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(The FASEB Journal. 1998;12:753-758.)
© 1998 FASEB

RESEARCH COMMUNICATION

DNA alterations in rat organs after chronic exposure to cigarette smoke and/or ethanol ingestion

Alberto Izzottia, Roumen M. Balanskyb, Penka M. Blagoevab, Zvetanka I. Mirchevab, Lucia Tulimieroa, Cristina Cartigliaa, and Silvio De Floraa,1

a Institute of Hygiene and Preventive Medicine, University of Genoa, Italy
b National Center of Oncology, Sofia 1756, Bulgaria


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In spite of the epidemiological evidence supporting a synergism between alcohol consumption and cigarette smoking in the pathogenesis of cancers of the aerodigestive tract, there is a paucity of experimental studies evaluating the effects of these agents under well-controlled conditions and exploring the mechanisms involved. We exposed groups of female BD6 rats, aged 8 months, to ethanol (5% in drinking water for 8 consecutive months) and/or whole-body to mainstream cigarette smoke (1 h/day, 5 days/week for 8 months). DNA was purified from different organs and analyzed for the presence of DNA–'otein crosslinks and 32P-postlabeled DNA adducts after butanol enrichment. No significant increase of DNA–protein crosslinks, compared to untreated controls, was induced by any treatment in liver, lung, or heart. `Spontaneous' nucleotidic modifications were detected by 32P-postlabeling in organs of untreated rats, with the highest levels occurring in the heart. Ingestion of ethanol did not affect DNA adduct levels in any of the organs examined: esophagus, liver, lung, and heart. Exposure to cigarette smoke induced formation of DNA adducts in the lung and heart, but not in the esophagus or liver. The combined ingestion of ethanol resulted in a significant formation of smoke-related DNA adducts in the esophagus and in their further, dramatic increase in the heart. It thus appears that ethanol consumption increases the bioavailability of DNA binding smoke components in the upper digestive tract and favors their systemic distribution. The mechanisms responsible for the interaction between ethanol and smoke and for the selective localization of DNA alterations in different organs are discussed. Formation of DNA adducts in the organs examined may be relevant in the pathogenesis of lung and esophageal cancers as well as in the pathogenesis of other types of chronic degenerative diseases, such as chronic obstructive pulmonary diseases and cardiomyopathies.—Izzotti, A., Balansky, R. M., Blagoeva, P. M., Mircheva, Z. I., Tulimiero, L., Cartiglia, C., De Flora, S. DNA alterations in rat organs after chronic exposure to cigarette smoke and/or ethanol ingestion. FASEB J. 12, 753–758 (1998)


Key Words: alcohol • synergisms • DNA adducts • lung cancer • esophageal cancer • heart diseases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TOBACCO SMOKING AND ALCOHOL CONSUMPTION constitute major risk factors that contribute to a substantial proportion of the overall cancer burden in humans (1, 2). Moreover, epidemiologic studies have shown that tobacco smoking and consumption of alcoholic beverages behave synergistically in the pathogenesis of epithelial cancers of the aerodigestive tract (1–3). In this domain, the best-documented effect is an increase in esophageal cancer, 75% of which is attributable in the U.S. to excessive alcohol intake (4).

The mechanisms responsible for the ethanol–smoke interaction have not been elucidated fully. Several questions could be addressed by using experimental test systems under well-controlled conditions, but unfortunately the literature on this subject is scanty. Indeed, in contrast to the large body of studies dealing with a number of the 3800 compounds identified in cigarette smoke, experimental data on the carcinogenic and genotoxic activity of this complex mixture are less abundant and conclusive than would be expected (5). This also reflects the problems encountered in evaluating the carcinogenicity of ethanol and cigarette smoke in animal models, which contrasts with the overwhelming evidence provided by epidemiologic studies.

We previously demonstrated that the subchronic treatment of rats with alcohol, combined with exposure to cigarette smoke, has contrasting effects on the genotoxicity of smoke in pulmonary alveolar macrophages and bone marrow polychromatic erythrocytes (6). In the present study, we evaluated the DNA alterations induced in the liver, esophagus, lung, and heart of rats chronically exposed to ethanol and cigarette smoke. Evidence is herein provided that significant levels of 32P-postlabeled DNA adducts are formed in the lung and heart of rats exposed whole-body to cigarette smoke only. The combined exposure to smoke and ethanol in drinking water caused a shift in organotropism, which resulted in the appearance of DNA adducts in the esophagus and in a further enhancement of their levels in the heart.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Female BD6 rats (Animal Laboratory, National Center of Oncology, Sofia, Bulgaria), aged 8 months and weighing 180–190 g, were used. The animals were housed in 51 x 35 x 21 cm plastic cages (three or four rats per cage) on sawdust bedding and maintained on standard rodent chow and tap water ad libitum. The animal room temperature was 25–27°C, with a relative humidity of 55% and a 12-h day–night cycle. The housing and treatment of rats were in accordance with national and institutional guidelines.

Treatments
After 10 days of acclimatization, the animals were randomly divided into four groups (seven rats each), including untreated rats and rats exposed for 8 months to either ethanol and/or cigarette smoke. Ethanol (Sigma Chemical Co., St. Louis, Mo.) was added to drinking water at a 5% concentration (v/v), accounting for an approximate intake of 1 ml ethanol (4.3 g/kg body weight) per day and of 240 ml (1038 g/kg body weight) throughout the entire study. Whole-body exposure to mainstream cigarette smoke was obtained, as described earlier (7), by using filter-tipped commercial cigarettes (Arda-Bulgartabac) that have a declared content of 31.5 mg tar and 1.6 mg nicotine. Briefly, each of the two groups of seven rats undergoing this treatment was placed in a 22.5-l sealed glass chamber that was subsequently filled by means of a 50-ml syringe with the mainstream smoke generated by one cigarette. The chamber was opened after 10 min and, after a 1–2 min interval needed to renew the air, filled again with fresh smoke for a total of six times a day, 5 days per week. Throughout the 8 months of treatment, each group was therefore exposed to the smoke generated by 1032 cigarettes. The concentration of total particulate matter in the exposure chamber was on average 533 mg/m3 air. Each rat was kept in an atmosphere containing the concentration of smoke-generated particulate matter described above for a total of 172 h.

After 8 months, all rats were anesthetized with diethyl ether and killed by cervical dislocation. Esophagus, liver, lung, and heart were removed from each animal, washed in 0.15 M NaCl, carefully inspected for the presence of gross lesions on their surface, frozen using liquid nitrogen, and stored at -80°C.

DNA extraction and 32P-postlabeling
The organs were thawed and homogenized in a Potter-Elvehjem apparatus at 4°C in 250 mM sucrose, 50 mM Tris-HCl, pH 7.6. Nuclear DNA was isolated from homogenates by solvent extraction, using an automatic DNA extractor (Genepure 341; Applied Biosystems, Foster City, Calif.) according to standard procedures (8), with some modifications as described previously (9).

Aliquots of 5 µg DNA were assayed for the presence of DNA adducts by 32P-postlabeling after enrichment with butanol (10, 11). Briefly, DNA was depolymerized by incubation with micrococcal nuclease (0.04 U/µg DNA) and spleen phosphodiesterase (1 mU/µg DNA) for 3.5 h at 37°C. Adducts were extracted by adding water-saturated butanol, tetrabutyl ammonium chloride, and ammonium formate, as described by Gupta (10). 32P-Postlabeling was performed by incubation, for 40 min at 24°C, with polynucleotide kinase (8 U) and 80 µCi of carrier-free [{gamma}-32P]ATP with a specific activity >= 7000 Ci/mmol. Thin-layer chromatography was carried out according to the standard procedure (10, 11) but using urea 7 M in D3 and D4 developments. Autoradiographs were exposed at -80°C for 18–72 h. The adducts were quantified by calculating relative adduct labeling values (10). Biochemicals were purchased from Sigma and Boehringer-Mannheim GmbH (Mannheim, Germany), polyethylenimine thin-layer chromatography sheets from Macherey-Nagel (Düren, Germany), and carrier-free [{gamma}-32P]ATP from ICN Biomedicals (Irvine, Calif.). The DNA from each tissue of each rat was tested by 32P-postlabeling at least twice and the average values of adducts/108 nucleotides were calculated for each sample. A 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 control in each labeling experiment.

DNA–'otein crosslinks
DNA–'otein crosslinks were analyzed in aliquots of lung, liver, and heart as described by Zhitkovich and Costa (12), with some modifications. Briefly, tissues were homogenized as described above. One volume of 2% sodium dodecyl sulfate (SDS) was added and the mixture was frozen at -20°C for 12 h. The samples were thawed and incubated at 65°C for 5 min, and one volume of 200 mM KCl was added by mixing carefully. The mixture was cooled on ice for 5 min and centrifuged; the upper phase containing free DNA was removed. The pellets, containing protein-bound DNA, were resuspended in K-SDS and precipitation was repeated three additional times. DNA protein complexes were digested by incubation with proteinase K (0.42 mg) for 3 h at 50°C; proteins were then removed by a final K-SDS precipitation. DNA was quantified both as free DNA and K-SDS precipitable protein-bound DNA by the addition of Hoechst 33258 and by fluorometric detection (12, 13). The results were expressed as percentage of protein-bound DNA on total DNA (free DNA plus protein-bound DNA). The ratio of K-SDS precipitable DNA in the variously treated groups as related to controls, also referred to as DNA protein crosslink coefficient (12), was calculated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The body weights of rats were not significantly affected by treatments throughout the duration of the experiment (data not shown). All organs examined were free of pathological lesions, detectable at macroscopic inspection immediately after the rats were killed.

32P-Postlabeling analyses detected the presence of radioactive spots in all organs and treatment groups. Diagonal radioactive zones were detectable only in the organs of smoke-exposed rats, irrespective of combination with ethanol. Figure 1 shows examples of autoradiographic images obtained by testing esophagus, liver, lung, and heart DNA from individual rats belonging to each one of the four treatment groups.



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  		Figure 1. Examples of autoradiographs obtained by 32P-postlabeling of DNA extracted from different organs of rats exposed whole-body to cigarette smoke and/or ingesting ethanol with drinking water for 8 months. Exposure times for development of autoradiographs were 18 h for heart samples, 24 h for liver samples, and 72 h for lung and esophagus samples.

Results of the 32P-postlabeling analyses are summarized in Table 1. As assessed by Kruskal-Wallis multivariate analysis, DNA adduct levels were significantly affected by treatments in all organs except the liver. In particular, individual comparisons by nonparametric Mann-Whitney U test showed no influence of ethanol administration in any organ, whereas a significant increase was induced by individual exposure to cigarette smoke in the lung (2.1-fold higher than in the controls) and in the heart (1.7-fold). The combined exposure to ethanol and cigarette smoke induced significant modifications in the esophagus (2.8-fold higher than in controls), lung (3.0-fold), and heart (3.7-fold). Nucleotidic modifications in the esophagus and heart of rats receiving the combined treatment were significantly higher compared not only to untreated rats, but also to rats exposed individually to either ethanol or cigarette smoke (see Table 1).


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  		Table 1. 32P-postlabeled nucleotidic modifications and DNA–'otein crosslinks in different tissues of rats exposed, either individually or in combination, to ethanol and/or cigarette smokea

DNA protein crosslinks could not be examined in the esophagus because the available tissue had already been used up for DNA adduct analyses. As shown in Table 1, there was no significant difference among treatment groups regarding the proportion of DNA bound to protein as related to total DNA in the liver, lung, and heart. When DNA–'otein crosslinking coefficients were calculated by relating the values recorded in treated animals to those recorded in controls within each tissue, all ratios were lower than 1.0, with the exception of those in the lung and heart of smoke-exposed rats and in the lung of rats exposed to both ethanol and cigarette smoke.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Of the two molecular endpoints under study, the analysis of DNA–'otein crosslinks was unable to detect alterations induced in the liver, lung, and heart of rats exposed to ethanol and/or cigarette smoke. A moderate (1.5-fold) increase over controls was recorded only in the lung of rats exposed to cigarette smoke. Although levels higher than 1.0 have been suggested to be biologically relevant (12), the above increase was not statistically significant. Accordingly, the evaluation of this kind of lesion, whose biological significance in vivo is poorly understood (12), was not sufficiently informative.

In contrast, the assessment of DNA alterations by 32P-postlabeling was highly efficient in revealing the tissue-specific effects produced by cigarette smoke and its combination with ethanol. DNA adducts were detected after butanol extraction, which (at variance with nuclease P1 digestion) enriches the adducts formed not only by polycyclic aromatic hydrocarbons, but also by aromatic amines present in cigarette smoke (10). No such DNA alterations were induced by the chronic consumption of ethanol alone, which is consistent with the recognized lack of genotoxicity of this agent. In fact, despite some contrasting data, ethanol per se was mostly negative in several in vitro and in vivo genotoxicity test systems (2, 14). 32P-Postlabeled DNA adducts in exfoliated oral mucosa cells were even found to be significantly lower in drinkers than in nondrinkers (15). The evaluation of ethanol genotoxicity and carcinogenicity in humans is complicated, however, by the dietary deficiency associated with alcohol abuse and by the fact that alcoholic beverages are complex mixtures that may contain low levels of various carcinogens (16).

On the other hand, the genotoxicity of cigarette smoke and its condensates in a variety of in vitro test systems is well established (1, 5, 17, 18). Most studies in animal models have shown the ability of cigarette smoke to induce clastogenic damage (1, 6, 7, 18–20). Formation of adducts to nuclear DNA in different organs of smoke-exposed rodents and humans who smoke is also well documented: 32P-postlabeling reveals the appearance of diagonal radioactive zones typically induced by complex mixtures (21). The levels of adducts to mitochondrial DNA of smoke-exposed rats are even higher (22).

As discussed extensively in a recent review article (21), several factors account for the selective localization of DNA adducts in different tissues and for the possible pathological consequences associated with these promutagenic lesions. Accumulation of DNA adducts is influenced by 1) toxicokinetics, with particular reference to the so-called first-pass effect, 2) metabolism, resulting from the balance between activating and detoxifying pathways, 3) efficiency and fidelity of DNA repair and chiefly of DNA excision repair, which can remove DNA adducts, and 4) cell proliferation rate, which affects the persistence of DNA adducts and at the same time is essential for a possible evolution toward a proliferative disease. These factors can explain the DNA alterations produced by the chronic exposure of rats to cigarette smoke as well as further changes, especially in organotropism, induced by its combination with ethanol consumption.

Of the four organs investigated, the liver was the only one in which 32P-postlabeling analyses failed to detect any effect of the treatments. This is not surprising since the first-pass effect of cigarette smoke in the liver, even after whole-body exposure, is relatively modest. Metabolic activity is particularly high not only in the sense of activation, but also with regard to detoxification; in any case, it is counterbalanced by efficient DNA repair. Proliferation of hepatocytes under normal conditions is poor; accordingly, evolution toward primary hepatocellular carcinoma requires the occurrence of proliferative stimuli. As a matter of fact, this cancer may occur in alcohol abusers as a consequence of cirrhosis, but ethanol per se is generally assumed to be devoid of carcinogenicity (23). The role of tobacco smoke in liver carcinogenesis is controversial (1). Even the combination of these risk factors did not result, under our experimental conditions, in an enhanced frequency of DNA adducts in the liver.

The lung is the most important target of cigarette smoke carcinogenicity (1). DNA adducts are localized in high amounts in lung cells of smokers (24–26), of rats exposed to cigarette smoke (9, 22, 27), and of mice receiving topical applications of cigarette smoke condensate (28). The first-pass effect after exposure to cigarette smoke is particularly intense in the lung, where both metabolism and DNA repair are appreciable, yet less efficient than in the liver (21). Formation of DNA adducts in the lung is related to initiation of lung cancer and especially of bronchogenic carcinoma (29), since the bronchial epithelium is composed of actively proliferating cells (21). The hypothesis was raised that the occurrence of DNA adducts in nonproliferating lung cells, such as type I pneumocytes, may contribute to the pathogenesis of chronic obstructive pulmonary diseases, particularly emphysema (21). Our data indicate that ethanol consumption does not further enhance the levels of DNA adducts induced by cigarette smoke in lung cells; therefore, it is not expected to worsen the potential health effects resulting from DNA alterations in the lung of smokers.

Neither exposure to cigarette smoke nor ethanol consumption alone affected the background levels of DNA adducts in the esophagus, but their combination was highly effective in producing these molecular lesions. The first-pass effect is modest in the esophagus of rats exposed whole-body to cigarette smoke. Although some ingestion of smoke components cannot be ruled out in this administration route, the respiratory tract is the main target, as shown by induction of arylhydrocarbon hydroxylase in the lung (but not in the liver) under the same experimental conditions as in the present study (30). Therefore, a likely interpretation, as will be discussed later, is that ethanol may have solubilized water-insoluble smoke components in the upper aerodigestive tract, thereby determining a first-pass effect in the esophagus. Also taking into account the high proliferation rate of esophageal mucosa cells, the results of molecular dosimetry analyses in the investigated animal model are perfectly in line with the conclusions of analytical epidemiology studies, showing a synergism between alcohol consumption and smoking habits in inducing esophageal cancer (2, 4).

DNA alterations in the heart are of particular interest. In fact, several studies in humans and rodents provided evidence that the heart is a preferential target for the localization of both `spontaneous' and smoke-related DNA adducts (reviewed in ref 21). A relatively high first-pass effect is provided by coronary circulation, and the poor metabolism of xenobiotics in the heart tissue (30) is presumably accompanied by a modest DNA repair. At variance with the situation during fetal life, cardiac myocytes in adults are perennial and fully differentiated cells. This results in the accumulation of DNA adducts with age, as has been documented in the heart of mice (31) and rats (A. Izzotti and R. Balansky, unpublished data). In the present study, it is noteworthy that the highest DNA adduct levels in untreated rats, which were aged 16 months at the time of death, were recorded in the heart where these DNA lesions were twice as high as in the lung and esophagus. The lack of proliferation of cardiac myocytes is incompatible with a neoplastic evolution in the adult heart, but suggests a possible association of DNA adducts with nonproliferative degenerative diseases, particularly smoke-related cardiomyopathies (9, 21). The results of this study confirmed the massive formation of DNA adducts in the heart of smoke-exposed rats and showed that the combined ethanol intake further increases adduct levels to a significant and dramatic extent. These experimental data may be important in the pathogenesis of heart diseases as well in light of the frequent association of alcohol drinking and smoking among the general population (32).

From a mechanistic point of view, it appears that solubilization by ethanol of smoke components determines their availability in the upper digestive tract, as demonstrated by the formation of DNA adducts in the esophagus, and their systemic distribution, as demonstrated by the formation of DNA adducts in the heart. A previous study using the same rat model but for shorter periods of exposure (10–30 days) showed that ethanol consumption decreases the smoke-induced cytogenetic damage in pulmonary cells (alveolar macrophages), but enhances it in bone marrow erythrocytes (6), which again reflects a systemic distribution of ethanol-soluble smoke clastogens. We recently reproduced in vitro the synergism of ethanol and cigarette smoke in the proximity of target cells. Amounts of ethanol as low as 5 µl, incorporated in 2.5 ml of top agar containing S9 mix and highly sensitive bacteria (Salmonella typhimurium YG1024 and YG1029), were in fact sufficient to produce a dramatic increase of the mutagenic response induced by cigarette smoke in an exposure chamber similar to the one used in the present in vivo study (5). The selective localization patterns of DNA adducts in different organs, as observed in this study, are thus consistent with the hypothesis regarding the ethanol's solvent effect.

Another important mechanism is that ethanol induces CYP2E1-dependent microsomal monooxygenases, which catalyze the metabolism of a variety of xenobiotics, including ethanol itself (33, 34). Pretreatment of rodents with ethanol resulted in an enhancement of the ability to metabolize typical components of cigarette smoke (such as polycyclic aromatic hydrocarbons, aromatic amines, and nitrosamines) to mutagenic metabolites (16, 35). The inducing effects of ethanol are more pronounced in extrahepatic tissues (e.g., in the esophagus) than in the liver (36, 37); it has also been shown that ethanol ingestion can change the organotropism of the carcinogenic effects of nitrosamines (35, 38), which are the only carcinogens to show specific induction of esophageal tumors in rodents (39). Moreover, ethanol-inducible CYP2E1 favors the microsomal generation of free radicals (40), and the resulting oxidative stress is potentiated when ethanol is administered together with xenobiotics (16). This may be accompanied by a depletion of reduced glutathione (GSH) (41). In this respect, it is important that cigarette smoke is a major indirect or direct source of chemical oxidants and free radicals (1, 42) and that 32P-postlabeling is efficient in revealing nucleotidic modifications induced by reactive oxygen species (21, 43), which can be inhibited by N-acetylcysteine, a GSH precursor (44).

We cannot rule out that the effects of ethanol on DNA repair may have contributed to its synergism with cigarette smoke in certain organs. For instance, ethanol has been shown to inhibit O6-methyl transferase activity (45). Therefore, different mechanisms can explain, concurrently and not alternatively, the influence of the combination of ethanol ingestion and exposure to cigarette smoke on the intensity and localization of the molecular alterations investigated in the present study.


   ACKNOWLEDGMENTS
 
This study was supported by the Bulgarian Ministry of Education and Science and by the Italian Ministry of University and Scientific-Technologic Research.


   FOOTNOTES
 
1 Correspondence: Institute of Hygiene and Preventive Medicine, University of Genoa, via A. Pastore 1, I-16132 Genoa, Italy.

2 Abbreviations: GSH, glutathione; SDS, sodium dodecyl sulfate

Received for publication November 21, 1997. Accepted for publication February 19, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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