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 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.     

Cytogenetic effects of cigarette smoke condensates in vitro and in vivo

Summary Various cigarette smoke condensates (CSC) were analyzed with respect to the induction of sister-chromatid exchanges (SCE) in human lymphocytes in vitro. CSC from a reference cigarette, from three different tobaccos of the reference cigarette, and from a British cigarette induced similar SCE frequencies. CSC from the reference cigarette did not induce SCE in Chinese hamster bone marrow cells in vivo.     

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.     

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in V79 cells by enantiomeric epoxides.

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate the influence of the stereoisomeric forms of epoxides in mammalian genotoxicity tests. The SCE-inducing potency of 12 pairs of (R)- and (S)-enantiomeric epoxides which differed in the degree of substitution of the oxirane ring was determined. Of these, 2 pairs of epoxides failed to induce SCE. Different SCE-inducing potencies between the (R)- and (S)-enantiomers were shown for 5 epoxides. This study demonstrates that stereoselectivity might play an important role in genotoxicity testing of chemicals with asymmetric C atoms.     

Mutagenicity of polycyclic hydrocarbons. V. Induction of sister-chromatid exchanges in vivo.

The potency of polycyclic hydrocarbons to induce SCEs in vivo was analysed. The most potent SCE-inducing compound was benzo[a]pyrene. Benzanthracene, benzo[b]fluoranthene, benzo[e]pyrene, phenanthrene, chrysene and dibenzanthracene enhanced the SCE frequency to a smaller extent. The number of anthracene-induced SCEs per metaphase was not increased as compared with the controls.     

Genotoxic properties of 4-hydroxyalkenals and analogous aldehydes

4-Hydroxynonenal (HNE), one of the major products of lipid peroxidation, has been demonstrated to induce genotoxic effects in the micromolar range. HNE has too structural domains, a lipophilic tail and a polar head with three functional groups: the aldehyde and hydroxy groups and the trans CC double bond. To evaluate their relative importance, the genotoxic effects of HNE were compared with those of the homologous aldehydes 4-hydroxyhexenal and 4-hydroxyundecenal (different lengths of the lipophilic tail), and the analogous aldehydes 2-trans-nonenal (lacking the OH group) and nonanal (lacking the OH group and the trans CC double bond). This investigation was carried out on primary cultures of adult rat hepatocytes in order to further determine the influence of biotransformation- and/or detoxification reactions.

A 3-h treatment with HNE induces statistically significant levels of SCE at concentrations ≥0.1 μM, micronuclei at concentrations ≥ 1 μM and chromosomal aberrations at a concentration of 10 μM. Compared to HNE the homologous aldehydes induced a significant genotoxic effect at higher concentrations. Statistically significant increases in SCE frequency were obtained at concentrations ≥ 1 μM for 4-hydroxyundecenal and at a concentration of 10 μM for 4-hydroxyhexenal. The induction of chromosomal aberrations was significantly elevated at concentrations of ≥ 10 μM and 10 μM for 4-hydroxyhexenal and 4-hydroxyundecenal, respectively. Except for a 4-hydroxyhexenal concentration of 1 μM, both aldehydes did not induce statistically significant levels of micronucleis.

The HNE analogous aldehydes 2-trans-nonenal and nonanal induced statistically significant frequencies of SCE at concentrations of ≥ 1 μM (nonanal) and ≥ 10 μM (2-trans-nonenal). No significant induction of chromosomal aberrations or micronuclei could be demonstrated.

The structure of the aldehydes investigated appears to influence the cyto- and genotoxic potential in the following ways. (1) The lenght of the lipophilic tail has no influence on chromosomal aberration induction, but appears to determine the yield of SCE and micronuclei, and the cytotoxic potential. (2) The lack of the OH group (2-trans-nonenal) reduces the SCE-inducing potential of the aldehyde shifting the dose-effect curve to higher concentrations. The similar shape compared to SCE induction by HNE indicates that possibly the same active metabolite is formed. (3) The lack of both the OH group and the CC double bond (nonanal) does not result in a complete loss of the SCE-inducing activity. The different shape of the dose-response curve suggests a different metabolism and/or a different mode of interaction with DNA.     

Effects of cigarette smoke on the immune system

Although the health risks of tobacco smoking are well documented, there is increasing evidence that smokers have a lower incidence of some inflammatory and neurodegenerative diseases. Many of the adverse and beneficial effects of smoking might result from the ability of cigarette smoke to suppress the immune system. Nicotine, which is one of the main constituents of cigarette smoke, suppresses the immune system but might have therapeutic potential as a neuroprotective and anti-inflammatory agent.     

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in Chinese hamster V79 cells

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate structure-activity relationships of epoxides in mammalian cells. For this purpose the SCE-inducing potency of 58 epoxides was determined. Of these, 16 failed to induce SCE in V79 cells. According to the substitution of the oxirane ring the results show general agreement with results obtained in the Ames test. Mono-substituted epoxides had the highest genotoxic potency compared to di- and tri-substituted epoxides. In detail, there are differences in genotoxic potency between bacteria and mammalian cells which can be explained by differences in the cellular uptake of the compounds and by detoxification reactions.     

Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with transient receptor potential melastatin 8 (TRPM8) ion channels in cold-sensitive sensory neurons, and transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by smoke irritants (acrolein, acetic acid, and cyclohexanone). Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did eucalyptol (460 ppm), another TRPM8 agonist. Menthol's effects were reversed by a TRPM8 antagonist, AMTB. Menthol's effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counterirritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote nicotine addiction and smoking-related morbidities.     

Changes measured in arterial blood following a single breath of cigarette smoke in rabbits.

This paper describes a method for monitoring short term changes in arterial blood in rabbits in response to a single breath of cigarette smoke. The method was developed to investigate the observation that neutrophil transit times through the lung are extended during acute exposures to cigarette smoke (1). In this model, we sought to monitor the time course of appearance of diffusible gas from smoke to the blood stream, the appearance of lipid peroxidation products and the activation of neutrophils. New Zealand white rabbits were anesthetized and fitted with a tracheostomy tube and an aortic catheter. Smoke was collected in a syringe from a non-filtered cigarette and injected immediately via the tracheostomy tube. Blood samples were collected at 1 second intervals. Carboxyhemoglobin levels increased 108% over pre-smoke levels, peaking at 5-7 seconds after the start of smoke exposure. Serum conjugated dienes, as measured by change in absorbance of lipid extracts at 234 nm, increased 40%, peaking at 10-11 seconds. Thiobarbituric acid (TBA) reactive material exhibited a variable response, with a statistically insignificant maximum at 12 seconds. Serum myeloperoxidase activity was not affected by smoke inhalation. This method provides a model for studying the acute effects of smoke inhalation and provides some evidence for oxidant stress following a single breath of cigarette smoke.     

Combined effect of alcohol and cigarette smoke on lipid peroxidation and antioxidant status in rats

The effect of long-term administration of alcohol and cigarette smoke independently and both in combination on lipid peroxidation and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) was studied in liver, kidney, heart and lungs of albino rats. The levels of peroxidation products viz., malondialdehyde, hydroperoxides and conjugated dienes were increased in all the tissues of alcohol administered and smoke-exposed rats. Activities of SOD and CAT were decreased in alcohol-treated and alcohol and smoke combination groups, but increased in smoke-exposed group. Activities of GPx and GST have shown an increase, while concentration of reduced glutathione was found decreased in all the three groups.     

Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells

Cigarette smoking is ranked among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanisms, however, that link cigarette smoking to increased incidence of atherosclerosis are not understood. The adherence of circulating monocytes to the endothelium, migration into the subendothelium, and subsequent formation of foam cells are principal initial events in the development of atherosclerosis. We therefore determined whether cigarette smoke caused increased adherence of monocytes to endothelial cells and the cellular mechanism of this increased adherence. Cigrette smoke condensate (CSC), the particulate fraction of cigarette smoke derived from 2R1 standard research cigarettes, at a concentration of 25–30 μg/ml (average yield of CSC is 26.1 mg/cigarette), augmented (70–90%) basal adherence of human peripheral blood monocytes to a cultured monolayer of endothelial cells derived from bovine aorta (BAEC) and human umbilical vein (HUVEC). There was a concomitant increase in the expression of CD11b ligand on the surface of monocytes as determined by flow cytometry, utilizing FITC conjugated Mab MO-1 (CD11b). However, nicotine (1–15 μg/ml) and cadmium sulfate (10 μg/ml), constituents of CSC, individually or in combination had no effect either on CD11b expression or adherence of monocytes to endothelial cells. Treatment of HUVEC with CSC for 60 min also resulted in an increased expression of ICAM-1 and ELAM-1 as determined by mean fluorescence intensity of ICAM-1 and ELAM-1 labeled cells in flow cytometric analysis. The CSC induced expression of CD11b in monocytes was optimal at 25–30 min and was inhibited by protein kinase C inhibitors, staurosporine and H-7, and also by baicalein, a lipoxygenase inhibitor. Similarly, CSC induced ICAM-1 and ELAM-1 expression in HUVEC was inhibited by protein kinase C inhibitors. CSC stimulated the adherence of human monocytes but not the monocytic cell lines HL-60, U937, and THP-1 to endothelial cells. The CSC stimulated adherence of human monocytes was inhibited (80%) by MAb to CD11b and 50% by Mab to ICAM-1 and ELAM-1. These results suggest that cigarettee smoke particulate constituents activate protein kinase C, leading to increased surface expression of adhesive ligand CD11b on peripheral blood monocytes and counter receptor(s) ICAM-1 and ELAM-1 in endothelial cells. The expression of ligand and counter receptor leads to potentiated adherence of monocytes to endothelial cells, an initial event in the pathogenesis of cigarette smoke induced inflammatory response in the vessel wall. © 1994 Wiley-Liss, Inc.     

A protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking (5,6,7,12). Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens(9,10). Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung(3). Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke(4). A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke(14). However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine(8). In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy(1,2,14). We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively(11,13).     

Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione.

Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.     

Mutagenicity of polycyclic hydrocarbons: V. Induction of sister-chromatid exchanges in vivo

The potency of polycyclic hydrocarbons to induce SCEs in vivo was analysed. The most potent SCE-inducing compound was benzo[a]pyrene. Benzathracene, benzo[b]fluoranthene, benzo[e]pyrene, phenanthrene, chrysene and dibenzanthracene enhanced the SCE frequency to a smaller extent. The number of anthracene-induced SCEs per metaphase was not increased as compared with the controls.     

Chemiluminescence of cigarette smoke.

Chemiluminescence from cigarette smoke (aerosol) and smoke "extracts" (suspensoids) are described. The emissons from aqueous and organic suspensoids persist for hours, are proportional to oxygen solubilities, possess energy of at least 1.8 electron volts, and display characteristics which suggest that the emissions may be partially sensitized by singlet oxygen.     

Thirdhand smoke: a new dimension to the effects of cigarette smoke on the developing lung

The underlying mechanisms and effector molecules involved in mediating in utero smoke exposure-induced effects on the developing lung are only beginning to be understood. However, the effects of a newly discovered category of smoke, i.e., thirdhand smoke (THS), on the developing lung are completely unknown. We hypothesized that, in addition to nicotine, other components of THS would also affect lung development adversely. Fetal rat lung explants were exposed to nicotine, 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA), or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the two main tobacco-specific N-nitrosamine constituents of THS, for 24 h. We then determined key markers for alveolar paracrine signaling [epithelial differentiation markers surfactant phospholipid and protein synthesis; mesenchymal differentiation markers peroxisome proliferator-activated receptor γ (PPAR-γ), fibronectin and calponin], the BCL-2-to-Bax ratio (BCL-2/Bax), a marker of apoptosis and the involvement of nicotinic acetylcholine receptors (nAChR)-α3 and -α7 in mediating NNA's and NNK's effects on the developing lung. Similar to the effects of nicotine, exposure of the developing lung to either NNK or NNA resulted in disrupted homeostatic signaling, indicated by the downregulation of PPAR-γ, upregulation of fibronectin and calponin protein levels, decreased BCL-2/Bax, and the accompanying compensatory stimulation of surfactant phospholipid and protein synthesis. Furthermore, nAChR-α3 and -α7 had differential complex roles in mediating these effects. NNK and NNA exposure resulted in breakdown of alveolar epithelial-mesenchymal cross-talk, reflecting lipofibroblast-to-myofibroblast transdifferentiation, suggesting THS constituents as possible novel contributors to in utero smoke exposure-induced pulmonary damage. These data are particularly relevant for designing specific therapeutic strategies, and for formulating public health policies to minimize THS exposure.     

Comparison of the induction by cigarette smoke condensates of sister-chromatid exchanges in Chinese hamster cells and of mutations in Salmonella typhimurium.

Three cigarette smoke condensates were tested for the induction of sister-chromatid exchanges in ovary cells of the Chinese hamster and for mutations in Salmonella typhimurium. In the sister-chromatid exchange test an effect was obtained that was not enhanced by the inclusion of a system for metabolic activation. In the Salmonella test, an effect was only obtained by including rat-liver homogenates derived from rats treated with inducers of the enzyme systems necessary for metabolic activation. It appears that the SCE test and the Salmonella test are sensitive to different components of cigarette smoke condensates.     

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.