ICANCER RESEARCH 56. 1642-1647. April 1, 1996|

Induction by Carcinogens and Chemoprevention by /V-Acetylcysteine of Adducts toMitochondrial DNA in Rat Organs1

Roumen Balansky,2 Alberto Izzotti, Leonardo Scatolini, Francesco D'Agostini, and Silvio De Flora3

Institute of Hygiene ami Preventive Medicine, University of Genoa, Via A. Pastore I, 1-16132 Genoa, Italy

ABSTRACT

Damage to mitochondria! DNA (mtDNA) has been postulated to beassociated with aging, cancer, and other chronic degenerative diseaseswhich are the predominant causes of death in the population. We usedmolecular dosimetry techniques, i.e., 32P postlabeling and synchronous

fluorescence spectrophotometry, in order to evaluate the formation ofadducts to both mtDNA and nuclear DNA (nDNA) in different organs ofrats exposed to genotoxic carcinogens. Adducts to mtDNA were detectedfollowing administration of benzo(a)pyrene i.p. or 2-acetylaminofluorene

by gavage as well as following exposure of animals to cigarette smoke.mtDNA adduci levels were consistently higher than those to nDNA in thesame cells, and qualitative differences were also pointed out in the case ofthe aromatic amine. The oral administration of the thiol ;V-acetylcv steine,

one of the most promising cancer chemopreventive agents endowed withnucleophilic and antioxidant properties, produced a significant decreaseof mtDNA adducts in the liver of 2-acetylaminofluorene-treated rats and,

sharply, in the lung and liver of rats exposed to cigarette smoke.

INTRODUCTION

mtDNA4 is a compact (16,569 bp), double-stranded, closed circular

molecule (1) that is inherited maternally (2). mtDNA is more vulnerable than nDNA for a number of reasons, including the lack ofprotective histones and non-histone proteins, its proximity to oxidants

generated during phosphorylative oxidation, a lower efficiency inDNA repair mechanisms, and a high rate of mitochondria! replication,which presumably removes the damaged organelles but rendersmtDNA more susceptible to genotoxic agents (3, 4).

The findings that mitochondria are altered in cancer cells and thatgenotoxic carcinogens effectively bind mtDNA led to the hypothesisthat mutations of this nucleic acid may be involved in the carcino-

genesis process (3, 5). Moreover, a myriad of mutations and rearrangements in mtDNA have been implicated as a cause of mitochondria! dysfunctions, mainly as a result of defects in phosphorylativeoxidation. These have been associated with aging processes as well aswith a variety of chronic degenerative diseases, including some formsof ischemie heart disease, cardiomyopathies, adult-onset diabetes,Parkinson's disease, Alzheimer's disease, Huntington's disease, and

several other neurological disorders (6-10).

Due to the crucial role of mtDNA alterations in the pathogenesis ofthose clinical problems that are the predominant causes of death indeveloped countries, it is important to explore the mechanisms leadingto mtDNA damage and to develop suitable preventative measures.With this aim, we used modern molecular dosimetry techniques, i.e..32P postlabeling and SFS, for comparatively evaluating the formation

Received 10/24/95; accepted 1/25/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This study was supported by the Italian National Research Council (CNR) Targeted

Project on Prevention and Control of Disease Factors (FATMA) and by the BulgarianMinistry of Science, Education and Technology.

2 Present address: National Center of Oncology, Sofia-1756. Bulgaria.' To whom requests for reprints should be addressed. Phone: (39) 10-353-8500; Fax:

(39) 10-353-8504.4 The abbreviations used are: mtDNA. mitochondria! DNA; nDNA, nuclear DNA;

SFS, synchronous fluorescence spectrophotometry; BP, benzo(a)pyrene; 2AAF,2-acetylaminofluorene; NAC, W-acetylcysteine.

of adducts to both mtDNA and nDNA in different organs of laboratory animals exposed to genotoxic carcinogens. These included two ofthe most extensively studied carcinogens, i.e., BP and 2AAF, whichare typical representatives of the chemical families of polycyclicaromatic hydrocarbons and aromatic amines, respectively. Thesecompounds are also typical components of complex mixtures, including cigarette smoke. Moreover, for the first time, we investigatedmolecular alterations of mtDNA following exposure to cigarettesmoke itself. A major goal of the present study was to assess theprotective effects of a pharmacological agent on the in vivo formationof DNA adducts. We used NAC, an analogue and precursor ofreduced glutathione, the clinical efficacy and safety of which havebeen established in more than 30 years of experience as a mucolyticdrug, in the therapy of respiratory diseases and, more recently, as anantidote toward several acute intoxications (11). Based on the experimental data generated by preclinical studies carried out during thepast 12 years, NAC is now considered to be one of the most promisingcancer chemopreventive agents and is currently being tested in severalcancer chemoprevention clinical trials (12-15). NAC works through a

variety of coordinated mechanisms, the most important of whichdepend on its remarkable antioxidant and nucleophilic properties,which warrant possible applications in the prevention of cancer andother pathological conditions associated with damage by free radicals(14, 15).

MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats (Morini strain), 3-4 months of ageand weighing 220-260 g, were acclimatized for 7-14 days, maintained on astandard rodent chow (Morini, San Polo d'Enza, Italy), and given drinking

water ad libitum. The animals were housed in a climatized environment at atemperature of 22 ± 1°C,relative humidity of 50 ±5%, and ventilation

accounting for 15 air renewal cycles/h, and with a 12-h light/dark cycle.

Animal care was in accordance with our institutional guidelines.Treatments. Each experimental group was composed of five rats. BP

(Sigma Chemical Co., St. Louis, MO) was dissolved in DMSO and administered in a single i.p. injection (100 mg/kg body weight). Sham-exposed rats

received an i.p. injection of the solvent. After 48 h, the animals were anesthetized with ethyl ether and killed by decapitation. Livers, lungs, and kidneyswere removed.

2AAF (Sigma) was dissolved in olive oil and administered daily by gavage(100 mg/kg body weight) for 8 consecutive days. Sham-exposed rats received

olive oil by gavage. After 48 h since the last treatment, the animals were killed,and the livers were removed.

A whole-body exposure of rats to cigarette smoke was obtained, as described previously (16), using filter-tipped commercial cigarettes having a

declared content of 14 mg tar and 0.9 mg nicotine. The animals were exposeddaily for 100 consecutive days to the mainstream smoke generated by eightcigarettes, accounting for a cumulative exposure of the rats composing thisgroup to the smoke of 800 cigarettes. Sham-exposed rats were placed for the

same time periods in an identical chamber but in the absence of cigarettesmoke. After 24 h since the last exposure, the animals were killed, and liversand lungs were removed.

Additional groups of rats treated with 2AAF or exposed to cigarette smokewere co-treated with NAC in the form of a water-soluble commercial prepa

ration containing 200 mg of this thiol (Fluimucil; Zambón Group, Vicenza,Italy). The drug was dissolved daily in drinking water at the concentration of11 mg/ml, which was calculated to yield an average dose of l g NAC/kg body

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CHEMOPREVENTION OF ADDUCTS TO MITIX'HONDRIAL DNA

weight. Administration of NAC started 3 days before the first treatment with2AAF or exposure to cigarette smoke and continued daily until the end of theexperiment.

Extraction of nDNA and mtDNA. The rat organs, kept until use at-80°C, were thawed and homogenized in ice-cold 0.25 M sucrose solution

supplemented with IÜmM EDTA (pH 7.2-7.4; l g wet tissue/5 ml solution) byusing a Potter-Elvehjem apparatus (400-600 rpm). These homogenates, constantly kept at 0-4°C, were centrifuged at 500 x g for 2-3 min to eliminate

cell debris. The supematants were further centrifuged at 1000 x ¡>for 15 min

to sediment the nuclei. The pellets and the supematants from each organ werepooled within the five rats composing each experimental group and were usedfor nDNA and mtDNA extractions, respectively. Isolation of nDNA was

obtained by solvent extraction, using an automatic DNA extractor (Genepure341; Applied Biosystems, Foster City. CA), according to standard procedures(17), with some modifications as described previously (18).

The mitochondria were pelleted by supernatant centrifugation at 9500 X g

for 15 min. The pellets were washed twice with 0.25 M sucrose solutionsupplemented with 10 mM EDTA (pH 7.2-7.4) and resuspended in 2 ml of abuffer composed of 50 mM glucose, 10 mM EDTA, and 25 mM Tris-HCl (pH

8.0). mtDNA was purified by using an alkaline extraction procedure, asdescribed previously (19). As assessed by densitometric analysis of ethidiumbromide-stained electrophoregrams. the yield of mtDNA. expressed as jug/gwet organ, was 4.1 for the kidney, 2.7-4.4 for the liver, and 1.2-1.9 for the

lung. According to the electrophoretic patterns on agarose gel, all three formsof mtDNA molecule, i.e.. supercoiled. relaxed circular, and linear, werepresent in the isolates from liver, lung, and kidney. The 260:280 nm ratio of thespecimens tested was consistently higher than 1.7. No contamination by nDNAwas detected when mtDNA preparations were tested by agarose gel electro-

phoresis of EcoRl. BumH\. Hindlll, Msp\, and Hpall restriction endonucleasedigests. Furthermore. CsCl-ethidium bromide gradients provided further evi

dence that mtDNA was not contaminated with nDNA. DNA samples werestored at —¿�80°Cuntil use.

Detection of DNA Adducts. All samples were assayed in triplicate by 32P

postlabeling and additionally, in the case of the BP experiment, by SFS.Chemicals were purchased from Sigma and Boehringer Mannheim GmbH(Mannheim. Germany), polyethylenimine TLC sheets were from Macherey-Nagel (Doren. Germany), and carrier-free (y-i2P]ATP, having a specific

activity S7.000 Ci/mmol. was from ICN Biomedicals (Irvine, CA).Aliquots of 5 fig DNA were analyzed for the presence of DNA adducts by

<2Ppostlabeling. following enrichment with butanol. Standard procedures (20)

were followed, except that the urea used as a solvent in TLC (D3 and D4) was7.0 M for cigarette smoke and 8.5 M for 2AAF. As specified in the legends toFigs. 2-4, autoradiography was performed at -80°C for 6 to 72 h, depending

on the intensity of radioactivity. DNA adducts were quantified by scintillationcounting in a Tri-Carb 1600 TR analyzer (Packard. Meriden. CT), followed by

calculation of relative adduci levels (20). Tridimensional image analyses ofautoradiographic spots were obtained by using the program NIH Image 1.57

running on a Macintosh Quadra 700.For SFS analyses, 50-;ng aliquots of DNA were hydrolyzed in 0.1 M HC1 at

90°Cfor 4 h in sealed glass vials. Synchronous scanning was performed with

a fixed AA of 34 nm between excitation and emission, using a Hitachi F-3000fluorescence spectrophotometer. Peaks in the 345-351 nm excitation range

were recorded (18). The intensity of peaks was expressed in arbitrary fluorescence units, indicating the difference between the intensity of the signalyielded by the peak and the baseline, which was further subtracted by a fixedvalue of 10. This was assumed as an arbitrary threshold of sensitivity of themethod to distinguish specific fluorescence signals from background noise.

RESULTS

Exposure to BP. The i.p. administration of BP to rats resulted inthe formation of adducts to both mtDNA and nDNA, which werequantified by SFS (Table 1). The rank of adduci levels waskidney>liver>lung, with a mtDNA:nDNA ratio in these three organsof 1.4, 1.7, and 1.9, respectively. No fluorescent signal was detectedwhen analyzing mtDNA or nDNA purified from the liver, lung, orkidney of sham-exposed rats receiving an i.p. injection of solvent. Fig.

1 shows an example of the results obtained at SFS by testing the livermtDNA from sham-exposed rats or liver nDNA and mtDNA fromBP-treated rats, displaying peaks of fluorescence with a maximum

excitation peak at 351 nm.When liver mtDNA from BP-treated rats was assayed by 32P

postlabeling, three distinct adducts were detected, the patterns ofwhich were identical to those produced by the corresponding nDNAsamples, although the latter yielded a weaker signal (Fig. 2). Assuggested by its Chromatographie mobility (21), the major adduci(Fig. 2, no. 2) is likely to correspond to N2-[\Oß-(lß.&a,9a-tf\\iy-

droxy-7,8,9,10-telrahydrobenzo(£í)pyrene)yl]deoxyguanosine, whereasadduci no. 1 is presumably due to adduction to DNA of 4,5-epoxides of

monohydroxylated metabolites in position 9 or 12 of the BP molecule(21). An adduci having the same Chromatographie mobility as adduci no.3 has been reported to be formed only in nDNA after in vivo exposure toBP and lo be enhanced by ireaimenl wilh cylochrome P-450 inducers

(22).Exposure to 2AAF and Co-Treatment with W-Acetylcysteine.

Administraron of 2AAF by gavage resulted in ihe formation of DNAadducls in rat liver, which were not deteclable in sham-exposed

Table 1 Adducls la nDNA and mtDNA in organs oj rats treated ivii/i three carcinogens and effect of co-trealment with NAC

Administered Co-treatment

agent with NAC RatorganBP

LiverLungKidney2AAF

Liver+LiverLiver+

LiverLiver+

LiverLiver+

LiverCigarette

smoke Liver+LiverLung+

LungMethodSFS6SFS11SFS*32P

postlabeling''(Total

adducts)Adduci1(dG-C8-AF)Adduci

2(dG-N2-AAF)Adduci

3(unknown)"P

postlabeling''(Total

adducts)"Ppostlabeling''(Tola!

adducts)Adduci

levels"nDNA47.1

±7.631.7±5.773.7

±6.2404

±29Noitested288

±37Nottested116±28Not

testedUndeteclableNot

tested0.

1 ±0.05Undeteclable4.8

±1.83.4±1.6mtDNA78.3

±19.661.8±6.4C102.4

±21.5856±177'626

±12/532±68'430

±126147±29127±47125±4897±191

.5 ±0.4'0.5±O.I711.

7±3.3'2.2±0.9^

" The results are means ±SD of three separate analyses.* Results expressed as fluorescence unils. dG-C8-AF, deoxyguanosine-CS-aminofluorene; dG-N2-AAF, deoxyguanosine-W2-acetylaminofluorene.' P < 0.01. mtDNA versus nDNA.''Results expressed as adducls/108 nucleotides.' P < 0.05. mtDNA versus nDNA.fP < 0.05. NAC+ versus NAC- (Student's / test).

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CHEMOPREVENTION OF ADDUCTS TO MITOCHONDRIA!. DNA

340 350 360 340 350 360 340 350 360

Excitation wavelength (nm)Fig. I. Examples of SFS spectra recorded with a sample of liver mtDNA from

sham-exposed rats M) and wilh samples of nDNA (B) or mtDNA (C) from BP-treated

rats.

animals. As shown in the example reported in Fig. 3, in addition totwo common adducts, 12P postlabeling analyses consistently detected

the presence of a third unidentified adduci to mtDNA that was absentin nDNA. According to their Chromatographie mobility (23), adducino. I is likely to correspond to deoxyguanosine-C8-AF, and adducino. 2 lo deoxyguanosine-A^-AAF. In addition to ihis qualitative

difference, the levels of adducls lo mlDNA were higher than those tonDNA, i.e., 1.9-fold higher for adduci no. 1 and 1.4-fold for adduci

no. 2. The lolal adduci levels were more lhan iwice in mlDNA.compared lo nDNA (Table 1).

The levels of DNA adducls were also measured in ihe liver mlDNAof 2AAF-treated rals receiving NAC wilh drinking waler. All ihree

adducls were decreased by adminislration of ihis ihiol, the prolectiveeffecl being slatistically significant when comparing total adducilevels and adduci no. 2 (Table 1).

Exposure to Cigarette Smoke and Co-Treatment with NAC.The whole-body exposure of rals lo mainslream cigarelte smoke for

100 conseculive days resulled in the appearance of adducts to liverand lung (Table 1), which were detectable by Ì2Ppostlabeling in Ine

form of lypical diagonal radioactive zones (Fig. 4). The presence ofadducls to liver nDNA was borderline to the sensitivity of the method.Adduci levels were considerably and significantly higher (P < 0.05)in the lung than in ihe liver for both nDNA (48-fold) and mtDNA(7.8-fold). Adducts to mtDNA were significantly higher (P < 0.05)than adducts to nDNA both in the liver (15-fold) and in the lung(2.4-fold).

Administration of NAC with drinking water attenuated the formation of smoke-related DNA adducts (Table 1; Fig. 4). In fact, adducls

to nDNA were no longer detectable in the liver and were decreased inthe lung, although such a difference did noi allain the statislicalsignificance threshold. Adducts to mtDNA were significantly lower(P < 0.05) in the liver (about 3-fold) and even lower in the lung(about 5-fold) of NAC-lreated rals, as compared lo the animals ex

posed to cigarette smoke without a chemoprevenlive agenl.

DISCUSSION

The preseni sludy led to several new findings, which may bearrelevance to the pathogenesis of cancer and other chronic degenera-

live diseases as well as to their prevenlion. As assessed by moleculardosimetry techniques, which can be also applied to human biomonitoring, adducts to mlDNA were found lo be formed in different organsof laboratory animals exposed lo individual DNA-binding carcino

gens of different chemical classes. Moreover, adducts to mtDNA weredetected in organs of rats exposed to cigarette smoke, a complexmixture associated with a variety of important degenerative diseases,which is the leading cause of death in developed countries. In allmonitored organs, adduci levels lo mlDNA were consistency higherthan those to nDNA of the same cells. Note that adduction to livermtDNA was not accompanied by changes in cytosine mcthylationpatterns, as shown by previously reported restriclion analyses of Ihesame specimens (19). Anolher novel finding of ihe present sludy waslhat the oral adminislration of a chemopreventive agenl was successful in attenualing ihese molecular biomarkers of biologically effectivedose.

In particular, adducts to both mtDNA and nDNA were higher in thelung than in the liver of rats exposed to cigarelte smoke, as docu-

Fig. 2. Example of 32P postlabeling of adducts to mtDNA

enriched by butanol extraction in the liver of rats receiving ani.p. injection of BP. The panel shows a tridimensional imageanalysis of an autoradiograph exposed for 8 h at -80°C. Thepossible nature of adducts /-.Õis specified in the text.

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CHEMOPREVENTION OF ADDUCTS TO MITOCHONDRIAL DNA

Fig. 3. Examples of '2P posllabeling of adducts to either

nDNA or mtDNA enriched by butano] extraction in the liver ofrats receiving 2AAF by gavage for S consecutive days. Thepanels show tridimensional image analyses of auloradiographsexposed for 6 h at —¿�80°C.The possible nature of adducts 1-3

is specified in the text.

merited by the formation of diagonal radioactive zones of differentintensity. These adduci patterns are typically detected by 12P postla-

beling in the nDNA of organs from humans and laboratory animalsexposed to complex mixtures, including cigarette smoke (24, 25). Inrats receiving an i.p. injection of BP. the rank of adduci levels waskidney>liver>lung, as assessed by SFS. a technique that specificallyevaluates the adduction of benzo(a)pyrene diolepoxide to DNA (18,26. 27). The selective localization of DNA adducts in different organsis the result of several factors including: (a) pharmacokinetics and. inparticular, "first-pass" effects of xenobiotics or their metabolites: (b)

local metabolism: (c) efficiency and fidelity of DNA repair; and (d)cell proliferation rate (14. 26). Thus, first-pass effects are likely to

account for the high levels of DNA adducts in the lung of rats exposedwhole-body to cigarette smoke, which mainly involves inhalation of

this complex mixture (27). It should be additionally noted that, compared to the lung, the liver has an outstanding metabolic capacity but.at the same time, is quite efficient at DNA excision repair, whichremoves DNA adducts (28).

32P postlabeling autoradiographs revealed that formation of adducts

to mtDNA and nDNA can differ not only quantitatively but alsoqualitatively. In fact, in addition to deoxyguanosine-C8-aminoflu-orene and deoxyguanosine-/V2-acetylaminofluorene, an unidentified

adduci very close to the latter one was detected in the liver mtDNAbut not in nDNA of 2AAF-treated rats. This mtDNA-specific adducimay be tentatively ascribed to some short-lived 2AAF metabolite

formed in proximity of mitochondria or even within these organdíes.The adduci levels were consistently higher to mtDNA than to

nDNA. in most cases, to a statistically significant extent, but theywere never dramatic, varying between a minimum of 1.4-fold (kidneyof BP-treated rats) and a maximum of 15-fold (liver of rats exposed to

cigarette smoke). To this respect, the data available in the literatureare rather controversial, depending on the experimental conditions andon the method used for the assessment of DNA binding, although theygenerally point to a greater capacity of genotoxic agents to bindmtDNA rather than nDNA. In fact, the earliest studies testing tritium-

labeled BP and other polycyclic aromatic hydrocarbons in cultured

mammalian cells showed that covalent binding was greater to mtDNAthan to nDNA by a factor ranging between 4 to over 500 (29-31 ). In

rats exposed to tritiated aflatoxin B,. adduction to liver mtDNA was3-4 times higher than adduction to liver nDNA (32), and the local

ization of the aflatoxin Brguanosine adduci was also found to beseveralfold greater in liver mitochondria than in nuclei, as evalualedby morphomelric analyses (33). Binding of radioactive /V-methyl-yV-niirosurea was, on ihe average, 5-fold higher than binding to nDNA.

both in vitro and in vivo (34). Moreover, liver mtDNA was alkylaledmore extensively than nDNA following administration of dimethylni-

trosamine (34, 35). whereas alkylation of mtDNA and nDNA wassimilar following administration of methyl methanesulfonate (36). Byusing 12Ppostlabeling, adducts to mtDNA in the liver of rats receiving

the food-borne heterocyclic amines IQ and PhIP by gavage were not

higher than those to nDNA. excepting the group of animals receivingmultiple doses of PhIP (37). In mice receiving i.p. injections of theheterocyclic aromatic carcinogen 7//-dibenzo[i-,^]carbazole or its me

tabolites, the mtDNA:nDNA binding index was lower than 1 (38). Apreferential binding of cisplatin to the mtDNA of CHO cells, accounting for a 6-fold higher incorporation compared to nDNA, was as

sessed by using a fluoroimmunoassay procedure (39).Under our test conditions, no mtDNA or nDNA adduci was de

tected in the organs of sham-exposed rats. Of particular interest is thepossibility of revealing, by means of suitable modifications of 12P

posllabeling, ihe so-called I-compounds (40). which are likely to be

associated with DNA damage caused by reactive oxygen species (41,42) and accumulate in an age-dependent manner in both nDNA andmtDNA (41). Most I-compounds. possibly originated from extrami-

lochondrial sources, are common lo mtDNA and nDNA, but a clusterof compounds, referred to as M-compounds, are mitochondria specific, presumably arising from DNA-reactive electron carriers of themitochondria! electron-transport chain (41).

In spite of its small size, mtDNA is a highly critical target becauseof the extraordinary importance of ihe biochemical processes accomplished in these organelles. Therefore, covalent modifications of mtDNAare likely lo be an important step as a mechanism involved in aging.

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CHHMOPREVENTION OF ADDUCTS TO MITOCHONDRIA!, DNA

Fig. 4. Examples of 12Ppostlabeling of adducls lo either

nDNA or mtDNA enriched by hutanol extraction in thelung and liver of rats exposed whole-body for 100 consecutive days to mainsiream cigarette smoke, as related to theadministration of the chemopreventive agent NAC withdrinking water. The panels show tridimensional imageanalyses of autoradiographs exposed for 48 h (lung) or 12h (liver) at -80°C. As discussed in the text, the adducts

tend to form diagonal radioactive zones.

cancer, and a variety of other chronic degenerative diseases (see "Introduction"). Primary prevention of these modifications can be pursued

primarily by avoiding exposure to genotoxic agents as much as possible.Besides this prescriptive approach, growing attention is paid to thepossibility of preventing chronic degenerative diseases via a prescriptiveapproach, i.e., by favoring the intake of protective factors and by fortifying the physiological defense mechanisms of the host organism (14).Previous studies performed in our laboratory showed that the oral administration of NAC can significantly decrease the formation of adductsto nDNA and specifically: (a) in the liver of rats receiving 2AAF with thediet; (b) in the liver, lung, heart, and testis of rats receiving intrathecal

instillations of BP; (c) in the liver, lung, trachea! epithelium, heart, aorta,testis. and kidney of rats exposed to cigarette smoke (14. 18. 26,43); and(d) in the lung of rats receiving intratracheal instillations of air paniculateextracts from a polluted environment (44).

Adducts to nDNA are usually associated with the earliest steps of thecarcinogenesis process, but according to our working hypothesis theyalso may be associated with other chronic degenerative diseases, e.g.,with cardiomyopathies when they are localized in the heart and with theatherogenetic process when they are locali/ed in the aorta, as inferredfrom studies not only in rodents (14, 18. 26) but also in humans (45). Theherein-reported decrease by NAC of adducts to mtDNA in the liver of

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CHEMOPREVEN'TION OF ADDUCTS TO \IITOCHONDRIAL DNA

rats treated with 2AAF and the sharp decrease in the lung and liver ofsmoke-exposed rats confirm the broad-spectrum protective potentiality of

this chemopreventive agent and provide evidence that it is feasible toattenuate chemically induced molecular alterations of mtDNA by meansof a pharmacological agent.

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1996;56:1642-1647. Cancer Res   Roumen Balansky, Alberto Izzotti, Leonardo Scatolini, et al.   -Acetylcysteine of Adducts to Mitochondrial DNA in Rat Organs

NInduction by Carcinogens and Chemoprevention by

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