Effects of Flavoring and Casing Ingredients on the Toxicity of Mainstream Cigarette Smoke in Rats

A series of in vitro and in vivo studies evaluated the potential effects of tobacco flavoring and casing ingredients. Study 1 utilized as a reference control cigarette a typical commercial tobacco blend without flavoring ingredients, and a test cigarette containing a mixture of 165 low-use flavoring ingredients. Study 2 utilized the same reference control cigarette as used in study 1 and a test cigarette containing eight high-use ingredients. The in vitro Ames Salmonella typhimurium assay did not show any increase in mutagenicity of smoke condensate from test cigarettes designed for studies 1 and 2 as compared to the reference. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure. Exposure to smoke from reference or test cigarettes in both studies induced increases in blood carboxyhemoglobin ((COHb)) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period. Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes.     

Effects of flavoring and casing ingredients on the toxicity of mainstream cigarette smoke in rats

A series of in vitro and in vivo studies evaluated the potential effects of tobacco flavoring and casing ingredients. Study 1 utilized as a reference control cigarette a typical commercial tobacco blend without flavoring ingredients, and a test cigarette containing a mixture of 165 low-use flavoring ingredients. Study 2 utilized the same reference control cigarette as used in study 1 and a test cigarette containing eight high-use ingredients. The in vitro Ames Salmonella typhimurium assay did not show any increase in mutagenicity of smoke condensate from test cigarettes designed for studies 1 and 2 as compared to the reference. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure. Exposure to smoke from reference or test cigarettes in both studies induced increases in blood carboxyhemoglobin ((COHb)) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period. Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes.     

Influences of exposure to cigarette smoke on concentration of nicorandil in plasma of rats.

Influences of acute exposure to cigarette smoke on plasma concentrations of nicorandil administered orally and parenterally were investigated in rats by HPLC. The animals were exposed to tobacco smoke of two kinds of cigarettes using a smoking machine (i.e., the cigarette smoke contained either low or high nicotine and tar). The plasma concentration of nicorandil administered orally at a dose of 10 mg/kg had a lower absorption phase in two cigarette smoke-exposed groups, particularly in the high nicotine and tar-containing cigarette smoke-exposed group, compared with the nonsmoking control group. The AUC and MRT values in a high nicotine and tar-containing cigarette smoke-exposed group were lower and higher, respectively, than in the nonsmoking control group. However, there was no marked difference in nicorandil plasma concentrations between the cigarette smoke-exposed group and the nonsmoking control group when nicorandil was administered ip or iv at a dose of 5 mg/kg. These results suggest that cigarette smoke exposure causes the suppression or delay of absorption of nicorandil from the gastrointestinal tract.     

Ninety-Day Inhalation Study in Rats, Comparing Smoke from Cigarettes That Heat Tobacco with Those That Burn Tobacco

Eight groups of 30 male and 30 female rats were exposed 1 hrper day, 5 days per week for 13 weeks, to smoke from reference(tobacco burned) or test (tobacco only heated) cigarettes, atnicotine concentrations of 5, 15, or 30µ/liter, of air.Similar smoke concentrations of wet total particulate matterand carbon monoxide were produced in each of the test/referencecomparisons. There was a pronounced depression of minute ventilationof animals in the reference groups, but not in the test animals.Blood carboxyhemoglobin concentrations were similar in animalsexposed to smoke from test and reference cigarettes. Plasmaconcentrations of nicotine and cotinine in the test groups werehigher than in the reference groups. There were no differencesbetween the smoke-exposed groups in terms of body weight orfeed consumption. At necropsy, an increase in heart weight wasnoted in both high exposure groups. There were notable differencesin histopathology, with fewer and less-pronounced changes inthe test groups than in the reference groups. Many of the histopathologicalresponses induced in the reference groups were absent in thetest groups. Overall, the study demonstrated a substantial reductionin the biological activity of smoke from the test cigarettewhen compared with the reference.     

Development and Characterization of an Elisa for Cotinine in Biological Fluids

Abstract

Cotinine is a major metabolite of nicotine and serves as an important biomarker of tobacco smoke exposure. To monitor exposure to tobacco smoke or nicotine, a sensitive enzyme-linked immunosorbent assay (ELISA) for cotinine was developed. The test had an 1–50 of between 0.5 and 1.0 ng/ml for cotinine and about 500-fold less affinity for nicotine. Few matrix effects were not detectable in human saliva, although relatively small matrix effects (1–50 for cotinine, 2.8 ng/ml) were observed in human serum and urine. The test accurately measured the levels of cotinine in NI5T standards in freeze-dried human urine derivative material (r = .9999) indicating its reliability for measurement of cotinine. The test readily detected low levels (5–500 nglml) of cotinine in human saliva and serum samples. Also, the levels of cotinine in plasma and urine samples from smoke-exposed mice and rats could be rapidly analyzed for cotinine. This ELISA is therefore a sensitive and accurate test for the determination of cotinine in plasma, serum, saliva, and urine samples from humans and animals, and can be successfully used for monitoring and quantifying exposure to tobacco smoke or nicotine.     

Urinary excretion of mutagens in passive smokers

Six healthy young volunteers with no history of active smoking were asked to keep on their Western diets avoiding the consumption of alcoholic beverages, excess coffee, any sort of medicament, and the known pro- and/or anti-mutagen-containing foods and drinks, 24 h before and during the experiments. They were exposed passively to cigarette smoke produced by 4 habitual smokers in an unventilated 48.6 m3 room for 8 h. The carbon monoxide concentration was 18.85 +/- 7.3 ppm during the 8-h exposure. Frameshift mutagens were isolated from 10-h urine samples using chloroform and were tested for mutagenicity in the Salmonella/mammalian microsome assay employing Salmonella typhimurium TA98. Although clearly enhanced, no significant mutagenic activity could be found with 25 ml equivalent urine/plate after passive exposure to cigarette smoke. The weak mutagenicities found were highly significant when 50 ml equivalent urine/plate was tested. No direct correlation was observed between urine mutagenicity and the urinary cotinine concentration. The results obtained are discussed with reference to inconsistent reports in the literature concerning the mutagenicity of urine after passive smoking.     

Subchronic inhalation by rats of mainstream smoke from a cigarette that primarily heats tobacco compared to a cigarette that burns tobacco

A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smoke-exposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.     

Development of a commercial cigarette “market map” comparison methodology for evaluating new or non-conventional cigarettes

A “market map” comparison methodology for cigarette smoke chemistry yields is presented. Federal Trade Commission machine-method smoke chemistry was determined for a range of filtered cigarettes from the US marketplace. These data were used to develop illustrative market maps for each smoke constituent as analytical tools for comparing new or non-conventional cigarettes to a sampling of the broader range of marketplace cigarettes. Each market map contained best-estimate “market-means,” showing the relationship between commercial cigarette constituent and tar yields, and yield “market ranges” defined by prediction intervals. These market map means and ranges are the basis for comparing new cigarette smoke yields to those of conventional cigarettes. The potential utility of market maps for evaluating differences in smoke chemistry was demonstrated with 1R4F and 2R4F Kentucky reference cigarettes, an Accord™ cigarette, and an Advance™ cigarette. Conventional cigarette tobacco nicotine, nitrate, soluble ammonia, and tobacco specific nitrosamine levels are reported. Differences among conventional cigarette constituent yields at similar tar levels were explained in part by the chemical composition range of those cigarette tobaccos. The study also included a comparison of smoke constituent yields and in vitro smoke cytotoxicity and mutagenicity assay results for the 1R4F Kentucky reference cigarette and its replacement 2R4F. Significant smoke yield differences were noted for lead, NNK, and NNN. The majority of their smoke constituent yields were within the market range developed from the sampled conventional cigarettes. Within the sensitivity and specificity of the in vitro bioassays used, smoke toxic activity differences for the two reference cigarettes were not statistically significant. These results add to the limited information available for the 2R4F reference cigarette.     

Toxicologic evaluation of humectants added to cigarette tobacco: 13-week smoke inhalation study of glycerin and propylene glycol in Fischer 344 rats

Glycerin (CAS no. 56-81-5) and propylene glycol (CAS no. 57-55-6) are commonly used as humectant ingredients in manufactured cigarettes to control and maintain the moisture content of the cut tobacco filler. The potential of these added humectants to affect the toxicity of cigarette smoke was investigated in a subchronic nose-only smoke inhalation study in rats. Filtered test cigarettes were prepared from an American-style tobacco blend containing either glycerin added at 5.1% w/w tobacco, propylene glycol at 2.2% w/w tobacco, or combinations of these humectants totaling 2.3%, 3.9%, and 7.2% w/w tobacco. Other groups of animals were exposed similarly to the smoke of reference cigarettes without added humectants, or to filtered air (sham control). Smoke exposures were conducted for 1 h/day, 5 days/wk for 13 wk, at target smoke particulate concentrations of 350 mg/m(3). All smoke-exposed groups had equivalent increases in blood carboxyhemoglobin, serum nicotine, and serum cotinine relative to the air controls. Smoke-associated reductions in body weights and occasional increases in heart and lung weights were generally similar among the various exposure conditions at necropsy. Increases in serum alkaline phosphatase and decreases in serum glucose and cholesterol were observed among smoke-exposed females relative to air controls. However, no significant differences in these parameters were evident between the humectant-containing and reference cigarette smoke exposure groups. Assessments of respiration conducted after 3, 6, 9, and 12 wk of smoke exposure indicated an initial smoke-related but not humectant-related decrease in respiratory rate, tidal volume, and minute volume during the first 20 min of each smoke exposure. Respiratory-tract histopathology was consistent across sexes and smoke groups, comprising (1) diffuse and focal alveolar pigmented macrophages and chronic interstitial inflammation in the lung, (2) laryngeal epithelial hyperplasia, squamous metaplasia, and scab formation, and (3) epithelial hyperplasia in the anterior nose. Smoke-related histopathology resolved substantially during a 6-wk postexposure recovery period. Addition of the tested humectants to cigarettes, singly or in combination, had no meaningful effect on the site, occurrence, or severity of respiratory-tract changes or on the measured indices of pulmonary function. It was concluded that the addition of glycerin and propylene glycol to cigarettes does not significantly affect the biological activity of inhaled cigarette smoke in this rat model.     

Development of a commercial cigarette "market map" comparison methodology for evaluating new or non-conventional cigarettes

A "market map" comparison methodology for cigarette smoke chemistry yields is presented. Federal Trade Commission machine-method smoke chemistry was determined for a range of filtered cigarettes from the US marketplace. These data were used to develop illustrative market maps for each smoke constituent as analytical tools for comparing new or non-conventional cigarettes to a sampling of the broader range of marketplace cigarettes. Each market map contained best-estimate "market-means," showing the relationship between commercial cigarette constituent and tar yields, and yield "market ranges" defined by prediction intervals. These market map means and ranges are the basis for comparing new cigarette smoke yields to those of conventional cigarettes. The potential utility of market maps for evaluating differences in smoke chemistry was demonstrated with 1R4F and 2R4F Kentucky reference cigarettes, an Accord cigarette, and an Advance cigarette. Conventional cigarette tobacco nicotine, nitrate, soluble ammonia, and tobacco specific nitrosamine levels are reported. Differences among conventional cigarette constituent yields at similar tar levels were explained in part by the chemical composition range of those cigarette tobaccos. The study also included a comparison of smoke constituent yields and in vitro smoke cytotoxicity and mutagenicity assay results for the 1R4F Kentucky reference cigarette and its replacement 2R4F. Significant smoke yield differences were noted for lead, NNK, and NNN. The majority of their smoke constituent yields were within the market range developed from the sampled conventional cigarettes. Within the sensitivity and specificity of the in vitro bioassays used, smoke toxic activity differences for the two reference cigarettes were not statistically significant. These results add to the limited information available for the 2R4F reference cigarette.     

TOXICOLOGIC EVALUATION OF HUMECTANTS ADDED TO CIGARETTE TOBACCO: 13-WEEK SMOKE INHALATION STUDY OF GLYCERIN AND PROPYLENE GLYCOL IN FISCHER 344 RATS

Glycerin (CAS no. 56-81-5) and propylene glycol (CAS no. 57-55-6) are commonly used as humectant ingredients in manufactured cigarettes to control and maintain the moisture content of the cut tobacco filler. The potential of these added humectants to affect the toxicity of cigarette smoke was investigated in a subchronic nose-only smoke inhalation study in rats. Filtered test cigarettes were prepared from an American-style tobacco blend containing either glycerin added at 5.1% w/w tobacco, propylene glycol at 2.2% w/w tobacco, or combinations of these humectants totaling 2.3%, 3.9%, and 7.2% w/w tobacco. Other groups of animals were exposed similarly to the smoke of reference cigarettes without added humectants, or to filtered air (sham control). Smoke exposures were conducted for 1 h/ day, 5 days/wk for 13 wk, at target smoke particulate concentrations of 350 mg/m 3. All smoke-exposed groups had equivalent increases in blood carboxyhemoglobin, serum nicotine, and serum cotinine relative to the air controls. Smoke-associated reductions in body weights and occasional increases in heart and lung weights were generally similar among the various exposure conditions at necropsy. Increases in serum alkaline phosphatase and decreases in serum glucose and cholesterol were observed among smoke-exposed females relative to air controls. However, no significant differences in these parameters were evident between the humectant-containing and reference cigarette smoke exposure groups. Assessments of respiration conducted after 3, 6, 9, and 12 wk of smoke exposure indicated an initial smoke-related but not humectant-related decrease in respiratory rate, tidal volume, and minute volume during the first 20 min of each smoke exposure. Respiratory-tract histopathology was consistent across sexes and smoke groups, comprising (1) diffuse and focal alveolar pigmented macrophages and chronic interstitial inflammation in the lung, (2) laryngeal epithelial hyperplasia, squamous metaplasia, and scab formation, and (3) epithelial hyperplasia in the anterior nose. Smoke-related histopathology resolved substantially during a 6-wk postexposure recovery period. Addition of the tested humectants to cigarettes, singly or in combination, had no meaningful effect on the site, occurrence, or severity of respiratory-tract changes or on the measured indices of pulmonary function. It was concluded that the addition of glycerin and propylene glycol to cigarettes does not significantly affect the biological activity of inhaled cigarette smoke in this rat model.     

Comparative 13-Week Inhalation Study of Cigarette Smoke from Cigarettes Containing Cast Sheet Tobacco

A subchronic, nose-only inhalation study was conducted to compare the effects of mainstream smoke from a reference cigarette containing conventional reconstituted tobacco sheet at 30% of the finished blend to mainstream smoke from cigarettes containing 10% or 15% cast sheet (a specific type of reconstituted tobacco sheet) substituted for part of the conventional reconstituted tobacco. Male and female Sprague-Dawley rats were exposed for 1 h/day, 5 d/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin (COHb), serum nicotine, plethysmography, gross pathology, and histopathology were determined. Exposure to cigarette smoke induced a number of changes in respiratory physiology, histopathology, and serum nicotine and COHb levels when compared to sham animals. When corresponding dose groups of reference and cast sheet mainstream smokes were compared, no biological differences were noted. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the reference and either cast sheet cigarette. Substitution of 10% or 15% cast sheet tobacco for conventional reconstituted tobacco sheet does not alter the inhalation toxicology of the mainstream smoke when compared to mainstream smoke from a reference cigarette containing conventional reconstituted tobacco sheet.     

Comparative 13-week inhalation study of cigarette smoke from cigarettes containing cast sheet tobacco

A subchronic, nose-only inhalation study was conducted to compare the effects of mainstream smoke from a reference cigarette containing conventional reconstituted tobacco sheet at 30% of the finished blend to mainstream smoke from cigarettes containing 10% or 15% cast sheet (a specific type of reconstituted tobacco sheet) substituted for part of the conventional reconstituted tobacco. Male and female Sprague-Dawley rats were exposed for 1 h/day, 5 d/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin (COHb), serum nicotine, plethysmography, gross pathology, and histopathology were determined. Exposure to cigarette smoke induced a number of changes in respiratory physiology, histopathology, and serum nicotine and COHb levels when compared to sham animals. When corresponding dose groups of reference and cast sheet mainstream smokes were compared, no biological differences were noted. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the reference and either cast sheet cigarette. Substitution of 10% or 15% cast sheet tobacco for conventional reconstituted tobacco sheet does not alter the inhalation toxicology of the mainstream smoke when compared to mainstream smoke from a reference cigarette containing conventional reconstituted tobacco sheet.     

SUBCHRONIC INHALATION BY RATS OF MAINSTREAM SMOKE FROM A CIGARETTE THAT PRIMARILY HEATS TOBACCO COMPARED TO A CIGARETTE THAT BURNS TOBACCO

A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smokeexposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.     

Effects of chronic smoke exposure on the cardiovascular responses to acute nicotine infusion in the rat

Rats were exposed daily to cigarette smoke for 17-22 weeks in order to characterize mean arterial pressure and regional hemodynamic effects of chronic smoke exposure and to determine if cardiovascular reactivity to acute nicotine infusions is altered by chronic smoke exposure. Urethane-anesthetized animals were instrumented with miniaturized pulsed-Doppler flow probes on the iliac and mesenteric vascular beds. Under resting conditions sham-smoked and smoke-exposed animals had similar levels of mean arterial pressure and mesenteric blood flow; however, resting heart rate was lower in the smoke-exposed group, while iliac blood flow was elevated in the smoke-exposed group. Acute nicotine infusion (6.25, 12.5 and 25 micrograms/kg per min) produced equivalent, dose-dependent pressor effects as well as increases in iliac and mesenteric resistance in sham and smoke-exposed groups. Thus, chronic cigarette smoke-exposure in rats may exert significant cardiovascular effects other than on arterial pressure such as lowered heart rate and elevated blood flow to skeletal muscle beds, while cardiovascular responses to nicotine are not altered by chronic smoke-exposure.     

Effects of whole cigarette smoke on human gingival fibroblast adhesion, growth, and migration

The aim of this study was to investigate the effects of a single exposure to whole cigarette smoke on human gingival fibroblast behavior. Normal oral mucosa fibroblasts were exposed once to whole cigarette smoke for 5, 15, or 30 min, and then were used to analyze cell adhesion, β1-integrin expression, cell growth and viability, cell capacity to contract collagen gel, and cell migration following wound infliction. Our findings showed that when gingival fibroblasts were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell adhesion, a decrease in the number of β1-integrin-positive cells, increased LDH activity in the target cells, and reduced growth. The smoke-exposed fibroblasts were also not able to contract collagen gel matrix and migrate following insult. Overall results demonstrate that a single exposure to whole cigarette smoke produced significant morphological and functional deregulation in gingival fibroblasts. This may explain the higher predisposition of tobacco users to oral infections and diseases such as cancer.     

Cigarette smoking, metabolic activation and carcinogenesis

Epidemiologically, it has been suggested that cigarette smoking is closely associated with an increased risk of cancers in various organs such as the lung, oropharynx, stomach, pancreas, liver and colon. Nevertheless, influences of cigarette smoking on experimental tumorigenesis in organs other than the respiratory tract remain to be elucidated. In our experimental studies, it has been shown that cigarette smoke exposure induces hepatic CYP enzymes, especially CYP1A2, in both rats and hamsters, and S9 fraction from their livers exposed to cigarette smoke specifically increases the mutagenicity in Ames assay of various heterocyclic amines (HCAs) contained in cigarette smoke and cooked food, which is in good agreement with the fact that HCAs are principally activated by CYP1A2 to proximate carcinogens. In fact, cigarette smoke exposure enhanced liver carcinogenesis in rats induced by 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (MeIQx), a major HCA. Furthermore, in our recent study, it was also shown that cigarette smoke exposure induces hepatic CYP2A8 in hamsters, which is homologous to CYP2A6 in human, and hepatic S9 fraction exposed to cigarette smoke increases the mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco specific nitrosamine, which is in line with the fact that NNK is metabolically activated by CYP2A6. Keeping these data, the aim of this review is to discuss any relevancy of modulated metabolic activation by cigarette smoking to cancer risk in human.     

The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields

Tobacco smoke contains more than 5600 constituents, of which approximately 150 are toxicants. This paper describes the activities in the Neutral Red uptake (NRU) assay, the Salmonella mutagenicity test (SAL), the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT) of Particulate Matter (PM) obtained from experimental cigarettes (ECs), designed to produce reduced levels of toxicants. The designs included tobacco substitute sheet (TSS) containing glycerol, which dilutes toxicants in smoke, or the incorporation of blend-treated (BT) tobacco to reduce the levels of nitrogenous toxicant precursors and some polyphenols. All samples were cytotoxic in the NRU, however TSS reduced PM cytotoxicity in this assay. All PMs were mutagenic in the SAL, MLA and IVMNT. Reductions in bacterial mutagenicity were observed in the SAL, for cigarettes with BT tobacco, compared with their respective controls. The quantitative changes in bacterial mutagenicity could be explained by analytical chemistry data on smoke generated from the ECs used in the study. These observations, and the absence of consistent qualitative differences in the activities of the experimental, control and reference cigarettes, suggest that reduced toxicity cigarettes, as measured by the tests described in this paper, may be developed without introducing any additional cytotoxic or genotoxic hazards, but the impact of this on human health risks remains unknown.     

Prenatal exposure to cigarette smoke enhances oxidative stress in astrocytes of neonatal rat

Oxidative stress is involved in the pathogenesis of smoking-related disease. Protection of astrocytes from oxidative insult appears essential to maintain brain function. In this study, we have investigated the effect of gestational cigarette exposure on astrocyte survival. Pregnant female were randomly allocated to the control group or to the cigarette smoke group in which they were placed in an exposure chamber and inhale three cigarettes smoke twice a day for a period of 20 days. The control group was kept in the exposure chamber for the same duration, but without exposure to cigarette smoke. Newborn rats from both groups were weighed 24?h after birth and then cerebral hemispheres were collected for astrocyte culture. Incubation of astrocytes isolated from animals exposed to cigarette smoke with 300?μM H₂O₂ for 1?h induced a significant decrease of the proportion of surviving cells compared to cells isolated form control animals. We have observed that H₂O₂-treated astroglial cells derived from cigarette smoke exposure showed more reduced superoxide dismutase and catalase activities than H₂O₂-treated astroglial cells from control animals. In conclusion, this study indicates that astroglial cells derived from newborn rats exposed in utero to cigarette smoke are more vulnerable to oxidative assault than cultured astrocytes obtained from control animals. These results point out the existence of excitotoxic lesions in newborn exposed in utero to cigarette smoke and suggest that despite their high antioxidative activities, astrocytes cannot survive and protect neurons under massive oxidative stress.     

Perinatal sidestream cigarette smoke exposure and the developing pulmonary surfactant system in rats.

1. Epidemiological studies have strongly implicated passive smoking with increased incidence of various respiratory diseases in children. Our earlier studies have shown that chronic exposure to tobacco smoke significantly changes the composition and the surface activity of the pulmonary surfactant in adult rats. The aim of the present study was to determine if perinatal exposure to sidestream cigarette smoke influences the composition and function of pulmonary surfactant system in developing rat pups. 2. Pregnant Sprague Dawley rats were exposed to sidestream cigarette smoke in a whole body exposure chamber for a total of 6 h each day and the pups born to these dams were further exposed to sidestream smoke for 2 h/day during postnatal period. Exposure of animals to smoke was ascertained by measuring their plasma cotinine. Surfactant analysis was performed on bronchoalveolar lavage fluids (BALF) collected from pups on postnatal day 1, 3, 7, 14, 21 and 35. The phospholipid (PL) content, percentage disaturated phosphatidylcholine (DSPC) and surfactant proteins (SP-A and SP-B) in BALF surfactant preparations from sham and smoke-exposed pups were compared. 3. Significant differences between the two groups were observed at two exposure points: a reduced level of SP-A on day 1, and, a higher level of SP-A and PLs on day 21, in smoke-exposed pups. Surface activity analysis of the surfactant films by pulsating bubble surfactometer did not show any significant differences between the sham and smoke-exposed groups at any exposure point. 4. The results indicate that perinatal exposure to sidestream smoke is capable of producing biochemical changes in the composition of pulmonary surfactant at different stages of development but these changes do not influence surface tension reducing properties of the surfactant.