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.
A review of almost 2000 studies showed that the large majority of 39 putative cancer chemopreventive agents induced "spontaneous" apoptosis. Inhibition of the programmed cell death triggered by a ...variety of stimuli was consistently reported only with ascorbic acid, alpha-tocopherol, and N-acetylcysteine (NAC). We performed experimental studies in rodents exposed to cigarette smoke, either mainstream (MCS) or environmental (ECS), and UV-A/B-containing light. The nonsteroidal anti-inflammatory drug sulindac did not affect the apoptotic process in the skin of light-exposed mice and in the lungs of ECS-exposed mice. Likewise, 5,6-benzoflavone, indole-3-carbinol, 1,2-dithiole-3-thione and oltipraz failed to modulate apoptosis in the respiratory tract of ECS-exposed rats. Phenethyl isothiocyanate further enhanced the frequency of apoptosis in pulmonary alveolar macrophages and bronchial epithelial cells, and upregulated several genes in the lung of ECS-exposed rats. Both individually and in combination with oltipraz, NAC inhibited apoptosis in the respiratory tract of rats exposed either to MCS or ECS. Moreover, NAC attenuated the ECS-related overexpression of proapoptotic genes and normalized the levels of proapoptotic proteins in rat lung. The transplacental administration of NAC to mice considerably attenuated gene overexpression in the liver of fetuses exposed to ECS throughout pregnancy. Inhibition of apoptosis by chemopreventive agents reflects their ability to counteract certain upstream signals, such as genotoxic damage, redox imbalances, and other forms of cellular stress that trigger apoptosis. On the other hand, enhancement of apoptosis is a double-edged sword, since it represents a protective mechanism in carcinogenesis but may contribute to the pathogenesis of other degenerative diseases. We suggest that stimulation of apoptosis by so many chemopreventive agents, as reported in the literature, may often reflect the occurrence of toxic effects at high doses.
ABSTRACT
We previously showed that sunlight‐mimicking light induces genotoxic damage not only in skin but also even in lung, bone marrow, and peripheral blood of hairless mice. Moreover, light and ...smoke acted synergically in the respiratory tract. To clarify the mechanisms involved, we investigated by cDNA‐arrays the expression of 746 toxicologically relevant genes in skin and lungs of mice exposed for 28 days to light and/or environmental cigarette smoke. Glutathione‐S–transferase‐Pi and catalase were overexpressed in the lungs of mice exposed to light only. Moreover, the light induced in skin the expression of genes involved in carcinogenesis, photoaging, and production of genotoxic and oxidizing derivatives traveling at a distance. Smoke induced the expression of multiple genes in both skin and lung, which reflect adaptive responses and mechanisms related to cancer and, possibly, to emphysema and stroke. As shown in mice exposed to both light and smoke, the light tended to increase smoke‐induced gene expression in lungs, while smoke tended to attenuate light‐induced gene expression in skin. The oral administration of the nonsteroidal anti‐inflammatory drug sulindac inhibited the light‐induced overexpression of cyclooxygenase‐2 and oxidative stress‐related genes in skin, and down‐regulated smoke‐induced genes involved in oxidative stress, removal of damaged proteins, inflammation, and immune response in lung. These results provide a mechanistic insight explaining the systemic alterations induced by both light and smoke in mouse skin and lungs.
Chromium(VI) compounds are genotoxic in a variety of cellular systems. Their potential carcinogenicity is affected by toxicokinetic patterns restricting bioavailability to certain targets, and by ...metabolic pathways affecting interaction of chromate-derived reactive species with DNA. Epidemiological data indicate that chromium(VI) can be carcinogenic to the human respiratory tract following inhalation at doses that are only achieved in certain occupational settings. However, concern has been raised that adverse effects may also result from oral intake. In order to further explore this issue, we performed studies in BDF1 and Swiss mice of both genders and various age. Sodium dichromate dihydrate and potassium dichromate were administered either with the drinking water, up to a concentration of 500
mg chromium(VI)/l for up to 210 consecutive days, or in a single intragastric dose of 17.7
mg/kg body weight. Under these conditions, no increase of the micronucleus frequency was observed in either bone marrow or peripheral blood erythrocytes. Conversely, the same compounds induced a clastogenic damage following intraperitoneal injection, which by-passes detoxification mechanisms. In addition, due to the hypothesis that susceptibility may be increased during the period of embryogenesis, we treated pregnant mice, up to a concentration of 10
mg chromium(VI)/l drinking water. There was no effect on the numbers of fetuses/dam and on body weight of fetuses. Again, no toxic or genotoxic effect was observed either in bone marrow of pregnant mice or in liver and peripheral blood of their fetuses. Thus, even at doses that largely exceed drinking water standards (up to 10,000 times) or by massive intragastric administration, chromium(VI) is not genotoxic to hematopoietic cells of either adult mice or transplacentally exposed fetuses. These conclusions are consistent with the poor toxicity and lack of carcinogenicity of oral chromium(VI), and are mechanistically explained by the high efficiency of chromium(VI) detoxification processes in the gastrointestinal tract.
The light emitted by halogen quartz bulbs contains a broad spectrum of UV wavelengths, is strongly genotoxic and is a potent inducer of skin tumors in hairless mice. By using a UVC filter, this light ...mimics solar radiation and induces a variety of genomic and transcriptional alterations in mouse skin. UV-related carcinogenesis involves depletion of antioxidants and glutathione in skin cells. On this basis, we evaluated modulation of carcinogenicity of UVC-filtered halogen lamps in SKH-1 hairless mice by the antioxidants N-acetyl-l-cysteine (NAC) and ascorbic acid (AsA). Both agents were given in the drinking water, either individually or in combination. The earliest skin lesions were detected after 300 days' exposure to light and became confluent in a number of mice after 480 days. NAC administration prolonged the latency time by 90 days. Moreover, NAC considerably and significantly decreased both incidence and multiplicity of light-induced skin tumors, prevented the occurrence of malignant lesions (squamocellular carcinomas) and reduced the tumor size. In contrast, AsA, which may behave as a prooxidant rather than an antioxidant, increased the multiplicity of total skin tumors, carcinomas in situ and squamocellular carcinomas. Co-administration of NAC with AsA significantly attenuated the negative effect of AsA, presumably due to the ability of this thiol to maintain a reduced environment. Therefore, in agreement with our previous in vitro findings, oral NAC is able to attenuate the detrimental effects of AsA.
ABSTRACT
The transplacental exposure of fetuses to maternal cigarette smoke may increase the risk of developmental impairments, congenital diseases, and childhood cancer. The whole‐body exposure of ...Swiss mice to environmental cigarette smoke (ECS) during pregnancy decreased the number of fetuses per dam, placenta weight, and fetus weight. ECS increased DNA adducts, oxidative nucleotide alterations, and cytogenetic damage in fetus liver. Evaluation by cDNA array of 746 genes showed that 61 of them were expressed in fetus liver under basal conditions. The oral administration of N‐acetylcysteine (NAC) during pregnancy enhanced the expression of three genes only, including two glutathione S‐transferases and α1‐antitrypsin precursor, whose deficiency plays a pathogenetic role in congenital emphysema. Transplacental ECS upregulated the expression of 116 genes involved in metabolism, response to oxidative stress, DNA and protein repair, and signal transduction. NAC inhibited the ECS‐related genetic damage and upregulation of most genes. ECS stimulated pro‐apoptotic genes and genes downregulating the cell cycle, which may justify growth impairments in the developing fetus. Thus, both genetic and epigenetic mechanisms were modulated by ECS. Moreover, hypoxia‐related genes and several oncogenes and receptors involved in proliferation and differentiation of leukocytes were induced in the fetal liver, which also bears hematopoietic functions.
Postgenomic methodologies have provided novel tools for evaluating safety and efficacy of cancer chemopreventive agents. We exposed rats to environmental cigarette smoke (ECS) for 28 days, with or ...without oral administration of
N-acetylcysteine (NAC). As assessed by
32P-postlabelling, ECS caused a 10-fold increase of DNA adduct levels, which were significantly reduced by NAC. Of 518 proteins tested by antibody microarray, ECS stimulated 56 activities involved in stress response, protein removal, cell replication, apoptosis, phagocytosis, and immune response. NAC alone did not change the amounts of any protein, whereas it significantly decreased the amounts of 6 ECS-induced proteins. The intensity of expression of 278 related genes, assessed by cDNA microarray, was significantly correlated with protein amounts. These observed molecular alterations, which can be attenuated by NAC, represent in part adaptive responses and in part reflect mechanisms contributing to the pathogenesis of smoke-related diseases, including lung cancer, asthma, chronic bronchitis, and emphysema.
Mutations and deletions in p53 are the most common genetic lesions in human cancer,and an extraordinarily high incidence of lung cancer occurs in smokers suffering from Li-Fraumeni syndrome, which is ...characterized by germ-line inactivation of one p53 allele. In contrast, p53 mutations are infrequent in lung tumors formed in A/J mice. Moreover, despite the dominant role of cigarette smoke in the epidemiology of human lung cancer, it is very difficult to reproduce the lung tumorigenicity of this complex mixture in animal models. We used a transgenic mouse with a dominant-negative p53 mutation to examine the effects of a mutant p53 on smoke-induced lung carcinogenesis in mice. p53 mutant (UL53-3 x A/J)F(1) mice of both genders and their wild-type (wt) littermate controls were exposed whole-body to environmental cigarette smoke (ECS) for up to 9.5 months. Untreated mutant mice of both genders underwent an early stimulus of bronchial cell proliferation, and an age-related formation of DNA adducts in lung and heart. In males, there was an age-related increase of micronucleated normochromatic erythrocytes in peripheral blood and an impairment of body weight gain. These findings underscore a physiological protective role of p53 in wt A/J mice. The response of wt and mutant mice to ECS was similar in terms of oxidative DNA damage in lung and heart, proliferation of the bronchial epithelium, and levels of p53 oncoprotein, as assessed after exposure for 28 days. In contrast, ECS-exposed mutant mice underwent a lower induction of apoptosis in bronchial epithelium, a greater formation of DNA adducts in lung and heart, and a more intense cytogenetic damage, shown by a higher frequency of micronuclei in pulmonary alveolar macrophages and in peripheral blood normochromatic erythrocytes. Interestingly, at the end of the experiment, DNA adducts were not repaired in either wt or mutant mice after discontinuing exposure to ECS for 1 week. A weak but significant increase of lung tumor incidence and multiplicity was induced in p53 mutant (UL53-3 x A/J)F(1) mice after exposure to ECS for either 5 months, followed by recovery in air for 4.5 months, or 9.5 continuative months. Conversely, no tumorigenic effect was observed in their wt littermate controls, carrying a 99.9% A/J background and 5% FVB genome. This contrasts with the weakly positive results obtained in previous studies using wt A/J mice. Thus, in agreement with the results of previous lung tumorigenicity studies performed with the smoke carcinogens benzo(a)pyrene and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, (UL53-3 x A/J)F(1) mice carrying a mutant p53 transgene appear to be more sensitive to ECS than the corresponding wt littermate controls. These findings provide evidence that p53 mutations play a role in smoke-related carcinogenesis not only in humans but also in A/J mice.
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.
Birth is characterized by a sudden transition from the maternal-mediated respiration to the autonomous pulmonary respiration. Notwithstanding the importance of the involved functional and metabolic ...changes, little is known about possible DNA alterations occurring in the lung during the perinatal period. We comparatively evaluated genomic and transcriptional changes in the lung of fetuses and newborn Swiss albino mice, whose dams had either been untreated or treated with oral
N-acetyl-
l-cysteine (NAC) throughout the pregnancy period. In the less than 24
h period elapsing between the end of fetal life and the start of post-natal life, nucleotide alterations occurred in mouse lung, as shown by a significant increase of both bulky DNA adducts and 8-hydroxy-2′-deoxyguanosine levels, detected by
32
P
post-labeling procedures. The frequency of micronuclei in peripheral blood erythrocytes was not significantly increased after birth. Multigene expression analysis of 746 selected genes, by cDNA arrays, showed that 33 of them (4.4%) were upregulated in the lung of newborn mice, as compared with fetuses. The overexpressed genes were mainly involved in protective mechanism as a response to oxidative changes, alterations of glutathione metabolism, cellular stress, and damage to DNA and proteins. The transplacental treatment with NAC totally prevented birth-related genomic alterations in lung DNA. NAC did not change the basal gene expression in mouse fetal lung, but attenuated the upregulation of most genes involved in oxidative stress, stress response, and DNA repair in the lung of newborn mice. In fact, only 13 genes (1.7%) were overexpressed in newborns from NAC-treated dams. It therefore appears that administration of NAC during pregnancy is beneficial not only to counteract the adverse effects of toxic agents, as supported by previous studies, but also to attenuate birth-related DNA alterations.
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