular dynamics in the microcirculation.

The purpose of these studies is to examine the early effects of chronic tobacco smoke exposure on vascular dynamics in the mesenteric microcirculation. Female rats were exposed daily to tobacco smoke from five reference cigarettes for a period of 2 mo. At the end of this period the smoke-treated rats had gained 12 g less than sham-treated controls, and arterial blood pressure in the smoke-treated animals was slightly less than pressure in the sham-treated animals. These are characteristic effects of tobacco smoke exposure on rats. Following the treatment period, red blood cell (RBC) velocity in single mesenteric capillaries and microvascular pressures in arterioles and venules were measured in accordance to established methods. There was no significant difference in pressure distribution on the arterial side of the mesenteric vascular network, but pressure in the venules of the smoke-treated animals was significantly higher than that of the sham-treated group. In association with the higher venular pressure in the smoke-treated animals, capillary RBC velocity (an index of capillary flow) was significantly lower. The reduction in velocity was in proportion to the decrease in pressure drop (arteriole-venule) across the capillary network.