ICANCERRESEARCH(SUPPL.)54. . April1.1994)

Chemoprevention of Carcinogen-DNA Adducts and Chronic Degenerative Diseases1

Alberto Izzotti, Francesco D'Agostini, Maria Bagnasco, Leonardo Scatolini, Alessandra Rovida,Roumen M. Balansky,2 Carmelo F. Cesarone, and Silvio De Flora3

Inclinile of Hygiene ami Preventive Medicine ¡A.I., F. D'A., M. B., L. S., A. R., R. M. B., S. D. F./ anil Institute of General Physiology ¡C.F. C.¡.University of Genoa, Via

Paslore I, ¡-Ì6132Genoa, Italy

Abstract

Molecular dosimetry techniques were exploited in order to assess theefficacy of experimental chemoprevention assays and to evaluate theinvolvement of DNA alterations, not only in cancer but also in otherchronic degenerative diseases. In agreement with other protective effectspreviously observed in the same animal models, the thiol /V-acetylcysteine(NAC) totally prevented or significantly reduced the formation of carcin-ogen-DNA adducts in three experimental systems in rats. Thus, as assessed by -"P postlabeling, supplement of the diet with NAC decreasedboth deo\yguanosine-C'K-aminofluorene adducts (butano! enrichment)

and deoxyguanosine-N,-acetylaminofluorene adducts (nuclease P, enrich

ment) formed in rat liver following dietary administration of2-acetyIaminofluorene for 3 weeks. DNA adducts were detected by syn

chronous fluorescence spectrophotometry in rat liver, lung, heart, andtestis following a daily i.t. instillation of benzo(a)pyrene for 3 consecutivedays. The whole-body exposure of rats to mainstream cigarette smoke for

40 consecutive days resulted in the appearance of DNA adducts in heart,lung, and aorta, whereas no adduci was detected by synchronous fluorescence spectrophotometry in liver, brain, and testis. Multiple DNA adductsin the aorta were also measured by '"P postlabeling. Administration of

NAC by gavage inhibited the formation of DNA adducts in all organs ofrats treated with benzol«Ipyrene or exposed to cigarette smoke. It is ofinterest that a single chemopreventive agent can display a broad-spectrum

protective ability. The selective localization of DNA adducts in differentorgans depends on pharmacokinetics, metabolic capacity, DNA repairefficiency, and cell proliferation rate. Whereas inhibition by NAC of DNAadducts in testis can be correlated with its demonstrated ability to preventdominant lethal mutations, we raise the hypothesis that DNA adducts inlung, heart, and aorta may be pathogenetically associated with lungcancer, cardiomyopathies, and arteriosclerosis, respectively. In order toexplore the involvement of molecular and biochemical alterations in human arteriosclerosis, we started an extensive collaborative project andreport here preliminary data showing the presence of DNA adducts inaorta smooth muscle cells obtained from arteriosclerotic patients.

Introduction

The thiol NAC4 has been extensively used over the past 30 years as

a mucolytic and untioxidant drug in the therapy of respiratory diseasesand. more recently, as an antidote towards certain acute intoxications.Studies performed in this laboratory and elsewhere have shown thatNAC exerts antimutagenic effects against a variety of mutagenicchemicals and complex mixtures (for recent reviews, see Refs. 1-3).

Moreover, NAC displayed anticarcinogenic effects in various organsof rodents, including skin, mammary glands, Zymbal glands, trachea,lung, liver, colon, and urinary bladder (4-9) (see Table 1). Based on

this experimental background and on its proven lack of toxicity, NACis at present considered one of the most promising chemopreventiveagents. It is currently under Phase I clinical trial in the United States

' Prcscnlcd at the 4th International Conference on Anticarcinogenesis & RadiationProtection. April 18-23. 1W3. Baltimore. MD. This work was supported by the NationalResearch Council (CNR-ENEL Project on "Interaction of Energetic Systems with Humanand Environmental Health" and CNR-targeted project "Prevention and Control of DiseaseFactors—FATMA"). Dr. R. M. Balansky is a recipient of a research fellowship in

toxicology provided by the European Science Foundation.-Permanent address: National Centre of Oncology. Sofia-1756, Bulgaria.1To whom requests for reprints should be addressed.4 The abbreviations used are: NAC. iV-acctylcysteinc; GSH. reduced glutathione; SFS.

synchronous fluorescence spectrophotometry: 2AAF. 2-acetvlaminofluorene: BP. ben«>-

(a)pyrene.

(6) and, alone or in combination with retinol palmitate, is assayed inan European Phase III trial (Euroscan) for the prevention of secondprimary tumors in patients previously treated for squamous cancer ofthe oral cavity or larynx or for non-small cell lung cancer (10).

The mechanisms responsible for the antimutagenic and anticarcinogenic properties of NAC have been extensively investigated in ourlaboratory (for recent reviews, see Refs. 1-3). In synthesis, in the

extracellular environment NAC works as a nontoxic analogue ofcysteine. It detoxifies direct-acting mutagens due to its nucleophilicand antioxidant activity, inhibits the nitrosation products of amin-

ocompounds formed in an acidic environment, and enhances thiolconcentrations in intestinal bacteria. Inside cells, NAC is readilydeacetylated to yield cysteine, which is the rate-limiting amino acid in

the intracellular synthesis of GSH. As a result, NAC enhances thedetoxification of carcinogens which are entrapped in nontarget cells,such as long-lived transport cells (e.g., erythrocytes) and sweeping

cells (e.g., pulmonary alveolar macrophages). In target cells, NACreplenishes GSH stores, which is particularly important in case ofdepletion due either to toxic stress or to cancer-associated viral

infections, such as hepatitis B and acquired immunodeficiency syndrome. The metabolism of xenobiotics is influenced by the modulation of Phase II enzymes, the GSH cycle, and the hexose monophos-

phate shunt. The intracellular derivatives of NAC block electrophiliccompounds and metabolites and efficiently scavenge reactive oxygenspecies. They protect against the down-regulation of nuclear enzymesproduced by carcinogens, decrease the carcinogen-induced DNAdamage, and prevent the formation of carcinogen-DNA adducts. All

of these mechanisms contribute to inhibit the initiation of cancer andare likely to affect later stages of the carcinogenesis process as well.

We describe here the results of experimental studies investigatingthe ability of NAC to reduce in vivo the formation of DNA adducts indifferent organs of rats exposed to environmental carcinogens. Alldata reported are either original or represent the expansion of previously published studies (11-13). In addition, we report here the

preliminary results of a study on the detection of DNA adducts inhuman aorta smooth muscle cells from arteriosclerotic patients, whichwas stimulated by the results of animal chemoprevention assays. Twodifferent molecular dosimetrv methods were used, i.e., the 32P post-

labeling technique, which detects the DNA adducts formed by avariety of carcinogenic metabolites (14), and SFS, which detects theDNA adducts formed by metabolites of polycyclic aromatic hydrocarbons, mainly benzo(«)pyrene diolepoxide-DNA adducts (15).

Materials and Methods

Treatment of Rats with 2-Acetylaminofluorene. Male Wistar rats(Morirti strain), weighing 80-100 g, were given a standard diet and drinking

water ad libitum and divided into three groups, each composed of five animals.The first group received a standard diet, the second group received the samediet supplemented with 0.05% 2AAF (Sigma Chemical Co., St. Louis, MO),and the third group received a diet supplemented with 0.05% 2AAF plus 0.1%NAC (a gift from Zambón Group. Bresso. Milan. Italy). After 21 days, theanimals were anesthetized with ethyl ether and killed by cervical dislocation.The livers were removed, washed with 10 mM Tris-0.15 M KC1, pH 7.4, andstored at -80°C.

Exposure of Rats to Mainstream Cigarette Smoke. Three groups of sixmale Sprague-Dawley rats (Morini strain), weighing 180-200 g, were used in

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CHEMOPREVENTION Oí-C'ARCINíXÕI-N-DNAAPDIKTS

Table I Protective effects of NAC in animal carcinogenic itv studies

Rodentspecies TestcarcinogenMouse

UrethanDMBA" + TRA''

OH-BBN'Rat

1.2-DimcthylhydrazineAzoxvmethiincAzoxymcthane2-Acetylaminonuorenc2-Acetylaminofluorcne

McthylnitrosurcaHamster

MethylnitrosureaNeoplastic

orpreneoplasticlesionsLung

adenomasSkin papillomasTransitional cell bladdercarcinomaIntestinal

tumorsColon aberrant crypt fociColon carcinomaZymbal gland squamous cell carcinomasLiver GGT4- foci

MammaryadenocarcinomasTrachea!

squamous cell carcinomaReferenceDe

Flora et al. 1986 (4)Rotstein and Slaga, 1988 (5)Kelloff el al. 1992(6)Wilpart

et al. 1986 (7)Pereira and Khourv, 1991 (8)Kelloff <•(«/.1992(6)

Cesarone ci al. 1987 (9)Ccsarone el al. 1987 (9)Kelloff el al. 1992(6)Kelloff

cía/. (1 "92 (6)

" 7.12-Dimethylbcnz(« (anthracene.h l2-O-Tctradecanoyl-13-phorbol acetate.' W-Butyl-W-(4-hydroxybutyl)nitrosamine, GOT. •y-glutamyltranspeptidase.

this study as described previously (12). Briefly, a whole-body exposure to

cigarette smoke was obtained by placing a group of six animals in a sealed20-liter glass chamber, which was filled by means of a 50-ml syringe with the

mainstream smoke (720 ml) generated by a commercial tiller cigarette containing 14 mg tar and 0.4 mg nicotine. After IO min. the chamber was openedand, after a 2-min interval needed for renewing the air. filled again with fresh

smoke 11 times (the first day) or 8 times (all subsequent days). Such anexposure was performed daily for 40 consecutive days, thereby accounting foran overall exposure to the smoke generated by 363 cigarettes. Sham-exposed

rats were kept in the same type of chamber for the same time periods but in theabsence of cigarette smoke. Solutions of NAC were prepared daily in 0.2 Mphosphate-buffered saline, pH 7.4. Due to their acidity, the final pH was

adjusted to 5.0 by adding 0.4 N sodium hydroxide. NAC was administered bygavage in aliquots containing l g of the thiol per kg body weights h before and5 h alter each daily exposure to cigarette smoke, starting 2 days before time (Iof the experiment. At the end of the experiment, all rats were anesthetized andkilled. Liver, lungs, heart, aorta, hrain. and testis were removed, washed, andstored at -8()°C.

Treatment of Rats with HP. Male Sprague-Dawley rats (Morini strain),weighing 320-360 g. were divided into three groups, each composed of fiveanimals. C'ontrol rats received daily i.t. instillations of 2% Tween 80 (Sigma)

in distilled water (2 ml/kg body weight) for 3 consecutive days. The secondgroup was treated i.t. with BP (25 mg/kg body weight; Sigma) in 2% Tween«SOaqueous vehicle, again for 3 consecutive days. The third group receivedNAC (I g/kg body weight) by gavage 5 h before each BP instillation. Threedays after the last treatment, all animals were anesthetized and killed. Liver,lungs, heart, and testis were removed, washed, and stored at -80°C.

Human Aorta Specimens. Blind-coded specimens of human abdominal

aorta, taken at surgery from arteriosclerotic patients, were kindly supplied byProf. G. L. Petrilli (Department of Vascular Surgery, E. O. Galliera. Genoa.Italy). The specimens were immediately transferred to the laboratory, wherethe three aorta layers were dissected, washed, and separately stored at -8()°C.

DNA Extraction. The rat organs were thawed and homogenized in aPotter-Elvehjem apparatus at 4°Cin 250 mM sucrose-50 mM Tris-HCl, pH 7.6.

DNA was isolated by solvent extraction using an automatic extractor (Gene-

pure 341; Applied Biosystems, Foster City. CA) according to Gupta (16) withsome modifications as described previously (12). The specimens of aortamedium layer, mainly containing smooth muscle cells, were thawed andprocessed like the rat organs except that homogenization was carried out in aPolytron apparatus and, after lysis and digestion with RNases and proteinaseK, the homogenate was centrifuged al XOOX g for 30 min in order to removecalcified deposits.

Detection of DNA Adducts by SFS. Aliquots of 50-100 ^g DNA were

assayed by SFS in duplicate or, when the amounts were sufficient, in triplicatefor the presence of DNA adducts. The samples were hydrolyzed in 0.1 M HC1at WC for 4 h in sealed glass vials. Synchronous scanning was performed witha fixed \\ of 34 nm between excitation and emission using a Hitachi F-3000fluorescence spectrophotometer. Peaks in the 379-385 nm emission range

were recorded. The mean levels of DNA adducts from each sample werequantified by using suitable standards (15). The thresholds of positivity corresponded to 1.6 adducts/107 nucleotides in the study with cigarette smoke andto 1.2 adducts/107 nucleotides in the study with BP.

Detection of DNA Adducts by 32P Postlabeling. Aliquots of 5 /xg DNAwere assayed for the presence of DNA adducts by **P postlabeling. following

enrichment with either nuclease PI or butanol. Standard procedures (14, 17)were followed, except that the urea used as a solvent in thin layer chromatog-

raphy (D3 and D4) was 6.5 M rather than 8.5 M. Autoradiography wasperformed at -80°Cfor 24-76 h. The identification of adducts in 2AAF-treated

rats was achieved according to Gupta and Earley (IK), based on the enrichmentprocedure and Chromatographie localization of spots. The adducts were quantified by calculating relative adduci labeling values (17). Biochemicals werepurchased from Sigma and Boehringer Mannheim GmbH (Mannheim, Germany), polyethylenimine thin layer chromatography sheets from Maeherey-Nagel (Doren. Germany), and carrier-free [-y-12P]ATP from ICN Biomedicals

(Irvine, CA) having a specific activity =Ã7̄,000 Ci/mmol.

Results

Chemoprevention of DNA Adducts in Rats Receiving2-Acetylaminofluorene with the Diet. No evident spot was detectedby 32P postlabeling in the liver DNA of untreated rats (not shown).

Administration of 2AAF with the diet resulted in the formation ofadducts in rat liver DNA. As shown in the example reported in Fig. 1(panels on the left), different types of adducts were detected followingenrichment with butanol or nuclease PI. In particular, the levelsrecorded in the 5 rats treated with 2AAF (means ±SD) were 118.2 ±21.3 dG-C,s-AF adducts/10* nucleotides and 10.9 ±2.8 dG-N2-AAFadducts/l()x nucleotides. An evident and significant (P < 0.001)

decrease of both types of DNA adduci levels was observed in the liverof rats cotreated with 2AAF and NAC with the diet (Fig. 1, panels onthe right), with mean levels of 63.9 ±10.7 and 2.6 ±0.9 dG-Cx-AFand dG-Ni-AAF adducts/10* nucleotides. thereby accounting for 46

and 76% decreases, respectively.

2AAF 2 AAF + NAC Knrichmenl

Nuclease PIdG-Nï-AAF

Fig. I. Formation of DNA adducts in the liver of Wistar rats after 3 weeks ol dietaryadministration of 2AAF (0.05CÕ)and the protective effect exerted by the concomitantdietary administration of NAC (0. I<i). DNA adducts were detected by *'P postlabeling

assay following enrichment with either hutanol or nuclease PI.

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Liver Brain Testis Lung Heart Aorta

••••••••••••»III»! I I »»I I888118o888o.0

e»e»

O Controls •Cigarelte smoke (CS) 9 CS + NAC (gavage)

Fig. 2. Formation of DNA adducts, as detected hy SFS, in different organs of Sprague-Dawley rats with whole-body exposure for 40 consecutive days to mainstream cigarettesmoke (accounting for a total of 363 cigarettes), and the protective effect exerted hy thegavage administration of NAC (1 g/kg body weight). Each symbol refers to a singleanimal, except that for aorta, for which samples were pooled from two animals each. Thesoliti horizontal lines and the corresponding dashed areas indicate the mean ±SD withineach group of rats. The dolled box (s l .6) includes all results lower than the threshold ofscnsilivily of the assay.

Fig. 3. Examples of SFS spectra with the DNA extracted from different organs of a ratwith whole-body exposure to mainstream cigarette smoke. The three notches on eachabscissa indicate the emission wavelength at 374. 384. and 3U4 nm. respectively.

Chemoprevention of DNA Adducts in Rats Receiving i.t. Instillations of BP. Decreasing levels of DNA adducts were formed in theliver, lung, and heart of all five rats and in the testis of three of fiverats treated i.t. with BP (Fig. 5). Of five rats additionally receivingNAC by gavage, four exhibited DNA adducts in the lung, one in theliver and heart, and none in testis. The decrease by NAC of the meanlevels of SFS-positive DNA adducts consequent to BP administration

was significant in all monitored organs, i.e., liver (P < 0.01), lung (P< 0.05), heart (P < 0.05), and testis (P < 0.05).

Detection of Carcinogen-DNA Adducts in Human Atherosclerotic Aorta—Preliminary Data. The analysis by SFS of 23 samplesof smooth muscle cells of human abdominal aorta from arterioscle-

rotic patients showed typical fluorescence peaks in 16 subjects(69.6%), with levels ranging between 2.5 and 15.5 adducts/107 nucle-otides (data not shown). The 32P postlabeling analysis (nuclease PI

enrichment) of four aorta samples, all of them SFS-positive, led to the

detection of several DNA adducts (Fig. 6).

Discussion

The data reported in this article provide examples of application ofmolecular dosimetry techniques in experimental studies on chemo-

prevention of cancer. In addition, they suggest that the same methodological tools can be exploited for assessing the involvement ofmolecular lesions, not only in cancer, but also in other chronicdegenerative diseases.

The findings concerning inhibition of carcinogen-DNA adducts, as

observed in three separate studies, are consistent with other protectiveeffects exerted by this thiol in the same experimental models. Thus,

Cigarette Smoke (CS) CS + NACFig. 4. Formation of DNA adducts. as detected hy 32P postlabeling assay, following

nuclease PI enrichment in the aorta of rats exposed to mainstream cigarette smoke and theprotective effect exerted by the gavage administration of NAC' under the same conditions

indicated in Fig. 2. Each sample was pooled from six animals.

Chemoprevention of DNA Adducts in Rats Exposed Whole-

Body to Mainstream Cigarette Smoke. No DNA adduci was detected by SFS in organs of sham-exposed rats (Fig. 2, O). Fig. 3

provides examples of SFS spectra recorded in six different organs ofa single smoke-exposed rat. As shown in Fig. 2 (•),no SFS-detect-

able adduci was revealed in liver, brain, or testis, whereas DNAadducts occurred in decreasing rank of levels in the heart, lung, andaorta of all smoke-exposed rats. Following administration of NAC bygavage (Fig. 2, semi-full circles), DNA adducts could be only detectedin the lung and heart of two of six smoke-exposed animals and in two

of three aorta pools. The mean levels of DNA adducts were significantly reduced by NAC treatment in heart and lung (both at P < 0.01)as well as in the aorta (P < 0.05). The appearance of several smoke-

related DNA adducts in the aorta and inhibition of their formation byNAC were also confirmed by 12P postlabeling (Fig. 4). In particular,total levels of 20.3 adducts/10K nucleotides were recorded in smoke-exposed rats and of 8.3 adducts/108 nucleotides in rats additionally

treated with NAC by gavage, thereby accounting for an overall 59.2%decrease.

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O Controls •B[a]P(i.t.) 3 B[a]P (i.t.) + NAC (gavage)

Fig. 5. Formation of DNA adducts, as detected by SFS, in different organs of Sprague-Dawley ruts receiving daily i.t. instillations of BP (25 mg/kg body weight) for 3consecutive days and the protective effect exerted hy the gavage administration of NAC(I g/kg body weight). The soliti, horizontal lines and the corresponding dashed areasindicate the mean ±SD within each group of rats. The dotted box (^ 1.2) includes resultslower than the threshold of sensitivity of the assay.

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CHEMOPRËVENTION OF CARCINOGEN-DNA ADDUCTS

Fig. 6. Detection by ';P postlaheling (nuclease PI enrichment) of adducts in the DNA

extracted from the medium layer (smooth muscle cells) of human abdominal aortaspecimens taken at surgery from four arteriosclerotic patients.

the herein described decrease of both dG-NU-AAF and dG-CK-AF

adducts to rat liver DNA occurred after 3 weeks of coadministrationof NAC with 2AAF in the diet. When followed by a week of standarddiet, this treatment corresponds to the first cycle of the Teebor andBecker (19) hepatocarcinogenesis model. Using a four-cycle protocol,

NAC had been shown to delay by at least one cycle, i.e., 4 weeks, theonset of significant alterations induced by 2AAF in the liver, including changes in the GSH cycle and other cytosolic activities (20),down-regulation of the nuclear enzyme poly(ADP-ribose) polymerase(21), DNA fragmentation, and formation of y-glutamyltranspeptidase-

positive foci (9). Moreover, both NAC and GSH had been successfulin totally preventing the formation of Zymbal gland carcinomas,which were conversely detected in all animals treated with the carcinogen alone 6 weeks after completion of the four-cycle treatment.

The observed ability of NAC to inhibit the formation of SFS-positive DNA adducts in liver, testis, heart, and lung of BP-treated rats

fully confirms and broadens the results of a previous study, which hadbeen carried out under identical experimental conditions (13). In thatstudy, NAC had significantly inhibited the formation of DNA adductsin liver and lung and, in addition, had prevented clastogenic damagein pulmonary alveolar macrophages, in spite of the unfavorable phar-

macokinetic conditions resulting from the i.t. administration of thecarcinogen and administration by gavage of the chemopreventiveagent (13). In reference to the protective effect observed in the testis,it is noteworthy that recent studies performed in another laboratoryhave shown that administration of NAC to the same rat strain used inour experiments enhances GSH concentrations in testis and epididy-

mis and inhibits chemically induced dominant lethal mutations (22).Previous studies demonstrated that exposure of rats to mainstream

cigarette smoke results in severe histológica!, cytological, cytogenetic,and metabolic alterations (23, 24). NAC efficiently prevented smoke-

induced emphysema and inflammatory, hyperplastic, and dysplasticchanges of bronchial and bronchiolar mucosae, alterations in thecellularity of bronchoalveolar lavage cells, enhancement of the frequency of micronucleated alveolar macrophages, and toxicity in bonemarrow erythroblasts (23). Moreover, NAC hampered the smoke-

induced stimulation of the liver activation of several promutagens(24). Evidence was also provided for a selective time-course appearance of SFS-positive DNA adducts in heart and lungs, which was

prevented by NAC (12). The present study additionally shows theabsence of this type of DNA adduci, not only in the liver, but also inbrain and testis, and their presence in appreciable amounts in the aorta

of the same smoke-exposed animals. Again, NAC significantly pre

vented this effect.The selective localization of DNA adducts in different organs

depends on several factors including pharmacokinetics ("first-pass"

effects), local metabolism, efficiency and fidelity of DNA repair, andcell proliferation rate. For instance, the liver has a high metaboliccapacity but also is efficient at DNA repair and removal of adducts.Both lungs and heart are highly exposed due to "first pass" effects,

and a poor local metabolism of xenobiotics, especially in the heart(24), should be compensated by the low efficiency of DNA repair,which will result in a longer persistence of adducts. An importantdifference between these two organs is that certain bronchopulmonarycells are actively proliferating, which favors their transformation intoneoplastic cells. Conversely, heart muscle cells are perennial, which isnot compatible with an evolution into a neoplastic mass but may leadto other degenerative diseases, such as cardiomiopathies. A similarsituation is envisaged in the aorta, where proliferation of smoothmuscle cells only occurs during formation of the atheroscleroticplaque.

Therefore, our working hypothesis is that the formation of smoke-related DNA adducts in the lung may be associated with lung carci-

nogenesis, in the heart with cardiomiopathies, and in the aorta witharteriosclerosis. Several lines of evidences already exist on the role ofsomatic mutations in arterial smooth muscle cells as one of themechanisms contributing to the pathogenesis of atheroscleroticplaques (25). In order to further substantiate this hypothesis, weimplemented an extensive project aimed at exploring a number ofmolecular and biochemical end points in human arteriosclerosis. Theresults which are reported here show the presence of SFS-positive

DNA adducts in the majority of the human aorta samples so faranalyzed and the occurrence of multiple spots detectable by 32P

postlabeling. These are very preliminary data and will have to beconfirmed on a larger scale, compared with several other parameterswhose evaluation is now in progress in our laboratory and otherlaboratories collaborating in this project, and be related to clinical,laboratory, and lifestyle characteristics of the patients under study. Inany case, the finding that a chemopreventive agent like NAC iscapable of inhibiting the formation of DNA adducts in differentorgans, where they possibly bear a distinctive pathogenetic meaning,deserves attention and warrants further studies in chronic degenerativediseases other than cancer.

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