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.     

Comparison of mouse strains and exposure conditions in acute cigarette smoke inhalation studies

Cigarette smoke exposures in mice have been conducted under various exposure conditions using different strains as animal models of smoke-related diseases. We exposed cigarette smoke to two strains of mice [C57BL/6J (C57) and AKR/J (AKR)] under two different exposure regimens (1?h or 4?h/day) at equivalent daily exposure amount (concentration?×?time). After 2 weeks exposure, mice were evaluated using exposure markers and biological responses. Smoke exposure suppressed respiratory parameters dependent on exposure concentration. The 1-h regimen groups generally showed a greater degree of respiratory suppression and relatively lower exposure markers of urinary nicotine metabolites than the corresponding 4-h regimen groups. Tidal volume was more suppressed in AKR compared to C57, while respiratory rate was more suppressed in C57. Plasma exposure markers and respiratory parameters suggested that C57 inhaled more volume of smoke than AKR. Changes in bronchoalveolar lavage fluid (BALF) cytology and enzyme parameters were most noticeable in the 1?h AKR groups. In BALF cytokine concentration, TARC concentration in C57 was higher than AKR, while KC and MCP-1 in AKR were higher than C57. Relative lung/body weight ratio in smoke-exposed C57 was generally higher, as well as the incidence and severity of lesions in respiratory organs compared to AKR. In summary, C57 appeared to inhale relatively more smoke and displayed greater inflammatory changes in respiratory tract than AKR. Comparison of exposure regimens suggests that a longer exposure duration at lower WTPM concentration might deliver a larger dose of smoke than a shorter exposure duration at higher WTPM concentration.     

Comparison of mouse strains and exposure conditions in acute cigarette smoke inhalation studies

Cigarette smoke exposures in mice have been conducted under various exposure conditions using different strains as animal models of smoke-related diseases. We exposed cigarette smoke to two strains of mice [C57BL/6J (C57) and AKR/J (AKR)] under two different exposure regimens (1?h or 4?h/day) at equivalent daily exposure amount (concentration?×?time). After 2 weeks exposure, mice were evaluated using exposure markers and biological responses. Smoke exposure suppressed respiratory parameters dependent on exposure concentration. The 1-h regimen groups generally showed a greater degree of respiratory suppression and relatively lower exposure markers of urinary nicotine metabolites than the corresponding 4-h regimen groups. Tidal volume was more suppressed in AKR compared to C57, while respiratory rate was more suppressed in C57. Plasma exposure markers and respiratory parameters suggested that C57 inhaled more volume of smoke than AKR. Changes in bronchoalveolar lavage fluid (BALF) cytology and enzyme parameters were most noticeable in the 1?h AKR groups. In BALF cytokine concentration, TARC concentration in C57 was higher than AKR, while KC and MCP-1 in AKR were higher than C57. Relative lung/body weight ratio in smoke-exposed C57 was generally higher, as well as the incidence and severity of lesions in respiratory organs compared to AKR. In summary, C57 appeared to inhale relatively more smoke and displayed greater inflammatory changes in respiratory tract than AKR. Comparison of exposure regimens suggests that a longer exposure duration at lower WTPM concentration might deliver a larger dose of smoke than a shorter exposure duration at higher WTPM concentration.     

In utero exposure to 1R4F reference cigarette smoke: evaluation of developmental toxicity.

The potential developmental effects of 1R4F reference cigarette smoke were examined using Sprague-Dawley rats exposed for 2 h/day, 7 days/week, by nose-only inhalation at target mainstream smoke concentrations of 150, 300, and 600 mg/m3 total particulate matter (TPM). Males were exposed 4 weeks prior to and during mating, with females exposed 2 weeks prior to mating and during mating, and through gestation day (GD) 20. Sham controls received filtered air to simulate nose-only exposure, while cage controls were maintained untreated. Smoke exposure was confirmed through biomarker evaluation (parental: carboxyhemoglobin, nicotine, and cotinine; fetal: nicotine and cotinine). Characteristic cigarette smoke-related histopathologic changes including nasal epithelial hyperplasia and squamous metaplasia and pigmented macrophages in the lung were observed in all exposed parental groups. Maternal toxicity during gestation was indicated at smoke concentrations of 300 and 600 mg TPM/m3, where corrected total body weight gain was significantly (p 相似文献   

Augmentation of elastase-induced emphysema by cigarette smoke: effects of reducing tar and nicotine content

The effects of reducing the tar and nicotine concentration of cigarette smoke were examined in a rat model of smoke-augmented, porcine pancreatic elastase- (PPE-) induced, pulmonary emphysema. Sixty-eight female Long-Evans rats were divided approximately evenly into seven groups: control, PPE, PPE plus sham smoke, high-tar/nicotine cigarette smoke (2R1; 38.8 mg total particulate matter and 2.2 mg nicotine per cigarette), low-tar/nicotine cigarette smoke (1R4F; 10.8 mg total particulate matter and 0.8 mg nicotine per cigarette), PPE + 2R1, and PPE + 1R4F. Three days after intratracheal administration of PPE (400 IU/kg), animals in the smoke-treated groups were exposed to 8-10 puffs of cigarette smoke daily, 7 d/wk for 12 wk. Sham-treated animals received room air in place of cigarette smoke. At the conclusion of the exposures, pulmonary function tests were performed under general anesthesia. Cigarette-smoke exposure alone did not produce significant changes in pulmonary function. Elastase-treated groups demonstrated significant increases in total lung capacity, functional residual capacity, and dynamic and static compliance, as well as significant decreases in carbon monoxide (CO) diffusing capacity and CO diffusion coefficient. Morphometric measurements of mean linear intercept demonstrated a loss of alveolar fine structure with enlargement of distal airspaces in PPE-treated rats. Exposure to either 2R1 or 1R4F cigarette smoke significantly enhanced many of the emphysematous changes produced by PPE, but there were no significant differences between the effects of the two smokes. These data indicate that reducing the tar and nicotine concentration of cigarette smoke does not lessen its ability to augment PPE-induced pulmonary emphysema in the rat.     

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 subchronic inhalation study of smoke from the 1R4F and 2R4F reference cigarettes

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a 1R4F research cigarette to that of a 2R4F research cigarette. Male and female rats were exposed for 1 h/day, 5 days/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, serum nicotine, pulmonary plethysmography, gross pathology, and histopathology were determined. When histological changes resulting from exposure to smoke from the two types of cigarettes were compared, no biologically significant differences were observed. 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 1R4F and the 2R4F cigarettes. The complete toxicological assessment in this comparative inhalation study of 1R4F and 2R4F cigarettes suggests no overall biologically significant differences between the rats exposed to the two cigarettes.     

Comparison of the physiological and morphological effects of cigarette smoke exposure at comparable weekly doses on Sprague-Dawley rats

A variety of dose x duration exposure regimens have been used in inhalation toxicity studies using rodents. We evaluated the effects of differences in smoke concentration and daily exposure duration under similar weekly cumulative exposures in rats to determine potential variation in type and severity of adverse effects in 13-week exposure studies. The weekly cumulative dosages were 2100 and 4200?μg wet total particle matter (WTPM)/L, and the daily exposure durations were 1 and 6?h. Weekly exposure duration was 5 or 7 days/week for groups exposed 1?h/day and 7 days/week for groups exposed 6?h/day. Recovery duration was 6 and 13 weeks. Mainstream smoke exposure suppressed body weight (BW) gain in both regimens. Lower dose groups exposed 1?h/day had a consistently greater of BW gain compared with corresponding 6?h/day groups. Respiratory rate, tidal volume, and minute volume (MV) were suppressed in a dose-dependent manner in both regimens. Higher MV in rats exposed for 6?h/day compared with rats exposed 1?h/day suggested that a lower concentration for longer duration resulted in a greater total inhaled mass (TIM) in rats exposed 6?h/day. Groups exposed for 6?h/day had lower blood carboxyhemoglobin and plasma nicotine levels than groups exposed 1?h/day, reflecting the lower carbon monoxide (CO) and WTPM concentrations in the 6?h/day groups. Data from examination of bronchoalveolar lavage fluids (BALF) and respiratory tract tissues indicated comparable effects between both regimens. Exposure-induced histopathological changes regressed similarly for both regimens after the recovery periods.     

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.     

Histopathology,Urine Mutacenicity,and Bone Marrow Cytocenetics of Mice Exposed Nose-Only to Smoke from Cigarettes that Burn or Heat Tobacco

Abstract

Male and female B6C3F₁ mice were exposed nose-only to smoke from a test cigarette that heats tobacco, or from a reference cigarette that burns tobacco. Cigarette smoke was generated by a smoking machine, and the concentrations of wet total particulate matter (WTPM) were adjusted to 0, 0.16, 0.32, or 0.64 mg/l. Exposures were performed 1 h/day for 14 consecutive days. Urine mutagenicity was assessed by a modified Ames bacterial assay Clastogenesis (sister-chromatid exchanges, chromosome aberrations, and micronuclei) was evaluated in bone marrow cells. Respiratory rate was depressed significantly by exposure to smoke from the reference cigarette, but not the test. Blood carboxyhemoglobin, plasma nicotine, and plasma cotinine showed exposure-dependent increases in the smoke-exposed animals. Histopathological changes similar to those noted previously in smoke-exposed rats were noted, with fewer and less pronounced changes in the animals exposed to smoke from the test cigarette when compared with the reference. Positive urine mutagenicity and clastogenic responses were observed in the animals treated with positive control chemicals. However the urine mutagenicity and clastogenic responses of smoke-exposed animals (both cigarette types) were not different from those of sham-exposed animals, except for the micronucleus assay where animals exposed to high concentrations of reference cigarette smoke showed a significant increase over controls.     

Evidence for carbon monoxide as the major factor contributing to lower fetal weights in rats exposed to cigarette smoke.

One of the major effects of cigarette smoking during pregnancy is bearing a child with lower birth weight. It has previously been demonstrated under experimental conditions in rats that exposure to reference cigarette smoke results in reduced birth weight (E. L. Carmines et al., 2003, Toxicol. Sci. 75, 134-147; C. L. Gaworski et al., 2004, Toxicol. Sci. 79, 157-169). The role of various smoke constituents on lower birth weight was evaluated by exposing time-pregnant Sprague-Dawley rats at the concentrations found in cigarette smoke. The rats were exposed for 2 h/day 7 days/week by nose-only inhalation. The target concentrations were designed to produce the same plasma levels of biomarkers as exposure to 2R4F reference cigarette smoke at a concentration of 600 mg/m(3) total particulate matter. The smoke constituents evaluated included carbon monoxide (CO), nicotine, and a mixture of aldehydes (acrolein, acetaldehyde, and formaldehyde). The smoke constituents were tested individually as well as in mixtures to evaluate potential interactions. Exposure to cigarette smoke during gestation produced a reduction in both maternal body weight gain and fetal weights. Exposure to nicotine reduced maternal body weight gain but had no effect on fetal weight. Exposure to CO had no effect on maternal body weight gain but reduced fetal weight to a degree comparable to cigarette smoke. Exposure to a mixture of aldehydes (acrolein, acetaldehyde, and formaldehyde) had no effect on either maternal body weight gain or fetal weight. Exposure to mixtures of nicotine and CO or nicotine, CO, and aldehydes did not demonstrate any interactions. The results of this study suggest that the observed reduction in fetal weight after exposure to cigarette smoke in rats is due to CO toxicity and not nicotine toxicity.     

Cytotoxicity and gene expression profiles in cell cultures exposed to whole smoke from three types of cigarettes.

The purpose of this study was to evaluate and compare the cytotoxicity and gene expression profiles in cell cultures exposed to whole smoke generated from a full flavor cigarette (Test 1), a low tar cigarette (Test 2), and an ultra-low tar cigarette (Test 3). In addition, a reference cigarette 2R4F was evaluated for cytotoxicity. Neutral red (NR) cytotoxicity assay was performed to determine relative cell death at each exposure concentration (n = 6). LC(50) was generated using wet total particular matter (WTPM), cigarette number, or nicotine concentrations. The overall order of cytotoxicity was Test 1 > 2R4F approximately Test 2 > Test 3. Cell culture samples were collected for RNA extraction at WTPM concentrations of each cigarette that gave similar nicotine concentrations. Affymetrix mouse whole genome 430 2.0 array was used to characterize the gene expression profiles for each cigarette. A total of 598 genes in Test 1, 176 genes in Test 2, and 234 genes in Test 3 samples were differentially expressed compared to the concurrent sham controls. The major biological processes associated with the changed genes in Test 1 samples were down-regulated DNA replication and cell proliferation; the same biological processes were much less affected in Test 2 and Test 3 samples. The common findings in all three cigarettes types were increased glutathione biosynthesis/consumption and inflammatory response, which are known biological effects caused by smoke exposure. The most significantly up-regulated genes were CYP1A1, GSTs, Hmox1, and Procr in smoke-exposed samples, which are either related to well-studied mechanisms of smoke exposure-related diseases or potential new biomarkers for assessing and monitoring biological effects of cigarette smoke exposure in vivo and in smokers. In summary, both the NR cytotoxicity assay and gene expression profiling were able to differentiate the three types of test cigarettes, and the results demonstrated reduced biological effects for the Test 2 and Test 3 cigarettes compared to the Test 1 cigarette in BALB/c-3T3 Cells.     

Pulmonary inflammation in mice exposed to mainstream cigarette smoke

Male C57Bl/6 (C57) and ICR mice were exposed by nose-only inhalation to mainstream cigarette smoke (MS) from 2R4F reference cigarettes, at concentrations of 75, 250, and 600 microg of total particulate matter (TPM) per liter, for up to 6 mo. Respiratory-tract tissue (nose, larynx, and lung), blood, and bronchoalveolar lavage fluid (BALF) samples were collected and analyzed at several time points. Blood samples were analyzed for biomarkers of exposure (COHb and nicotine). BALF was analyzed for biomarkers of cell injury, inflammation, oxidative stress, enzyme activity, and cytokines. Blood COHb and plasma nicotine concentrations increased in a dose-dependent manner, confirming smoke exposure. Mild emphysema was observed following 28 wk of exposure. Macrophage accumulation and inflammatory infiltrates were observed around the alveolar ducts and adjacent vasculature. There was an approximately 13% increase in mean linear intercept (Lm) only in ICR mice exposed to 600 microg/L TPM. There were no significant changes in biomarkers of oxidative stress secondary to smoke exposures; however, 8-isoprostane significantly increased following the 13-wk post-inhalation period. BALF macrophage and neutrophil counts were rapidly and consistently elevated, while lymphocyte counts gradually increased over time. MS-induced inflammatory responses observed in this study are comparable to changes reported in chronic smokers, supporting the role of chronic inflammation in the pathogenesis of emphysema. However, mild emphysema in minimal numbers of mice suggests that MS exposure concentration and/or duration in the current study were not sufficient to induce a definitive emphysema phenotype.     

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.     

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.     

Comparison of the inhalation toxicity of kretek (clove cigarette) smoke with that of American cigarette smoke. II. Fourteen days,exposure

The comparative repeat dose toxicity of American cigarettes and kreteks (Indonesian cigarettes containing approximately 60% tobacco and 40% shredded clove buds) was assessed by exposure of groups of five male and five female rats to equivalent (approximately 2 mg/l in terms of total particulate matter) concentrations of smoke from each type of cigarette over 15 consecutive days. The smoke was delivered nose only using an HRC Rodent Smoking Machine (Mark IV). For each type of cigarette, three doses were used. These were achieved by regulating the daily total duration of exposure to smoke. The different doses used were 2x, 4x and 6x, 15-min exposures, presented daily over a period of approximately 6 h. Intergroup comparisons were made between American and kretek groups which received the same daily durations of smoke exposure. Higher doses of smoke resulted in reduced bodyweight gains and food consumption in male groups; the response of female groups was not as clear. At the highest dose, male rats exposed to kretek smoke gained significantly more weight by comparison with males exposed to American smoke. Higher doses of smoke tended to increase water consumption in both sexes of groups exposed to American smoke; kretek smoke produced no obvious effect. Smoke exposures produced the expected responses in certain haematological and blood biochemical parameters attributed to exposure to CO and the irritants present in cigarette smoke. Such responses were, however, confined largely to the groups exposed to American smoke. Macroscopic pathological findings attributed to smoke inhalation were confined to the lungs, and consisted of minimal to moderate discolouration and incomplete collapse of the lung. The latter finding occurred in 60% of males and 60% of females in the American high dose group and 20% of females in the kretek intermediate dose group. The weights of several organs appeared to have been affected by smoke exposures. Of these, only the increase in lung weight associated with exposure to American smoke was considered remarkable. In females exposed to American smoke, the group mean lung weights were significantly higher than the corresponding kretek group lung weights. In males, the difference was significant at the intermediate dose level only. The histopathological lesions seen in the lungs were typical of the lesions encountered in inhalation studies with tobacco smoke and included increased numbers, vacuolation and brown pigmentation of macrophages, acute alveolitis, bronchiolar epithelial hyperplasia and cuboidal ciliated cell metaplasia of alveolar duct; the two latter findings were present in some American high dose rats only. Occasional foci of alveolar haemorrhage were seen in odd rats but the incidence was not clearly dose related for either type of cigarette; the highest incidence, 30%, was seen in the high dose American group. At all dose levels, the lesions encountered were more severe with American smoke. No unique lesions were detected with kretek smoke.     

Effect of cigarette smoking on hepatic biotransformations in rats

Adult male rats were nose-only exposed to cigarette smoke for 20 min each day for 6 months. Smoke inhalation was confirmed by urinary excretion of nicotine (4.5 μg/rat/day) and elevated blood carboxyhemoglobin (13.3%). Phenobarbital- and 3-methylcholanthrene-treated rats were included as positive controls for assessing hepatic enzyme induction. Cigarette smoke did not induce statistically significant alterations in hepatic enzyme activity when measured in vivo (pentobarbital sleep time and zoxazolamine paralysis time) or in vitro (oxidation, hydrolysis, glucuronidation, and glutathione conjugation). In some cases, the smoke-exposed rats did exhibit higher microsomal enzyme activity than did the controls, but this increase was also evident in the sham-control group. Therefore, these increases were attributed to stress and not to smoking per se. Additional evidence of stress associated with manipulation of the animals was the smaller percentage weight gains of the smoke-exposed and sham control rats over the 6-month period as compared to the controls (12, 35, and 50%, respectively). Smoking reduced hepatic glutathione by as much as 15%, but the glutathione transferase activity was not affected. These studies showed that chronic exposure of rats to cigarette smoke did not alter hepatic biotransformation processes, but do suggest that smokers may be less efficient than nonsmokers in the deactivation of xenobiotics by glutathione conjugation mechanisms.     

Inhalation bioassay of cigarette smoke in rats

Groups of 80 female rats were exposed to cigarette smoke from three types (code 13 = high tar, low nicotine; code 27 = low tar, medium nicotine; code 32 = high tar, high nicotine) of cigarettes in Maddox-ORNL smoking machines, eight cigarettes per day, 7 days per week, for up to 24 months. An additional group received sham exposures and a fifth group served as untreated controls. The sham-exposed animals had significantly lower body weights than the untreated controls. The smoke-exposed animals had significantly lower weights than the sham-exposed controls; the weights were lower for the code 27 and code 32 animals than for the code 13 animals during the second year of exposure. The survival of the code 13 animals was similar to that for the sham-exposed and untreated control group; survival times of the code 27 and code 32 animals were shorter. Body weight and survival reflected the high- and low-nicotine dose groups indicated by in vivo dosimetry measurements. Smoke-induced histopathologic lesions consisted primarily of pulmonary smoke granulomas; the smoke granulomas were less severe in the code 27 exposure group than in the groups exposed to smoke from code 13 or code 32 cigarettes. Additional changes included pulmonary alveolar epithelial hyperplasia, and squamous metaplasia and basal cell hyperplasia of laryngeal and tracheal epithelium. One primary epidermoid carcinoma was found in the lung of a code 27 rat. The rats tolerated the chronic exposures relatively well and certain of the smoke-induced lesions allowed differentiation between the different types of cigarettes.     

Fourteen-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette: I. Inhalation Toxicology and Histopathology

Sprague-Dawley rats were exposed 6 hr per day for 14 consecutivedays to aged and diluted sidestream smoke (ADSS), used as asurrogate for Environmental Tobacco Smoke (ETS), at concentrationsof 0.1 (typical), 1 (extreme), or 10 (exaggerated) mg of particulatesper cubic meter. Animals were exposed nose-only, inside whole-bodychambers, to ADSS from the 1R4F reference cigarette. End-pointsincluded histopathology, CO-ox-imetry plasma nicotine and cotinine,clinical pathology, and organ and body weights. The only pathologicalresponse observed was slight to mild epithelial hyperplasiaand inflammation in the most rostral part of the nasal cavity,in the high-exposure group only. No effects were noted at mediumor low exposures. The minimal changes noted were reversible,using a subgroup of animals kept without further treatment foran additional 14 days. Overall, the end-points used in the studydemonstrated that there was no detectable biological activityof ADSS at typical or even 10-fold ETS concentrations and thatthe activity was only minimal at very exaggerated concentrations(particle concentrations 100 times higher than typical real-worldconcentrations).     

Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke

Electronic cigarettes (E-cigarettes) are a potential means of addressing the harm to public health caused by tobacco smoking by offering smokers a less harmful means of receiving nicotine. As e-cigarettes are a relatively new phenomenon, there are limited scientific data on the longer-term health effects of their use. This study describes a robust in vitro method for assessing the cytotoxic response of e-cigarette aerosols that can be effectively compared with conventional cigarette smoke. This was measured using the regulatory accepted Neutral Red Uptake assay modified for air–liquid interface (ALI) exposures. An exposure system, comprising a smoking machine, traditionally used for in vitro tobacco smoke exposure assessments, was adapted for use with e-cigarettes to expose human lung epithelial cells at the ALI. Dosimetric analysis methods using real-time quartz crystal microbalances for mass, and post-exposure chemical analysis for nicotine, were employed to detect/distinguish aerosol dilutions from a reference Kentucky 3R4F cigarette and two commercially available e-cigarettes (Vype eStick and ePen). ePen aerosol induced 97%, 94% and 70% less cytotoxicity than 3R4F cigarette smoke based on matched EC₅₀ values at different dilutions (1:5 vs. 1:153 vol:vol), mass (52.1 vs. 3.1?μg/cm²) and nicotine (0.89 vs. 0.27?μg/cm²), respectively. Test doses where cigarette smoke and e-cigarette aerosol cytotoxicity were observed are comparable with calculated daily doses in consumers. Such experiments could form the basis of a larger package of work including chemical analyses, in vitro toxicology tests and clinical studies, to help assess the safety of current and next generation nicotine and tobacco products.