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.     

Mutagenicity of marijuana and Transkei tobacco smoke condensates in the salmonella/microsome assay

Extracts and smoke condensates of marijuana, Transkei home-grown tobacco and also commercial cigarette tobaccos were assayed for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation. No mutagenic activity was detected in dichloromethane extracts of marijuana and tobacco per se, but all the smoke condensates exhibited mutagenicity with metabolic activation. The only strain not mutated by any of the pyrolyzates was TA1535. Transkei tobacco pyrolyzate proved to be the most mutagenic, followed by marijuana, pipe and cigarette tobacco. Mutagenicity was positively associated with the nitrogen content of the various products. The potent mutagenic action of marijuana smoke condensate, coupled with a condensate yield of more than 50% higher than that of cigarette and pipe tobacco, indicates a high carcinogenic risk associated with marijuana smoking.     

Mutagenicity of smoke condensates from joss sticks

Smoke condensates obtained from 8 Japanese brands of joss sticks were assayed for mutagenicity on Salmonella typhimurum TA100 and TA98 with and without metabolic activation by S9 mix. An average of 22 mg of smoke condensate was obtained per joss stick weighing about 0.3 g. Smoke condensates obtained from all the joss sticks tested showed definite mutagenicity with a linear dose response on both tester strains of bacteria with metabolic activation. Without S9 mix, the smoke condensates from some of the joss sticks also showed positive mutagenicity on TA100 at 0.15 or 0.3 mg/plate. The revertant numbers over the background counts induced by 1-mg samples of the smoke condensates from joss sticks with S9 mix were 140–310 with TA100 and 90–200 with TA98. These values were one-fifth to half that of cigarette smoke condensate with TA100 and one-fourteenth to one-fifth that with TA98. However, it was calculated that, when joss sticks are burnt continuously in a closed space under the usual conditions, the mutagenicity of their smoke per unit volume of air is similar to that of the smoke produced by the smoking of two cigarettes per hour.Extracts of unburnt joss sticks with methanol, chloroform or dimethyl-sulfoxide had no mutagenicity, showing that the burning process produces the mutagenic substance(s).     

Reduction in mutagenicity of cigarette smoke condensate by added sugars.

The effects of adding sugars to high- and low-tar cigarettes on the mutagenicity of their smoke condensates were studied using Salmonella typhimurium TA100 and TA98 with and without metabolic activation. The sugars tested were glucose, fructose, galactose, sorbitol, sucrose and lactose. The lowest mutagenicities observed with these sugars per mg of smoke condensate assayed on TA98 with metabolic activation were 37% (high-tar cigarettes) and 22% (low-tar cigaretts) of that of smoke condensate from untreated cigarettes. Addition of sugars increased the total amounts of smoke condensates, but the mutagenicities of the total condensates were also decreased by all the sugars, the lowest values being 35% (high-tar cigarettes) and 36% (low-tar cigarettes) of that of smoke condensates from cigarettes without added sugar. On assay with TA100 with metabolic activation, decreases in both specific and total mutangenicities of condensates of high-tar cigarettes were observed with all the sugars tested except galactose and sucrose. Treatment with glucose, fructose or sorbitol decreased the specific mutagenicity of condensates of low-tar cigarettes and glucose and fructose reduced also their total mutagenicity. The effects of added sugars were more marked when assayed on TA98 than on TA100 and of the sugars tested fructose and sorbitol had the greatest effects. Addition of sugars had no effect of the mutagenicity of cigarette-smoke condensate without metabolic activation.     

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.     

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.     

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.     

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.     

Mutagenicity testing of condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures

Condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures were investigated for their potential to produce genetic damage in the tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and in the mouse bone marrow micronucleus assay. Both smoke condensates contained several chlorinated hydrocarbons among which tetrachloroethylene, hexachloroethane, hexachlorobutadiene and hexachlorobenzene were identified by GC/MS. Condensate of smoke from titanium dioxide/hexachloroethane showed a dose-related positive response in the Salmonella assay with strains TA98 and TA100 in the absence of metabolic activation from rat liver S9 fraction. Both smoke condensates were negative in the micronucleus assay but produced a small but significant depression of erythropoietic activity. The results indicate that smoke condensate from titanium dioxide/hexachloroethane mixtures contains unidentified compound(s) that may be considered mutagenic in the Salmonella assay.     

Mutagenicity of wood smoke condensates in the Salmonella/microsome assay

Smoke condensates of woods used for food preservation and aromatization in Nigeria were tested for mutagenic activity using Salmonella typhimurium TA98 and TA100. The woods were: white mangrove (Avicennia nitida), red mangrove (Rhizophora racemosa), mahogany Khaya sp.), abura (Mitragyna ciliata), alstonia (Alstonia boonei) and black afara (Terminalia ivorensis). Cigarette tar was tested for comparison. The condensates induced dose-dependent increases in the number of His+ revertants mainly with S9 mix. With the exception of mahogany and cigarette smoke condensate, the smoke condensates induced more revertants/microgram condensate in TA100 than in TA98. The number of revertants/microgram condensate ranged between 0.04 and 0.9 for the wood smoke condensates and was 0.12 for the cigarette smoke in TA100. The range was between 0.1 and 0.30 for the wood smoke condensates and 0.18 revertants/microgram condensate for cigarette smoke condensate in TA98. Concentrations of 7 polycyclic aromatic hydrocarbons (PAHs) in the condensates were determined namely, pyrene, benzo[a]pyrene, benz[a]anthracene, benzo[k]fluoranthene, benzo[b]chrysene, benzo[g,h,i]perylene and dibenzo[a,e]pyrene. The condensates contained varying concentrations of the individual PAHs and those with higher concentrations generally showed greater mutagenic activities. However, the order of mutagenic potency in the bacterial strains differed from the order of PAH concentrations, which were lower than the concentrations at which they are reported to induce mutations. When 6 of the PAHs were mixed in the concentrations in which they were found in the individual condensates, the mixtures did not induce mutation so that the contribution of the PAHs to the mutagenic activities of the condensates could not be determined.     

Genetic toxicology studies comparing the activity of sidestream smoke from cigarettes which burn or only heat tobacco

The results of in vitro genetic toxicology studies of sidestream cigarette smoke (SSCS) from cigarettes which heat but do not burn tobacco were compared to those of sidestream smoke from cigarettes which burn tobacco. SSCSs from 5 cigarettes were compared. Three of the cigarettes, the Kentucky reference research cigarette (1R4F), a commercially available ultra-low-tar brand (ULT) and a commercially available ultra-low-tar menthol brand (ULT-menthol) burn tobacco while two of the cigarettes, a regular (TEST) and a menthol (TEST-menthol) heat tobacco. SSCSs from all cigarettes were prepared by identical techniques, which involved collecting sidestream smoke particulate matter on Cambridge filter pads and combining the particulate matter with the vapor-phase materials collected by bubbling the smoke exiting the Cambridge pad through DMSO. The SSCSs obtained (equivalent to 0.4 cigarettes/ml DMSO) were evaluated at identical concentrations in an in vitro genetic toxicology test battery. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in Ames bacterial strains TA98, TA100, TA1537 and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative results in strain TA1535. In the absence of metabolic activation, 1R4F, ULT and ULT-menthol SSCSs were not significantly mutagenic. TEST and TEST-menthol SSCSs produced negative results in all 5 bacterial strains, both with and without metabolic activation. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in the CHO chromosomal aberration assay and in the CHO sister-chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol SSCSs produced negative results in both assays, either with or without metabolic activation. The SSCSs from 1R4F, ULT and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol SSCSs were negative in this assay. All 5 SSCSs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. SSCSs from the 1R4F, ULT and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol SSCSs were not cytotoxic under either condition. These results demonstrate that sidestream smoke from cigarettes which heat but do not burn tobacco (TEST and TEST-menthol) was neither genotoxic nor cytotoxic under conditions where sidestream smoke from cigarettes which burn tobacco (1R4F, ULT and ULT-menthol) was genotoxic and/or cytotoxic in a concentration-dependent manner.     

Genotoxicity of heated cooking oil vapors.

Epidemiological studies of lung cancer in Chinese women indicated that factors other than cigarette smoking are related to lung cancer risk. A case-control study suggested that indoor air pollution, particularly from cooking oil emissions, may be involved. Condensates of volatile emissions from rapeseed and soybean cooking oils were prepared and found to be genotoxic in short-term tests including the Salmonella mutation assay, SV50 forward-mutation assay, and sister-chromatid exchange assay, as well as the micronucleus assay in mouse bone marrow. In contrast, condensates from rapeseed oil with butylated hydroxyanisole or hydrogenated rapeseed oil were not mutagenic, implicating oxidation products as the cause for mutagenicity. Peanut oil and lard condensates were not mutagenic in any assay. The association of exposure to Chinese rapeseed cooking-oil emissions and lung-cancer risk may be related to the mutagenic component of these condensates.     

Induction of chromosome damage by methylene chloride in CHO cells

The genotoxicity of methylene chloride was determined using sister-chromatid exchange (SCE) and chromosome aberration assays in cultured Chinese hamster ovary (CHO) cells. Methylene chloride caused extensive chromosome aberrations both with and without metabolic activation. However, the results of the SCE assay were negative for methylene chloride. These results agree with previously observed genotoxic effects of methylene chloride in Salmonella typhimurium and Saccharomyces cerevisiae. The fact that methylene chloride causes chromosome aberrations without increasing the SCE level indicates that complete reliance on the induction of SCE as a test system for assessing chromosomal effects is not valid.     

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.     

Dose-responsive increase in sister-chromatid exchanges in bone-marrow cells of mice exposed nose-only to whole cigarette smoke

The effect of whole cigarette smoke exposure on bone-marrow sister-chromatid exchanges (SCEs) was studied in B6C3F1 mice. Animals were exposed nose-only to 10% (v/v) cigarette smoke 5 days/week for 2 weeks. Four dose levels of cigarette smoke (1, 4, 9 and 18 exposures/day) were studied using 2 cigarette types, Kentucky reference 3A1 (3A1) and American Blend (AB). A single exposure represented approximately 1 cigarette. A dose-dependent increase in SCEs was observed for both the 3A1 and AB cigarettes at dose levels which had no effect on bone-marrow cell-replication kinetics. These findings represent the first demonstration of a dose-responsive increase in cigarette smoke-induced SCEs in a rodent model system.     

Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

Background

Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β₂-integrin activation and function in neutrophilic transmigration through endothelium.

Methods and results

Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β₂-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β₂-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.

Conclusion

This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β₂-integrins on the cell surface. Blocking this activation of β₂-integrins might be an important target in cigarette smoke induced neutrophilic diseases.     

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.     

Genotoxicity and genotoxic enhancing effect of tetrandrine in Salmonella typhimurium

Tetrandrine has been used for the treatment of silicosis in China. The potential genotoxic and carcinogenic hazards of this drug were studied using the Salmonella/histidine reversion assay and the SOS/Umu test. The results show that tetrandrine was weakly mutagenic to Salmonella typhimurium TA98 with metabolic activation and did not induce SOS response. However, tetrandrine increased the mutagenic activity of benzo[alpha]pyrene, trinitrofluorenone (TNF), 2-aminoanthracene (2AA), diesel emission particles, airborne particles, and cigarette smoke condensate by more than 100%; the activity of aflatoxin B1 and fried beef was increased by over 75%. It also increased the 2AA and TNF-induced SOS response by more than 300%. These results indicated that tetrandrine was a weak promutagen inducing frameshift mutations and was a potent genotoxic enhancer. The mechanism for the genotoxic enhancement is not known. However, the fact that the increase in mutagenicity was noted only in TA98 and not in TA1538 suggested that the enhancement of genotoxicity by tetrandrine may result from an increase in error-prone DNA repair.     

Isolated hamster brown fat cells: A bioassay for toxicity tests at the cellular level. Effect of cigarette smoke condensates from high and low tar yield cigarettes

A test system for the detection of substances with cell-irritating or cell-damaging effects using isolated brown fat cells is presented and the effects of cigarette smoke condensates (CSC) from high and low tar yield cigarettes on this system examined. The condensates inhibited noradrenaline stimulated respiration in a dose-dependent manner. Ethanol solutions of the condensates had a stronger inhibitory effect than DMSO solutions. The CSC strongly inhibited the mitochondrial function but other cellular functions were also impaired.