[show abstract][hide abstract] ABSTRACT: Cardiovascular diseases and cancer are the leading causes of death in most countries. These diseases share many common risk factors as well as pathogenetic determinants, and their incidence is related to age in an exponential manner. Furthermore, it has become apparent that several treatments used in therapy or even in prevention of cancer can impair the structural and functional integrity of the cardiovascular system, giving rise to an interdisciplinary field: cardio-oncology. However, tumors and cardiovascular diseases also share common protective factors: they can be prevented either by avoiding exposure to recognized risk factors, and/or by favoring the intake of protective compounds and by modulating the host defense machinery. These latter approaches are generally known as chemoprevention. A great variety of dietary and pharmacological agents have been shown to be potentially capable of preventing cancer in preclinical models, most of which are of plant origin. Phytochemicals, in particular diet-derived compounds, have therefore been proposed and applied in clinical trials as cancer chemopreventive agents. There is now increasing evidence that some phytochemicals can be also protective for the heart, having the potential to reduce cancer, cardiovascular disease and even anticancer drug-induced cardiotoxicity. We introduce the concept that these compounds induce pre-conditioning, a low level cellular stress that induces strong protective mechanisms conferring resistance to toxins such as cancer chemotherapeutics. Cancer cells and cardiomyocytes have fundamental differences in their metabolism and sensitivity to preconditioning, autophagy and apoptosis, so that dosage of the prevention compounds is important. Here we discuss the mechanisms responsible for the cardiotoxicity of anticancer drugs, the possibility to prevent them and provide examples of diet-derived phytochemicals and other biological substances that could be exploited for protecting the cardiovascular system according to a joint cardio-oncological preventative approach.
Current drug targets 12/2010; 12(13):1909-24. · 3.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) is of particular interest due to the very high production and widespread environmental contamination. We recently demonstrated that the oral administration of BPA to mice results in the formation of DNA adducts not only in liver but also in mammary tissue. The present study aimed at evaluating the modulation of BPA-related DNA adducts and proteome alterations by the chemopreventive agents budesonide (BUD) and phenethyl isothiocyanate (PEITC). Swiss ICR (CD-1) mice received, for 8 days, BPA with the drinking water and either chemopreventive agent with the diet. We measured DNA adducts by 32P postlabeling and 656 proteins by antibody microarray. BPA induced the formation, with similar patterns, of DNA adducts in liver and in mammary tissue. Moreover, BPA dysregulated 13 proteins in mammary tissue, mostly in the sense of upregulation, including estrogen receptor-β and proteins involved in cell proliferation, inhibition of apoptosis, tissue remodeling, inflammation, stress response, and glutathione synthesis. PEITC significantly inhibited the formation of BPA-induced DNA adducts, but only at the highest dose tested, and BUD was totally ineffective. The chemopreventive agents modulated a variety of BPA-induced changes in proteome profiles. However, as shown by both hierarchical cluster analysis and principal component analysis, BUD and especially PEITC were not able to restore the physiological situation in BPA-treated mice. Therefore, the in vivo use of proteome analysis proves to be a sensitive tool for the early prediction not only of protective effects but also of adverse effects of chemopreventive agents.
Current Cancer Drug Targets 02/2010; 10(2):147-154. · 4.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: The thiol N-acetyl-L-cysteine (NAC), an analogue and precursor of reduced glutathione, has cancer chemopreventive properties attributable to its nucleophilicity, antioxidant activity, and a variety of other mechanisms. We demonstrated recently that NAC has anti-invasive, antimetastatic, and antiangiogenic effects in in vitro and in vivo test systems. In the present study, s.c. transplantation of KS-Imm cells in (CD-1)BR nude mice resulted in the local growth of Kaposi's sarcoma, a highly vascularized human tumor. The daily administration of NAC with drinking water, initiated after the tumor mass had become established and detectable, produced a sharp inhibition of tumor growth, with regression of tumors in half of the treated mice along with a markedly prolonged median survival time. The production of vascular endothelial growth factor (VEGF) and certain proliferation markers (proliferating cell nuclear antigen and Ki-67) were significantly lower in Kaposi's sarcomas from NAC-treated mice than from control mice. Treatment of KS-Imm cells with NAC in vitro resulted in a dose-dependent inhibition of chemotaxis and invasion through inhibition of gelatinase-A (matrix metalloproteinase-2, MMP-2) activity without altering MMP-2 or MMP-9 mRNA levels. NAC also significantly inhibited VEGF production but did not affect proliferation markers in vitro. Reverse transcription-PCR analysis indicated that total VEGF mRNAs were reduced by 10 mM NAC. Taken together, these findings provide evidence that NAC, the safety of which even at high doses has been established in almost 40 years of clinical use, in addition to its chemopreventive action, has a strong antiangiogenic potential that could be exploited for preventing cancer progression as well as used in cancer adjuvant therapy.
Cancer Research 12/2001; 61(22):8171-8. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/2001; 480-481:9-22. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Our previous studies showed that nucleotide alterations, evaluated by (32)P postlabeling, are systematically detected in smooth muscle cells of atherosclerotic lesions localized in the aorta of surgical patients. The level of these molecular lesions was correlated with the occurrence of known atherogenic risk factors, among which the number of currently smoked cigarettes, and was significantly enhanced in individuals having a null GSTM1 genotype as compared to individuals carrying the GSTM1 genotype. The present study had the dual objective of evaluating the formation of DNA adducts in the whole thoracic aorta of Sprague-Dawley rats, exposed whole-body to cigarette smoke for 28 consecutive days, and of investigating the effects of chemopreventive agents given orally during the same period. High levels of (32)P postlabeled DNA adducts were formed in the aorta of smoke-exposed rats, with an overall 11 times increase over the total levels observed in sham-exposed rats, and with increases ranging between three and 63 times for seven individual DNA adducts. Supplement of the diet with either 1,2-dithiole-3-thione, phenethyl isothiocyanate or 5,6-benzoflavone had no or poor effects on the smoke-related formation of nucleotide alterations in the aorta. In contrast, oltipraz, given with the diet, N-acetyl-L-cysteine, given with drinking water and, even more potently, their combination exerted remarkable protective effects. The results of this experimental study, together with the previous findings in humans, suggest that DNA alterations may contribute to the atherogenic process, clarify a possible mechanism of cigarette smoke, a well known atherogen, and show the potential protective effects of certain drugs towards these alterations.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 08/2001; 494(1-2):97-106. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: A Phase II chemoprevention trial was carried out in Qidong, Jiangsu Province, People's Republic of China. The recruited subjects, all of whom were positive for serum aflatoxin-albumin adducts, were divided into three treatment arms: placebo; oltipraz ([5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione]) given daily at 125 mg p.o.; and oltipraz given once per week at 500 mg p.o. Besides biomarkers related to aflatoxin B(1) exposure, the genotoxicity of blind-coded urine XAD-2 concentrates was evaluated in 201 subjects on the fifth and seventh week of intervention. Genotoxicity was assessed both in the Ames reversion test in strain YG1024 of Salmonella typhimurium, in the presence of an exogenous metabolic system (S9 mix), with or without beta-glucuronidase, and in a DNA repair test in Escherichia coli. Heating of concentrated urine samples or of cigarette smoke condensates was discovered to result in a significant enhancement of their mutagenicity. It was also found that the mutagenicity of condensates from the most extensively used brands of cigarettes in Qidong was much lower than that of Western cigarette brands. Urine mutagenicity was unrelated to treatment with oltipraz, intervention time, gender, and supplement of S9 mix with beta-glucuronidase. Mutagenicity was significantly but variably higher in cigarette smokers than in nonsmokers, which suggests that the urinary excretion of mutagens in the examined population was not exclusively attributable to smoking. Nevertheless, within smokers (28% of the recruited subjects; 67% of all males), the mutagenic potency was significantly correlated with the self-reported number of cigarettes smoked per day and, even more sharply, with the cotinine concentrations in urines. In conclusion, this study demonstrated the validity of urine mutagenicity assays as a biomarker of tobacco smoke exposure that can be investigated on a relatively large scale in chemoprevention trials and provided evidence that oltipraz treatment had no influence on this parameter in the examined population.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Reduced glutathione (GSH) plays a critical role as an intracellular defense system providing detoxification of a broad spectrum of reactive species and their excretion as water-soluble conjugates. Conjugation of GSH with electrophiles is catalyzed by GSH S-transferases (GST), which constitute a broad family of phase II isoenzymes. Two of the GST encoding genes, GSTM1 (mu) and GSTT1 (theta), have a null genotype due to their homozygous deletion that results in lack of active protein. Polymorphisms within GSTT1 and especially GSTM1 have often been associated with cancer in various organs as well as with elevated levels of DNA adducts in various cell types. We recently demonstrated that DNA adducts are consistently detectable in smooth muscle cells (SMC) of human abdominal aorta affected by atherosclerotic lesions. Here we provide evidence that levels of adducts to SMC DNA from atherosclerotic lesions are consistently increased in individuals having the null GSTM1 genotype, whereas no association was established with the GSTT1 polymorphism. The influence of GSTM1 deletion was better expressed in never-smokers and ex-smokers than in current smokers. These findings bear relevance to the epidemiology of atherosclerosis and suggest that metabolic polymorphisms may contribute to the interindividual variability in susceptibility not only to carcinogens, but also to DNA binding atherogens.
The FASEB Journal 04/2001; 15(3):752-7. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Oncology 04/2001; 18(3):607-15. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Cancer 01/2001; 88(5):702-7. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Ten years have elapsed since the International Agency for Research on Cancer (IARC) evaluated the carcinogenicity of chromium and chromium compounds. Further studies performed during the last decade have provided further epidemiological, experimental and mechanistic data which support the IARC conclusions. A wealth of results indicate that, at variance with chromium(0) and chromium(III), chromium(VI) can induce a variety of genetic and related effects in vitro. The lack of carcinogenicity of chromium(0) and chromium(III) compounds in experimental animals is well established, and only a minority of animal carcinogenicity data with chromium(VI) compounds were positive (30 out of 70, i.e. 42.9%). Moreover, most positive studies used administration routes which do not mimic any human exposure and by-pass physiological defense mechanisms. Typically, positive results were only obtained at implantation sites and at the highest dose tested. Exposure to chromium(VI) has been known for more than a century to be associated with induction of cancer in humans. Carcinogenicity requires massive exposures, as is only encountered in well defined occupational settings, and is site specific, being specifically targeted to the lung and, in some cases, to the sinonasal cavity. Increased death rates for cancers at other sites, which were occasionally reported in some epidemiological studies, were almost invariably not statistically significant, and inconsistent (being counterbalanced by other studies which apparently showed decreased rates for the same cancers). As we recently quantified in human body compartments, chromium(VI) can be reduced in body fluids and non-target cells, which results in its detoxification, due to the poor ability of chromium(III) to cross cell membranes. In target cells, chromium(VI) tends to be metabolized by a network of mechanisms leading to generation of reduced chromium species and reactive oxygen species, which will result either in activation or in detoxification depending on the site of the intracellular reduction and its proximity to DNA. When introduced by the oral route, chromium(VI) is efficiently detoxified upon reduction by saliva and gastric juice, and sequestration by intestinal bacteria. If some chromium(VI) is absorbed by the intestine, it is massively reduced in the blood of the portal system and then in the liver. These mechanisms explain the lack of genotoxicity, carcinogenicity, and induction of other long-term health effects of chromium (VI) by the oral route. Within the respiratory tract, chromium(VI) is reduced in the epithelial-lining fluid, pulmonary alveolar macrophages, bronchial tree and peripheral lung parenchyma cells. Hence, lung cancer can only be induced when chromium(VI) doses overwhelm these defense mechanisms. The efficient uptake and reduction of chromium(VI) in red blood cells explains its lack of carcinogenicity at a distance from the portal of entry into the body. All experimental and epidemiological data, and the underlying mechanisms, point to the occurrence of thresholds in chromium(VI) carcinogenesis.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2000; 446(2):215-23. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: 7,12-Dimethylbenz(a)anthracene (DMBA) is a prototype carcinogen that induces a high yield of mammary tumors in rats after a single feeding. We investigated the induction and chemoprevention of DNA adducts in female Sprague Dawley rats receiving DMBA by gavage according to a variety of treatment schedules. The patterns of 32P-postlabeled DNA adducts in liver and mammary epithelial cells were similar to those produced by the in vitro reaction of metabolically activated DMBA with calf thymus DNA. There was a high and statistically significant correlation between dose of DMBA administered to rats (0, 0.6, 2.4, and 12 mg/kg body weight) and levels of DNA adducts in both types of cells. The regression lines relating DMBA doses to total DNA adduct levels were significantly divergent and crossed at 1.5 mg/kg body weight, indicating that, at lower doses, the formation of DNA adducts is more intense in target mammary cells, whereas at higher doses, DNA adduct levels are more elevated in liver cells, presumably due to the greater metabolic capacity of this organ. When the rats were sacrificed 7 days rather than 2 days after DMBA administration, DNA adduct levels were approximately halved in both liver and mammary cells. The observed patterns can be interpreted based on toxicokinetic factors, local and distant metabolism, removal of DNA adducts by excision repair, and cell proliferation rate. Of three chemopreventive agents given with the diet to rats treated with 12 mg of DMBA, 5,6-benzoflavone (1650 ppm) was the most effective, inhibiting DNA adduct formation in liver and mammary cells by 96.5 and 83.5%, respectively. Feeding of 1,2-dithiole-3-thione (600 ppm) inhibited this biomarker by 68.5 and 50.2%, whereas butyl hydroxyanisole (BHA; 5000 ppm) showed a significant inhibition in the liver (46.5%) but was ineffective in mammary cells (29.0%, not significant). These data correlate nicely with the results of a parallel study in which 5,6-benzoflavone, 1,2-dithiole-3-thione, and BHA inhibited formation of hemoglobin adducts by 80.0, 44.0, and 0%, respectively; the incidence of mammary tumors by 82.4, 47.1, and 5.9%, respectively; and their multiplicity by 92.6, 80.0, and 7.4%, respectively. Therefore, biomarkers of biologically effective dose are highly predictive of the efficacy of chemopreventive agents in the DMBA rat mammary model. The selective inhibition by BHA of DNA adducts in the liver but not in mammary cells is consistent with the finding that this phenolic antioxidant stimulated phase II activities in the liver but not in the mammary gland (L. L. Song et al., manuscript in preparation). In any case, the broad-spectrum inducer 5,6-BF appears to be more effective than the two monofunctional phase II inducers, presumably because an enhanced activation of DMBA to reactive metabolites is coordinated with their blocking, detoxification, and excretion.
Cancer Research 10/1999; 59(17):4285-90. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The thiol N-acetylcysteine (NAC) is a chemopreventive agent that acts through a variety of mechanisms and can prevent in vivo carcinogenesis. We have previously shown that NAC inhibits invasion and metastasis of malignant cells as well as tumor take. Neovascularization is critical for tumor mass expansion and metastasis formation. We investigated whether a target of the anti-cancer activity of NAC could be the inhibition of the tumor angiogenesis-associated phenotype in vitro and in vivo using the potent angiogenic mixture of Kaposi's sarcoma cell products as a stimulus. Two endothelial (EAhy926 and human umbilical vein endothelial [HUVE]) cell lines were utilized in a panel of assays to test NAC ability in inhibiting chemotaxis, invasion, and gelatinolytic activity in vitro. NAC treatment of EAhy926 and HUVE cells in vitro dose-dependently reduced their ability to invade a reconstituted basement membrane, an indicator of endothelial cell activation. Invasion of HUVE cells was inhibited with an ID50 of 0.24 mM NAC, whereas inhibition of chemotaxis required a 10 fold higher doses, indicating that invasion is a preferential target. NAC inhibited the enzymatic activity and conversion to active forms of the gelatinase produced by endothelial cells. The matrigel in vivo assay was used for the evaluation of angiogenesis; NAC strongly inhibited neovascularization of the matrigel sponges in response to Kaposi's sarcoma cell products. NAC prevented angiogenesis while preserving endothelial cells, implying that it could be safely used as an anti-angiogenic treatment.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The assessment of pathological effects produced by environmental tobacco smoke in humans is controversial in epidemiological studies. On the other hand, animal models are poorly sensitive to smoke carcinogenicity. We designed an experimental study assessing the tissueselective formation and persistence of DNA adducts in smoke-exposed rats. Sprague-Dawley rats were exposed for 6 h per day, 5 days per week, to environmental smoke resulting from a mixture of sidestream and mainstream smoke generated from Kentucky 2R1 reference cigarettes. The total particulate matter was in the range of 73-93 mg/m(3). DNA adducts were measured by (32)P-post-labelling in rat organs (lung, heart, liver, bladder and testis), tissues (dissected tracheal epithelium) and cells [isolated bronchoalveolar lavage (BAL) cells]. A time-related increase of (32)P-post-labelled DNA modifications was detectable by autoradiography, in the form of massive diagonal radioactive zones and individual spots. Top levels were reached after 4-5 weeks of exposure. The ratio of smoke-induced DNA adducts to the background levels detected in sham-exposed rats was 11.2 in the tracheal epithelium, 10.4 in BAL cells, 7.3 in the heart, 6.3 in the lung, 5.1 in the bladder, 1.9 in the testis and 1. 1 in the liver. Appearance of DNA adducts in the lung was also revealed by synchronous fluorescence spectrophotometry. Smoke-related oxidative damage was demonstrated by a significant enhancement of 8-hydroxy-2'-deoxyguanosine in lung DNA. In parallel, there was a time-related induction of lung microsomal arylhydrocarbon hydroxylase activity, an elevation in cytosolic glutathione S-transferase activity, and a moderate but progressive and significant depletion of reduced glutathione. After discontinuing exposure to environmental cigarette smoke for 1 week, DNA adduct levels significantly dropped in the lung, tracheal epithelium, heart and bladder. The decrease was evident but not statistically significant in BAL cells, and was negligible in the heart. The selective localization and the differential persistence of these promutagenic nucleotide modifications in rat organs, tissues and cells suggest that exposure to environmental cigarette smoke, at least under the high exposure regimens used in experimental studies, may pose a potential risk of developing mutation-related diseases.