Department of Health Sciences, Section of Hygiene and Preventive Medicine, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy and
1 National Cancer Institute, Rockville, MD 20892, USA
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Abstract |
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Abbreviations: 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; AHH, arylhydrocarbon hydroxylase; BAL, bronchoalveolar lavage; DRZ, diagonal radioactive zone; ETS, environmental tobacco smoke; GSH, glutathione; GST, glutathione S-transferase; PAM, pulmonary alveolar macrophages; SFS, synchronous fluorescence spectrophotometry; TPM, total particulate matter.
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Introduction |
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Animal models have provided a poor contribution to the assessment of health risks posed by tobacco smoke as a complex mixture. Indeed, it is very difficult to reproduce the occurrence of tumors in rodents exposed under controlled conditions to either mainstream smoke, sidestream smoke or ETS (3,4). The recent discovery of intermediate biomarkers provides a valuable tool for evaluating the actual exposure to carcinogens and/or the potential risks resulting thereof. In particular, DNA adducts and other nucleotide modifications detectable by 32P-post-labelling are indicators of biologically effective dose. Although DNA adduct formation does not necessarily imply an evolution towards the final pathological event, it certainly predicts the risk of developing a disease more reliably than the external exposure dose alone. These molecular lesions have been associated with cancer (5) as well as with other chronic degenerative diseases, such as cardiovascular diseases and chronic obstructive pulmonary diseases, which are the leading causes of death in the population (5,6).
We report here the results of an experimental study using rats exposed, for varying periods of time, to a mixture of sidestream smoke and mainstream smoke, thus mimicking exposure to ETS. The selective localization and the differential persistence of DNA adducts, observed in different organs, tissues and cells, suggests a potential role for ETS in mutation-related diseases.
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Materials and methods |
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Cigarettes
Kentucky 2R1 reference cigarettes, having a declared content of 44.6 mg total particulate matter (TPM) and 2.45 mg nicotine each, with a 23 mm butt remaining after smoking, were purchased from the Tobacco Research Institute (University of Kentucky, Lexington, KY). The cigarettes were kept for 48 h before use in a standardized atmosphere humidified with a mixture of 70% glycerol and 30% water.
Smoke exposure system
A whole-body exposure of rats to cigarette smoke was achieved by using a smoking machine (model TE-10; Teague Enterprises, Davis, CA). This machine (7) is designed to produce sidestream smoke or mainstream smoke, or a combination of the two, by burning either 2, 5 or 10 cigarettes at one time. We used a mixture of sidestream (89%) and mainstream smoke (11%), mimicking an exposure to ETS. Based on preliminary experiments, we decided to burn five 2R1 cigarettes at one time, 6 h per day divided in two 3 h rounds with a 3 h interval, 5 days per week. This accounted for a whole-body exposure to the smoke generated by 600 cigarettes per week. Under these conditions, the TPM in the exposure chambers was in the range of 7393 mg/m3. The average CO concentration in the exposure chamber, as assessed with a Dräger gas detector (Drägerwerk AG, Lübeck, Germany), was 350 p.p.m.
Treatments
The rats were divided into seven groups, each composed of five animals. One group (sham-exposed rats) was kept in a filtered air environment. Five groups were exposed to ETS for either 1, 2, 3, 4 or 5 weeks, and the rats were killed 16 h after the last treatment. An additional group was exposed for 4 weeks, and the rats were killed 1 week after the last treatment.
The rats were anaesthetized with ethyl ether and killed by cervical dislocation. Bronchoalveolar lavage (BAL) was performed by lavaging the left lung of each rat with five 10 ml aliquots of cold (4°C) 0.15 M NaCl infused via a cannula inserted into the trachea, while the right lung was excluded by ligating the main bronchus. BAL cells were washed twice and stored at 80°C. The right lung and the whole trachea, heart, liver, bladder and testes were collected from each animal. The lung was divided into two equal parts, the upper part to be used for biochemical analyses, and the lower part for DNA adduct determinations. The ciliated epithelium of the trachea was accurately scraped. All cells, tissues and organs were stored at 80°C until use.
Purification of DNA and detection of DNA adducts
Heart, liver, bladder, testes and a portion of right lung were thawed and homogenized in a Potter-Elvehjem apparatus at 4°C in 250 mM sucrose, 5 mM dithiothreitol, 50 mM TrisHCl (pH 7.6). DNA was isolated by solvent extraction using an automatic extractor (Genepure 341; Applied Biosystems, Foster City, CA) according to the method of Gupta (8), with some modifications as described previously (9). Due to the low amount of material available for each sample, DNA extraction from the tracheal epithelium and BAL cells was obtained by using the same procedure, except that 30 µl of Quik-Precip (Edge-Biosystems, Gaithersburg, MD) were added during precipitation with alcohol. DNA was eluted with water. Purity of DNA was checked by spectrophotometric analysis (9). Aliquots of 5 µg DNA were assayed for the presence of DNA adducts by butanol extraction, as described previously (10). Each sample was labelled with 60 µCi of carrier-free [-32]ATP (ICN Biochemicals, Irvine, CA), having a specific activity
7000 Ci/mmol. Thin-layer chromatography was carried out on sheets of polyethyleneimine (Macherey-Nägel, Düren, Germany) according to standard procedures (10). As specified in the Results, two different solvents in D3 were compared in preliminary experiments, one using 7 M urea and 3 M lithium formate (system A), and the other one using isopropanol:4 M ammonium hydroxide (1:1, v:v) (system B). Autoradiography was performed by using a 32P InstantImager Electronic Autoradiographic System equipped with InstantQuant software (model A2024; Packard, Meriden, CT). The relative adduct levels were calculated (11), and DNA adduct levels in each sample were expressed as DNA adducts/108 nucleotides. A 7R,8S,9S-trihydroxy-10R-(N2-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diolepoxide-N2-dGp) reference standard (National Cancer Institute Chemical Carcinogen Reference Standard Repository, Midwest Research Institute, Kansas City, MO) was used as a positive quality control in order to check the labelling efficiency in each experiment. The presence of ATP excess after the labelling step was evaluated by thin-layer chromatography in 0.12 M sodium phosphate, pH 6.8. Each sample was tested either in duplicate (BAL cells and tracheal epithelium) or triplicate (lung, heart, bladder, liver and testes).
Lung samples were also assayed for the presence of DNA adducts by synchronous fluorescence spectrophotometry (SFS). For this purpose, 50 µg aliquots of DNA were hydrolyzed in 0.1 M HCl at 90°C for 4 h in sealed glass vials. Synchronous scanning was performed with a fixed of 34 nm between excitation and emission, using a Hitachi F-3000 fluorescence spectrophotometer. Peaks in the 345351 nm excitation range were recorded (10). The intensity of peaks was expressed in arbitrary fluorescence units, indicating the difference between the intensity of the signal yielded by the peak and the baseline, which was further subtracted by a fixed value of 10. This was assumed as an arbitrary threshold of sensitivity of the method to distinguish specific fluorescence signals from the background noise.
Oxidative DNA damage
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) was measured in lung samples from sham-exposed rats and rats exposed to ETS for 4 weeks. A 32P-post-labelling procedure was used (12,13). In order to avoid artifacts which may result from radiation-induced oxidation of guanine, a selective hydrolysis of purines was achieved by incubating depolymerized DNA with 90% trifluoroacetic acid at room temperature for 10 min before the 32P-labelling reaction (14,15).
Biochemical analyses
The upper part of the right lung from each rat was thawed, washed in 10 mM Tris plus 0.15 M KCl, pH 7.4, finely minced and homogenized in 50 mM Tris plus 0.25 M sucrose, pH 7.4 (3 ml/g wet tissue) using a Potter-Elvehjem apparatus with teflon pestle. The homogenate was centrifuged at 12 000 g for a further 20 min. The pellet was discarded, and aliquots of the second supernatant were harvested at 80°C as post-mitochondrial (S12) fractions. The remaining supernatant was gauze filtered and further centrifuged at 105 000 g for 1 h. The resulting supernatant was harvested at 80°C in small aliquots as cytosolic (S105) fraction. The pellet was washed once and resuspended in 50 mM Tris plus 0.1 mM EDTA, pH 7.4, supplemented with 20% glycerol (0.5 ml/g of original tissue), and harvested at 80°C in small aliquots as microsomal fractions. All steps for preparing subcellular fractions were carried out at 4°C, using sterile materials and operating under aseptic conditions. In order to prevent sulfhydryl group oxidation, the S12 fractions used for glutathione (GSH) analyses were prepared separately by supplementing the cell homogenate with 20 mM EDTA. The samples were pooled within each experimental group and tested in triplicate.
The protein content of S12, S105 and microsomal fractions was determined by the colorimetric method of Lowry et al. (16), using bovine serum albumin as a standard. Aryl hydrocarbon hydroxylase (AHH) activity was measured in lung microsomes by means of a spectrofluorimetric method (17), evaluating the conversion of benzo[a]pyrene into fluorescent metabolites. The results were expressed as pmol 3-OH-benzo[a]pyrene/min/mg protein. Glutathione S-transferase (GST) was assayed in the cytosolic fractions by the spectrophotometric method of Habig et al. (18), using 1-chloro-2,4-dinitrobenzene as a substrate and expressing the results as mU (nmol/min)/mg protein. GSH was measured in cytosolic fractions according to the spectrofluorimetric method of Hissin and Hilf (19). The results were expressed as µmol GSH/g wet lung.
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Results |
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Formation of 32P-post-labelled DNA adducts in organs, tissues and cells of smoke-exposed rats
Figure 1 shows the time course formation of DNA adducts, and provides typical examples of autoradiographic patterns observed with lung, heart, bladder, tracheal epithelium and BAL cells from ETS-exposed rats. The autoradiographic patterns were consistent with the occurrence of massive DRZs, plus up to four major and two minor spots in the tracheal epithelium, up to three major spots in BAL cells and up to two spots, one major and one minor, in either lung, heart or bladder.
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No significant increase of DNA adduct levels was found in the liver, as assessed by comparing the samples obtained from the five sham-exposed rats (2.4 ± 1.1 adducts/108 nucleotides) and the five rats exposed whole-body to ETS for 4 weeks (2.6 ± 1.5 adducts/108 nucleotides). In the testis, the increase of DNA adducts after 4 weeks was moderate (3.9 ± 2.1 versus 2.1 ± 0.7 adducts/108 nucleotides) but statistically significant (P < 0.05 by Student's t-test, and P = 0.05 by non-parametric MannWhitney U test).
Accordingly, the ratio of top levels of DNA adducts in smoke-exposed rats to the background levels in sham-exposed rats was 11.2 in the tracheal epithelium, 10.4 in BAL cells, 7.3 in the heart, 6.3 in the lung, 5.1 in the bladder, 1.9 in the testis and 1.1 in the liver.
Detection of adducts to lung DNA by synchronous fluorescence spectrophotometry
SFS was additionally used to detect DNA adducts in the lung. No fluorescence signal was detected in DNA samples from either sham-exposed rats or rats exposed to ETS for 2 or 3 weeks. Three out of five rats exposed for 4 weeks had SFS-positive adducts, with a mean (± SD) level of 2.8 ± 2.8 fluorescence units. All rats exposed for 5 weeks were positive, with levels of 8.1 ± 2.7 fluorescence units.
Persistence of DNA adducts after discontinuation of exposure to cigarette smoke
One group of rats was treated for 4 weeks, and thereafter exposure to ETS was discontinued for an additional week before killing. As shown in Figure 1, the decrease of 32P-post-labelled DNA adduct levels was negligible in the heart, whereas in BAL cells the decrease was biologically appreciable but did not reach the statistical significance threshold. As compared with DNA adduct levels measured after either 4 or 5 weeks of continual exposure to smoke, the drop was statistically significant in the tracheal epithelium and bladder (P < 0.05 in both cases, as assessed by Student's t-test for unpaired data) and in the lung (P < 0.01). After discontinuing exposure, DNA adducts in the lung were no longer detectable by SFS, being below the threshold of sensitivity of this technique (data not shown).
In any case, even after discontinuing exposure to ETS for one week, the levels of DNA adducts were still appreciably and significantly higher than in sham-eposed rats, i.e. 6.2-fold in the heart (P < 0.001), 6.1-fold in BAL cells (P < 0.001), 5.3-fold in the tracheal epithelium (P < 0.01), 2.2-fold in the bladder (P < 0.01) and 2.0-fold in the lung parenchyma (P < 0.01).
Smoke-related oxidative damage in lung DNA
8-OH-dG was measured in duplicate, using a 32P-post-labelling procedure, in lung DNA from the five sham-exposed rats and the five rats exposed to ETS for 4 weeks. The means (± SD) were 1.1 ± 0.2 (range: 0.91.5) and 3.0 ± 1.8 (range: 1.86.2) 8-OH-dG/105 nucleotides, respectively. This difference was statistically significant when analysed by Student's t-test (P = 0.05) and even more evidently, due to the variability observed in the treated group, when analysed by non-parametric MannWhitney U test (P < 0.01).
Biochemical parameters
Three biochemical parameters were evaluated in lung preparations (Figure 2). Exposure to ETS resulted in a marked and significant induction of AHH activity in lung microsomal fractions, which progressively increased for 4 weeks, and thereafter reached a plateau. In particular, the increase over sham-exposed rats was 1.7-fold after 2 weeks, 2.1-fold after 3 weeks, 3.5-fold after 4 weeks and 3.4-fold after 5 weeks. The rapid reversibility of AHH induction was demonstrated by the finding that, 1 week after discontinuation of exposure to cigarette smoke, AHH activity dropped down to the same levels as in sham-exposed rats. The concentrations of reduced GSH, measured in lung post-mitochondrial (S12) fractions, tended to undergo a moderate but progressive and consistent decrease in smoke-exposed rats, which became statistically significant after 4 weeks of exposure. GSH concentrations were 71.4% of controls after 4 weeks of exposure, a figure which did not change (73%) after discontinuing exposure for 1 week. After 5 weeks of exposure, GSH concentrations were 66.7% of controls. GST activity was measured in lung cytosolic fractions using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. As shown in Figure 2
, there was a slight but progressive and consistent increase in GST, which became statistically significant after 5 weeks, when GST activity was 1.3 times higher than in controls.
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Discussion |
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High levels of DNA adducts were detected after whole-body exposure of rats to a mixture of sidestream smoke and mainstream smoke. The selective localization of these molecular lesions in different organs, tissues and cells has been postulated to depend on several factors, including: (i) toxicokinetics, and in particular the so-called first-pass effect; (ii) local metabolism and metabolism in distant organs; (iii) efficiency of DNA repair removing DNA adducts; and (iv) cell proliferation rate, influencing the replacement of cells carrying these promutagenic lesions with unaffected cells (5,27). The last two mechanisms also affect the persistence of DNA adducts, which was investigated in the present study by discontinuing exposure to ETS for 1 week.
The respiratory tract is the main target for ETS carcinogenicity in humans (3). A variety of cell types are localized in airways. The cellularity of BAL mainly consists of pulmonary alveolar macrophages (PAM), which can also be obtained in humans by means of a semi-invasive technique. The results obtained in ETS-exposed rats highlight the convenience of measuring DNA adducts in these cells as a biomarker of molecular dose related to pulmonary carcinogenesis. Indeed, smoke-related DNA adduct levels were particularly high in BAL cells, and tended to decrease after discontinuation of exposure to smoke, although the relative decrease was not as sharp as in the tracheal epithelium or in the lung mixed cell population. PAM are extremely long-lived cells, and their turnover is warranted by the intense removal from the alveolar spaces via the mucociliatory escalator, accounting for a daily removal of 0.75x106 PAM in rats (28) and 24120x106 PAM in humans (29). It is noteworthy that PAM not only have a very intense sweeping activity, but they are also equipped with the inducible metabolic machinery capable of activating procarcinogens (30) and detoxifying direct-acting carcinogens (31,32).
DNA adduct levels were also particularly high in the tracheal epithelium of ETS-exposed rats, which confirms our results obtained in a previous study carried out in SpragueDawley rats exposed whole-body to mainstream cigarette smoke (33). DNA adduct levels were much higher than in other studies evaluating the formation of DNA adducts in the whole trachea of smoke-exposed rats (25,26) rather than in the dissected tracheal epithelium. The rapid removal of DNA adducts after discontinuing exposure to ETS is consistent with the intense turnover of this epithelium. Interestingly, the histological structure of the rat trachea resembles that of the human bronchus, which is the major site of smoking-related cancer in humans (34), and the tracheal epithelial cells of rats exposed to cigarette smoke undergo preneoplastic changes (35).
32P-post-labelled DNA adduct levels were remarkably high in the lungs of ETS-exposed rats, but not as much as in the dissected tracheal epithelium or in isolated BAL cells. The time course formation and the persistence of these molecular lesions depend on the mixed nature of the cell populations represented in the lung parenchyma. After discontinuing exposure for an additional week, adduct levels decreased rapidly in the respiratory tract but, in any case, at that time they still were significantly higher than in sham-exposed rats. The observed drop of DNA adducts is consistent with the findings of a previous study in smoke-exposed rats, in which DNA adduct levels in the lung declined to near control levels in just 3 weeks (36). Certain studies on the kinetics of disappearance of DNA adducts suggest a diphasic course (37). Based on these patterns, and keeping in mind the occurrence of DRZ and the high levels of DNA adducts detected in our study, it is likely that our evaluation of DNA adduct persistence covers the period of fast removal. In humans, removal of smoke-related DNA adducts seems to be slower (3840).
Cigarette smoke is a rich source of oxidants (41), and nucleotide modifications produced by reactive oxygen species can be measured with high sensitivity using 32P-post-labelling procedures (1215). Our results support the contribution of this oxidative mechanism to DNA damage, since 8-OH-dG was significantly increased in lung DNA after 4 weeks of exposure to ETS. Lung samples were also assayed by SFS, which optimally detects the hydrolysis product of benzo[a] pyrene diolepoxide, i.e. benzo[a]pyrene tetrol, and, with lower efficiency, the derivatives of other polycyclic aromatic hydrocarbons (9). The results of these analyses indicated that exposure of rats to ETS induces the formation of SFS-positive DNA adducts in the lung. Due to the lower sensitivity (~100 times) as compared with 32P-post-labelling, appearance of a fluorescence signal in lung DNA, detectable by SFS, required 45 weeks of exposure. Again, this signal was rapidly lost after withdrawal of exposure to ETS.
Parallel biochemical analyses showed that exposure to ETS influences the pulmonary metabolism of xenobiotics. The most evident effect was a time-related induction of CYP1A1-mediated AHH activity in lung microsomes. This finding is consistent with a broad literature regarding the inducibility of pulmonary AHH by cigarette smoking in rodents (3,42,43) and humans (44). An interesting finding was the rapid reversibility of AHH inducibility following discontinuation of exposure to ETS. Another effect observed in ETS-exposed rats was a moderate but progressive and significant depletion of pulmonary GSH, which was accompanied, probably as a compensation mechanism, by an increase in cytosolic GST activity. The observed depletion of GSH is of some interest, since previous experimental studies in smoke-exposed rodents yielded conflicting findings. The hypothesis was raised that an early depletion of GSH due to reaction with cigarette smoke components may be followed by an increased synthesis of this tripeptide (45). These patterns are not substantiated by our data, which showed, under our experimental conditions, a time-related decrease of GSH, which was not reversed even after discontinuation of exposure to ETS.
Since DNA adducts are promutagenic lesions, their occurrence in the lung is likely to play a role in lung carcinogenesis. The poor persistence of smoke-related adducts in the lung suggests that a continuative exposure is needed for fixation of DNA damage. Together with the known reversibility of smoke-related promoting stimuli, the poor persistence of molecular lesions observed in our animal model is consistent with the beneficial effect of smoking cessation on lung cancer risk (3). Interestingly, lung tumors were induced in A/J mice exposed to ETS in the same type of smoking machine available to our laboratory, and under similar experimental conditions (4). It is reasonable to postulate that formation of DNA adducts and fixation of DNA damage may be associated with a neoplastic evolution when these alterations occur in proliferating lung cells. For instance, lung tumors in strain A/J mice are thought to originate from the non-ciliated bronchiolar Clara cells or from type II alveolar cells (4). A tentative hypothesis has been forwarded that the formation of DNA adducts in non-proliferating lung cells, such as type I alveolar cells, may play some role in the pathogenesis of other chronic degenerative pulmonary diseases (5). Emphysema has been demonstrated to occur in SpragueDawley rats exposed whole-body to mainstream cigarette smoke (46). Certainly, this hypothesis is not alternative but complementary to other well-known theories which do not imply a genotoxic mechanism in the pathogenesis of emphysema (47).
Cigarette smoking is an important cause of bladder cancer in humans, with an attributable proportion of the order of 50% in men and 25% in women (3). The main responsibility for the bladder carcinogenicity of cigarette smoke has been ascribed to aromatic amines (48). These compounds form DNA adducts which, as discussed previously, can be enriched by butanol extraction. In fact, the time-related increase of DNA adduct levels in rat bladder was well evident, although these molecular lesions tended to be removed quickly. It is likely that DNA adduct levels would have been even higher by examining the isolated bladder epithelium rather than the whole organ. However, in preliminary experiments we realized that it was not possible to scrape sufficient amounts of epithelium for DNA adduct analysis.
In a previous study, using SpragueDawley rats exposed for 40 consecutive days to massive doses of mainstream cigarette smoke, we found a remarkable formation of DNA adducts in testis (49). In the present study, after 4 weeks of exposure to ETS, the increase of DNA adducts was not striking although it was statistically significant. Again, a variety of cellular populations constitute this organ, and we cannot determine whether the observed molecular lesions may have affected either somatic cells and/or germ cells, with different pathological risks.
The lack of formation of DNA adducts in the liver of ETS-exposed rats is in agreement with a broad literature generated in animal models and human studies, which consistently showed an absence of ETS-related DNA adducts in the liver, contrasting with the high levels recorded in other organs (reviewed in ref. 5). In the liver, the first-pass effect for inhaled genotoxins is relatively modest. Moreover, as compared with other organs which are systemically exposed to smoke components, such as heart and bladder, an intense metabolic activation in hepatocytes is counterbalanced by quite efficient detoxification processes and DNA excision repair mechanisms (5,27). Indeed, also as a consequence of the modest cell proliferation rate under physiological conditions, the role of tobacco smoke in liver carcinogenesis is still uncertain (3).
Formation of ETS-related adducts in the heart deserves particular attention. In fact, several literature data show that the levels of DNA adducts, detected by various techniques, are at least as high in the heart as in the lung in both smoke-exposed rodents and smoking humans (5,50). The results obtained in ETS-exposed rats are in line with this general trend. They additionally show that DNA adducts in the heart are not significantly removed after exposure withdrawal. From a mechanistic point of view, an appreciable first-pass effect in the heart is provided by the coronary circulation. Metabolism of xenobiotics is poor (43) but, as shown in the present study, DNA repair is also ineffective. Adult cardiac myocytes do not replicate, which will result in an accumulation of DNA adducts with age (51). The lack of proliferation of cardiac myocytes, which are perennial and fully differentiated cells in adults, is incompatible with a neoplastic evolution. On the other hand, occurrence and accumulation of these promutagenic lesions are likely to be associated with non-proliferative degenerative heart diseases, and we proposed a possible role in the pathogenesis of smoke-related cardiomyopathies (5).
In conclusion, most epidemiological studies support the view that exposure to ETS involves a carcinogenic risk to humans. Due to the difficulties and problems encountered in this type of research, the epidemiological evidence is so far limited to lung cancer (3). Our results were generated by treating rats with very high doses of a mixture of sidestream smoke and mainstream smoke, mimicking exposure to ETS, over a relatively short period of time. This is a general drawback, which limits the extrapolation of experimental data to the human situation. Nevertheless, the observed patterns of DNA adduct distribution and persistence in different organs, tissues and cells of ETS-exposed rats suggest that this kind of exposure involves the risk of inducing molecular lesions which potentially play a role in carcinogenesis as well as in the pathogenesis of other chronic degenerative diseases.
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Acknowledgments |
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Notes |
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References |
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