Formation of DNA adducts in the aorta of smoke-exposed rats, and modulation by chemopreventive agents

Mutation Research 494 (2001) 97–106

Formation of DNA adducts in the aorta of smoke-exposed rats, and modulation by chemopreventive agents Alberto Izzotti, Anna Camoirano, Cristina Cartiglia, Elena Tampa, Silvio De Flora∗ Department of Health Sciences, University of Genoa, Via Pastore 1, I-16132 Genoa, Italy Received 16 February 2001; received in revised form 10 April 2001; accepted 17 April 2001

Abstract Our previous studies showed that nucleotide alterations, evaluated by 32 P postlabeling, are systematically detected in smooth muscle cells of atherosclerotic lesions localized in the aorta of surgical patients. The level of these molecular lesions was correlated with the occurrence of known atherogenic risk factors, among which the number of currently smoked cigarettes, and was significantly enhanced in individuals having a null GSTM1 genotype as compared to individuals carrying the GSTM1 genotype. The present study had the dual objective of evaluating the formation of DNA adducts in the whole thoracic aorta of Sprague-Dawley rats, exposed whole-body to cigarette smoke for 28 consecutive days, and of investigating the effects of chemopreventive agents given orally during the same period. High levels of 32 P postlabeled DNA adducts were formed in the aorta of smoke-exposed rats, with an overall 11 times increase over the total levels observed in sham-exposed rats, and with increases ranging between three and 63 times for seven individual DNA adducts. Supplement of the diet with either 1,2-dithiole-3-thione, phenethyl isothiocyanate or 5,6-benzoflavone had no or poor effects on the smoke-related formation of nucleotide alterations in the aorta. In contrast, oltipraz, given with the diet, N-acetyl-l-cysteine, given with drinking water and, even more potently, their combination exerted remarkable protective effects. The results of this experimental study, together with the previous findings in humans, suggest that DNA alterations may contribute to the atherogenic process, clarify a possible mechanism of cigarette smoke, a well known atherogen, and show the potential protective effects of certain drugs towards these alterations. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Cigarette smoke; Aorta; DNA adducts; Atherosclerosis; Chemoprevention

1. Introduction

Abbreviations: BF, 5,6-benzoflavone; CS, cigarette smoke; D3T, 1,2-dithiole-3-thione; ECS, environmental cigarette smoke; GSH, reduced glutathione; NAC, N-acetyl-l-cysteine; OPZ, oltipraz or 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione; PEITC: phenethyl isothiocyanate; SFS, synchronous fluorescence spectrophotometry; SMC, smooth muscle cells ∗ Corresponding author. Tel.: +39-10-353-8500; fax: +39-10-353-8504. E-mail address: [email protected] (S. De Flora).

Cardiovascular diseases and cancer represent by far the leading causes of death in the population of developed countries. Due to the fact that, according to the International Classification of Diseases [1], these pathological conditions embrace more than 1000 nosological entities, which often are differentiated from biological, clinical and epidemiological standpoints, any generalization should be banned. Nevertheless, it is noteworthy that, in the framework of the intricate network associating risk factors with

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chronic degenerative conditions, some of these diseases share common risk factors, either biological, physical or chemical, as well as protective factors supplied, e.g. with the diet or by administering suitable pharmacological agents [2]. In addition, certain mechanisms, such as oxidative stress and damage to DNA, either nuclear or mitochondrial, may play a role in the pathogenesis of different chronic degenerative diseases [2–4]. The DNA adducts represent biomarkers of biologically effective dose. Unless removed via DNA repair mechanisms, these molecular lesions will evolve into a mutation in proliferating cells thereby triggering the initiation of cancer and possibly of other chronic degenerative diseases [2]. A widely used technique for detecting adduction of electrophilic metabolites to DNA, namely 32 P postlabeling, also detects nucleotide alterations due to other mechanisms, such as oxidative DNA damage [5]. DNA adducts are detectable in the heart of both smokers [6,7] and rats exposed to cigarette smoke, either mainstream [8] or environmental [9]. Due to the lack of proliferation of cardiac myocytes in adults, these molecular lesions cannot evolve into tumors but, according to our hypothesis, could evolve into degenerative heart diseases, such as cardiomyopathies [2]. The role of DNA damage in atherosclerosis represents an important and controversial issue. The “response to injury” or “inflammation” mechanism is the prominent theory in the pathogenesis of atherosclerosis [10]. However, there are also several lines of evidence that the atherosclerotic plaque could be initiated by genotoxic damage in the smooth muscle cells (SMC) constituting the tunica media of arteries [2,4]. In any case, these theories are complementary rather than mutually exclusive [2,11,12]. In previous studies, we demonstrated that DNA adducts can be detected in aorta SMC from human atherosclerotic lesions, using various techniques including 32 P postlabeling, HPLC/fluorescence, synchronous fluorescence spectrophotometry (SFS) and three-dimensional SFS [11,13]. In particular, a molecular epidemiology study, evaluating SMC DNA from the atherosclerotic lesions of abdominal aorta taken at surgery from 84 patients, showed that the 100% of samples was positive at 32 P postlabeling [11]. Interestingly, DNA adduct levels were significantly correlated with known atherogenic risk factors including age, number of currently

smoked cigarettes, arterial pressure, blood cholesterol (total/HDL), triglycerides, and oxidative DNA damage in the same cells [11]. These conclusions were further supported by a study evaluating 30 thoracic aorta samples taken at autopsy, which showed a higher frequency of DNA adducts in subjects with frequent atherosclerotic lesions and a significant correlation between DNA adduct levels and blood cholesterol [14]. Furthermore, we demonstrated that DNA adduct levels in SMC from atherosclerotic lesions are significantly and consistently increased in individuals having a null GSTM1 genotype, i.e. the gene encoding for the detoxifying phase II enzyme glutathione S-transferase (GST) isoform ␮, compared to subjects carrying the GSTM1 genotype [15]. This finding provides evidence that there is an interindividual variability related to this metabolic polymorphism which affects the susceptibility to genotoxic atherogens [15]. Although less sensitive than humans, the rat has been proposed as a model for atherosclerosis [16] and has been extensively used in a number of studies, some of which evaluated the atherogenic response in cigarette smoke (CS)-exposed rats [see, e.g. 17,18]. We report here the results of an experimental study in rats exposed to CS, which had a dual goal. The first one was to assess whether exposure of rats to CS results in the formation of appreciable amounts of DNA adducts in the aorta, also as compared to other cells, tissues and organs of the same animals evaluated in a parallel study [19]. The second objective was to evaluate the ability of selected compounds, which are promising agents in cancer chemoprevention, to modulate the formation of smoke-related DNA adducts. The tested agents, all given orally, included 1,2-dithiole-3-thione (D3T) and its substituted analogue 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz or OPZ); phenethyl isothiocyanate (PEITC), which is found at relatively high levels in watercress; 5,6-benzoflavone (BF), a synthetic flavonoid which interacts with the Ah receptor; and N-acetyl-l-cysteine (NAC), an analogue and precursor of reduced glutathione (GSH). A combination of OPZ and NAC was also assayed. The results obtained provide evidence for the strong induction of DNA adducts in the aorta of smoke-exposed rats and for the possibility of attenuating these molecular alterations by means of certain chemopreventive agents.

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2. Materials and methods 2.1. Animals Male Sprague-Dawley rats, aged 8 weeks and weighing 270–290 g, were purchased from Harlan Italy (Corezzana, Milan, Italy). During acclimatization for 7 days, the rats were maintained on a Teklad IRM Rat/Mouse diet (Harlan Italy). Both drinking water and diet were given ad libitum. The rats were housed in a climatized environment at a temperature of 22 ± 1◦ C, relative humidity of 50 ± 5%, ventilation accounting for 15 air renewal cycles per hour, and with a 12 h light–dark cycle. Animal housing and treatments were in accordance with Italian and institutional guidelines. 2.2. Chemopreventive agents The BF was purchased from Sigma Chemical Co. (St. Louis, MO), OPZ, D3T and PEITC were supplied by the Division of Cancer Prevention Repository, National Cancer Institute (Rockville, MD). These agents were incorporated in Teklad diet (Harlan Teklad, Madison, WI). The diets were prepared and kindly supplied by Dr. C.J. Grubbs (Chemoprevention Center, University of Alabama, Birmingham, AL). NAC, used in the form of a commercial product (Fluimucil) supplied by Zambon (Vicenza, Italy), was dissolved and diluted in drinking water. 2.3. Exposure to cigarette smoke A whole-body exposure of rats to cigarette smoke was obtained by using a smoking machine (model TE-10, Teague Enterprises, Davis, CA) in which each smoldering cigarette is puffed for 2 s, once every minute for a total of eight puffs, at a flow rate of 1.05 l/min to provide a standard puff of 35 cm3 [20]. The 2R1 reference cigarettes, having a declared content of 44.6 mg total particulate matter and 2.45 mg nicotine each with a 23 mm butt remaining after smoking one cigarette, were purchased from the Tobacco Research Institute (University of Kentucky, Lexington, KY). The smoking machine was adjusted to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%), mimicking an exposure to environmental CS (ECS), by burning five cigarettes

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at one time, 6 h a day divided in two 3 h rounds with a 3 h interval, for 28 consecutive days. The cages were rotated daily in the four exposure chambers, where the total particulate matter was on an average 83 mg/m3 , and CO concentration was 350 ppm. 2.4. Experimental groups After acclimatization, when the weight was in the 290–315 g range, the rats were divided into eight experimental groups, each composed of eight animals. One group of sham-exposed rats, kept in filtered air and maintained in a standard diet, served as untreated controls. The rats belonging to the other seven groups were exposed to ECS. One of these groups did not receive any further treatment, whereas the other six groups of rats received chemopreventive agents, starting 3 days before the first day of exposure to ECS and continuing throughout the 28 days of exposure. In particular, four groups received diets supplemented with either OPZ (400 mg/kg diet), D3T (400 mg), PEITC (500 mg) or BF (500 mg). Another group received a standard diet, and NAC was given with drinking water at a concentration accounting for a calculated daily intake of approximately 1 g/kg body weight. The last group of ECS-exposed rats received a combined treatment with OPZ and NAC, with the same concentrations as given in the single treatments in diet and drinking water, respectively. The actual intake of chemopreventive agents was calculated by taking into account the concentration of compounds, either in the diet or in drinking water, the food and water consumption, and the body weight of rats (see Section 3). 2.5. Purification of aorta DNA At the end of the period of exposure to ECS, the rats were starved for 24 h, anesthesized with ethyl ether and killed by cervical dislocation. The thoracic aorta was collected from each animal, accurately washed in ice-cold 0.15 M NaCl, and stored at –80◦ C until use, together with other organs and blood from the same animals to be used in parallel studies [19,21]. The aorta samples were thawed and homogenized in a Potter–Elvehjem apparatus at 4◦ C in 250 mM sucrose, 5 mM dithiothreitol, 50 mM Tris–HCl, pH 7.6. DNA was isolated by solvent extraction using an automatic DNA extractor (Genepure 341, Applied

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Biosystems, Foster City, CA) [9]. A silica-based reagent (Quik-PrecipTM , Edge BioSystems Inc., Gaithersburg, MD) was used in order to bind DNA during alcohol precipitation. DNA was analyzed by spectrophotometric scanning. The 260/270 and 260/280 absorbance ratios were 1.2±0.1 and 1.7±0.1 (means± S.D. of 64 DNA samples), respectively. Absorbance at 230 nm could not be determined due to interference of the silica-based reagent at this wavelength. 2.6.

32 P

the value of 0.1/108 adducts/normal nucleotides was assumed as an arbitrary threshold of method sensitivity for adducts of unknown molecular structure. 2.7. Statistical analyses The differences between the experimental groups regarding food intake, body weight gain and DNA adduct levels were evaluated by Student’s t-test for unpaired data.

postlabeling analyses

DNA adducts were evaluated by 32 P postlabeling following butanol extraction [22]. Each DNA sample (6 ␮g) was depolymerized by micrococcal nuclease (0.04 U/␮g DNA) (Sigma Chemical Co.) and spleen phosphodiesterase (0.57 ␮g/␮g DNA) digestion. The adducts were enriched by water saturated butanol and labeled with 64 ␮Ci carrier free [␥-32 P]ATP (ICN Biochemicals, Irvine, CA) having a specific activity ≥7000 Ci/mmol. Multidirectional thin layer chromatography (TLC) was carried out on polyethyleneimine sheets (Macherey and Nägel, Düren, Germany) according to standard procedures. Based on previous comparative analyses [9], the chromatographic media were: D1, 1.0 M sodium phosphate, pH 6.0; D2, 7 M urea, 3 M lithium formate, pH 3.8; D3, 7 M urea, 0.6 M lithium chloride, Tris 0.5 M, pH 8.0; D4, 1.7 M sodium phosphate, pH 6.0. Autoradiographic images and quantification of spots were obtained by using a 32 P InstantImager Autoradiographic System equipped with InstantQuant software (model A2024, Packard, Meriden, CT). In each experiment we used a reference standard of 7R,8S,9S-trihydroxy-10R-(N2 -deoxyguanosyl-3 phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEN2 -dGp) (National Cancer Institute Chemical Carcinogen Reference Standard Repository, Midwest Research Institute, Kansas City, MO). DNA-free samples were used as negative controls. DNA adduct levels were quantified by calculating the relative adduct labeling index (cpm adducts/cpm normal nucleotides) [22]. The aorta DNA samples from each one of the 64 rats were tested in two separate experiments. The results were expressed as DNA adducts per 108 nucleotides. Although under our experimental conditions up to 1/1010 BPDE-N2 -dGp molecules are detectable,

3. Results 3.1. Food intake The daily food intake (mean ± S.D. of 31 days) was very similar in sham-exposed rats (19.5 ± 1.2 g per rat per day) and rats exposed to ECS (19.4 ± 0.9). The food intake in ECS-exposed rats was not changed by administering either NAC (19.5 ± 0.7), PEITC (19.5 ± 1.0) or BF (18.9 ± 1.2), whereas it was slightly but significantly reduced by administering either OPZ (18.5 ± 1.6, P < 0.01), D3T (18.0 ± 2.6, P < 0.01) or OPZ plus NAC (17.5 ± 2.2, P < 0.01). 3.2. Body weight gain After 4 weeks of exposure to ECS, the body weight gain (12.8%) was significantly (P < 0.01) lower as compared to sham-exposed rats (23.4%), and was not further modified by administration of either OPZ (11.7%), BF (10.0%), OPZ plus NAC (9.3%), NAC (7.7%) or PEITC (7.3%). In contrast, there was no body weight gain in ECS-exposed rats treated with D3T (P < 0.001, as compared not only to sham-exposed rats but also to ECS-exposed rats). 3.3. Intake of chemopreventive agents The actual intake of each agent was calculated by taking into account its concentration either in the diet (OPZ, D3T, PEITC and BF) or in drinking water (NAC), the daily consumption of food or water (not shown), and the body weights measured weekly (not shown). The actual intakes (means ± S.D. of the values recorded during the 4 weeks of treatment) were 23.1 ± 1.9 mg per day per kg body weight for OPZ,

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Fig. 1. Autoradiographic patterns of 32 P postlabeled DNA adducts in the thoracic aorta of a sham-exposed rat (SHAM) and of a rat exposed to environmental cigarette smoke (ECS). OR is the origin and D1, D2, D3 and D4 are the development directions of TLC.

24.9±2.3 for D3T, 1195.8±146.7 for NAC, 31.1±0.2 for PEITC, and 26.9 ± 5.2 for BF. When given in combination, the intakes of OPZ and NAC were very similar to those calculated when given individually, i.e. 22.0 ± 3.1 and 1190.1 ± 150.9, respectively. 3.4. Formation of DNA adducts in the aorta of ECS-exposed rats Up to seven discrete spots could be detected by analyzing aorta DNA samples from both sham-exposed and ECS-exposed rats (Fig. 1). In ECS-exposed rats there was a trend to formation of a diagonal radioactive zone (DRZ), but individual spots were always well distinguishable from the background (Fig. 1). DRZ was not per se included in the calculation of total DNA adducts since the seven individual spots, especially following long autoradiographic exposures, covered the whole DRZ area. Out of the seven adducts detected, spot 4 showed chromatographic properties resembling those of the BPDE-N2 -dGp standard, although this does not necessarily imply a structural characterization. From a quantitative point of view (Table 1), the signal was very weak in sham-exposed rats, being in some cases close to the sensitivity threshold assumed for the method (0.1 adducts per 108 nucleotides). Both individual spots and total DNA

adducts were remarkably increased in ECS-exposed rats. Only for spots 5 (four times increase) and 7 (three times increase) the differences were not statistically significant between ECS- and sham-exposed rats. Sharp and significant increases were recorded for spots 6 (4 times), 3 (9 times), 4 (10 times), 2 (36 times) and 1 (63 times). Note that spots 1 and 2, which were the major adducts in ECS-exposed rats, were barely detectable in sham-exposed rats. The increase of total DNA adducts in ECS-exposed rats was 11 times. 3.5. Effects of chemopreventive agents on ECS-related DNA adducts None of the tested agents significantly enhanced the levels of ECS-induced DNA adducts in the thoracic aorta. D3T was the only one which did not significantly affect any spot. BF and PEITC inhibited the formation of individual adducts (spots 2 and 3 for BF; spot 2 for PEITC) but failed to inhibit the levels of total adducts. Individually, OPZ and NAC approximately halved the levels of total ECS-induced DNA adducts and significantly decreased individual adducts (spots 2 and 7 for NAC; spots 2, 3, 6 and 7 for OPZ). Thus, formation of spot 2 was significantly inhibited by all tested chemopreventive agents with the exception of

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Table 1 Induction of DNA adducts in the thoracic aorta of rats exposed to environmental cigarette smoke and effect of chemopreventive agentsa DNA adducts Treatment groups SHAM Spot Spot Spot Spot Spot Spot Spot Total

1 2 3 4 5 6 7

0.1 0.1 0.4 0.2 0.3 0.3 0.3

± ± ± ± ± ± ±

0.1 0.1 0.5 0.3 0.4 0.4 0.5

ECS + BF

ECS 6.3 3.6 3.6 2.0 1.2 1.1 0.9

± ± ± ± ± ± ±

2.1c 1.8c 1.5c 1.8b 1.8 0.9b 0.6

6.4 1.6 3.8 2.1 0.7 1.8 1.2

± ± ± ± ± ± ±

2.6c 1.2c,d 0.8c,d 0.7c 0.4 0.7c 0.7b

1.7 ± 1.1 18.6 ± 5.2c 17.6 ± 5.8c

ECS + D3T ECS + PEITC ECS + NAC ECS + OPZ ECS + NAC + OPZ 6.6 1.8 3.7 1.7 0.6 1.4 1.0

± ± ± ± ± ± ±

2.3c 2.0b 1.3c 0.7c 0.4 1.1b 0.8b

16.8 ± 4.9c

4.3 1.4 2.8 2.2 0.7 1.8 1.0

± ± ± ± ± ± ±

2.1c 0.7c,d 1.3c 0.7c 0.5 1.1c 0.6b

14.1 ± 6.0c

2.9 0.7 2.9 2.0 0.3 0.7 0.3

± ± ± ± ± ± ±

1.1c 0.5c,e 0.9c 0.8c 0.2 0.4 0.2d

9.8 ± 3.6c,e

4.1 1.3 1.2 1.4 0.1 0.3 0.2

± ± ± ± ± ± ±

2.0c 1.3b,d 1.0e 0.9c 0.2 0.2d 0.1e

8.5 ± 4.5c,e

2.4 0.6 1.8 1.6 0.2 0.6 0.3

± ± ± ± ± ± ±

0.8c,e,f 0.3c,e 0.8c,e,g 1.1c 0.2 0.3 0.2d

7.4 ± 1.8c,e

a The data are expressed as DNA adducts per 108 nucleotides and are the means ± S.D. of the mean results obtained in eight rats per experimental group. 32 P postlabeling analyses were run by testing in duplicate the thoracic aorta DNA from each rat. b P ≤ 0.05, as compared to sham-exposed rats. c P ≤ 0.01, as compared to sham-exposed rats. d P ≤ 0.05, as compared to ECS-exposed rats, in the absence of chemopreventive agents. e P ≤ 0.01, as compared to ECS-exposed rats, in the absence of chemopreventive agents. f P ≤ 0.01, as compared to rats treated with ECS + OPZ. g P ≤ 0.01, as compared to rats treated with ECS + NAC.

D3T. The combination of OPZ and NAC was the most powerful treatment in inhibiting the formation of total DNA adducts in the aorta of ECS-exposed rats, with significant effects on spots 1, 2, 3 and 7. Furthermore, the combined treatment was significantly more effective than the individual treatment with OPZ for spot 1 and the individual treatment with NAC for spot 3. In all cases, however, the effects of OPZ and NAC were less than additive.

4. Discussion The first conclusion of the present study is that the whole-body exposure of rats to ECS results in a strong induction of DNA adducts in their thoracic aorta. Autoradiographic spots with identical chromatographic patterns were detected by analyzing the aorta DNA samples from sham-exposed and ECS-exposed rats, suggesting that, rather than determining the formation of new DNA adducts, exposure to smoke results in the amplification of pre-existing lesions. This is in agreement with the conclusion drawn by Gupta et al. [23], who analyzed several tissues (lung, trachea, larinx, heart and bladder) from smoke-exposed rats. However, we found a broad variability in the ECS-induced increase of individual DNA adducts, which ranged

between three and 63 times, with the maximum increase for two major spots which were barely detectable in sham-exposed rats. Total DNA adduct levels were particularly high in ECS-exposed rats, the increase over sham-exposed rats being 11-fold. By comparison, in the same animals the increase was 14.5-fold in the tracheal epithelium, 10.3-fold in bronchoalveolar lavage cells, 8.5-fold in the lung, and 6.5-fold in the heart [19]. The localization and accumulation of DNA adducts in different organs are the composite result of several mechanisms including toxicokinetics, metabolism, DNA repair, and cell proliferation [2]. Of these factors, toxicokinetics, and in particular the so-called first-pass effect, are relatively modest in the aorta SMC of smokers, at least as compared to respiratory tract cells. Metabolites of smoke components can be formed in distant organs, e.g. in lung and liver, and transported to the aorta tunica media, which is largely avascular, via the blood of adventitial vasa vasorum [24]. In addition, SMC are known to possess a complete set of activating and deactivating enzymes [2,4,18]. The proliferation rate of arterial SMC is usually low [25] but can be stimulated by certain factors, such as platelet-derived growth factor [26] and lipoprotein(a) [27]. SMC mitogens and chemotactic agents are released during the regenerative repair process of

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injured cells and, in addition, mutations in SMC could induce the constitutive production of growth factors resulting in autocrine stimulation of growth [28]. The cell replication rate is particularly important, since on one hand it produces a cell turnover which causes a loss of DNA adducts by dilution, but on the other hand, it hampers DNA repair processes and favors the development of a mass of initiated cells [2]. In the case of arterial SMC, this could result in the formation either of tumors, such as leiomyosarcomas [29], or of atherosclerotic plaques [2]. The CS is both a powerful atherogen and carcinogen, and tobacco-associated cancers and severe atherosclerosis tend to occur together in the same individual [30]. In spite of the strength of the association between smoking and atherosclerosis, the underlying cellular and molecular mechanisms have been so far essentially unknown [31]. In this light, the particularly high levels of DNA adducts detected in the aorta of ECS-exposed rats deserve particular interest. This experimental finding is in agreement with the consistent detection of DNA adducts in aorta SMC from human atherosclerotic lesions [11]. Also from a qualitative point of view, it is of interest that seven spots were detected by analyzing the aorta DNA of ECS-exposed rats (this study). Coincidentally, the levels of seven of the nine spots detected in human aorta SMC from atherosclerotic lesions were correlated with the number of currently smoked cigarettes. Identification of the chemical nature of unknown adducts detected by 32 P postlabeling would require huge amounts of DNA. The correspondence between the smoke-related DNA adducts detected in rat and human aorta will be evaluated in future experiments. The second objective of the present study was to evaluate the ability of agents which are frequently used in cancer chemoprevention research to interfere with the formation of DNA adducts in the aorta of ECS-exposed rats. Of the five investigated agents, no effect was produced by D3T, which at the tested dose exerted systemic toxicity, as inferred from the complete lack of body weight gain of the ECS-exposed rats treated with this compound. In the same animals, D3T was also found to be genotoxic by enhancing the frequency of micronucleated pulmonary alveolar macrophages [19]. BF and PEITC, which are broad-spectrum modulators of phase I and phase II enzyme activities involved in the metabolism of xeno-

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biotics [32], had limited effects on DNA adduct levels, which were significantly attenuated only for one or two spots. In contrast, both OPZ, a derivative of D3T, and NAC, an analogue and precursor of GSH, were successful in decreasing most individual adducts to a significant extent and halved the total levels of ECS-induced DNA adducts. Although the protective effect was less than additive, the combination of NAC and OPZ was the most powerful treatment in inhibiting the formation of DNA adducts. The selective effects of the five investigated agents in aorta are only partially coincident with those observed in broncholaveolar lavage cells, tracheal epithelium, lung and heart of the same animals, in which not only NAC but also PEITC, BF and, less effectively, D3T inhibited the formation of ECS-related DNA adducts, whereas OPZ was ineffective. Combination of NAC and OPZ was the most effective treatment in inhibiting the formation of both DNA adducts in the respiratory tract and the hemoglobin adducts of 4-aminobiphenyl and benzo(a)pyrene-7,8-diol-9,10-epoxide [19]. Of the two agents displaying protective effects towards aorta DNA, NAC has extensively been used as a mucolytic drug for almost four decades. Moreover, this thiol has been used as antidote in acute intoxications, and has been shown to exert antigenotoxic and anticarcinogenic effects in a variety of test systems [33,34]. This thiol works extracellularly per se and intracellularly as a precursor of cysteine and GSH [35]. Its antigenotoxic and anticarcinogenic effects have been ascribed to a variety of mechanisms, including trapping of electrophiles and scavenging of reactive oxygen species [35,36], inhibition of nitrosation [37], enhancement of thiol concentration in intestinal bacteria [38], enhanced detoxification in nontarget cells [35,36], stimulation of metabolic activation coordinated with enhanced detoxification and block of reactive metabolites [39], inhibition of spontaneous mutations related to DNA repair background [40], protection of nuclear enzymes and enhanced repair of DNA damaged by carcinogens [41], correction of hypomethylation [42], signal transduction modulation [43], inhibition of angiogenesis [44], inhibition of type IV collagenases involved in degradation of basement membranes, and inhibition of chemotaxis, invasion and metastasis of malignant cells [45]. NAC has also been shown to have protective effects in cardiovascular diseases. It has been demonstrated in isolated rabbit

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aorta that NAC attenuates the cigarette smoke induced inhibition of arterial relaxation [46]. Moreover, NAC has been used and/or proposed as an antidote to doxorubicin cardiotoxicity [47], for attenuating the myocardial damage induced by ischemia and reperfusion [48], and for potentiating the hemodynamic and antiplatelet effects of nitroglycerin in man [49]. The synthetic dithiolethione OPZ, which had originally been proposed as an antischistosomiasis agent, has also been shown to inhibit mutation and cancer in variety of experimental test systems [33,50–52]. The main mechanism of OPZ is enhancement of GSH and a potent induction of phase II metabolic enzyme expression, particularly of GSH S-transferases [50–52]. This is accompanied by a selective induction of those cytochrome P450 monooxygenases which enhance carcinogen detoxification by inhibiting phase I enzymes to retard metabolic activation and by trapping of electrophiles [52]. In conclusion, the results obtained point to the occurrence of DNA alterations, induced experimentally in the aorta of smoke-exposed rats, as a possible mechanism contributing, at least partially, to the multifaceted pathogenesis of the atherogenic process. Inhibition of this mechanism by certain pharmacological agents, such as OPZ, NAC and their combination, deserves further attention regarding their potential use for the chemoprevention of atherosclerosis.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

Acknowledgements We thank Dr. C.J. Grubbs from the University of Alabama at Birmingham for supplying the diets incorporating chemopreventive agents. This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC).

References [1] World Health Organization, International Statistical Classification of Diseases and Related Health Problems, 10th Revision, Vol. 1, World Health Organization, Geneva, Switzerland, 1992. [2] S. De Flora, A. Izzotti, K. Randerath, E. Randerath, H. Bartsch, J. Nair, R. Balansky, F.J. van Schooten, P. Degan, G. Fronza, D. Walsh, J. Lewtas, DNA adducts in chronic degenerative diseases. Pathogenetic relevance and

[11]

[12]

[13]

[14]

[15]

[16] [17]

implications in preventive medicine, Mutat. Res. 366 (1996) 197–238. J. Trosko, C. Chan, An integrative hypothesis linking cancer, diabetes, and atherosclerosis: the role of mutations and epigenetic changes, Med. Hypoth. 6 (1980) 455–468. B.A. Bridges, D.E. Bowyer, E.S. Hansen, A. Penn, K. Wakabayashi (Eds.), Report of the ICPEMC Subcommittee 7/1, The Possible Involvement of Somatic Mutations in the Development of Atherosclerotic Plaques (special issue), Mutat. Res. 239 (1990) 143–187. A. Izzotti, M. Orlando, L. Gasparini, L. Scatolini, C. Cartiglia, L. Tulimiero, S. De Flora, In vitro inhibition by N-acetylcysteine of oxidative DNA modifications detected by 32 P postlabeling, Free Rad. Res. 28 (1998) 165–178. J. Lewtas, J. Mumford, R.B. Everson, B. Hulka, T. Wilcosky, W. Kozumbo, C. Thompson, M. George, L. Dobias, R. Sram, X. Li, J. Gallagher, Comparison of DNA adducts from complex mixture exposures in various human tissues and experimental systems, Environ. Health Perspect. 99 (1993) 88–97. F.J. van Schooten, A. Hirvonen, L.M. Maas, B.A. de Mol, J.C.S. Kleinjans, D.A. Bell, J.D. Durrer, Putative susceptibility markers of coronary artery diseases: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue, FASEB J. 12 (1998) 1409–1417. A. Izzotti, R.M. Balansky, P.M. Blagoeva, Z.I. Mircheva, L. Tulimiero, C. Cartiglia, S. De Flora, DNA alterations in rat organs following chronic exposure to cigarette smoke and/or ethanol ingestion, FASEB J. 12 (1998) 753–758. A. Izzotti, M. Bagnasco, F. D’Agostini, C. Cartiglia, R.A. Lubet, G.J. Kelloff, S. De Flora, Formation and persistence of nucleotide alterations in rats exposed whole-body to environmental cigarette smoke, Carcinogenesis 20 (1999) 1499–1505. R. Ross, Atherosclerosis — an inflammatory disease, New Engl. J. Med. 340 (1999) 115–126. S. De Flora, A. Izzotti, D. Walsh, P. Degan, G.L. Petrilli, J. Lewtas, Molecular epidemiology of atherosclerosis, FASEB J. 11 (1997) 1021–1031. M.G. Andreassi, N. Botto, M.G. Colombo, A. Biagini, A. Clerico, Genetic instability and atherosclerosis: can somatic mutations account for the development of cardiovascular diseases? Environ. Mol. Mutagen. 35 (2000) 265–269. A. Izzotti, S. De Flora, G.L. Petrilli, J. Gallagher, M. Rojas, K. Alexandrov, H. Bartsch, J. Lewtas, Cancer biomarkers in human atherosclerotic lesions. I. Detection of DNA adducts, Cancer Epidem. Biom. Prev. 4 (1995) 105–110. B. Binkova, P. Strejc, O. Boulbel`ık, Z. Stàvkovà, I. Chvàtalova, R. Sram, DNA adducts and human atherosclerotic lesions, Homeostasis 39 (1999) 168–173. A. Izzotti, C. Cartiglia, J. Lewtas, S. De Flora, Increased DNA alterations in atherosclerotic lesions of individuals lacking the GSTM1 genotype, FASEB J. 15 (2001) 752–757. J. Ritskes-Hoitinga, A.C. Beynen, Atherosclerosis in the rat, Artery 16 (1988) 25–50. A.J. Sherrat, B.T. Culpepper, W.C. Lubawy, Relative participation of the gas phase and total particulate

A. Izzotti et al. / Mutation Research 494 (2001) 97–106

[18]

[19]

[20]

[21]

[22] [23]

[24]

[25]

[26] [27]

[28]

[29] [30]

[31]

[32]

[33]

matter in the imbalance in prostacyclin and thromboxane formation seen following chronic cigarette smoke exposure, Prostaglandins Leukot. Essent. Fatty Acids 34 (1988) 15–18. M.J. Thirman, J.H. Albrecht, M.A. Krueger, R.R. Erikson, D.I. Cherwitz, S.S. Park, H.V. Gelboin, J.L. Holtzman, Induction of cytochrome CYP1A1 and formation of toxic metabolites of benzo(a)pyrene by rat aorta: a possible role in atherogenesis, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 5397–5401. A. Izzotti, R.M. Balansky, F. D’Agostini, C. Bennicelli, S.R. Myers, C.J. Grubbs, R.A. Lubet, G.J. Kelloff, S. De Flora, Modulation of biomarkers by chemopreventive agents in smoke–exposed rats, Cancer Res. 61 (2001) 2472–2479. S.V. Teague, K.E. Pinkerton, M. Goldsmith, A. Gebremichael, S. Chang, R.A. Jenkins, J.H. Moneyhum, Sidestream cigarette smoke generation and exposure system for environmental tobacco smoke studies, Inhal. Toxicol. 6 (1994) 79–93. F. D’Agostini, R.M. Balansky, A. Izzotti, R.A. Lubet, G.J. Kelloff, S. De Flora, Modulation of apoptosis by cigarette smoke and cancer chemopreventive agents in the respiratory tract of rats, Carcinogenesis 22 (2001) 375–380. A. Izzotti, Detection of modified DNA nucleotides by post-labeling procedures, Toxicol. Meth. 8 (1998) 175–205. R.C. Gupta, J.M. Arif, C.G. Gairola, Enhancement of pre-existing DNA adducts in rodents exposed to cigarette smoke, Mutat. Res. 424 (1999) 195–205. D.W. Crawford, D.H. Blankenhorn, Arterial wall oxygenation, oxyradicals, and atherosclerosis, Atherosclerosis 89 (1991) 97–108. D. Gordon, M.A. Reidy, E.P. Benditt, S.M. Schwartz, Cell proliferation in human coronary arteries, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 4600–4604. R.E. Ross, The pathogenesis of atherosclerosis — an update, New Engl. J. Med. 314 (1986) 488–500. D.J. Grainger, H.L. Kirschenlohr, J.C. Metcaffe, P.L. Weissberg, D.P. Wade, R.M. Lawn, Proliferation of human smooth muscle cells promoted by lipoprotein(a), Science 260 (1993) 1655–1658. K.S. Ramos, E. Chacon, D. Acosta Jr., Toxic responses of the heart and vascular systems, in: C.D. Klaasen (Ed.), Casarett and Doull’s Toxicology, The Basic Science of Poisons, McGraw-Hill, New York, 1996, pp. 487–527. M.B. McManus, B. Toth, K.D. Patil, Aortic rupture and aortic smooth muscle tumors in mice, Lab. Invest. 57 (1987) 78–85. E.S. Hansen, Shared risk factors for cancer and atherosclerosis: a review of the epidemiological evidence, Mutat. Res. 239 (1990) 163–179. R. Ross, Rous–Whipple Award Lecture: Atherosclerosis: a defense maschanism gone awry, Am. J. Pathol. 143 (1993) 987–1002. A.R. Boobis, D.W. Nebert, J.S. Felton, Comparison of ␤-naphthoflavone and 3-methylcholanthrene as inducers of hepatic cytochrome(s) P448 and arylhydrocarbon-hydroxylase activity, Mol. Pharmacol. 13 (1978) 259–268. G.J. Kelloff, C.W. Boone (Eds.), Cancer chemopreventive agents: drug development status and future prospects, J. Cell. Biochem. 20 (Suppl.) (1994) 1–304.

105

[34] S. De Flora, A. Izzotti, F. D’Agostini, R.M. Balansky, Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoke- and oxidative stress-related end-points, Carcinogenesis 22 (2001), in press. [35] S. De Flora, R. Balansky, C. Bennicelli, A. Camoirano, F. D’Agostini, A. Izzotti, C.F. Cesarone, Mechanisms of anticarcinogenesis: the example of N-acetylcysteine, in: C. Ioannides, D.F.V. Lewis (Eds.), Drugs, Diet and Disease, Vol. 1, Mechanistic Approaches to Cancer, Hemel Hempstead, Ellis Horwood, UK, 1995, pp. 151–203. [36] S. De Flora, C.F. Cesarone, R.M. Balansky, A. Albini, F. D’Agostini, C. Bennicelli, M. Bagnasco, A. Camoirano, L. Scatolini, A. Rovida, A. Izzotti, Chemopreventive properties and mechanisms of N-acetylcysteine. The experimental background, J. Cell. Biochem. 58 (Suppl. 22) (1995) 33–41. [37] S. De Flora, C.F. Cesarone, C. Bennicelli, A. Camoirano, D. Serra, M. Bagnasco, A.I. Scovassi, L. Scarabelli, U. Bertazzoni, Antigenotoxic and anticarcinogenic effects of thiols. In vitro inhibition of the mutagenicity of drug nitrosation products and protection of rat liver ADP-ribosyl transferase activity, in: F. Feo, P. Pani, A. Columbano, R. Garcea (Eds.), Chemical Carcinogenesis: Models and Mechanisms, Plenum Press, New York, 1988, pp. 75–86. [38] A. Camoirano, G.S. Badolati, P. Zanacchi, M. Bagnasco, S. De Flora, Dual role of thiols in N-methyl-N-nitro-N-nitrosoguanidine genotoxicity, Life Sci. Adv. — Exp. Oncol. 7 (1988) 21–25. [39] S. De Flora, Mechanisms of inhibitors of mutagenesis and carcinogenesis, Mutat. Res. 402 (1998) 151–158. [40] S. De Flora, C. Bennicelli, A. Rovida, L. Scatolini, A. Camoirano, Inhibition of the spontaneous mutagenicity in Salmonella typhimurium TA102 and TA104, Mutat. Res. 307 (1994) 157–167. [41] C.F. Cesarone, M. Menegazzi, L. Scarabelli, A.I. Scovassi, P. Giannoni, R. Izzo, H. Suzuki, A. Izzotti, M. Orunesu, U. Bertazzoni, Protection of nuclear enzymes by aminothiols, in: F. Nygaard, A.C. Upton (Eds.), Anticarcinogenesis and Radiation Protection 2, Plenum Press, New York, 1991, pp. 261–268. [42] K. Lertratanangkoon, R.S. Orkiszewski, J.M. Scimeca, Methyl-donors deficiency due to chemically induced glutathione depletion, Cancer Res. 56 (1996) 995–1005. [43] S. Bergelson, R. Pinkus, V. Daniel, Intracellular glutathione levels regulate fos/jun induction and activation of glutathione S-transferase gene expression, Cancer Res. 54 (1994) 36–40. [44] T. Cai, G.F. Fassina, D. Giunciuglio, M. Morini, M.G. Aluigi, L. Masiello, S. Fontanini, F. D’Agostini, S. De Flora, D.M. Noonan, A. Albini, N-acetylcysteine inhibits endothelial cell invasion and angiogenesis while protecting from apoptosis, Lab. Invest. 79 (1999) 1151–1159. [45] A. Albini, F. D’Agostini, D. Giunciuglio, I. Paglieri, R. Balansky, S. De Flora, Inhibition of invasion, gelatinase activity, tumor take and metastasis of malignant cells by N-acetylcysteine, Int. J. Cancer 61 (1995) 121–129. [46] Y. Ota, K. Kugiyama, S. Sugiyama, M. Ohgushi, T. Matsumara, H. Doi, N. Ogata, H. Oka, H. Yasue,

106

A. Izzotti et al. / Mutation Research 494 (2001) 97–106

Impairment of endothelium-dependent relaxation of rabbit aortas by cigarette smoke extract. Role of free radicals and attenuation by captopril, Atherosclerosis 131 (1997) 195– 202. [47] J.H. Doroshow, J.Y. Locker, I. Ifirm, C. Myers, Doxorubicin cardiac toxicity, J. Clin. Invest. 68 (1981) 1053–1064. [48] M.B. Forman, D.W. Puett, C.U. Cates, D.E. McCroskey, J.K. Backman, H.L. Greene, R. Virmani, Glutathione redox pathway and reperfusion injury. Effect of N-acetylcysteine on infarct size and ventricular function, Circulation 78 (1988) 202–213. [49] J.D. Horowitz, E.M. Antman, B.H. Lorell, W.H. Barry, T.W. Smith, Potentiation of the cardiovascular effects of

nitroglycerin by N-acetylcysteine, Circulation 68 (1983) 1247–1253. [50] S.S. Ansher, P. Dolan, E. Bueding, Biochemical effects of dithiolthiones, Food Chem. Toxicol. 24 (1986) 405–415. [51] P.A. Egner, S.J. Gange, P.M. Dolan, J.D. Groopman, A. Munoz, T.W. Kensler, Levels of aflatoxin-albumin biomarkers in rat plasma are modulated by long-term and transient interventions with OPZ, Carcinogenesis 16 (1995) 1769– 1773. [52] V. Breinholt, L. Dragsted, Antitumorigenic Dithiolethiones, Nordic Council of Ministers, Copenhagen, Vol. 613, TemaNord, Ekspressen Tryk and Kopicenter, Copenhagen, 1997, pp. 1–73.