Transplacental antioxidants inhibit lung tumors in mice exposed to cigarette smoke after birth: a novel preventative strategy?

Birth is characterized by an intense oxidative stress resulting in nucleotide alterations and gene overexpression in mouse lung. We showed that cigarette smoke (CS) is carcinogenic when exposure starts soon after birth and applied this bioassay to evaluate the efficacy of chemopreventive agents. The present study evaluated whether administration of the antioxidants N-acetyl-L-cysteine (NAC) and vitamin C or ascorbic acid (AsA) during pregnancy can protect strain H Swiss mice exposed to CS after birth. Exposure to CS, for 4 months, of newborns from untreated mice resulted in significant alterations at 8 months of life, including alveolar epithelial hyperplasia, emphysema, blood vessel proliferation, microadenomas, adenomas, and malignant tumors in lung, liver parenchymal degeneration and urinary bladder epithelium hyperplasia. Treatment throughout pregnancy with either NAC, a scavenger of reactive oxygen species, or AsA, an electron donor, did not affect fertility, parity, and body weight of newborns. Prenatal antioxidants significantly inhibited most lesions in adult mice exposed to CS since birth. For instance, the incidence of emphysema was reduced from 27.5% in CS-exposed mice that were untreated during pregnancy to 7.1% and 14.0% in those treated prenatally with NAC and AsA, respectively. Lung adenomas were reduced from 34.8% to 16.7% and 9.3%, respectively. Malignant lung tumors were reduced from 13.0% to 4.7% by prenatal AsA. Liver parenchymal degeneration was reduced from 58.0% to 14.3% by prenatal NAC. These data mechanistically support a "transplacental chemoprevention" strategy, aimed at protecting the newborn from oxidative stress and the adult from CS-related diseases appearing later in life.

Multiple points of intervention in the prevention of cancer and other mutation-related diseases.

Multiple points of intervention are the target for dietary and pharmacological interventions aimed at preventing cancer and other diseases in which mutations in somatic cells play a pathogenetic role. For instance, our studies showed that DNA adducts can be consistently detected in arterial smooth muscle cells from human atherosclerotic lesions. Their levels were significantly correlated with the occurrence of atherogenic risk factors known from traditional epidemiology and were strikingly enhanced in atherosclerotic patients lacking the GSTM1 genotype. Cancer chemoprevention has a dual goal, i.e. prevention of occurrence of the disease (primary prevention) and early detection and reversion of tumors at a premalignant stage (secondary prevention). At a later stage, attempts can be made to prevent local recurrences as well as invasion and metastasis of malignant cells (tertiary prevention). For a rational use of chemopreventive agents it is essential not only to evaluate their efficacy and safety but also to understand the mechanisms involved. Sometimes it is difficult to discriminate whether modulation of a given end-point is actually a specific mechanism or rather the epiphenomenon of other events. For instance, we recently found that apoptosis is considerably stimulated in the respiratory tract of smoke-exposed rats; whereas certain chemopreventive agents work by further enhancing smoke-related apoptosis, other agents appear to downregulate apoptosis simply because they inhibit the genotoxic events signaling this process. We propose here a detailed, updated classification of the points of intervention exploitable in the prevention of mutation and cancer. The general outline includes a variety of extracellular and cellular mechanisms modulating the genotoxic response and tumor initiation as well as tumor promotion, progression, angiogenesis, invasion, and metastasis. This classification is not intended to provide a rigid scheme, since several intervention points are reiterated several times over different phases of the process. Moreover, some mechanisms are strictly interconnected or partially overlapping. Interestingly, a number of chemopreventive agents work through multiple mechanisms, which warrants a higher efficacy and a broader spectrum of action. It is also convenient to combine chemopreventive agents working through complementary mechanisms. In recent preclinical studies, we observed that combination of N-acetylcysteine with either oltipraz or ascorbic acid produces additive or more than additive protective effects towards early biomarkers and/or experimentally-induced tumors.

Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points.

Although smoking cessation is the primary goal for the control of cancer and other smoking-related diseases, chemoprevention provides a complementary approach applicable to high risk individuals such as current smokers and ex-smokers. The thiol N-acetylcysteine (NAC) works per se in the extracellular environment, and is a precursor of intracellular cysteine and glutathione (GSH). Almost 40 years of experience in the prophylaxis and therapy of a variety of clinical conditions, mostly involving GSH depletion and alterations of the redox status, have established the safety of this drug, even at very high doses and for long-term treatments. A number of studies performed since 1984 have indicated that NAC has the potential to prevent cancer and other mutation-related diseases. N-Acetylcysteine has an impressive array of mechanisms and protective effects towards DNA damage and carcinogenesis, which are related to its nucleophilicity, antioxidant activity, modulation of metabolism, effects in mitochondria, decrease of the biologically effective dose of carcinogens, modulation of DNA repair, inhibition of genotoxicity and cell transformation, modulation of gene expression and signal transduction pathways, regulation of cell survival and apoptosis, anti-inflammatory activity, anti-angiogenetic activity, immunological effects, inhibition of progression to malignancy, influence on cell cycle progression, inhibition of pre-neoplastic and neoplastic lesions, inhibition of invasion and metastasis, and protection towards adverse effects of other chemopreventive agents or chemotherapeutical agents. These mechanisms are herein reviewed and commented on with special reference to smoking-related end-points, as evaluated in in vitro test systems, experimental animals and clinical trials. It is important that all protective effects of NAC were observed under a range of conditions produced by a variety of treatments or imbalances of homeostasis. However, our recent data show that, at least in mouse lung, under physiological conditions NAC does not alter per se the expression of multiple genes detected by cDNA array technology. On the whole, there is overwhelming evidence that NAC has the ability to modulate a variety of DNA damage- and cancer-related end-points.

Modulation of biomarkers by chemopreventive agents in smoke-exposed rats.

Chemoprevention opens new perspectives in the prevention of cancer and other chronic degenerative diseases associated with tobacco smoking, exploitable in current smokers and, even more, in exsmokers and passive smokers. Evaluation of biomarkers in animal models is an essential step for the preclinical assessment of efficacy and safety of potential chemopreventive agents. Groups of Sprague Dawley rats were exposed whole body to a mixture of mainstream and sidestream cigarette smoke for 28 consecutive days. Five chemopreventive agents were given either with drinking water (N-acetyl-L-cysteine, 1 g/kg body weight/day) or with the diet (1,2-dithiole-3-thione, 400 mg; Oltipraz, 400 mg; phenethyl isothiocyanate, 500 mg; and 5,6-benzoflavone, 500 mg/kg diet). The monitored biomarkers included: DNA adducts in bronchoalveolar lavage cells, tracheal epithelium, lung and heart; oxidative damage to pulmonary DNA; hemoglobin adducts of 4-aminobiphenyl and benzo(a)pyrene-7,8-diol-9,10-epoxide; micronucleated and polynucleated alveolar macrophages and micronucleated polychromatic erythrocytes in bone marrow. Exposure of rats to smoke resulted in dramatic alterations of all investigated parameters. N-Acetyl-L-cysteine, phenylethyl isothiocyanate, and 5,6-benzoflavone exerted a significant protective effect on all alterations. 1,2-Dithiole-3-thione was a less effective inhibitor and exhibited both a systemic toxicity and genotoxicity in alveolar macrophages, whereas its substituted analogue Oltipraz showed limited protective effects in this model. Interestingly, combination of N-acetyl-L-cysteine with Oltipraz was the most potent treatment, resulting in an additive or more than additive inhibition of smoke-related DNA adducts in the lung and hemoglobin adducts. These results provide evidence for the differential ability of test agents to modulate smoke-related biomarkers in the respiratory tract and other body compartments and highlight the potential advantages in combining chemopreventive agents working with distinctive mechanisms.

Modulation of apoptosis by cigarette smoke and cancer chemopreventive agents in the respiratory tract of rats.

Preclinical studies may elucidate the meaning of biomarkers applicable to epidemiologic studies and to clinical trials for cancer prevention. No study has explored so far the effect of cigarette smoke on apoptosis in vivo. We evaluated modulation of apoptosis in cells of the respiratory tract of smoke-exposed Sprague-Dawley rats both by morphological analysis and TUNEL method. In a first study, exposure of rats to mainstream cigarette smoke for either 18 or 100 consecutive days produced a significant and time-dependent increase in the proportion of apoptotic cells in the bronchial and bronchiolar epithelium. Oral N:-acetylcysteine did not affect the background frequency of apoptosis but significantly and sharply decreased smoke-induced apoptosis. In a second study, exposure of rats to a mixture of sidestream and mainstream smoke for 28 consecutive days resulted in a >10-fold increase in the frequency of pulmonary alveolar macrophages undergoing apoptosis. Dietary administration of either 5,6-benzoflavone, 1,2-dithiole-3-thione or oltipraz did not affect the frequency of smoke-induced apoptosis, whereas phenethyl isothiocyanate produced a further significant enhancement. Again, N-acetylcysteine and its combination with oltipraz significantly decreased smoke-induced apoptosis. In both studies exposure to smoke resulted in a sharp increase of cells positive for proliferating cell nuclear antigen (PCNA), which was unaffected by the examined chemopreventive agents. These findings highlight the concept that modulation of apoptosis has diversified meanings. Different meanings (as explained in the following lines). First, the apoptotic process is triggered as a defense system against genotoxic agents, such as the components of cigarette smoke. The further induction produced by phenethyl isothiocyanate, favoring removal of damaged cells, represents an example of a detoxification mechanism. Inhibition of smoke-induced apoptosis by N:-acetylcysteine should be interpreted as an epiphenomenon of antigenotoxic mechanisms, as shown in parallel studies evaluating modulation of DNA alterations in the respiratory tract of the same animals. Thus, it is important to discriminate between whether the opposite modulation of apoptosis is per se a protective mechanism or the beneficial outcome of other mechanisms inhibiting genotoxicity.

Pilot studies evaluating the lung tumor yield in cigarette smoke-exposed mice.

In spite of the major role played by cigarette smoking in the epidemiology of lung cancer, it is very difficult to reproduce the carcinogenicity of this complex mixture in animal models. We implemented a series of pilot experiments in three mouse strains, exposed either to environmental cigarette smoke (ECS) or mainstream cigarette smoke (MCS) or its condensate (MCSC). The whole-body exposure of Aroclor-treated A/J mice to ECS resulted in a rapid and potent induction of micronuclei in peripheral blood erythrocytes. After 6 months of exposure, 6 h a day, followed by 4 months of recovery in filtered air, both lung tumor incidence and multiplicity were significantly increased as compared to sham-exposed mice (77.8% vs. 22.2%, and 1.11+/-0.26 vs. 0.22+/-0.15, means +/- SE). Multiple i.p. injections of butylated hydroxytoluene did not significantly enhance the tumor yield. Another experiment confirmed the responsiveness of A/J mice exposed to ECS for 5 months, followed by 4 months of recovery in air (75.0% vs. 25.0%, and 1.05+/-0.17 vs. 0.25+/-0.10). In contrast, the increase in lung tumor yield after exposure to ECS for 2 months, followed by recovery in air for 7 months, was not significant, and the continuous exposure to ECS for 9 months was totally ineffective. These data, in agreement with previous results of others, show that exposure of A/J mice to ECS for 5-6 months, followed by recovery in air for 4 months, is successful in inducing a weak but significant and reproducible increase in lung tumor yield. Furthermore, the simultaneous exposure to the light emitted by halogen quartz bulbs for 9 months and to ECS for 5 months, followed by 4 months in air, was again weakly tumorigenic (incidence of 55.0% and multiplicity of 0.75+/-0.19), whereas exposure to both ECS and light for 9 months was devoid of effect. The whole-body exposure of A/J mice to MCS, 1 h a day for 5 months, or weekly i.p. injections of MCSC for 5 months, followed in both cases by 4 months of recovery in air, failed to enhance the lung tumor yield. The whole-body exposure of SKH-1 hairless mice to ECS for 6 months, followed by exposure to halogen light for 8 months, resulted in the formation of multiple skin tumors but failed to produce lung tumors. The whole-body exposure of C57BL/6 mice to ECS for 6 months failed to induce any lung tumor but caused alopecia, gray hair, and hair bulb cell apoptosis, which were prevented by the oral administration of N-acetylcysteine.

Interactions between N-acetylcysteine and ascorbic acid in modulating mutagenesis and carcinogenesis.

Both ascorbic acid (AsA, vitamin C) and N-acetylcysteine (NAC), a precursor and analogue of glutathione, possess a broad array of biological properties underlying their protective role in a variety of pathophysiological conditions. However, under certain circumstances, AsA behaves as a pro-oxidant rather than an anti-oxidant and produces adverse effects. This prompted us to evaluate whether NAC could interact with AsA in preventing mutation and cancer. AsA significantly increased spontaneous revertants in the Salmonella typhimurium strains TA102 and TA104, which are sensitive to oxidative mutagens. In contrast, NAC lowered the spontaneous background in TA104 and neutralized the negative effects of AsA. Moreover, NAC and AsA showed additive effects in reducing chromium(VI) and in reverting its mutagenicity. A single i.p. injection of urethane (1 g/kg body weight) to 120 A/J mice resulted, after 4 months, in the formation of a total of 1,532 lung tumors, 425 in the 30 mice treated with the carcinogen only, 404 in those treated with urethane plus AsA, 365 in those treated with urethane plus NAC and 338 in those treated with urethane plus the combination of AsA and NAC (both given daily with drinking water at the dose of 1 g/kg body weight). Compared to positive controls, tumor multiplicity was poorly affected by AsA, whereas it was significantly decreased by NAC and even more so by its combination with AsA. The overall volumes of lung tumors in the 4 groups were 107.5, 89.3, 61.3 and 49.7 mm(3), respectively. Tumor sizes were slightly but significantly decreased in mice treated with AsA and more so in those treated with NAC and NAC plus AsA, their combination being significantly more effective than each individually. All protective effects elicited by combining the 2 drugs were additive. Therefore, NAC prevents the adverse effects of AsA on spontaneous mutagenicity; at the same time, this thiol behaves in an additive fashion with AsA, inhibiting the mutagenicity of chromium(VI) and the lung tumorigenicity of urethane in mice. These findings suggest that NAC and AsA could conveniently be combined in cancer chemoprevention and other pharmacological interventions.

Less than additive interaction between cigarette smoke and chromium(VI) in inducing clastogenic damage in rodents.

A combination of tobacco smoking with certain agents has been shown to exert synergistic carcinogenic effects. On the other hand, antagonism betweeen smoke and other pulmonary carcinogens has also been documented by both epidemiological and experimental data. In spite of a very large number of studies carried out for decades in workers exposed to hexavalent chromium, the influence of smoking habits on lung carcinogenesis induced by this metal has not been clarified. For this reason, we performed two studies evaluating clastogenic effects in rodents. In the first one, BDF(1) mice were exposed whole-body to mainstream cigarette smoke for 5 days and, on the last day, they received an i.p. injection of potassium dichromate. In the second study, Sprague-Dawley rats were exposed whole-body to environmental cigarette smoke for 18 consecutive days and for the same period of time they received daily intra-tracheal instillations of sodium dichromate. Individually, the two hexavalent chromium salts and cigarette smoke, either mainstream or environmental, enhanced the frequency of micronuclei in bone marrow polychromatic erythrocytes of both mice and rats. Moreover, individual exposure to either environmental cigarette smoke or sodium dichromate enhanced the frequency of micronuclei and multiple nuclei in pulmonary alveolar macrophages of rats. In both studies, combined exposure to cigarette smoke and hexavalent chromium produced less than additive clastogenic effects. These results are consistent with our previous data, showing that hexavalent chromium and either benzo[a]pyrene or cigarette smoke condensate behave antagonistically in in vitro mutagenicity test systems and that the chromium reducing capacity of human pulmonary alveolar macrophages and peripheral lung parenchyma is enhanced in smokers. Taken together, in the absence of any epidemiological evidence, these findings rule out any occurrence of synergism between cigarette smoke and hexavalent chromium, at least in certain stages of the carcinogenesis process.

Induction of alopecia in mice exposed to cigarette smoke.

Besides being responsible for a high proportion of those chronic degenerative diseases that are the leading causes of death in the population, tobacco smoking has been associated with skin diseases. Smoke genotoxicants are metabolized in hair follicle cells, where they form DNA adducts and cause DNA damage. The suspicion was raised that, in humans, a link may exist between smoking and both premature grey hair and hair loss. In order to check this hypothesis, we carried out a study in C57BL/6 mice exposed whole-body to a mixture of sidestream and mainstream cigarette smoke. After 3 months exposure, most mice developed areas of alopecia and grey hair, while no such lesions occurred either in sham-exposed mice or in smoke-exposed mice receiving the chemopreventive agent N-acetylcysteine with drinking water. Cell apoptosis occurred massively in the hair bulbs at the edge of alopecia areas. Smoke-exposed mice had extensive atrophy of the epidermis, reduced thickness of the subcutaneous tissue, and scarcity of hair follicles. On the whole, exposure to smoke genotoxic components appears to alter the hair cycle with a dystrophic anagen pattern. Although this mechanism is different from that of genotoxic cytostatic drugs, N-acetylcysteine appears to exert protective effects in both conditions.

Induction, persistence and modulation of cytogenetic alterations in cells of smoke-exposed mice.

In spite of the major role played by smoking tobacco in the epidemiology of chronic degenerative diseases, it is difficult to mimic the genotoxic and carcinogenic effects of this complex mixture in animal models. We undertook an experimental study evaluating the time-course induction, persistence and modulation of cytogenetic alterations induced in BDF(1) mice exposed whole-body to mainstream cigarette smoke. The animals were divided into five groups, including: (i) 72 sham-exposed mice; (ii) 72 mice exposed to smoke for up to 3 weeks; (iii) 72 mice treated daily with the thiol N-acetylcysteine (NAC, 0.5 g/kg body weight) with drinking water; (iv) 72 mice exposed to smoke and treated daily with NAC, starting 5 days before exposure to smoke; and (v) 48 mice exposed to smoke and treated daily with NAC, starting 1 day after discontinuation of exposure to smoke. After 1, 2, 3, 4, 5, 6, 7, 10 and 14 weeks since the start of exposure to cigarette smoke, eight mice per group were killed, and cytogenetic parameters were evaluated. Exposure to smoke induced a high frequency of micronucleated and binucleated (BN) pulmonary alveolar macrophages, which persisted for at least 14 weeks. The frequency of micronuclei increased early in bone marrow polychromatic erythrocytes, but declined to background levels upon discontinuation of exposure to smoke. By comparison, their induction in circulating normochromatic erythrocytes (NCE) was slightly delayed, less intense but still significant, and persisting for an additional 3 weeks. Administration of NAC, throughout duration of the experiment, strongly inhibited the smoke-induced formation of micronuclei in alveolar macrophages and had some transiently significant effect on the induction of BN macrophages. NAC did not significantly decrease the smoke-induced formation of micronuclei in bone marrow cells, whereas it attenuated the formation of micronuclei in peripheral blood NCE. When given after discontinuation of exposure to cigarette smoke, NAC did not affect the cytogenetic alterations but normalized the altered bronchoalveolar lavage cellularity. The present data provide a detailed analysis of time-related cytogenetic alterations in smoke-exposed mice, both in the respiratory tract and at a systemic level, and show the effects of NAC on these parameters and on the pulmonary inflammatory response.

Inhibitory effects of freeze-dried milk fermented by selected Lactobacillus bulgaricus strains on carcinogenesis induced by 1,2-dimethylhydrazine in rats and by diethylnitrosamine in hamsters.

Fermented milk products might be used for cancer chemoprevention due to their putative anticarcinogenic and antitumor activities. The diet was supplemented with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B 144 (product FFM.B 144) added throughout the experiment at doses of 1.3 g and 2.5 g per rat, 5 times a week starting 3 weeks before the first carcinogen injection. This treatment significantly inhibited, by 26.2-28.6% and by 34.2%, the total intestinal carcinogenesis induced by 1,2-dimethylhydrazine (DMH, 21 mg/kg, s.c., once per week for 20 weeks) in male and female BD6 rats, respectively. FFM.B144 decreased the tumor incidence and multiplicity in large bowel, caecum, and duodenum. Protective effects were better expressed in female animals, with exception of that observed in duodenum. Supplementation of diet with freeze-dried milk fermented by Lactobacillus bulgaricus strain LBB.B5 (product FFM.B5) inhibited DMH-induced carcinogenesis only in the large bowel, but had no significant protective effect when all intestinal tumors were taken into account. However, both freeze-dried products favorably shifted the differentiation of large bowel tumors by increasing the proportion of benign and highly differentiated malignant tumors and decreasing in parallel the number of poorly differentiated carcinomas without influencing the tumor size. A lower number of cases with visible mesenterial metastasis was also observed in FFM-treated rats. In addition, both FFM.B 144 and FFM.B5 significantly inhibited, by 26-33%, the induction in the same rats of ear-duct tumors. FFM.B144 but not FFM.B5 was also effective in inhibiting the tracheal carcinogenesis induced in Syrian golden hamsters by diethylnitrosamine (DEN, 100 mg/kg, two s.c. injections), the protective effect being better expressed in female animals. The anticarcinogenic potential of some fermented milk products might be exploited in chemoprevention of cancer in humans.

Age-related increases of 8-hydroxy-2'-deoxyguanosine and DNA-protein crosslinks in mouse organs.

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and DNA-protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by 32P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during 32P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8 +/- 0.7 8-OH-dG/10(5) normal nucleotides), brain (1.6 +/- 0.5) and heart (2.3 +/- 0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7 +/- 0.4; brain, 3.6 +/- 1.1; heart, 6.8 +/- 2.2 8-OH-dG/10(5) normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P < 0.01), and 3.0-fold increase in heart (P < 0.001). A similar trend was observed for DPXL levels, which were the 1.8 +/- 0.3%, 1.2 +/- 0.2%, and 2.2 +/- 0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9 +/- 0.4%, 2.0 +/- 0.4%, and 3.4 +/- 0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P < 0.01), and 1.5 (P < 0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r = 0.619, P < 0.001) and heart (r = 0.800, P < 0.0001), but not in liver (r = 0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.

Chemoprevention by N-acetylcysteine of urethane-induced clastogenicity and lung tumors in mice.

A major goal in pre-clinical cancer chemoprevention research is to assess the predictive value of intermediate biomarker modulation towards tumor prevention. With this aim, BALB/c mice were treated with 10 daily i.p. injections of urethane (ethyl carbamate), each of 400 mg/kg body weight. Groups of mice received with drinking water either a drug containing the thiol N-acetylcysteine (NAC), at 0.1 or 0.5 g/kg body weight, or its excipient, starting 27 days before the first injection of the carcinogen until the end of the experiment. Out of the 30 mice, 10 per group were identified and individually monitored for 8 sequential times in order to assess the course of micronucleated normochromatic erythrocytes in peripheral blood. This systemic genotoxicity biomarker increased during the 10-day period of treatment with urethane, reached a peak 2 to 6 days after the last injection, and was still significantly higher than the baseline after 10 additional days. Clastogenicity was significantly inhibited by NAC, with a dose-related effect, but not by the drug excipient. As evaluated 4 months after the first injection of urethane, most mice developed lung tumors, whose multiplicity was not affected by the drug excipient but was significantly decreased in the presence of NAC. Correlation between the frequency of micronucleated normochromatic erythrocytes at peak levels and lung-tumor multiplicity was highly significant when evaluated in the context of all 40 mice undergoing cytogenetic analyses (r = 0.561, p = 0.0002). It was similarly high, but did not reach the significance threshold, within each treatment group, due to the lower number of animals and some deviations from the regression line. Therefore, the prediction of lung-tumor yield based on the intensity of the early genotoxicity biomarker is justified when formulated within a sufficiently large group of animals, but is not absolute at individual level.

DNA alterations in rat organs after chronic exposure to cigarette smoke and/or ethanol ingestion.

In spite of the epidemiological evidence supporting a synergism between alcohol consumption and cigarette smoking in the pathogenesis of cancers of the aerodigestive tract, there is a paucity of experimental studies evaluating the effects of these agents under well-controlled conditions and exploring the mechanisms involved. We exposed groups of female BD6 rats, aged 8 months, to ethanol (5% in drinking water for 8 consecutive months) and/or whole-body to mainstream cigarette smoke (1 h/day, 5 days/week for 8 months). DNA was purified from different organs and analyzed for the presence of DNA-protein crosslinks and 32P-postlabeled DNA adducts after butanol enrichment. No significant increase of DNA-protein crosslinks, compared to untreated controls, was induced by any treatment in liver, lung, or heart. 'Spontaneous' nucleotidic modifications were detected by 32P-postlabeling in organs of untreated rats, with the highest levels occurring in the heart. Ingestion of ethanol did not affect DNA adduct levels in any of the organs examined: esophagus, liver, lung, and heart. Exposure to cigarette smoke induced formation of DNA adducts in the lung and heart, but not in the esophagus or liver. The combined ingestion of ethanol resulted in a significant formation of smoke-related DNA adducts in the esophagus and in their further, dramatic increase in the heart. It thus appears that ethanol consumption increases the bioavailability of DNA binding smoke components in the upper digestive tract and favors their systemic distribution. The mechanisms responsible for the interaction between ethanol and smoke and for the selective localization of DNA alterations in different organs are discussed. Formation of DNA adducts in the organs examined may be relevant in the pathogenesis of lung and esophageal cancers as well as in the pathogenesis of other types of chronic degenerative diseases, such as chronic obstructive pulmonary diseases and cardiomyopathies.

DNA adducts and chronic degenerative disease. Pathogenetic relevance and implications in preventive medicine.

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

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.

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.

No effect of treatment with carcinogens on cytosine methylation of mitochondrial DNA isolated from rat organs by phenol-free alkaline extraction.

Due to the postulated role of mitochondrial DNA damage in aging as well as in the pathogenesis of several chronic degenerative diseases, including tumors, there is a need for easy, safe, rapid and inexpensive methods for mitochondrial DNA isolation. We propose a simple, 1-day protocol based on alkaline extraction, which avoids time-consuming gradient centrifugations and use of toxic phenol. This procedure was used for the recovery of mitochondrial DNA from different rat organs (liver, kidney, heart, lung, brain and testis). The yield was quite high. Purity was sufficient enough to perform restriction analyses with several endonucleases. No changes were observed in the structure or methylation patterns of -CCGG- sites in liver mitochondrial DNA isolated from rats exposed whole-body to mainstream cigarette smoke or treated with the carcinogens benzo[alpha]pyrene, 2-acetylaminofluorene or diethylnitrosamine.

Effects of cigarette smoke and disulfiram on tumorigenicity and clastogenicity of ethyl carbamate in mice.

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 1.0 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.

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.