Time-dependent formation of 8-oxo-deoxyguanosine in the lungs of mice exposed to cigarette smoke

Active and passive smoking are major risk factors for lung cancer. Pro-oxidants in tobacco smoke have been implicated in smoking-associated disease development due to their potential role in inducing oxidative stress. Previous studies have failed to associate increased levels of oxidative damage to DNA with the formation of the potentially mutagenic lesion, 8-oxo-2-deoxyguanosine (8-oxodG), probably due to repair of this lesion. However, no systematic studies have been performed to assess the dose- and time-dependent formation and removal of this lesion by cigarette smoke exposure. In the present study, female A/J mice were exposed to side-stream cigarette smoke in a whole body exposure chamber for 6 h a day, 5 days a week for up to 6 weeks. Age-matched controls were maintained in filtered ambient air. Lung tissues were harvested from 2, 4, and 6 weeks smoke-exposed mice after 1, 3, 6, and 20 h, following the cessation of smoking. A significant increase in the levels of 8-oxodG in lung DNA was observed in 10 day smoke-exposed mice at 1 (11.5+/-1.1/10(6) nucleotides), 3 (20.2+/-2.7/10(6) nucleotides; p=0.0008), and 6 h (17.2+/-1.0/10(6) nucleotides; p<0.005) postcessation, as compared with age-matched sham treatment (8.8+/-2.3/10(6) nucleotides) (mean+/-SD). The levels significantly declined 20 h after the cessation of smoke exposure (14.0+/-1.6/10(6) nucleotides), although they were still higher than the control. Our results strongly suggest that there is a significant increase in the 8-oxodG levels immediately after the cessation of smoking, which is repaired over time. This initial increase in 8-oxodG levels may lead to gene mutations, and accumulation of such mutations over time can eventually lead to malignant transformation of the cells.     

Palladium alters cigarette smoke toxicological profile, but accumulates in the lungs of rats during inhalation exposure

The use of a palladium (Pd) catalyst has been proposed to promote combustion of tobacco, thereby reducing concentrations of certain toxic components of smoke, including polyaromatic hydrocarbons (PAHs). In the present work, toxicological comparisons were made using experimental cigarettes containing no added Pd, against otherwise similar cigarettes containing three different amounts of Pd as potassium tetrachloropalladate added to the tobacco. A full analysis of smoke chemistry was made, along with a subchronic 90-day inhalation study with mainstream smoke (rats exposed to 150 mg/m(3) of total particulate matter, 6 h/day for 90 consecutive days) and in vitro evaluations of Salmonella mutagenicity, cytotoxicity, and in vivo clastogenicity (micronucleus). Addition of Pd to the tobacco resulted in 20-30% reductions in the concentrations of 6 PAHs and 2 aromatic amines, but it also resulted in transfer of Pd to smoke and in 10-50% increases in concentrations of several tobacco-specific nitrosamines. Mutagenicity was reduced by about 50% in 2 of 5 strains of Salmonella (with S9 only), while the cytotoxicity and micronucleus assays showed no changes. Histopathology responses were similar across the four smoke inhalation groups. Smoke Cd was reduced by 40-70% in the smoke, leading to lower lung concentrations; however, the presence of Pd in smoke led to accumulation of Pd in the lungs increasing in both a dose-and an exposure-related manner. While catalysts such as Pd addition may alter the typical chemical/toxicological profile of smoke, a concern arises regarding the "risk-benefit" of the addition of such chemically active materials as Pd to cigarette tobacco, leading to potential pulmonary accumulation with unknown consequences.     

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.     

Immunohistochemical evaluation of oxidative stress in murine lungs after cigarette smoke exposure

Cigarette smoking generates an oxidative stress in the lung, which may contribute to the pathogenesis of chronic obstructive pulmonary disease. We performed an immunohistochemical study to evaluate oxidative stress in the lung after acute cigarette smoke (CS) exposure in mice. Paraffin-embedded lung tissue sections were prepared from mice exposed and unexposed to CS for 1 h. The sections were immunostained with antibodies against 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative DNA adduct, and 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation product. The bronchiolar and alveolar epithelium of mice unexposed to CS exhibited weak signals for 8-OHdG and 4-HNE, whereas by 1 h after CS exposure the signals in the bronchiolar epithelial cells and the alveolar epithelial cells, particularly type II cells, had increased dramatically. The increases in both were associated with increased 8-OHdG levels in bronchoalveolar lavage fluid as determined by enzyme-linked immunoassay. These results suggest that acute CS exposure imposes oxidative stress predominantly on bronchiolar epithelial and alveolar type II cells, confirming that cigarette smoking causes oxidative damage to the respiratory epithelium.     

Inhalation of cigarette smoke induces regions of altered DNA methylation (RAMs) in SENCAR mouse lung

The development of early biomarkers, both of exposure and effect, would substantially improve science-based risk assessment with regard to cigarette smoke (CS)-associated toxicity. Altered DNA methylation, an epigenetic mechanism, is linked to CS-induced lung tumorigenesis. We have taken an unbiased approach (i.e. genomic regions are not pre-selected) to assess early methylation changes within lung DNA from female SENCAR mice treated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), and then exposed to air alone, or CS for 4 or 8 weeks. Regions of altered DNA methylation (RAMs) were detected in mice treated with DMBA alone, or DMBA + 0.16, 0.32 or 0.48 mg wet total particulate matter per liter (WTPM/L) CS, using methylation-sensitive restriction digestion, arbitrarily primed PCR and capillary electrophoresis. Comparison of the RAMs that formed in different treatment groups revealed: (1) RAMs which “carried forward” across time (i.e. occurred at both 4 and 8 weeks) in a particular dose group, in addition to unique RAMs observed only at 8 weeks, and (2) RAMs which “carried forward” across dose (i.e. occurred in at least 2 dose groups at a particular time point), in addition to unique RAMs observed only in 1 dose group. Furthermore, a subset of RAMs was observed, at both 4 and 8 weeks, in DMBA-treated and DMBA + CS-exposed groups; the presence of unique RAMs in the latter suggest that combined DMBA + CS treatment more than just “magnifies” a subset of cell populations bearing the methylation changes induced by DMBA alone. Importantly, only minimal histopathological changes were observed in the lungs of CS-treated mice. This study is the first to demonstrate changes in lung DNA methylation at early times following exposure to CS, e.g., prior to overt histopathology. Thus, altered methylation might serve as a biomarker of CS exposure, and, in light of the fact that methylation changes are linked to CS-induced lung tumorigenesis, might also be useful as biomarkers of effect.     

Acute inhalation of ozone induces DNA methylation of apelin in lungs of Long-Evans rats

Apelin has cardiopulmonary protective properties that promote vasodilation and maintenance of the endothelial barrier. While reductions in apelin have been identified as a contributor to various lung diseases, including pulmonary edema, its role in the effect of air pollutants has not been examined. Thus, in the current study, we sought to investigate if apelin is a downstream target of inhaled ozone and if such change in expression is related to altered DNA methylation in the lung. Male, Long-Evans rats were exposed to filtered air or 1.0?ppm ozone for 4?h. Ventilation changes were assessed using whole-body plethysmography immediately following exposure, and markers of pulmonary edema and inflammation were assessed in the bronchoaveolar lavage (BAL) fluid. The enzymatic regulators of DNA methylation were measured in the lung, along with methylation and hydroxymethylation of the apelin promoter. Data showed that ozone exposure was associated with increased enhanced pause and protein leakage in the BAL fluid. Ozone exposure reduced DNA cytosine-5-methyltransferase (DNMT) activity and Dnmt3a/b gene expression. Exposure-induced upregulation of proliferating cell nuclear antigen, indicative of DNA damage, repair, and maintenance methylation. Increased methylation and reduced hydroxymethylation were measured on the apelin promoter. These epigenetic modifications accompanied ozone-induced reduction of apelin expression and development of pulmonary edema. In conclusion, epigenetic regulation, specifically increased methylation of the apelin promoter downstream of DNA damage, may lead to reductions in protective signaling of the apelinergic system, contributing to the pulmonary edema observed following the exposure to oxidant air pollution.     

No increase in carcinogen-DNA adducts in the lungs of monkeys exposed chronically to marijuana smoke.

Rhesus monkeys exposed to marijuana smoke either 7 or 2 days/weeks (HI and LO groups, respectively), or ethanol-extracted marijuana smoke for 7 days/week (EM) or sham treatment (SH) for 1 year were sacrificed 7 months following the last exposure. Pulmonary levels of carcinogen-DNA adducts were determined. Although mean or median adduct levels were not statistically different, 15 of 22 adduct measures were highest in the EM group and lowest 12 of 22 times in the SH group. The levels of aromatic carcinogen-DNA adducts seem no higher in the lungs of animals exposed to marijuana smoke than in untreated animals. Ethanol-extracted marijuana may have effects greater than marijuana itself.     

Effects of cigarette smoke on the metabolism of vasoactive hormones in rat isolated lungs.

1. The effects of exposure of rats to cigarette smoke have been studied on the metabolism of vasoactive hormones in isolated lungs from these animals. 2. Rats were exposed for 1 h per day to cigarette smoke for 1 day or for 10 days. 3. Angiotensin I conversion was increased after 1 day's exposure but after 10 days' exposure conversion returned to normal. 4. Inactivation of prostaglandin E2 was decreased after 1 day's exposure. After 10 days' exposure there was a further decrease which could not be attributed to smoke alone. 5. The inactivation of 5-hydroxytryptamine and bradykinin remained unchanged after both short and longer exposures to smoke. 6. The metabolic activity of the lung towards some vasoactive hormones in the pulmonary circulation is affected by exposure of the animal to cigarette smoke and such changes may be relevant to the initiation of cardiovascular changes consequent upon cigarette smoking.     

Pathological alterations in Syrian golden hamster lungs after passive exposure to cigarette smoke

The effects of 2 types of research cigarettes, differing in their total smoke delivery and condensate, were examined as to their histopathological effects on Syrian golden hamster lungs. The animals were passively exposed to the total smoke of the cigarettes once a day, 5 days/week for 1 year. Experimental and control animals were killed one day after termination of exposure. Varying effects on the macrophages of pulmonary alveolar tissue were observed. Infiltration of lung tissue by “Brown cells” was a common pathological alteration. Qualitative and quantitative differences existed between the two cigarette groups with respect to the occurrence of such “Brown cell” clumps. The response of the lung tissue to smoke exposure would appear to be dependent upon the amount of mainstream total particulate matter (TPM), the amount of condensate, the time exposed and the number of cigarettes.     

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.     

Brain and lungs of rats are differently affected by cigarette smoke exposure: Antioxidant effect of an organoselenium compound

Cigarette smoke exposure has been associated with oxidative stress in several organs. Antioxidant effect of diphenyl diselenide (PhSe)₂, an organoselenium compound, on oxidative damage induced by sub-chronic cigarette smoke exposure in brain and lungs of rats was investigated. Animals were exposed 5 times/week to one, two, three and four cigarettes for exposure periods of 15 min during the first, second, third and fourth weeks. Reactive species (RS) levels, enzymatic antioxidant defenses (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities) and non-enzymatic antioxidant defenses (ascorbic acid and non-protein thiol (NPSH) levels) were examined in brain and lungs of rats. An increase in RS levels induced by cigarette smoke in both tissues of rats was demonstrated. Cigarette smoke altered enzymatic antioxidant defenses (GST, CAT and SOD activities) in both tissues, and reduced the non-enzymatic antioxidant defenses in lungs. (PhSe)₂ (0.5 mg/kg/day, 5 times/week) restored RS levels and antioxidant defenses in brain of rats exposed to cigarette smoke. (PhSe)₂ treatment increased NPSH levels, GST and GR activities per se in lungs of rats. In conclusion, sub-chronic exposure to cigarette smoke caused alterations in antioxidant defense system and a tissue-specific oxidative stress in brain and lungs of rats. (PhSe)₂ restored antioxidant defenses in lungs and brain of rats.     

Testing of cigarette smoke inhalation for teratogenicity in rats.

The effect of cigarette smoke inhalation on the developing rat embryo was studied. Female rats were exposed to smoke prior to and after mating at miximal tolerated doses. Male rats were ezposed to smoke to prior to mating to investigate the influence upon sperm. On the 21st day of gestation pregnant females of all groups were killed and implantations, litter sizes, foetal resorptions and abortion, foetal weight and length recorded: no significant differences were observed for these parameters between smoke-exposed and control animals. Total maternal wight gain during gestation was lower for smoke-exposed animals than for non-smoke-exposed animals. Examination of foetuses stained according to the method of Frohberg and Oettle resulted in normal variation in the skeletal development in smoke-exposed and control groups as well as in placebo groups. No skeletal malformations could be found.     

The effect of cigarette smoke on the plasma piroxicam concentrations in rats

The plasma concentration of unchanged piroxicam has been determined at 15, 30, 60 and 90 min after 10 mg kg-1 oral administration of the drug to rats exposed to cigarette smoke or pretreated with phenobarbitone, 3,4-benzpyrene or ethanol. Plasma piroxicam concentrations decreased in rats pretreated with phenobarbitone, 3,4-benzpyrene and ethanol and in rats 24 h after exposure to cigarette smoke.     

Tobacco smoke exposure induces nicotine dependence in rats

Rationale

Tobacco smoke contains nicotine and many other compounds that act in concert on the brain reward system. Therefore, animal models are needed that allow the investigation of chronic exposure to the full spectrum of tobacco smoke constituents.

Objectives

The aim of these studies was to investigate if exposure to tobacco smoke leads to nicotine dependence in rats.

Methods

The intracranial self-stimulation procedure was used to assess the negative affective aspects of nicotine withdrawal. Somatic signs were recorded from a checklist of nicotine abstinence signs. Nicotine self-administration sessions were conducted to investigate if tobacco smoke exposure affects the motivation to self-administer nicotine. Nicotinic receptor autoradiography was used to investigate if exposure to tobacco smoke affects central α7 nicotinic acetylcholine receptor (nAChR) and non-α7 nAChR levels (primarily α4β2 nAChRs).

Results

The nAChR antagonist mecamylamine dose-dependently elevated the brain reward thresholds of the rats exposed to tobacco smoke and did not affect the brain reward thresholds of the untreated control rats. Furthermore, mecamylamine induced more somatic withdrawal signs in the smoke-exposed rats than in the control rats. Nicotine self-administration was decreased 1 day after the last tobacco smoke exposure sessions and was returned to control levels 5 days later. Tobacco smoke exposure increased the α7 nAChR density in the CA2/3 area and the stratum oriens and increased the non-α7 nAChR density in the dentate gyrus.

Conclusion

Tobacco smoke exposure leads to nicotine dependence as indicated by precipitated affective and somatic withdrawal signs and induces an upregulation of nAChRs in the hippocampus.     

Tail-tremor induced by exposure to cigarette smoke in rats.

Tremors appearing only in the tail (tail-tremor) induced by cigarette smoke and subcutaneous nicotine were investigated using a smoking machine and Wistar rats. Daily exposure (twice a day) to smokes of two commercial cigarettes (Mild-Seven Select for the first 7 days and Long-Peace for the next 6 days) caused the tail-tremor to appear even if it was slight. A single subcutaneous nicotine (0.5 mg/kg) administration to rats exposed to the cigarette smokes for 13 days markedly caused the tail-tremor. On the other hand, daily subcutaneous injection of nicotine (0.5 mg/kg/day) also caused the tail-tremor to appear beginning on the 4th day and the incidence of tremor increased to 100% by the 12th day. These results indicate that tail-tremor can be caused not only by daily subcutaneous administration of nicotine but also by daily exposure to cigarette smoke.     

Effects of standard cigarette smoke and nicotine-reduced cigarette smoke on plasma concentrations of indomethacin administered orally to rats.

The influence of acute exposures to standard (ST) and nicotine-reduced (NR) cigarette smokes on the plasma concentration of orally administered indomethacin (IM, 5 mg/kg) was investigated in rats. IM plasma concentrations in the ST- and NR-groups were lower than those in the non-smoking control group, while the lowered effect in the NR-group was slightly weaker than in the ST-group. These results suggest that the plasma concentrations of IM administered orally are lowered by the acute exposure of cigarette smoke, and this influence may be attributed largely to constituents other than nicotine in the cigarette smoke as well as slightly attributable to nicotine.     

Effects of standard cigarette and nicotine-less cigarette smoke inhalings on nicorandil plasma levels in rats

The influence of cigarette smoke on nicorandil plasma levels at a dose of 10 mg/kg administered orally was investigated in rats. The animals were exposed to standard and nicotine-less cigarette smoke for 8 min using a 'smoking machine'. In nonsmoking control rats, nicorandil plasma levels increased rapidly and reached the maximum (approx. 7.6 micrograms/ml) after 1 h and then decreased gradually. On the other hand, nicorandil plasma levels in the rats inhaling standard cigarette and nicotine-less cigarette smoke reached the maximum (approx. 4.7 and 4.9 micrograms/ml, respectively) after 1-2 h. These results suggest that nicorandil plasma levels after oral administration are influenced not only by standard cigarette smoke but also by nicotine-less cigarette smoke.     

New syntheses of DNA adducts from methylated anilines present in tobacco smoke

A new synthetic pathway for the formation of deoxyguanosine-monoarylamine adducts is described, involving the generation and use of arylnitrenes as electrophilic synthons. Photolysis of aryl azides, the most common method for generating arylnitrenes, was tested in the presence of 2'-deoxyguanosine. N-(2'-Deoxyguanosin-8-yl)monoarylamine (dG-C8-Ar) adducts were obtained, but the yields were typically low. Deoxygenation of nitro- and nitrosoarenes by triethyl phosphite in the presence of 2'-deoxyguanosine proved to be an effective method, by which dG-C8-Ar, (2'-deoxyguanosin-N1-yl)monoarylamine (dG-N1-Ar), and (2'-deoxyguanosin-O(6)-yl)monoarylamine (dG-O(6)-Ar) adducts were obtained in acceptable yields.     

Pharmacodynamic effects of chronic cigarette smoke exposure in spontaneously hypertensive rats

In investigating the influence of chronic cigarette smoke exposure on hypertension, we compared the pharmacodynamic effects of enforced exposure to smoke on spontaneously hypertensive rats (SHR) with those on Wistar-Kyoto (WKY) rats. Chronic cigarette smoke exposure for 8 weeks decreased the elevated heart rate of mature male SHR to approximately the rate in WKY rats 24 h after smoke exposure. Both systolic and diastolic blood pressures also decreased slightly. However, WKY rats showed a marked rise in heart rate soon after exposure to cigarette smoke began, with no change in blood pressure, while the heart rate of SHR in the early stage remained similar to that of animals without exposure, although their blood pressure was clearly reduced. The body weight of both strains tended to decrease during smoke exposure, but the effect was more severe in SHR. Moreover, the effects of chronic smoke exposure were observed using retired, aged female SHR breeders. A decrease in body weight and heart rate, but not in blood pressure, was also recognized even in these mature animals. These effects gradually recovered after withdrawal from exposure. On the basis of these results, a profile of chronic cigarette smoke exposure under hypertension is discussed in this study.