Fraction Of Cigarette Smoke Condensate Research Articles

Unscheduled DNA synthesis in human lymphocytes treated with cigarette smoke condensate and its semivolatile fraction.

A cigarette smoke condensate (CSC) and the semivolatile fraction (SVF) from the CSC were tested for their ability to induce unscheduled DNA synthesis (UDS) in human lymphocytes. Exposed cells were incubated with tritiated thymidine and the UDS was determined by liquid scintillation counting of precipitated DNA. The tests were performed with and without metabolic activation by liver fraction (S9) from rats induced with Aroclor 1254. No UDS activity was detected, neither for the CSC nor for the SVF. Two control substances, benzo(a)pyrene and methyl methanesulfonate, increased the UDS activity in tests with and without metabolic activation, respectively. The scintillometric method used for detection of UDS was not sufficiently sensitive to be used as a tool for genetic risk assessment of CSC fractions.

Selective clonal expansion and microenvironmental permissiveness in tobacco carcinogenesis.

Historically our knowledge about the direct carcinogenic activity of cigarette smoke and its constituents grew from painting experiments on the skin of mice to produce papillomas and carcinomas. The neutral fraction of cigarette smoke condensate had most of the carcinogenic activity in this test and was rich in carcinogenic polycyclic aromatic hydrocarbons (PAHs), the most abundant by far being BP. However, the concentration of BP in the condensate was only about 2% the amount of pure BP required to cause skin tumors. In other fractions there were non-carcinogenic constituents that promoted tumor formation when applied repeatedly to mouse skin that had been initiated by a single subcarcinogenic application of BP. There were also constituents of cigarette smoke that acted as co-carcinogens when applied simultaneously with repeated applications of BP. BP was effective as an initiator at lower concentrations than as a complete carcinogen, and some non-carcinogenic PAHs in the condensate were also active initiators. It was concluded from these studies that cigarette smoke condensate is primarily a tumor-promoting and co-carcinogenic agent with weak activity as a complete carcinogen. A major effect of promoters, and possibly of co-carcinogens, is a diffuse hyperplasia which includes selective expansion of clones carrying endogenous mutations and/or mutations induced by PAHs and other carcinogens such as NNK. The induced mutations as well as damaged cells would occur throughout the exposed region and, along with the hyperplasia, increase the permissiveness of the cellular microenvironment for neoplastic expression of any potential tumor cell in its midst. Since neither the promoters nor co-carcinogens in tobacco smoke are known to interact directly with DNA, their effects can be considered epigenetic processes that act upon genetically altered cells. Examples are cited from studies of experimental skin carcinogenesis, smoking-induced histopathological changes in human lung and spontaneous transformation in cell culture to illustrate the genetic and epigenetic interactions of neoplastic development in general and their significance for smoking-induced lung cancer in particular. Certain dietary modifications that appear to be effective in moderating the promotional phase of animal and human carcinogenesis are suggested for trial in managing lung cancer.

Inhibition of Endothelial Cell Migration by Cigarette Smoke Condensate

Cigarette smoking is among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanism, however, that links cigarette smoking to an increased incidence of atherosclerosis is poorly understood. Endothelial cell (EC) integrity is critical in preventing vascular lesion formation, and after a loss of EC integrity reendothelialization must be rapid and complete. We therefore investigated whether cigarette smoke affected the ECs ability to migrate or altered the intracellular signals generated during migration. The DMSO-soluble fraction of cigarette smoke condensate (CSC), derived from the standard research cigarette, was tested on cultured ECs (HUVEC) derived from human umbilical vein. The addition of CSC caused a dose-dependent decrease in the ability of EC to migrate as measured over a 24-h time period. Nicotine and cadmium sulfate, two constituents of cigarette smoke, individually or in combination, had no effect on migration. Examination of the tyrosine phosphorylation state of various intracellular proteins by Western blot analysis showed that CSC caused the hyperphosphorylation of a 130-kDa protein. In addition, other intracellular proteins showed changes in their phosphorylation states after CSC addition. These results support the hypothesis that CSC is detrimental to normal EC function in maintaining vascular integrity and suggest that smokers are more likely to develop complications of vascular disease due to delayed or incomplete reendothelialization as a consequence of decreased EC migration.

Basic fraction of cigarette smoke condensate enhances UV-induced mutagenesis due to the inhibition of DNA repair

Basic fraction of cigarette smoke condensate enhances UV-induced mutagenesis due to the inhibition of DNA repair

Detection of direct mutagenicity of cigarette smoke condensate in mammalian cells

Mutagenicity of cigarette smoke condensate (CSC) and the acidic, basic and neutral fractions of CSC was examined in the AL hybrid cell, a Chinese hamster ovary cell containing one human chromosome 11. Since the human chromosome 11 is not necessary for survival of the AL cells, mutations involving large deletions and chromosomal loss by non-dysjunction are non-lethal events that are detectable by loss of human cell surface antigens (a1, a2 and a3) encoded by genes on chromosome 11p (a1 and a3) and 11q (a2) through an antibody-complement lysis assay. Exposure of AL cells to CSC without exogenous metabolic activation caused a dose-dependent cytotoxicity and mutagenicity. Mutagenicity also increased with time of incubation up to 3 h with a maximum of 300 a1- mutants/10(5) survivors (250% above background; P less than 0.0005) after incubation with 100 micrograms/ml CSC. Cytotoxicity and mutagenicity of CSC were inversely proportional to cell density. Fifty percent lethal doses for the acidic, basic and neutral fractions of CSC after 3 h of incubation were 30, 100 and 240 micrograms/ml respectively, and the acidic fraction at a concentration of 25 micrograms/ml induced 350 a1- mutants/10(5) survivors (230% above background; P less than 0.0005); the basic and neutral fractions were less mutagenic. These results indicate that CSC and fractions of CSC can directly produce a spectrum of mutations, through both deletional and non-dysjunctional mechanisms of a kind known to lead to inactivation of tumor suppressor genes.

Further studies on the comutagenic activity of cigarette smoke condensate

The comutagenic effect exerted by cigarette smoke condensate (CSC) was investigated. In vitro experiments with Salmonella typhimurium strains TA98 and TA98/1,8DNP 6 indicated that CSC specifically enhances the mutagenicity of polyaromatic amines such as 2-aminofluorene, 2-acetylaminofluorene, 4-acetylaminofluorene and 2-aminoanthracene. The pattern of comutagenicity of CSC was shown to differ from that of norharman, a tobacco-related known comutagenic substance. Both black and blond tobacco CSCs proved to interact synergistically with 2-aminoanthracene mutagenicity. Chemical fractionation of CSC indicates the occurrence of comutagenic substance(s) in both neutral and basic components. Further in vitro experiments with 2-acetylaminofluorene metabolites and derivatives suggest that the comutagenic effect of CSC could involve later step(s) in the metabolic activation of fluorenylamines, i.e., the conversion of hydroxylamines into ultimate reactive species. The possible occurrence of a synergistic interaction of CSC with chemical mutagens in vivo was evaluated. Administration of 2-aminoanthracene/CSC mixtures, previously shown to be comutagenic in vitro, failed to demonstrate a synergistic effect in SCE induction in bone marrow cells of mice. This apparent discrepancy may rely on divergences in the activation pathways of polycyclic amines in vitro and in vivo.

Effects of cigarette smoke fractions on the chemotaxis of polymorphonuclear leukocytes.

The effects of cigarette smoking fractions on polymorphonuclear leukocyte (PMN) chemotaxis were determined using the 51Cr-assay. Water-soluble fractions (WSF) of cigarette smoke produced from several tobacco types differed in inhibitory potencies (i.e., flue cured greater than or equal to Maryland greater than or equal to blended greater than Burley greater than or equal to Turkish) corresponding to the respective unsaturated aldehyde content of the smoke from these tobaccos. Fractionation of cigarette smoke condensate (CSC) demonstrated that the more polar fractions were potent inhibitors of chemotaxis whereas those containing nicotine and the polycyclic hydrocarbons were weak inhibitors of chemotaxis. Unlike the inhibitory effects of WSFs, CSC fractions did not inhibit random migration and their inhibition of chemotaxis could not be completely prevented by reduced glutathione. These data suggest that while the alpha, beta-unsaturated aldehydes present in the vapor phase of smoke are among the most potent inhibitors of in vitro PMN chemotaxis, other polar, nonvolatile constituents of cigarette smoke also inhibit chemotaxis and by a mechanism which differs from that of the unsaturated aldehydes.

Identification of cembratriene-4,6-diol as antitumor-promoting agent from cigarette smoke condensate.

Cigarette smoke condensate (CSC) was separated into several fractions and each was tested for an inhibitory effect on the early antigen (EA) of Epstein-Barr virus (EBV) which can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Two diastereoisomers of 2,7,11-cembratriene-4,6-diol (alpha- and beta-CBT) were isolated from the neutral fractions of CSC and these showed potent inhibitory effects on the induction of EBV-EA by TPA. The doses of alpha- and beta-CBT required for 50% inhibition of EBV-EA induction by TPA were 7.7 and 6.7 micrograms/ml, respectively, which are comparable with those of retinoic acid, a potent inhibitor of induction of epidermal ornithine decarboxylase (ODC) activity and tumor promotion by TPA in mice. Application of alpha- and beta-CBT to mouse skin prior to treatment with TPA inhibited TPA-induced ODC activity. The degree of inhibition was dependent on the dose and application of 16.5 mumol/mouse of alpha- and beta-CBT resulted in a 50 and 40% reduction, respectively, of the maximum of the ODC activity induced as a result of treatment with TPA. In initiation-promotion experiments, alpha-CBT markedly inhibited the promoting effect of TPA on skin tumor formation in mice which were initiated with 7,12-dimethylbenz[a]anthracene, but beta-CBT was found to be less effective. Application of 3.3 mumol of alpha-CBT 40 min prior to treatment with TPA (1 microgram) resulted in a 53% reduction in the number of papillomas per mouse. Our present data suggest that EBV-EA inhibition assay using Raji cells is effective for the first screening of inhibitors of tumor promotion, and provide evidence that CSC contains antitumor-promoting agents in addition to carcinogenic and tumor-promoting agents already reported.

Isolation and ldentification of Aza-Arenes of Tobacco Smoke

Abstract The nitrogen analogues of polynuclear aromatic hydrocarbons (aza-arenes) were isolated and identified in a basic fraction of cigarette smoke condensate. Silicic acid chromatography removed the predominant nicotine alkaloids, while gel chromatography on Bio-Beads S-X12 in benzene effectively separated the aza-arenes from interfering aliphatic compounds. In addition, the gel columns separated the aza-arenes by ring number and degree of alkylation on the basis of an adsorption-type mechanism. These gel characteristics facilitated the identifications of a large number of isomeric aza-arenes. Compounds identified included 2-vinylpyridine, 3-vinylpyridine and 2-phenylpyridine as well as quinoline, isoquinoline, 4-azafluorene, benzoquinolines, benzoisoquinolines, 1-azafluoranthene, 7-azafluoranthene, 4-aza-pyrene, 7- azaindole, pyrroloquinoline and their mono-, di- and trimethyl derivatives. All eight possible isomers of benzoquinoline and benzoisoquinoline were found, four of which are being reported for the first time. Evidence was also found for the probable presence of 5,6-benzo-7-azaindole.

Mutagenicity of fractions of cigarette smoke condensate in Neurospora crassa and Salmonella typhimurium

The mutagenicities of selected fractions of cigarette smoke condensate (CSC) were studied in Neurospora crassa for the presence of direct-acting mutagens. CSCs from the University of Kentucky Reference Cigarette 1R1 were assayed in a forward-mutation test at the adenine-3 ( ad-3) region in resting conidia of a 2-component heterokaryon. Direct-acting mutagenic activity was found in an enriched polycyclic aromatic hydrocarbons (EPAH) fraction and in a basic fraction (Swain 5). No direct-acting mutagenic activity was detected in an acidic fraction (Swain 8), although it was highly toxic to resting conidia. The EPAH fraction also was tested in the presence of S9 mix prepared from Aroclor-1254-induced rat liver. It was found to be mutagenic, but higher doses were required than in the absence of S9 mix. In addition, the mutagenicities of CSC and 10 fractions of CSC were investigated in Salmonella typhimurium TA1538 by the incorporation and preincubation methods. In general, preincubation did not enhance the mutagenicities of the fractions, and the two rankings of mutagenic potency of the condensates that were obtained by the two methods were not significantly different. This is the first report of the presence of potent direct-acting mutagenicity in the EPAH and Swain 5 fractions of CSC.

A Study of Tobacco Carcinogenesis. XX. Role of Catechol as a Major Cocarcinogen in the Weakly Acidic Fraction of Smoke Condensate<xref ref-type="fn" rid="fn2">2</xref>, <xref ref-type="fn" rid="fn3">3</xref>, <xref ref-type="fn" rid="fn4">4</xref>

The weakly acidic fraction of cigarette smoke condensate was fractionated by preparative high-pressure liquid chromatography into major subfractions I-IV. Major subfractions II and III were fractionated further into subfractions A-J. Subfractions A-J were tested for cocarcinogenicity on the skin of noninbred Ha:ICR Swiss albino mice by application with 0.003% benzo[a]pyrene. Subfractions A-C and F-J showed significant cocarcinogenic activity; subfractions A, F, and H were the most active. Catechol was a major component of subfraction A and was also detected in subfractions B-D and F. Major components of the other subfractions included hydroquinone (B), coniferyl alcohol (C and H), hydroxyphenyl alcohols (D), alkyl-2-hydroxy-2-cyclopenten-1-ones (C, D, and F), hydroxyacetophenones (F), phenolic cyano compounds (F), and fatty acids (F). The results demonstrate the importance of catechol as a cocarcinogen in the weakly acidic fraction of cigarette smoke condensate and indicate the presence of other cocarcinogens.

Gel permeation chromatography of oxygenated components of cigarette smoke condensate

Oxygenated neutral constituents of a tumor-inhibiting fraction of cigarette smoke condensate were separated from interfering phenolic compounds by a combination of silicic acid and gel chromatography. Silicic acid adsorption and Bio-Beads gel chromatography separated aliphatic from aromatic ketones, and gel chromatography on Sephadex LH-20 resolved aromatic ketones from phenols. Identifications were achieved by studying individual fractions by g.c. and g.c.—m.s.

Characterization of catechols, resorcinols, and hydroquinones in an acidic fraction of cigarette smoke condensate

Characterization of catechols, resorcinols, and hydroquinones in an acidic fraction of cigarette smoke condensate

Fractionation of cigarette smoke condensate for chemical and biological testing

Large-scale fractionation of cigarette smoke condensate (CSC) was carried out by gel filtration and silicic acid column chromatography, and selected fractions and their subfractions were tested for tumorigenicity by mouse-skin bioassay. The weak-acid fraction was separated into four subfractions, the polynuclear aromatic hydrocarbon (PAH) fraction into two subfractions, and the polar neutral lipid fraction into three subfractions. Also, combinations of subfractions were examined for synergic effects and portions of all active material were subjected to chemical analyses by gel filtration, column, thinlayer and gas chromatography, and ultraviolet and mass spectrometry. A weak-acid subfraction (F-63) in which catechol was concentrated, and which comprized 3.27% CSC was shown to be tumorigenic, as were combinations of this sub fraction with the active PAH (F-67) sub fraction and a polar, neutral lipid (F-70) sub fraction, representing 0.01 and 0.05% CSC, respectively. The results indicated that catechol may be a potent cocarcinogen and that the PAH in CSC interact with other components to exert a tumorigenic effect.

Analysis of a Tumor-Inhibiting Fraction of Tobacco Smoke Condensate

A tumor-inhibiting neutral fraction of cigarette smoke condensate was partially characterized by use of a combination of gel filtration and gas chromatography. This fraction contained solanesol, sterols, and other neutral oxygenated compounds, such as acetophenones and fluorenones. The compound, 1,3-1ndandlone, was Identified for the first time In cigarette smoke. I ntroduction A step-wise process for the fractionation of cigarette smoke condensate (CSC) has been developed at this laboratory to separate and then to identify those components with biological activity (1,2). The procedure involves fractionation of CSC into acidic, basic, and neutral fractions by acid-base extractions. Subsequently, the are further separated by column chromatography on silicic acid with increasingly polar solvent mixtures. The fraction eluted by diethyl and called ether soluble neutrals (ESN) was recently shown to inhibit the tumor-promoting effect of 12-0-tetradecanoyl phorbol-13-acetate when applied concurrently with it on the backs of mice (3). Various compounds in CSC have been shown to inhibit tumors (4,5). Oxygenated neutral components of polluted air have also been shown to inhibit tumors (6). The present report describes a preliminary study on the composition of the ESN fraction of CSC.

Squamous lesions in lungs of rats exposed to tobacco-smoke-condensate fractions by repeated intratracheal instillation.

Twice-weekly intratracheal instillations in rats of up to 24 mg of Fraction (R + P)G suspended in either infusine (I) or buffered saline/gelatine (BS/G) gave rise to foci of squamous metaplasia of alveolar epithelium (SqM) and squamous neoplasms (SqN). Fraction (R + P)G, which is a fraction of cigarette-smoke condensate almost as tumorigenic for mouse skin as the nearly 30 × larger mass of condensate from which it is derived, could be given in this way for up to 40 weeks without excessive mortality or any marked effect on the rate of body-weight gain. By contrast, similar treatment with Fraction N(QG), a fraction having very low tumorigenic activity for mouse skin, induced no SqN and barely any excess of SqM over that induced by either vehicle alone.The effects of Fraction (R + P)G on the incidence of SqM and SqN were both time and dose related, the effect on SqM incidence being already evident after 10 weeks of treatment. No SqN seen were unequivocally malignant, though, due to the design of the experiment, only 5 rats exposed to Fraction (R + P)G were observed more than 60 weeks after the start of the experiment.Other changes in the lung, including aggregates of alveolar macrophages laden with golden-brown pigment (GBM) and foci of cuboidal/columnar metaplasia of alveolar epithelium (CCM), were frequently seen in response to both fractions. Fraction (R + P)G administered in I was more effective in causing SqM and SqN than the same fraction administered in BS/G. The implications of the findings are discussed, particularly the possibility that the intratracheal/instillation technique might be useful as a rapid bioassay for comparing the tumorigenicity of different cigarette-smoke condensates.

Chemical Studies on Tobacco Smoke-LIV Determination of Hydroxybenzyl Alcohols and Hydroxyphenyl Ethanols in Tobacco and Tobacco Smoke

p-hydroxyphenyl)-ethanol (PHPE) end m-hydroxybenzyl alcohol (MHBA) were major components of subfractions of the tumor-promoting fractions of cigarette smoke condensate (CSC). Tofurther Investigate the possible role of these and related compounds in tumor promotion and cocarclnogenesls by CSC, an analytical method was developed for hydroxyphenyl ethanols and hydroxybenzyl alcohols In tobacco and tobacco smoke. The method consisted of a series of solvent partitions followed by preparative thin-layer chromatography and gas-liquid chromatography of the silylated hydroxyphenyl alcohol fraction, p-Hydroxybenzyl alcohol (PHBA) was used as Internal standard. The main- stream smoke of a typical 85-ram U.S. non-filter cigarette contained 3.3---0.3/~g/cig of PHPE and 1.0  0.1 /Jg/clg of MHBA; that from filter cigarettes contained from 1.2-2.8 /Jg/cig PHPE and 0.28-0.66 Hg/cig MHBA. PHPE, but not MHBA, was detected in unburned tobacco (0.96-15.0/Jg/g). The major source of PHPE In mainstream smoke was apparently transfer from tobacco, while MHBA was formed, at least partially, from cellulose. PHPE levels in cigarette tobacco and smoke were reduced by extraction of the tobacco with the hexane-ethanol azeotrope.

Monitoring Phenolics in Cigarette Smoke Condensate Fractions by Micro-Polyamide Thin-Layer Chromatography*

A rapid, sensitive, and convenient thin-layer chromatographic method was developed for the detection of phenolic compounds--particularly catechol (1,2-dlhydroxybonzene) in cigarette smoke. Micro-polyamide plates are used with a benzene, methanol, and acetic acid mixture (45:8:4) as developing solvent and dlazotlzed benzldine as a spray reagent to produce vivid, characteristically colored spots. Data on Rf values and colors for some phenolics In cigarette smoke condensate are given.

The ldentification of High Molecular Weight Polynuclear Aromatic Hydrocarbons in a Biologically Active Fraction of Cigarette Smoke Condensate

AbstractA gel filtration chromatography method was developed for the isolation and concentration of the high molecular weight polynuclear aromatic hydrocarbons (PAH) contained in the most biologically active fraction of cigarette smoke condensate (CSC). The unusually complex mixture of large PAH found in CSC necessitated the use of preparative gas chromatography followed by high-pressure liquid chromatography to achieve separation and identification. Mass spectral, ultra-violet absorption, and chromatographic retention data were needed for the comprehensive identification of the large molecular weight PAH components of CSC. The majority of the more than 200 isolated compounds were identified. Compounds newly identified in CSC included 3,4-dimethylenepyrene, 3,4-trimethylenepyrene, cyclopenta(c,-d)pyrene, 4,5-methylenetriphenylene, benzo[b]perylene, and several dibenzofluoranthenes.

Fractionation of synthetic crude oils from coal for biological testing

A separations procedure that has been used extensively for the reproducible fractionation of cigarette smoke condensate in order to test this material systematically for its carcinogenic properties has been applied to coal liquefaction products. Two types of product oils, one a light oil and the other a heavy tar, have been processed successfully by this procedure. Reproducibility was similar to that reported for condensate fractionations. Proportions, by weight, of fourteen major fractions and some subfractions of the two products were determined. Mutagenicity of the major fractions of the light oil product was tested by microbiological techniques using several strains of Salmonella typhimurium and some mutagenic effect was shown by four of the fractions. The possible carcinogenicity and mutagenicity of the individual fractions are discussed in relation to the reported effects of corresponding cigarette smoke condensate fractions.