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.
Exposure of male Balb/C mice to mainstream cigarette smoke for 4 months, starting 10 or 30 days before the administration of ethyl carbamate (0.3% in drinking water for 3 weeks), resulted in an up to ...57.6% (
P < 0.05) decrease of lung adenoma multiplicity. However, the number of ethyl carbamate-induced lung tumors was not significantly affected by exposure to cigarette smoke when ethyl carbamate was injected i.p. in single doses of 0.5 or l.0 g/kg, irrespective of the different treatment schedules used, i.e. (a) 10 days before and 4 days after the ethyl carbamate injection; (b) throughout the experiment starting 10 days before the ethyl carbamate injection, and (c) until the end of the experiment, starting 30 days after the ethyl carbamate injection. Disulfiram (500 mg/kg), given by gavage 24 h and 1 h before the ethyl carbamate injection, decreased by 88.5% (
P < 0.001) the multiplicity of lung adenomas but had no effect on tumorigenesis when administered after the carcinogen injection. Proadifen (SKF-525 A, 50 mg/kg) injected i.p. 24 h and 1 h before and 24 h and 48 h after the injection with ethyl carbamate tended to decrease the multiplicity of lung adenomas, but not to a significant extent. Furthermore, disulfiram given 24 h and 1 h before the i.p. administration of ethyl carbamate completely prevented its clastogenicity in mouse bone marrow. On the other hand, cigarette smoke, which was per se a weak clastogen in bone marrow erythroblasts, synergistically potentiated the clastogenic response to ethyl carbamate in a more than additive fashion.
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.
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 benzoapyrene 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 benzoapyrene, 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.
In addition to possessing an extraordinary sweeping activity, pulmonary alveolar macrophages (PAM) are equipped with the biochemical mechanisms involved in the metabolism of carcinogens, which were ...found to be inducible in humans by cigarette smoke. Moreover, several defense processes were stimulated in rat PAM after in vivo administration of the anticarcinogen N-acetylcysteine (NAC). Benzoapyrene diol epoxide (BPDE)-DNA adducts, as revealed by synchronous fluorescence spectrophotometry, were selectively detected in PAM of smokers and persisted up to 6 months. The amount of adducts was significantly correlated with the number of currently smoked cigarettes but not with the cigarettes smoked in a lifetime (pack-years). Nevertheless, deviations from the regression line pointed out the role of interindividual variability factors in adduct formation. Probably due to the low mitotic rate of PAM in the respiratory lumen, the frequency of micronuclei was not enhanced in smokers. However, parallel assays in rats showed that micronuclei can be enhanced after massive intratracheal administration of benzoapyrene or whole-body exposure to high amounts of mainstream cigarette smoke, which also induced BPDE-DNA adducts in lung cells and other organs, including the heart. All these adverse effects were markedly inhibited by the oral administration of NAC, which provides the premise and rationale for a future study on the protective effects of oral NAC in heavy smokers.
A series of experiments was carried out to assess cytotoxic and cytogenetic effects in bone marrow polychromatic erythrocytes (PCE) and pulmonary alveolar macrophages (PAM) resulting from individual ...or combined exposure of male BD6 rats to ethanol, cigarette smoke and Aroclor 1254. Addition of 5% ethanol to drinking water did not affect the micronucleus frequency but consistently enhanced the proportion of polynucleated PAM. Moreover, the higher concentration used (10%) was cytotoxic in the bone marrow. Whole-body exposure to cigarette smoke elevated the micronucleus frequency in both PCE (4.0-4.4-fold) and PAM (2.0-3.6-fold) and enhanced the frequency of polynucleated PAM. After 3 weeks of combined exposure, ethanol produced contrasting effects in smoke-exposed rats, i.e. an increase of micronuclei in PCE and a decrease in PAM. An i.p. injection of Aroclor 1254 was per se devoid of any influence on the monitored parameters but tended to attenuate the cytotoxic and cytogenetic changes produced by cigarette smoke or ethanol in both types of cell.
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