RESUMO
Epidemiological studies show that there is limited evidence that tobacco smoking causes breast cancer in humans. In rodents, many tobacco smoke chemicals cause mammary gland tumors. This study evaluated the mammary gland differentiation in mice exposed to environmental cigarette smoke (ECS), using 3R4F Kentucky reference cigarettes, starting after birth and continuing daily for 10 weeks (total particulate exposure 95 mg/m3; CO 610 ppm). We also analyzed the effects of oral administration of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin (1600 mg/kg) or naproxen (320 mg/kg), on mammary gland differentiation, either in unexposed or ECS-exposed mice. The ECS exposure caused delay of mammary glands development. We speculate that this delay may result from aryl hydrocarbon receptor (AHR) signaling activation, which has an antiestrogenic effect and crosstalk to the estrogen metabolism pathway. Similarly, naproxen impaired gland differentiation in unexposed and ECS-exposed mice, while aspirin hindered its development only in unexposed mice. The lack of differentiation induced by the NSAIDs could be explained by their antiestrogenic effect through inhibition of aldo-keto reductases. In ECS-exposed animals, aspirin induced intense lobular formation, which could indicate that aspirin is counteracting the AHR signaling induced by ECS. Based on the differentiation induced by aspirin in ECS-exposed animals, we postulate that these mice would be less susceptible to mammary carcinogenesis. Our results suggest that exposure to smoke at an early age impairs the development of the mammary gland, thus resulting in a longer period of susceptibility and increased risk of breast cancer. However, addition of aspirin can abrogate this effect.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Aspirina/administração & dosagem , Glândulas Mamárias Animais/efeitos dos fármacos , Neoplasias Mamárias Experimentais/prevenção & controle , Poluição por Fumaça de Tabaco/efeitos adversos , Administração Oral , Animais , Carcinogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Suscetibilidade a Doenças/etiologia , Feminino , Masculino , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Naproxeno/administração & dosagem , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fumaça/efeitos adversos , Nicotiana/efeitos adversosRESUMO
Evaluation of the reducing capacity of human gastric fluid from healthy individuals, under fasted and fed conditions, is critical for assessing the cancer hazard posed by ingested hexavalent chromium [Cr(VI)] and for developing quantitative physiologically-based pharmacokinetic models used in risk assessment. In the present study, the patterns of Cr(VI) reduction were evaluated in 16 paired pre- and post-meal gastric fluid samples collected from 8 healthy volunteers. Human gastric fluid was effective both in reducing Cr(VI), as measured by using the s-diphenylcarbazide colorimetric method, and in attenuating mutagenicity in the Ames test. The mean (±SE) Cr(VI)-reducing ability of post-meal samples (20.4±2.6µgCr(VI)/mL gastric fluid) was significantly higher than that of pre-meal samples (10.2±2.3µgCr(VI)/mL gastric fluid). When using the mutagenicity assay, the decrease of mutagenicity produced by pre-meal and post-meal samples corresponded to reduction of 13.3±1.9 and 25.6±2.8µgCr(VI)/mL gastric fluid, respectively. These data are comparable to parallel results conducted by using speciated isotope dilution mass spectrometry. Cr(VI) reduction was rapid, with >70% of total reduction occurring within 1min and 98% of reduction is achieved within 30min with post-meal gastric fluid at pH2.0. pH dependence was observed with decreasing Cr(VI) reducing capacity at higher pH. Attenuation of the mutagenic response is consistent with the lack of DNA damage observed in the gastrointestinal tract of rodents following administration of ≤180ppm Cr(VI) for up to 90days in drinking water. Quantifying Cr(VI) reduction kinetics in the human gastrointestinal tract is necessary for assessing the potential hazards posed by Cr(VI) in drinking water.
Assuntos
Cromo/química , Suco Gástrico/química , Poluentes Químicos da Água/química , Adulto , Cromo/toxicidade , Jejum , Histidina/genética , Humanos , Concentração de Íons de Hidrogênio , Mutagênese , Testes de Mutagenicidade , Oxirredução , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Poluentes Químicos da Água/toxicidadeRESUMO
Chemoprevention provides an important strategy for cancer control in passive smokers. Due to the crucial role played by smoke-related chronic inflammation in lung carcinogenesis, of special interest are extensively used pharmacological agents, such as nonsteroidal anti-inflammatory drugs (NSAIDs). We evaluated the ability of aspirin and naproxen, inhibitors of both cyclooxygenase-1 and cyclooxygenase -2, to modulate environmental cigarette smoke (ECS)-induced lung carcinogenesis in A/J mice of both genders. Based on a subchronic toxicity study in 180 postweaning mice, we used 1600 mg/kg diet aspirin and 320 mg/kg diet naproxen. In the tumor chemoprevention study, using 320 mice, exposure to ECS started soon after birth and administration of NSAIDs started after weaning. At 10 weeks of life, the NSAIDs did not affect the presence of occult blood in feces. As assessed in a subset of 40 mice, bulky DNA adducts and 8-hydroxy-2'-deoxyguanosine levels were considerably increased in ECS-exposed mice and, irrespective of gender, both NSAIDs remarkably inhibited these nucleotide alterations. After exposure for 4 months followed by 5 months in filtered air, ECS induced a significant increase in the yield of surface lung tumors, the 43.7% of which were adenomas and the 56.3% were adenocarcinomas. Oct-4 (octamer-binding transcription factor 4), a marker of cell stemness, was detected in some adenocarcinoma cells. The NAIDs attenuated the yield of lung tumors, but prevention of ECS-induced lung adenomas was statistically significant only in female mice treated with aspirin, which supports a role for estrogens in ECS-related lung carcinogenesis and highlights the antiestrogenic properties of NSAIDs.
Assuntos
Anticarcinógenos/farmacologia , Aspirina/farmacologia , Neoplasias Pulmonares/prevenção & controle , Naproxeno/farmacologia , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Anticarcinógenos/uso terapêutico , Aspirina/uso terapêutico , Dano ao DNA , Avaliação Pré-Clínica de Medicamentos , Feminino , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Masculino , Camundongos , Naproxeno/toxicidade , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
Lapatinib, a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), is prescribed for the treatment of patients with metastatic breast cancer overexpressing HER-2. Involvement of this drug in pulmonary carcinogenesis has been poorly investigated. We used murine models suitable to evaluate cigarette smoke-related molecular and histopathological alterations. A total of 481 Swiss H mice were used. The mice were exposed to mainstream cigarette smoke (MCS) during the first four months of life. After 10 weeks, MCS caused an elevation of bulky DNA adducts, oxidative DNA damage and an extensive downregulation of microRNAs in lung. After four months, an increase in micronucleus frequency was observed in peripheral blood erythrocytes. After 7.5 months, histopathological alterations were detected in the lung, also including benign tumors and malignant tumors, and in the urinary tract. A subchronic toxicity study assessed the non-toxic doses of lapatinib, administered daily with the diet after weaning. After 10 weeks, lapatinib significantly attenuated the MCS-related nucleotide changes and upregulated several low-intensity microRNAs in lung. The drug poorly affected the MCS systemic genotoxicity and had modest protective effects on MCS-induced preneoplastic lesions in lung and kidney, when administered under conditions that temporarily mimicked interventions either in current smokers or ex-smokers. On the other hand, it caused some toxicity to the liver. Thus, on the whole, lapatinib appears to have a low impact in the smoke-related lung carcinogenesis models used, especially in terms of tumorigenic response.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Pulmão/efeitos dos fármacos , Quinazolinas/farmacologia , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Peso Corporal/efeitos dos fármacos , Adutos de DNA , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Eritrócitos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Lapatinib , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , MicroRNAs , Inibidores de Proteínas Quinases/farmacologia , Testes de Toxicidade SubcrônicaRESUMO
Assessing the correlation between molecular endpoints and cancer induction or prevention aims at validating the use of intermediate biomarkers. We previously developed murine models that are suitable to detect both the carcinogenicity of mainstream cigarette smoke (MCS) and the induction of molecular alterations. In this study, we used 931 Swiss mice in two parallel experiments and in a preliminary toxicity study. The chemopreventive agents included vorinostat, myo-inositol, bexarotene, pioglitazone and a combination of bexarotene and pioglitazone. Pulmonary micro-RNAs and proteins were evaluated by microarray analyses at 10 weeks of age in male and female mice, either unexposed or exposed to MCS since birth, and either untreated or receiving each one of the five chemopreventive regimens with the diet after weaning. At 4 months of age, the frequency of micronucleated normochromatic erythrocytes was evaluated. At 7 months, the lungs were subjected to standard histopathological analysis. The results showed that exposure to MCS significantly downregulated the expression of 79 of 694 lung micro-RNAs (11.4%) and upregulated 66 of 1164 proteins (5.7%). Administration of chemopreventive agents modulated the baseline micro-RNA and proteome profiles and reversed several MCS-induced alterations, with some intergender differences. The stronger protective effects were produced by the combination of bexarotene and pioglitazone, which also inhibited the MCS-induced clastogenic damage and the yield of malignant tumors. Pioglitazone alone increased the yield of lung adenomas. Thus, micro-RNAs, proteins, cytogenetic damage and lung tumors were closely related. The molecular biomarkers contributed to evaluate both protective and adverse effects of chemopreventive agents and highlighted the mechanisms involved.
Assuntos
Aberrações Cromossômicas , Neoplasias Pulmonares/etiologia , MicroRNAs/genética , Nicotiana/efeitos adversos , Proteoma , Fumaça/efeitos adversos , Animais , Anticarcinógenos/administração & dosagem , Anticarcinógenos/farmacologia , Peso Corporal/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , CamundongosRESUMO
Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m(3) of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were measured by (32)P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , MicroRNAs/metabolismo , Nicotiana/toxicidade , Fumaça/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Análise por Conglomerados , Adutos de DNA/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Relação Dose-Resposta a Droga , Feminino , Camundongos , MicroRNAs/genética , Análise em Microsséries , Gravidez , Análise de Componente Principal , Ratos , Fumar/efeitos adversos , Abandono do Hábito de FumarRESUMO
Endocrine disruptors (EDs) represent a major toxicological and public health issue, and the xenoestrogen bisphenol A (BPA) has received much attention due to its high production volume and widespread human exposure. Also, due to its similarity to diethylstilbestrol, a known human carcinogen, BPA has been investigated for its genotoxic and carcinogenic properties, but the results have been either inconclusive or controversial. Metabolically activated BPA has previously been shown to form DNA adducts both in vitro and in rat liver. The present study was designed (a) to assess the sensitivity threshold of DNA-adduct detection by 32P-postlabelling in an acellular system and (b) to evaluate the formation of DNA adducts in both liver and mammary cells of female CD-1 mice receiving BPA in their drinking water (200 mg/kg body weight) for eight consecutive days. The reaction of BPA with calf thymus DNA, in the presence of S9 mix, resulted in a dose-dependent formation of multiple DNA adducts, with a detection limit of approximately 10 ng of this ED under our experimental conditions. Administration of BPA to mice confirmed that DNA adducts are formed in liver (3.4-fold higher levels than in controls). In addition, new evidence is provided that DNA adducts are formed in target mammary cells (4.7-fold higher than in controls). Although DNA adducts do not necessarily evolve into tumours or other chronic degenerative diseases, the formation of these molecular lesions in target mammary cells may bear relevance for the potential involvement of BPA in breast carcinogenesis.
Assuntos
Adutos de DNA , Disruptores Endócrinos/farmacologia , Fígado/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Fenóis/toxicidade , Animais , Compostos Benzidrílicos , Feminino , Técnicas In Vitro , CamundongosRESUMO
Our recent studies have shown that both cigarette smoke and UV-containing light, which are the most widespread and ubiquitous mutagens and carcinogens in the world, cause systemic genotoxic damage in hairless mice. Further studies were designed with the aim of evaluating the induction of genotoxic and carcinogenic effects in Swiss albino mice exposed to smoke and/or light since birth. We observed that a 4-month whole-body exposure of mice to mainstream cigarette smoke, starting at birth, caused an early and potent carcinogenic response in the lung and other organs. Our further experiments showed that exposure of mice to environmental cigarette smoke, during the first 5 weeks of life, resulted in a variety of significant alterations of intermediate biomarkers, including cytogenetic damage in bone marrow and peripheral blood, formation of lipid peroxidation products, increase of bulky DNA adduct levels, induction of oxidative DNA damage, and overexpression of OGG1 gene in lung, stimulation of apoptosis, hyperproliferation and loss of Fhit protein in pulmonary alveolar macrophages and/or bronchial epithelial cells, and early histopathological alterations in the respiratory tract. Moreover, exposure of mice to UV-containing light, mimicking solar irradiation, significantly enhanced oxidative DNA damage and bulky DNA adduct levels in lung, and synergized with smoke in inducing molecular alterations in the respiratory tract. The baseline OGG1 expression in lung was particularly high at birth and decreased in post-weanling mice. Oxidative DNA damage and other investigated end-points exhibited differential patterns in post-weanling mice and adult mice. The findings of these studies provide a mechanistic clue to the general concept that the neonatal period and early stages of life are critical in affecting susceptibility to carcinogens.
Assuntos
Animais Recém-Nascidos , Dano ao DNA , Luz/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Carcinógenos/toxicidade , Predisposição Genética para Doença , Camundongos , Mutagênicos/toxicidade , Raios Ultravioleta/efeitos adversosRESUMO
Purpose: MicroRNAs are small non-coding RNAs that regulate gene expression, thereby playing a role in a variety of physiological and pathophysiological states. Exposure to cigarette smoke extensively downregulates microRNA expression in pulmonary cells of mice, rats, and humans. Cellular microRNAs are released into body fluids, but a poor parallelism was previously observed between lung microRNAs and circulating microRNAs. The purpose of the present study was to validate the application of this epigenetic biomarker by using less invasive collection procedures. Experimental design: Using microarray analyses, we measured 1135 microRNAs in 10 organs and 3 body fluids of mice that were either unexposed or exposed to mainstream cigarette smoke for up to 8 weeks. The results obtained with selected miRNAs were validated by qPCR. Results: The lung was the main target affected by smoke (190 dysregulated miRNAs), followed by skeletal muscle (180), liver (138), blood serum (109), kidney (96), spleen (89), stomach (36), heart (33), bronchoalveolar lavage fluid (32), urine (27), urinary bladder (12), colon (5), and brain (0). Skeletal muscle, kidney, and lung were the most important sources of smoke-altered microRNAs in blood serum, urine, and bronchoalveolar lavage fluid, respectively. Conclusions: microRNA expression analysis was able to identify target organs after just 8 weeks of exposure to smoke, well before the occurrence of any detectable histopathological alteration. The present translational study validates the use of body fluid microRNAs as biomarkers applicable to human biomonitoring for mechanistic studies, diagnostic purposes, preventive medicine, and therapeutic strategies.
Assuntos
Líquidos Corporais/metabolismo , MicroRNAs/metabolismo , Especificidade de Órgãos , Fumar/efeitos adversos , Animais , Peso Corporal , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos Endogâmicos ICR , MicroRNAs/genética , Análise de Componente Principal , RNA/isolamento & purificação , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
We recently showed that nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the lung tumors induced by cigarette smoke, either mainstream (MCS) or environmental (ECS), in female mice. We used subsets of mice to analyze the expression of 1135 microRNAs in both lung and blood serum, as related to the whole-body exposure to smoke and/or oral administration of either aspirin or naproxen. In a first study, we evaluated early microRNA alterations in A/J mice exposed to ECS for 10 weeks, starting at birth, and/or treated with NSAIDs for 6 weeks, starting after weaning. At that time, when no histopathological change were apparent, ECS caused a considerable downregulation of pulmonary microRNAs affecting both adaptive mechanisms and disease-related pathways. Aspirin and naproxen modulated, with intergender differences, the expression of microRNAs having a variety of functions, also including regulation of cyclooxygenases and inflammation. In a second study, we evaluated late microRNA alterations in Swiss H mice exposed to MCS during the first 4 months of life and treated with NSAIDs after weaning until 7.5 months of life, when tumors were detected in mouse lung. Modulation of pulmonary microRNAs by the two NSAIDs was correlated with their ability to prevent preneoplastic lesions (microadenomas) and adenomas in the lung. In both studies, exposure to smoke and/or treatment with NSAIDs also modulated microRNA profiles in the blood serum. However, their levels were poorly correlated with those of pulmonary microRNAs, presumably because circulating microRNAs reflect the contributions from multiple organs and not only from lung.
RESUMO
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.
Assuntos
Genes p53/fisiologia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Nicotiana , Fumaça/efeitos adversos , Animais , Apoptose/fisiologia , Divisão Celular/fisiologia , Cocarcinogênese , Adutos de DNA/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Feminino , Genes p53/genética , Predisposição Genética para Doença , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Transgênicos , Mutação , Miocárdio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Aumento de PesoRESUMO
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 alpha1-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.
Assuntos
Feto/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Exposição Materna , Fumar , Acetilcisteína/farmacologia , Animais , Anticarcinógenos/farmacologia , Apoptose , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Hipóxia Celular , Adutos de DNA/metabolismo , Reparo do DNA , Feminino , Feto/anatomia & histologia , Genoma , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fígado/embriologia , Fígado/metabolismo , Camundongos , Oxirredução , Estresse Oxidativo , Paridade/efeitos dos fármacos , Placenta/anatomia & histologia , Placenta/efeitos dos fármacos , Gravidez , Fumar/efeitos adversos , Transcrição GênicaRESUMO
Analysis of transcriptome and proteome profiles by microarray technologies provides a formidable, new tool in cancer chemoprevention research. An ideal chemopreventive agent should not excessively alter per se the basal make-up of multigene expression and protein synthesis and should at the same time be able to attenuate alterations induced by risk factors. In order to validate this working hypothesis, we previously performed a series of studies in animal models using the thiol N-acetyl-l-cysteine (NAC) and the nonsteroidal antiinflammatory drug sulindac. We report herein the results of new studies evaluating modulation of DNA adduct levels and expression of 4858 genes in lung and liver of Sprague-Dawley rats, either unexposed or exposed to environmental cigarette smoke (ECS). The tested chemopreventive agents included NAC, oltipraz (OPZ), 5,6-benzoflavone (5,6-BF), phenethyl isothiocyanate (PEITC), and indole 3-carbinol (I3C). Combinations of OPZ with NAC and of PEITC with I3C were also assayed. Excepting OPZ, all treatments inhibited by at least 50% the formation of bulky DNA adducts in the lung of ECS-exposed rats. Hierarchical cluster analysis and principal component analysis allowed us to classify the agents according to their influence on basal gene expression and their ability to attenuate ECS-induced transcriptome alterations. PEITC and I3C were the most effective but the least safe agents. 5,6-BF displayed intermediate patterns. OPZ was poorly effective in lung and considerably altered the basal gene expression in liver. NAC had a medium efficacy and was the safest agent, as also supported by the analysis of 518 proteins in rat lung.
Assuntos
Anticarcinógenos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteoma , Acetilcisteína/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Análise por Conglomerados , Sequestradores de Radicais Livres/farmacologia , Perfilação da Expressão Gênica , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/análise , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fumaça , Sulindaco/farmacologia , NicotianaRESUMO
N-Acetyl-L-cysteine (NAC) has been shown to exert cancer-protective mechanisms and effects in experimental models. We report here the results of a randomized, double-blind, placebo-controlled, Phase II chemoprevention trial with NAC in healthy smoking volunteers. The subjects were supplemented daily with 2 x 600 mg of oral tablets of NAC (n = 20) or placebo (n = 21) for a period of 6 months, and internal dose markers [plasma and bronchoalveolar lavage (BAL) fluid cotinine, urine mutagenicity], biologically effective dose markers [smoking-related DNA adducts and hemoglobin (Hb) adducts], and biological response markers (micronuclei frequency and antioxidants scavenging capacity) were assessed at both pre- and postsupplementation times (T(0) and T(1), respectively). Overall, the internal dose markers remained unchanged at T(1) as compared with T(0) in both NAC and placebo groups. When quantifying the biologically effective dose markers, we observed an inhibitory effect of NAC toward the formation of lipophilic-DNA adducts (5.18 +/- 0.73 versus 4.08 +/- 1.03/10(8) nucleotides; mean +/- SE; P = 0.05) as well as of 7,8-dihydro-8-oxo-2'-deoxyguanosine adducts in BAL cells (3.9 +/- 0.6 versus 2.3 +/- 0.2/10(5) nucleotides; P = 0.003). There was no effect of NAC on the formation of lipophilic-DNA adducts in peripheral blood lymphocytes or polycyclic aromatic hydrocarbon-DNA adducts in mouth floor/buccal mucosa cells or 4-aminobiphenyl-Hb adducts. Likewise, quantification of the biological response markers showed an inhibitory effect of NAC on the frequency of micronuclei in mouth floor and in soft palate cells (1.3 +/- 0.2 versus 0.9 +/- 0.2; P = 0.001) and a stimulating effect of NAC on plasma antioxidant scavenging capacity (393 +/- 14 versus 473 +/- 19 microM Trolox; P = 0.1) but not on BAL fluid antioxidant scavenging capacity. We conclude that NAC has the potential to impact upon tobacco smoke carcinogenicity in humans because it can modulate certain cancer-associated biomarkers in specific organs.
Assuntos
Acetilcisteína/uso terapêutico , Anticarcinógenos/uso terapêutico , Antioxidantes/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Neoplasias/prevenção & controle , Fumar/metabolismo , Acetilcisteína/administração & dosagem , Administração Oral , Adulto , Anticarcinógenos/administração & dosagem , Antioxidantes/administração & dosagem , Biomarcadores/análise , Cotinina/metabolismo , Adutos de DNA , Dano ao DNA , Método Duplo-Cego , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Humanos , Masculino , Testes de Mutagenicidade , Neoplasias/etiologia , Fumar/efeitos adversosRESUMO
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 24h 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 32P 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.
Assuntos
Acetilcisteína/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Pulmão/fisiologia , Transcrição Gênica/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/farmacologia , Genômica , Pulmão/efeitos da radiação , Troca Materno-Fetal , Camundongos , GravidezRESUMO
We monitored the mutagenicity of extracts of sediment fine particles collected, both in the cold season and in the hot season, from 10 reaches along the Po River, the main Italian watercourse. Each sample was representative of several kilometers of river stretch. At sub-toxic doses, the samples were not mutagenic to the Salmonella typhimurium his(-) strains TA98, TA100 and TA102, irrespective of the presence of S9 mix. However, they induced a mutagenic response in YG1024, which is typically reverted by frameshift mutagens that are metabolized in bacteria via acetyl-CoA:N-hydroxylamine O-acetyltransferase. Mutagenicity of sediments was higher during the cold season and had a spatial distribution consistent with the occurrence of pollution sources and confluence with polluted tributaries. Nevertheless, in the final stretch, near the Po delta into the Adriatic Sea, mutagenicity of sediments was low, comparable to that detected in the Po proximal reach, not far away from its springs. Genotoxicity biomarkers were evaluated in three cyprinid species, the "Italian nase" (Chondrostoma söetta), chub (Leuciscus cephalus), and barbel (Barbus plebejus), captured upstream and downstream of the confluence of a polluted tributary (Lambro River) with the Po River. There was no difference between the two areas concerning concentrations of fluorescent aromatic compounds in fish bile while, after metabolic activation, the bile of fish caught from the more polluted area became mutagenic to YG1024. Moreover, the levels of adducts to liver DNA were significantly higher in L. cephalus caught from the more polluted area, and the increase of micronucleated erythrocyte frequency was borderline to statistical significance, but only in C. söetta. Thus, certain biomarkers of exposure and effect in fish, as assessed under field conditions, correlate with the pollution of river sediments by mutagenic compounds.
Assuntos
Cyprinidae/genética , Sedimentos Geológicos , Mutagênicos/toxicidade , Poluentes do Solo/toxicidade , Animais , Bile/metabolismo , Biomarcadores , Biotransformação , Adutos de DNA/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Testes para Micronúcleos , Testes de Mutagenicidade , Mutagênicos/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genéticaRESUMO
The epidemic of lung cancer and the increase of other tumours and chronic degenerative diseases associated with tobacco smoking have represented one of the most dramatic catastrophes of the 20th century. The control of this plague is one of the major challenges of preventive medicine for the next decades. The imperative goal is to refrain from smoking. However, chemoprevention by dietary and/or pharmacological agents provides a complementary strategy, which can be targeted not only to current smokers but also to former smokers and passive smokers. This article summarises the results of studies performed in our laboratories during the last 10 years, and provides new data generated in vitro, in experimental animals and in humans. We compared the ability of 63 putative chemopreventive agents to inhibit the bacterial mutagenicity of mainstream cigarette smoke. Modulation by ethanol and the mechanisms involved were also investigated both in vitro and in vivo. Several studies evaluated the effects of dietary chemopreventive agents towards smoke-related intermediate biomarkers in various cells, tissues and organs of rodents. The investigated end-points included metabolic parameters, adducts to haemoglobin, bulky adducts to nuclear DNA, oxidative DNA damage, adducts to mitochondrial DNA, apoptosis, cytogenetic damage in alveolar macrophages, bone marrow and peripheral blood erytrocytes, proliferation markers, and histopathological alterations. The agents tested in vivo included N-acetyl-L-cysteine, 1,2-dithiole-3-thione, oltipraz, phenethyl isothiocyanate, 5,6-benzoflavone, and sulindac. We started applying multigene expression analysis to chemoprevention research, and postulated that an optimal agent should not excessively alter per se the physiological background of gene expression but should be able to attenuate the alterations produced by cigarette smoke or other carcinogens. We are working to develop an animal model for the induction of lung tumours following exposure to cigarette smoke. The most encouraging results were so far obtained in models using A/J mice and Swiss albino mice. The same smoke-related biomarkers used in animal studies can conveniently be applied to human chemoprevention studies. We participated in trials evaluating the effects of N-acetyl-L-cysteine and oltipraz in smokers from Italy, The Netherlands, and the People's Republic of China. We are trying to develop a pharmacogenomic approach, e.g. based on genetic metabolic polymorphisms, aimed at predicting not only the risk of developing cancer but also the individual responsiveness to chemopreventive agents.
Assuntos
Carcinógenos/análise , Mutagênicos/análise , Fumaça/efeitos adversos , Fumar , Animais , Regulação da Expressão Gênica , Humanos , Neoplasias Pulmonares/epidemiologia , Modelos AnimaisRESUMO
Autophagy is an intracellular lysosomal degradation process induced under stress conditions. Autophagy also plays a major role in ocular patho-physiology. Molecular aging does occur in the trabecular meshwork, the main regulator of aqueous humor outflow, and trabecular meshwork senescence is accompanied by increased oxidative stress. However, the role of autophagy in trabecular meshwork patho-physiology has not yet been examined in vivo in human ocular tissues. The purpose of the herein presented study is to evaluate autophagy occurrence in ex-vivo collected human trabecular meshwork specimens and to evaluate the relationship between autophagy, oxidative stress, and aging in this tissue. Fresh trabecular meshwork specimens were collected from 28 healthy corneal donors devoid of ocular pathologies and oxidative DNA damage, and LC3 and p62 protein expression analyzed. In a subset of 10 subjects, further to trabecular meshwork proteins, the amounts of cathepesin L and ubiquitin was analyzed by antibody microarray in aqueous humor. Obtained results demonstrate that autophagy activation, measured by LC3II/I ratio, is related with. oxidative damage occurrence during aging in human trabecular meshwork. The expression of autophagy marker p62 was lower in subjects older than 60 years as compared to younger subjects. These findings reflect the occurrence of an agedependent increase in the autophagy as occurring in the trabecular meshwork. Furthermore, we showed that aging promotes trabecular-meshwork senescence due to increased oxidative stress paralleled by autophagy increase. Indeed, both oxidative DNA damage and autophagy were more abundant in subjects older than 60 years. These findings shed new light on the role of oxidative damage and autophagy during trabecular-meshwork aging.
Assuntos
Envelhecimento/genética , Autofagia/genética , Glaucoma de Ângulo Aberto/genética , Estresse Oxidativo , Proteínas de Ligação a RNA/biossíntese , Malha Trabecular/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Envelhecimento/patologia , Feminino , Glaucoma de Ângulo Aberto/patologia , Humanos , Lisossomos/genética , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxirredução , Proteínas de Ligação a RNA/genética , Malha Trabecular/patologiaRESUMO
The anti-diabetic drug metformin is endowed with anti-cancer properties. Epidemiological and experimental studies, however, did not provide univocal results regarding its role in pulmonary carcinogenesis. We used Swiss H mice of both genders in order to detect early molecular alterations and tumors induced by mainstream cigarette smoke. Based on a subchronic toxicity study, oral metformin was used at a dose of 800 mg/kg diet, which is 3.2 times higher than the therapeutic dose in humans. Exposure of mice to smoke for 4 months, starting at birth, induced a systemic clastogenic damage, formation of DNA adducts, oxidative DNA damage, and extensive downregulation of microRNAs in lung after 10 weeks. Preneoplastic lesions were detectable after 7.5 months in both lung and urinary tract along with lung tumors, both benign and malignant. Modulation by metformin of 42 of 1281 pulmonary microRNAs in smoke-free mice highlighted a variety of mechanisms, including modulation of AMPK, stress response, inflammation, NFκB, Tlr9, Tgf, p53, cell cycle, apoptosis, antioxidant pathways, Ras, Myc, Dicer, angiogenesis, stem cell recruitment, and angiogenesis. In smoke-exposed mice, metformin considerably decreased DNA adduct levels and oxidative DNA damage, and normalized the expression of several microRNAs. It did not prevent smoke-induced lung tumors but inhibited preneoplastic lesions in both lung and kidney. In conclusion, metformin was able to protect the mouse lung from smoke-induced DNA and microRNA alterations and to inhibit preneoplastic lesions in lung and kidney but failed to prevent lung adenomas and malignant tumors induced by this complex mixture.
Assuntos
Adutos de DNA/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Metformina/administração & dosagem , Fumar/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Camundongos , MicroRNAs/biossíntese , MicroRNAs/efeitos dos fármacosRESUMO
Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be involved in breast and prostate carcinogenesis, which share similar estrogen-related mechanisms. We previously demonstrated that administration of BPA to female mice results in the formation of DNA adducts and proteome alterations in the mammary tissue. Here, we evaluated the ability of BPA, given with drinking water, to induce a variety of biomarker alterations in male Sprague-Dawley rats. In addition, we investigated the formation of DNA adducts in human prostate cell lines. In BPA-treated rats, no DNA damage occurred in surrogate cells including peripheral blood lymphocytes and bone marrow erythrocytes, where no increase of single-strand DNA breaks was detectable by comet assay and the frequency of micronucleated cells was unaffected by BPA. Liver cells were positive at transferase dUTP nick end labeling assay, which detects both single-strand and double-strand breaks and early stage apoptosis. BPA upregulated clusterin expression in atrophic prostate epithelial cells and induced lipid peroxidation and DNA fragmentation in spermatozoa. Significant levels of DNA adducts were formed in prostate cell lines treated either with high-dose BPA for 24 h or low-dose BPA for 2 months. The BPA-related increase of DNA adducts was more pronounced in PNT1a nontumorigenic epithelial cells than in PC3 metastatic carcinoma cells. On the whole, these experimental findings support mechanistically the hypothesis that BPA may play a role in prostate carcinogenesis and may, potentially, affect the quality of sperm.