In vitro studies of biological effects of cigarette smoke condensate. II. Induction of sister-chromatid exchanges in human lymphocytes by weakly acidic, semivolatile constituents

Cigarette smoke condensate is known to enhance the frequency of sister-chromatid exchanges (SCE) in human lymphocytes in vitro and some of the activity has been found in the most volatile part of the particulate phase, the semivolatile fraction. In this study we have investigated the chemical composition and the SCE-inducing activity of the weakly acidic, semivolatile fraction of a cigarette smoke condensate. A number of individual weakly acidic compounds were also tested for their SCE-inducing effects. The weakly acidic fraction was separated by preparative gel chromatography into 11 subfractions (F1-F11). The chemical composition was determined by gas chromatography and gas chromatography-mass spectrometry. Measurements of the effects on SCE in human lymphocytes were used to evaluate the genotoxic effects. All fractions except F11 induced SCE in a dose-dependent way. The most active fraction was F4 which contained mainly alkyl-2-hydroxy-2-cyclopenten-1-ones. The individual compounds to be tested for induction of SCE were selected on the basis of their abundance in the weakly acidic subfractions and on the basis of their occurrence in the environment. Of 23 tested compounds, most of which were alkylphenols, 7 induced SCE, i.e., catechol, 2-(1-propenyl)phenol, cyclotene, maltol, isoeugenol, 2-methoxyphenol (guaiacol) and vanillin. Many of these are important flavor components that occur not only in tobacco and tobacco smoke but also in food, candies, beverages and perfumes.     

In vitro studies of the biological effects of cigarette smoke condensate. III. Induction of SCE by some phenolic and related constituents derived from cigarette smoke. A study of structure-activity relationships

Since our earlier studies of 23 individual weakly acidic constituents of cigarette smoke indicated that benzenes having vicinal oxygenation or a conjugated double bond induce sister-chromatid exchanges (SCE), we have now selected and examined a complementary set of 27 smoke constituents for their SCE-inducing properties. Of the 50 compounds tested in all, 23 were found to induce SCE, and these include all benzaldehydes but one and the majority of the compounds having a conjugated carbon-carbon double bond as well as several of the guaiacols. These groups of active compounds comprise important flavourants such as vanillin, ethylvanillin, isoeugenol and guaiacol. The structure-activity relationships encountered here may be useful in predicting the SCE-inducing activity of related compounds.     

In vitro studies of biological effects of cigarette smoke condensate. I. Genotoxic and cytotoxic effects of neutral, semivolatile constituents

Much of the biological activity of cigarette smoke resides in the neutral fraction of the particulate phase. Since the volatile constituents of this material, the semivolatiles, are accessible to selective filtration, some of the biological activity of cigarette smoke might be reduced. In view of this, the genotoxic and cytotoxic effects and the chemical composition of the semivolatile neutral material of a cigarette smoke condensate was investigated. Cigarette smoke condensate obtained from domestic American blend type cigarettes, was separated into a volatile, a nonvolatile and a semivolatile fraction. The semivolatile constituents were fractionated by liquid-liquid extraction into 4 subfractions: acids, phenols, bases and neutrals. The neutral material was separated further by silica gel chromatography into 7 subfractions of varying polarity. The major components of these were identified by gas chromatography-mass spectrometry. These fractions were studied using 4 in vitro short-term tests, of which 2, the Ames test and induction of sister-chromatid exchanges, provided information on their genotoxicity and the other 2 provided information on their cytotoxicity by measuring inhibition of cell growth and inhibition of oxidative metabolism. Sister-chromatid exchanges were induced by the neutral fraction and the 7 subfractions, the activities of which increased with increasing polarity. Neither the total neutral material, nor the subfractions showed any mutagenic activity in the Ames test. The cytotoxic effect of the fractions of medium polarity, was greater than that of the total neutral material, while the most and the least polar fractions were less toxic.     

Comparison of Phenols in Smokes of Lamina and Midrib of Flue-cured Tobacco

In order to reveal the difference of the main stream smoke composition between lamina and midrib of flue-cured tobacco, the yields of neutral, basic, acidic and phenolic fractions of the smoke condensates of lamina and midrib cigarettes were compared. The composition of the phenolic fractions were also compared by glass capillary gas chromatography. Neutral and basic fractions were dominant in lamina smoke condensate, while ether insoluble fraction was dominant and formed about a half in midrib smoke condensate. For the semi-quantitative analysis of phenols with gas chromatography mono- and dihydroxybenzenes were extracted from the smoke condensate and missing of these compounds by oxidation was prevented by addition of dl-ascorbic acid. After trimethylsilylation they were simultaneously examined with gas chromatography. It was found that 4-methyl, 4-ethyl and 4-vinylcatechol in lamina smoke were much more rich than those in midrib smoke, while coniferylalcohol (a compound found first in cigarettes smoke condensates) was much more rich in midrib smoke.     

Genotoxicity and PAC analysis of particulate and vapour phases of environmental tobacco smoke

Samples of indoor air were collected from an office room (88 m3) both before smoking and during experimental smoking of 96 cigarettes by 10 persons within 6 h. The particulates were collected on glass-fibre filters and the vapour-phase compounds on XAD-2 resin. The samples were extracted with acetone and analysed quantitatively for polycyclic aromatic compounds and qualitatively with GC-MS. The extracts of filters and XAD-2 resins were fractionated into neutral/acidic and 2 basic (strong and weak bases) fractions; all these fractions were tested with the sister-chromatid exchange (SCE) assay in Chinese hamster ovary (CHO) cells and with the Salmonella/microsome test (strain TA98). Total concentrations of PAC were 205 ng/m3 in the background sample and 1207 ng/m3 after contamination by cigarette smoking. The total PAC concentrations were 4-6 times higher in the vapour phase than in the particulate phase. The fractions of the particulate samples collected before smoking showed mainly marginal genotoxic activity, whereas after smoking their genotoxicity increased dramatically. The fractions of the vapour phase samples were not genotoxic before smoking, but after smoking the neutral/acidic and strong basic fractions induced responses in both assays. The SCE assay was more sensitive towards the vapour-phase mutagens of environmental tobacco smoke (ETS). The relative responses of the two basic fractions, whereas the fraction containing neutral and acidic compounds was the most potent in the SCE assay. In the Salmonella test, the mutagenic activity was mainly detected with metabolic activation, while the induction of SCE in CHO cells was also seen without an exogenous metabolic activation system.     

Effects of UV-irradiation on the SCE frequency in human lymphocyte cultures

UV-irradiation (254 nm) was found to induce a smaller increase of SCE in human lymphocytes than in human fibroblasts and CHO cells. The UV-induced SCE frequency in human lymphocytes was not influenced by the duration between irradiation and the subsequent S-phase. UV-irradiated lymphocytes showed a slightly more than additive response to the SCE-inducing effect of HN2 and acetaldehyde in comparison with non-irradiated cells. The UV-induced SCE frequency was similar in lymphocyte cultures containing 20 and 100 microM of BrdUrd. The results suggest that human lymphocytes are relatively insensitive to the SCE-inducing effect of UV-irradiation, and that SCE-inducing damage caused by UV is not removed during the G1 phase in these cells.     

Inhibition of human blood aldehyde dehydrogenase activity by cigarette-smoke condensate.

The effect of a cigarette-smoke condensate (CSC) and three CSC subfractions on the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity in human blood cells was examined under physiological conditions in vitro. Incubation of intact or sonicated cells with different concentrations of crude CSC resulted in a dose-dependent reduction of the ALDH activity. The inactivation was only restored in part after extensive washing of the cells, indicating that the inhibition observed was mainly irreversible. The nonvolatile (NV) subfraction of the CSC caused a reduction in ALDH activity similar to that obtained with crude CSC, while the semivolatile (SV80) and volatile (SV20) subfractions did not significantly affect ALDH. The present results, showing that the human blood cell ALDH is inactivated by constituents of cigarette smoke in vitro, suggest that the blood ALDH activity reduction found in habitual smokers is also caused by components formed during the combustion of tobacco.     

Cigarette smoke: in vitro effects of condensate fractions on mitochondrial respiration

Various fractions and subfractions (I₁, I₂, I₃, II₁, II₂, and II₃) isolated by the counter current distributions of tobacco smoke condensates (TSC) inhibited the State 3 respiration of rat liver mitochondria. All the fractions except fraction I₁ which contained a large portion of the water soluble components of TSC, stimulated the State 4 respiration. Respiratory control and phosphorylative activity of mitochondria were also impaired upon treatment with TSC fractions. It is concluded that inhibitors and uncouplers of respiratory chain are present in various fractions of tobacco smoke condensate.     

Diurnal Changes in Uric Acid Metabolism of Rats Fed a Casein Diet

Direct gas chromatographic determination of tobacco smoke was developed. Tobacco smoke condensate was collected on a glass fiber filter and the components were converted into their trimethylsilyl derivatives and then subjected to glass capillary column gas chromatography. By this method, all tobacco smoke components, including unstable phenolic substances and water-soluble polyhydroxy compounds, were simultaneously determined. Compositional differences between lamina and midrib smoke of flue-cured tobacco leaves were also clarified by the method. The results indicate that there are quantitative differences in nicotine, phenols, levoglucosan, quinic acid γ-lactone and the other major components between lamina and midrib smoke.     

Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods

Antimicrobial activity of seven commercial smoke preparations (four liquid and three solid) was studied. The minimum inhibitory concentration (MIC) was determined against a selection of food spoilage and pathogenic micro-organisms. The main smoke components were identified and quantified by gas chromatography/mass spectrometry. The most effective condensate was S2. All strains except Salmonella enteritidis were inhibited by S2 with an MIC <0·5–1·5%. Smoke extract L2 inhibited growth of Vibrio vulnificus, Yersinia enterocolitica, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, L. inocua, Brochothrix thermosphacta and Lactococcus lactis ssp. lactis with an MIC of <0·2–0·8%. The condensate L3 inhibited effectively V. vulnificus, B. subtilis, L. innocua and Staph. aureus. L1, L4, S1 and S3 had no inhibitory effects at levels tested against most micro-organisms. Vibrio vulnificus was the most susceptible micro-organism to test compounds. The antimicrobial activity of smoke preparations was related to the concentration of phenols.     

Induction of H2AX Phosphorylation in Pulmonary Cells by Tobacco Smoke: A New Assay for Carcinogens

DNA double strand breaks (DSBs) are potentially carcinogenic lesions. The induction of DSBs triggers phosphorylation of histone H2AX. Phosphorylated H2AX, denoted p-H2AX, may be detected immunocytochemically and the intensity of p-H2AX immunofluorescence (IF) reveals the frequency of DSBs. Using this assay we tested whether the exposure of A549 human pulmonary adenocarcinoma cells to tobacco smoke, and normal human bronchial epithelial cells (NHBE) to tobacco smoke condensate, induces DSBs. Cellular p-H2AX IF and DAPI fluorescence of individual cells were measured by laser scanning cytometry (LSC). Exposure of A549 cells to tobacco smoke and NHBE cells to smoke condensate led to H2AX phosphorylation in both a time and dose dependent manner. The maximal rate of H2AX phosphorylation was seen during the initial 4h of cell treatment. At high doses (50 _g/ml of smoke condensate), H2AX phosphorylation continued to increase for up to 24h. No differences in the level of H2AX phosphorylation were apparent between cells in G1 vs S vs G2/M phase of the cell cycle in response to treatment with smoke condensate. The data provide strong evidence that exposure of A549 cells to tobacco smoke or NHBE cells to smoke condensate rapidly induces DSBs in these cells. The present assay to detect and measure DSBs induced by tobacco products complements other mutagenicity assays and may be applied to test potential carcinogens in other products.     

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.     

Reaction of tobacco smoke aldehydes with human hemoglobin

Formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, and acrolein, all of which are constituents of tobacco smoke, were reacted in 5 mM concentration with the purified major fraction of normal adult human hemoglobin (hemoglobin Ao) in 1 mM concentration. A cigarette smoke condensate, diluted to contain 5 mM total aldehydes, was also reacted with 1 mM hemoglobin Ao. Cationic exchange high-performance liquid chromatography (HPLC) showed that the products formed from simple aliphatic aldehydes, with the exception of formaldehyde, were analogues of those formed from acetaldehyde, earlier shown by us to be imidazolidinone derivatives, that is, cyclic addition products of the N-terminal aminoamide function of α and β chains. Formaldehyde and acrolein produced a heterogeneous mixture of derivatives including crosslinked hemoglobin dimers. The greater proportion of modified hemoglobins produced by condensate aldehydes resembled those formed from acetaldehyde, the most abundant aldehyde in the condensate. A smaller fraction consisted of crosslinked hemoglobin dimers, presumably due to the action of formaldehyde. Mass spectrometric and HPLC analyses of the 2,4-dinitrophenylhydrazones precipitated from the condensate documented the presence of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, furfsral, and methylfurfural. The toxicity of aldehydes is briefly discussed in the context of the findings of this study.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate.

Mutagenic activities of cigarette smoke condensate were assayed in the presence of S-9 Mix using Salmonella typhimurium TA 98. The results were examined in relation to chemical data of tobacco leaves. Among the nitrogenous constituents examined, the contents of total nitrogen and protein nitrogen and the soluble nitrogenous fraction were positively and significantly related to an increase in mutagenic activity of the smoke condensate, whereas nicotine and nitrate were not important in contributing to mutagenic potency of such condensates. The age of tobacco leaves influenced the mutagenic potency of the condensate, which was lowest in leaves from the lower stalk position and increased with ascending leaf position on the stalk. Smoke condensate from tobacco with higher sugar content resulted in lower mutagenic activity. The present results, together with the previous study on the mutagenicity of the amino acid pyrolyzates, suggest that potent mutagens in cigarette smoke condensate are nitrogen-containing compounds, which may be formed from proteins and amino acids during the burning of a cigarette.     

Fecapentaene causes sister-chromatid exchanges in human lymphocytes

Fecapentaenes are potent mutagenic compounds found in human feces that are considered as potential colon carcinogens. It is demonstrated that a synthetic racemic all-trans fecapentaene-12 (fec-12) causes a strong dose-dependent increase in the frequency of sister-chromatid exchanges (SCE) in human lymphocytes exposed at different stages of the cell cycle. The SCE-inducing capacity is consistent with published results on the DNA-damaging activity of fec-12 such as formation of DNA single-strand breaks and interstrand cross-links.     

Cytogenetic effects of tobacco smoke exposure among involuntary smokers

Tobacco smoke is highly genotoxic and produces chromosomal damage in several experimental systems. Active smokers have been shown to have an increased prevalence of somatic chromosome damage in their peripheral blood lymphocytes: this is seen in most cases as an increased sister-chromatid exchange (SCE) frequency and often also as increased structural chromosome aberrations (CAs). Among passive smokers, in association with exposure to environmental tobacco smoke, no such induction of chromosomal damage has been documented. In the present paper we report negative results on induction of chromosomal damage in 2 separate groups of intensive involuntary exposure to tobacco smoke, non-smoking restaurant personnel and newborn children of smoking mothers. While significant exposure in these groups is clearly seen in biochemical intake markers, e.g. cotinine and thiocyanate values in plasma, the conventional cytogenetic parameters, structural chromosome aberrations and sister-chromatid exchanges, are unable to detect the low exposures of involuntary smokers.     

Factors affecting mutagenic activity of cigarette smoke condensate in Salmonella typhimurium TA 1538.

Smoke condensates from Burley tobacco, bright-type tobacco and various brands of commercial cigarettes were tested for mutagenicity by using a microsomal test system with Salmonella typhimurium TA 1538. Smoke condensate from Burley tobacco had much higher mutagenic activity than that from bright-type tobacco. Increased mutagenic activity was observed with smoke condensates from Burley tobacco grown with increasing amounts of nitrogen fertilizer, and from commercial cigarettes blended with Burley tobacco. There was a significant correlation between nitrate content of cigarette and mutagenic activity of the resulting smoke condensate. The results suggest that nitrate in cigarettes may influence the formation of potential mutagens during the burning of a cigarette.     

Analysis of Tobacco and Smoke Condensate for Penicillic Acid

Gas chromatographic analyses of smoke condensate from commercial, unfiltered cigarettes spiked with penicillic acid (500 or 1,000 ppm), a reported carcinogenic substance from certain fungi, indicated approximately 3% of unchanged compound was transported in the smoke. Analysis of tobacco on which either Aspergillus ochraceus or Penicillium cyclopium was grown revealed microgram quantities of the compound. Small amounts of the material were also found in moldy tobacco from commercial storage. The results of these investigations suggest that fungi may be a source of carcinogenic compounds in tobacco and tobacco smoke.     

Different SCE-inducing effects of HN2 and MMS in early and late G1 in human lymphocytes

The induction of SCE was studied in PHA-stimulated human lymphocytes exposed to nitrogen mustard (HN2) or methyl methanesulfonate (MMS) for various time periods in the G1 phase. HN2 was found to induce about 10 times more SCE when cells were exposed in late G1 (24 h after PHA) as compared to early G1 (immediately after PHA). In contrast, only a small difference was observed between cells exposed to MMS in late or early G1. The results suggest that different types of SCE-inducing alkylating damage agents are removed at widely different rates in human G1-lymphocytes.