Induction by carcinogens and chemoprevention by N-acetylcysteine of adducts to mitochondrial DNA in rat organs.

Damage to mitochondrial DNA (mtDNA) has been postulated to be associated with aging, cancer, and other chronic degenerative diseases which are the predominant causes of death in the population. We used molecular dosimetry techniques, i.e., 32P postlabeling and synchronous fluorescence spectrophotometry, in order to evaluate the formation of adducts to both mtDNA and nuclear DNA (nDNA) in different organs of rats exposed to genotoxic carcinogens. Adducts to mtDNA were detected following administration of benzo(a)pyrene i.p. or 2-acetylaminofluorene by gavage as well as following exposure of animals to cigarette smoke. mtDNA adduct levels were consistently higher than those to nDNA in the same cells, and qualitative differences were also pointed out in the case of the aromatic amine. The oral administration of the thiol N-acetylcysteine, one of the most promising cancer chemopreventive agents endowed with nucleophilic and antioxidant properties, produced a significant decrease of mtDNA adducts in the liver of 2-acetylaminofluorene-treated rats and, sharply, in the lung and liver of rats exposed to cigarette smoke.

Chemoprevention of carcinogen-DNA adducts and chronic degenerative diseases.

Molecular dosimetry techniques were exploited in order to assess the efficacy of experimental chemoprevention assays and to evaluate the involvement of DNA alterations, not only in cancer but also in other chronic degenerative diseases. In agreement with other protective effects previously observed in the same animal models, the thiol N-acetylcysteine (NAC) totally prevented or significantly reduced the formation of carcinogen-DNA adducts in three experimental systems in rats. Thus, as assessed by 32P postlabeling, supplement of the diet with NAC decreased both deoxyguanosine-C8-aminofluorene adducts (butanol enrichment) and deoxyguanosine-N2-acetylaminofluorene adducts (nuclease P1 enrichment) formed in rat liver following dietary administration of 2-acetylaminofluorene for 3 weeks. DNA adducts were detected by synchronous fluorescence spectrophotometry in rat liver, lung, heart, and testis following a daily i.t. instillation of benzo(a)pyrene for 3 consecutive days. 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 adduct was detected by synchronous fluorescence spectrophotometry in liver, brain, and testis. Multiple DNA adducts in the aorta were also measured by 32P postlabeling. Administration of NAC by gavage inhibited the formation of DNA adducts in all organs of rats treated with benzo(a)pyrene or exposed to cigarette smoke. It is of interest that a single chemopreventive agent can display a broad-spectrum protective ability. The selective localization of DNA adducts in different organs depends on pharmacokinetics, metabolic capacity, DNA repair efficiency, and cell proliferation rate. Whereas inhibition by NAC of DNA adducts in testis can be correlated with its demonstrated ability to prevent dominant lethal mutations, we raise the hypothesis that DNA adducts in lung, heart, and aorta may be pathogenetically associated with lung cancer, cardiomyopathies, and arteriosclerosis, respectively. In order to explore the involvement of molecular and biochemical alterations in human arteriosclerosis, we started an extensive collaborative project and report here preliminary data showing the presence of DNA adducts in aorta smooth muscle cells obtained from arteriosclerotic patients.

In vitro inhibition by N-acetylcysteine of oxidative DNA modifications detected by 32P postlabeling.

Reactive oxygen species are involved in the pathogenesis of cancer and other chronic degenerative diseases through a variety of mechanisms, including DNA damage. We investigated by 32p and 33P postlabeling analyses the nucleotidic modifications induced in vitro by treating calf thymus DNA with H2O2 and CuSO4, interacting in a Fenton type reaction. Six different enrichment procedures and three chromatographic systems were comparatively assayed. The chromatographic system using phosphate/urea, which is more suitable for detecting bulky DNA adducts, was rather insensitive. In contrast, the system using acetic acid/ammonium formate revealed high levels of mononucleotidic modifications. In terms of ratio of adduct levels in treated and untreated DNA, the enrichment procedures ranked as follows: nuclease P1 (19.6), no enrichment (18.3), digestion to trinucleotides (17.6), digestion to monophosphate mononucleotides (8.4), digestion to dinucleotides (3.4), and extraction with butanol (<1.0). The system using formic acid/ammonium formate was quite efficient in detecting 8-hydroxy-2'-deoxyguanosine. Labeling with 33p further enhanced the sensitivity of the method. The oxidative damage was so intense to produce a strong DNA fragmentation detectable by agarose gel electrophoresis, and nucleotidic modifications were more intense when DNA fragmentation was greater. The DNA alterations produced by H2O2 alone were significantly lower than those produced following reaction of H2O2 with CuSO4. The thiol N-acetylcysteine (NAC) was quite efficient in inhibiting both nucleotidic modifications and DNA fragmentation produced in vitro by either H2O2 or the .OH generating system. These results support at a molecular level the findings of previous studies showing the ability of NAC to inhibit the genotoxicity of peroxides and of reactive oxygen species generated by electron transfer reactions.

Enhanced levels of DNA adducts in the liver of woodchucks infected with hepatitis virus.

Liver DNA specimens from woodchucks kept in captivity, 10 naturally infected with hepatitis virus (WHV) and five WHV-free, were examined for the presence of carcinogen-DNA adducts by 32P-postlabeling. The number of adducts was significantly higher in WHV carriers than in uninfected animals, and the total amounts of adducts per 10(9) nucleotides were also considerably enhanced by WHV infection, when using both butanol extraction (22.2 +/- 7.1 vs. 12.6 +/- 2.8, means +/- S.D.) and nuclease P1 enrichment (8.5 +/- 5.9 vs. 2.8 +/- 1.7). Two individual adducts were also significantly higher in WHV carriers. No significant variation occurred as related to age, sex or time length of captivity. These findings are consistent with our previous studies supporting an enhanced metabolism of chemical hepatocarcinogens in both human and woodchuck hepadnavirus infections. Several significant and remarkable correlations were pointed out by relating DNA adduct data to more than 30 virological, histopathological and metabolic parameters which had been previously evaluated in the same animals. For instance, numbers and/or levels of adducts were positively related to the amounts of virus present in hepatocytes, to cell damage (gamma-glutamyltranspeptidase activity), to the severity of the liver histopathological picture, and to monooxygenase activities, while they were inversely related to cellular glutathione concentrations and to detoxification of the direct-acting mutagen 4-nitroquinoline 1-oxide. The major adduct significantly correlated with the metabolic activation of the aromatic amine 2-aminofluorene and of the heterocyclic amines 3-amino-1-methyl-5H-pyrido(4,3)indole (Trp-P-2) and 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ), whereas another adduct significantly correlated with the metabolic activation of the mycotoxin aflatoxin B1. Thus, the enhanced metabolism of chemical hepatocarcinogens and the increased formation of carcinogen-DNA adducts in the liver of WHV carriers appear to represent one of the mechanisms contributing to the association between chronic hepadnavirus infection and development of primary hepatocellular carcinoma.

Inhibition of the 'spontaneous' mutagenicity in Salmonella typhimurium TA102 and TA104.

Thirty-four compounds belonging to various chemical classes were assayed for the ability to modulate the 'spontaneous' mutagenicity in strain TA104 of S. typhimurium, and 17 of them were also assayed in TA102. All test agents, many of which were already known or suspected to act as inhibitors of induced mutagenicity, had been previously monitored in our laboratory for antimutagenicity towards either 4-nitroquinoline 1-oxide in TA100 and/or cigarette smoke in TA98 with S9 mix. A considerable proportion of test compounds decreased the number of spontaneous revertants in TA104 (44.1%) and/or TA102 (41.2%) to a significant extent, with dose-related and reproducible effects. In almost all cases the antimutagenic effect was genuine and not related to bacterial killing or growth inhibition. The results obtained suggest that the DNA repair background plays a prominent role in the genesis of spontaneous mutations in these strains, containing the hisG428 mutation which is typically reverted by oxidative mutagens. Due to its theoretical and practical implications, the finding that several chemopreventive agents can attenuate the rate of spontaneous reversion deserves attention.

[Research on adducts of benzo(a)pyrene and DNA in different cells using synchronous fluorescence spectrophotometry]. / Ricerca di addotti fra benzo(a)pirene diolepossido e DNA in differenti cellule mediante spettrofotometria a fluorescenza sincrona.

The levels of benzo(a)pyrene diolepoxide(BPDE)-DNA adducts were measured in rat and human cells by synchronous fluorescence spectrophotometry. BPDE-DNA adducts detected in human pulmonary alveolar macrophages were related to current smoking habits, in contrast to the adducts found in peripheral blood leukocytes. BP administration to rats produced BPDE-DNA adducts in both liver and lungs. Although small yet repeatable signals were also detected in lung DNA from rats treated for 3 days with tobacco smoke. None of the samples obtained from untreated animals was positive. The detection of BPDE-DNA adducts may be used in biomonitoring and experimental studies for determining of exposure to BP even when applied as a constituent of complex mixtures.

[Chemoprevention of genotoxic damage in lung cells of rats exposed to cigarette smoke]. / Chemioprevenzione del danno genotossico in cellule polmonari di ratti esposti a fumo di sigaretta.

Male Sprague-Dawley rats were exposed to cigarette smoke (CS) according to a complex protocol which lasted 40 days. Some of the groups were pre-treated with N-acetyl-L-cysteine (NAC) by gavage (1 g/kg b.w.) 5 h before each exposure. Bronchoalveolar lavage was performed in each animal at the end of each exposure period in order to recover pulmonary alveolar macrophages (PAM). Cells were identified and counted under the microscope, and the number of micronucleated (MN) and binucleated (BN) PAM was registered. The results showed an increase in the number of MN PAM, which was already evident after 8 days of CS exposure; this increase remained constant after 28 and 40 days of exposure. A significant decrease in the number of MN PAM was observed in the animals pre-treated with NAC. BN PAM were significantly increased after 28 and 40 days of exposure; again, a slight yet not significant decrease was detected in NAC-pre-treated animals. On the whole, this study demonstrates that CS is clearly clastogenic to alveolar macrophages and that NAC can efficiently prevent this cytogenetic damage.

Post-mortem stability of benzo[a]pyrene diolepoxide--DNA adducts in rat organs.

In order to evaluate the stability of benzo[a]pyrene diolepoxide--DNA adducts two separate studies were carried out in rats, either treated i.p. with benzo[a]pyrene (100 mg/kg body wt) or sham-exposed. The measurement of DNA adducts in 155 samples of liver, lung or heart, each of them tested in duplicate, was performed by synchronous fluorescence spectrophotometry. In the first study fragments of rat liver or lung were stored for varying times at varying temperatures. No decrease of adduct levels occurred at 4 degrees C for at least 72 h, whereas a significant decrease was recorded in both liver and lung after 48 h at 20 degrees C or 24 h at 37 degrees C. In the second study liver, lungs and heart were collected from rats either immediately after killing or after storage of cadavers for 16 h at 20 degrees C or 16 h at 20 degrees C plus 24 h at 4 degrees C, thereby mimicking typical storage conditions of human cadavers before autopsy. Under these conditions no significant variation of fluorescent adducts was observed in any organ. In conclusion, at least for this kind of adduct, the use of autopsy samples following proper storage of cadavers seems to be acceptable.

Inhibition by N-acetylcysteine of carcinogen-DNA adducts in the tracheal epithelium of rats exposed to cigarette smoke.

The ability of the aminothiol N-acetylcysteine (NAC) to prevent the formation of carcinogen-DNA adducts in tracheal epithelial cells was investigated in Sprague-Dawley rats exposed whole-body to mainstream cigarette smoke for either 40 or 100 consecutive days. 32P-Postlabelling analyses showed the occurrence of DNA adducts (12.49 adducts/10(8) nucleotides) after 40 days of exposure, with a trend to formation of characteristic diagonal radioactive zones. Total adduct levels were not further enhanced after 100 days of exposure to smoke, although significant changes occurred in the amounts of individual adducts. NAC, given by gavage in the 40 day study and in drinking water in the 100 day study, significantly inhibited the formation of smoke-related carcinogen-DNA adducts in the tracheal epithelium, to such an extent that adduct levels were not significantly higher than those detected in sham-exposed control rats. Together with a variety of other molecular, clastogenicity, metabolic, cytological and histopathological end-points investigated in rodents and with the preliminary evidence arising from a study in humans, these results document the considerable efficacy of oral NAC in inhibiting smoke-related carcinogen-DNA adducts.

Chemoprevention of smoke-related DNA adduct formation in rat lung and heart.

The formation of smoke-related DNA adducts and their chemoprevention were investigated in tissues of male Sprague-Dawley rats, by testing a total of 132 DNA samples by synchronous fluorescence spectrophotometry (SFS), which mainly detects benzo[a]pyrene diolepoxide (BPDE)-DNA adducts. Groups of six animals each were exposed whole-body to mainstream cigarette smoke, once daily, for up to 40 consecutive days. No adduct was revealed in liver DNA, whereas smoke-related DNA adducts were detectable in the lung from the 8th day of exposure and continued to increase until the 40th day. Adducts to heart DNA, which were monitored after 28 and 40 days of exposure, attained even higher levels than those detected in the lungs of the same animals. A high correlation existed between the amounts of smoke-related DNA adducts measured in these two organs. The daily administration by gavage of the thiol N-acetyl-L-cysteine (NAC), an effective mutation and cancer chemopreventive agent, which had been previously shown to inhibit the formation of SFS-positive DNA adducts in rats receiving intratracheal instillations of benzo[a]pyrene, significantly prevented occurrence of the same adducts in both heart and lungs of smoke-exposed rats. No fluorescence signal was observed in liver, lung, or heart DNA of sham-exposed animals. The findings of this molecular dosimetry study complement the results of parallel histopathologic, cytogenetic, biochemical and metabolic analyses of tissues and cells from the same rats, providing evidence for a variety of significant alterations produced by exposure to cigarette smoke and for the specific protective role of NAC.

Adducts to nuclear DNA and mitochondrial DNA as biomarkers in chemoprevention.

DNA adducts are biomarkers evaluating the biologically effective dose of carcinogens, which reflects, more realistically than the external exposure dose, an enhanced risk of developing cancer. Likewise, inhibition of DNA adduct formation can be assumed as an indicator of decreased risk. Molecular dosimetry techniques can be exploited in anticarcinogenicity studies in animal models as well as in Phase II clinical chemoprevention trials. We have extensively used these end points in animal studies using individual carcinogens and complex mixtures. As assessed by 32P-postlabelling, DNA adducts were formed in the liver of rats fed a diet supplemented with 2-acetylaminofluorene. DNA adducts were detected by synchronous fluorescence spectrophotometry (SFS) in rat liver, lung, heart and testis following intratracheal (l.t) instillations of benzo[a]pyrene. The whole-body exposure of rats to mainstream cigarette smoke resulted in the appearance of DNA adducts in lung, heart, aorta and kidney, whereas adducts were not detected by SFS in liver, brain, oesophagus and testis. Moreover, typical diagonal radioactive zones and multiple DNA adducts were revealed by 32P-postlabelling in the tracheal epithelium, nasal mucosa, aorta and testis of smoke-exposed rats. Formation of adducts to lung DNA, as assessed by both 32P-postlabelling and SFS, also occurred in rats receiving i.t. instillations of air particulate extracts from polluted urban and rural areas. The oral administration of the thiol N-acetylcysteine (NAC) significantly inhibited the formation of DNA adducts in all organs of the rats exposed to the aforementioned carcinogens, which correlated with the parallel inhibition of biochemical, cytogenetic and histopathological alterations as well as with inhibition of preneoplastic and neoplastic lesions in rodents. Our working hypothesis is that DNA adducts in trachea/lung, heart and aorta may be associated with lung cancer, cardiomyopathies and atherosclerosis, respectively. DNA adducts were consistently detectable in the DNA of smooth muscle cells from abdominal aorta specimens taken at surgery from atherosclerotic patients. Even broader are the consequences of mitochondrial (mt) DNA impairment, which has been associated with aging, cancer, and other degenerative diseases. Our data show that adduct levels are consistently higher in mtDNA than in the nuclear DNA in different organs of rats exposed either to benzo[a]pyrene, 2-acetylaminofluorene or cigarette smoke. NAC significantly decreased the formation of adducts to mtDNA when administered with drinking-water. Inhibition of adducts to nuclear DNA is one of the end points evaluated in ongoing Phase II chemoprevention trials in high-risk individuals.

Chemopreventive properties and mechanisms of N-Acetylcysteine. The experimental background.

The thiol N-acetylcysteine (NAC), now under clinical trial for cancer chemoprevention both in Europe (project Euroscan) and in the US (National Cancer Institute), has been shown during the past decade to exert protective effects in a variety of experimental test systems. NAC inhibited spontaneous mutagenicity and that induced by a number of chemical compounds and complex mixtures. Moreover, NAC significantly decreased the incidence of neoplastic and preneoplastic lesions induced by several chemical carcinogens in rodents (mice, rats, hamsters), e.g., in lung, trachea, colon, liver, mammary gland, Zymbal gland, bladder and skin. Our studies provided evidence that multiple mechanisms contribute to NAC antimutagenicity and anticarcinogenicity. They include extracellular mechanisms, such as detoxification of reactive compounds due to the nucleophilic and antioxidant properties of NAC, inhibition of nitrosation products, and enhancement of thiol concentration in intestinal bacteria; trapping and enhanced detoxification of carcinogens in long-lived non-target cells, such as erythrocytes and bronchoalveolar lavage cells; mechanisms working in the cytoplasm of target cells, such as replenishment of GSH stores, modulation of metabolism of mutagens/carcinogens, blocking of electrophiles, and scavenging of reactive oxygen species; and nuclear effects, such as inhibition of DNA adduction by metabolites of carcinogens, inhibition of "spontaneous" mutations, attenuation of carcinogen-induced DNA damage, and protection of nuclear enzymes, such as poly(ADP-ribose) polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)