Basal levels of inorganic elements, genetic damages, and hematological values in captive Falco peregrinus

It is essential to determine the basal pattern of different biomarkers for future evaluation of animal health and biomonitoring studies. Due to their great displacement capacity and to being at the top of their food chains, birds of prey are suitable for monitoring purposes. Furthermore, some birds of prey are adapted to using resources in urban places, providing information about this environment. Thus, this study determined the basal frequency of micronuclei and other nuclear alterations in peripheral blood erythrocytes of Falco peregrinus. Hematological and inorganic elements analysis were also performed. For this purpose, 13 individuals (7 females and 6 males) were sampled in private breeding grounds. Micronucleus, nuclear buds, nucleoplasmic bridges, notched nuclei, binucleated cells and nuclear tails were quantified. Inorganic elements detected included the macro-elements Ca, P, Mg, Na, Cl, S and K as well as the micro-elements Fe, Al and Zn. Our study found similar values compared to previous studies determining the reference ranges of hematologic parameters in falcons. The only different value was observed in the relative number of monocytes. Thus, this study is the first approach to obtaining reference values of cytogenetic damage in this species and could be useful for future comparisons in biomonitoring studies

Systems chemo-biology analysis of DNA damage response and cell cycle effects induced by coal exposure

Cell cycle alterations are among the principle hallmarks of cancer. Consequently, the study of cell cycle regulators has emerged as an important topic in cancer research, particularly in relation to environmental exposure. Particulate matter and coal dust around coal mines have the potential to induce cell cycle alterations. Therefore, in the present study, we performed chemical analyses to identify the main compounds present in two mineral coal samples from Colombian mines and performed systems chemo-biology analysis to elucidate the interactions between these chemical compounds and proteins associated with the cell cycle. Our results highlight the role of oxidative stress generated by the exposure to the residues of coal extraction, such as major inorganic oxides (MIOs), inorganic elements (IEs) and polycyclic aromatic hydrocarbons (PAH) on DNA damage and alterations in the progression of the cell cycle (blockage and/or delay), as well as structural dysfunction in several proteins. In particular, IEs such as Cr, Ni, and S and PAHs such as benzo[a]pyrene may have influential roles in the regulation of the cell cycle through DNA damage and oxidative stress. In this process, cyclins, cyclin-dependent kinases, zinc finger proteins such as TP53, and protein kinases may play a central role

In vitro genotoxic effect of secondary minerals crystallized in rocks from coal mine drainage

Coal processing generates a large volume of waste that can damage human health and the environment. Often these wastes produce acid drainage in which several minerals are crystallized (evaporites). This study aimed to identify secondary minerals, as well as the genotoxic potential of these materials. The samples were collected at two sites along the Rocinha River in Santa Catarina state (Brazil): (1) directly from the source of the acid drainage (evaporite 1), and (2) on the river bank (evaporite 2). The samples were characterized by X-ray diffraction and by particle-induced X-ray emission techniques. In vitro genotoxicity testing using Comet assay and Micronucleus test in V79 cells was used to evaluate evaporite samples. Our study also used System Biology tools to provide insight regarding the influence of this exposure on DNA damage in cells. The results showed that the samples induced DNA damage for both evaporites that can be explained by high concentrations of chromium, iron, nickel, copper and zinc in these materials. Thus, this study is very important due to the dearth of knowledge regarding the toxicity of evaporites in the environment. The genetic toxicity of this material can be induced by increased oxidative stress and DNA repair inhibition

Obese rats are more vulnerable to inflammation, genotoxicity and oxidative stress induced by coal dust inhalation than non-obese rats

Obesity is an important nutritional disorder worldwide. Its association with environmental pollution may trigger an increase in oxidative stress and inflammatory parameters. Coal is a resource used throughout the world as an important fuel source for generating electricity. The ashes released by the coal combustion cause serious problems for human health due to their high toxicity and their capacity to bioaccumulate. The aim of this work was to investigate the effects of coal dust inhalation in the organs of obese and non-obese Wistar rats. Pro-inflammatory cytokines, oxidative stress, oxidative damage, histological analysis, comet assay, and micronuclei were investigated. Both obesity and coal dust inhalation increased the pro-inflammatory cytokines IL-1β and TNF-α and decreased HSP70 levels in serum, however, in obese animals that inhaled coal dust these changes were more pronounced. Liver histological analysis showed severe microvesicular steatosis in obese animals that inhaled coal dust. Lung histologic investigation showed abnormalities in lung structure of animals exposed to coal dust and showed severe lung distensibility in obese animals exposed to coal dust

Risco ocupacional em fumicultores : genotoxicidade associada à suscetibilidade genética

Agricultores envolvidos no cultivo do tabaco estão constantemente expostos a uma grande variedade de químicos. O uso de agrotóxicos em larga escala tem provocado danos à saúde destes trabalhadores assim como a manipulação das folhas de fumo úmidas, pois além de substâncias antropogênicas persistentes outros compostos orgânicos com potencial pesticida estão presentes nas folhas do tabaco. A nicotina, através do contato dermal com as folhas do fumo, tem causado um envenenamento agudo nos trabalhadores da lavoura. O conjunto de sintomas desta exposição é conhecido como doença da folha verde. Este estudo teve como objetivo avaliar o efeito genotóxico em fumicultores expostos ocupacionalmente a agroquímicos e nicotina. A instabilidade genômica, a morte celular, a dosagem de colinesterase, cotinina e marcadores de estresse oxidativo bem como o conteúdo de elementos traço foram analisados nas células destes trabalhadores. Para verificar a possível modulação de genes de suscetibilidade com os resultados dos biomarcadores, os polimorfismos dos genes GSTT1, GSTM1, GSTP1, CYP2A6, PON, OGG1, RAD51, XRCC1 e XRCC4 foram avaliados. No primeiro momento, amostras de sangue periférico foram coletadas de 30 indivíduos expostos e de 30 indivíduos não expostos com o intuito de investigar o risco ocupacional em três diferentes períodos da safra de fumo (entresafra, aplicação intensa de pesticidas e na colheita da folha). Neste estudo foi observado aumento significativo de dano ao DNA, avaliado pelo ensaio Cometa, em fumicultores comparados ao grupo não exposto em todos os períodos da safra e, um significante aumento nas frequência de células micronucleadas na entresafra. Correlação entre idade e tempo de exposição em relação aos resultados do ensaio Cometa e do teste de MN não foi encontrado. O dano ao DNA foi maior em homens comparado a mulheres, mas diferença significativa foi observada apenas na entresafra. Não houve diferença na atividade da colinesterase entre os grupos estudados. Elevado nível de cotinina foi observado na colheita da folha de fumo, demonstrando exposição à nicotina. Em um segundo momento, amostras de sangue periférico e de mucosa oral foram coletadas de aproximadamente 111 agricultores em dois períodos da safra (na aplicação intensa de pesticidas e na colheita) e de 56 indivíduos não expostos. Os resultados destes testes demonstraram anomalias nucleares nas células de fumicultores expostas a misturas de substâncias com potencial genotóxico e citotóxico. Diferença nos resultados foi percebida entre os dois momentos da safra de fumo investigados. Modulação no dano ao DNA e na morte celular em células da mucosa oral foi observada nos genes PON1 e CYP2A6. Diferença entre gênero e tempo de exposição não foi encontrada nos diferentes parâmetros analisados. Houve correlação entre idade e os resultados obtidos no teste de MN em linfócitos no grupo não exposto e no momento da aplicação de pesticidas. Em relação ao uso do equipamento de proteção individual, diferença na frequência de MN no momento da aplicação de pesticidas foi observada. Dos nove marcadores de suscetibilidade estudados, somente GSTM1 nulo e CYP2A6*9, demonstraram associação com os resultados obtidos pelo teste de MN em linfócitos. Diferença na atividade da colinesterase e nos níveis de cotinina não foi observada em relação aos diferentes genótipos de PON1 e de CYP2A6, respectivamente. Aumento de cromo, magnésio, alumínio, cloro, zinco e potássio foram percebidos no momento da aplicação de pesticidas em relação ao grupo não exposto e à colheita. Maior atividade da superóxido dismutase no grupo exposto em relação ao grupo não exposto foi observada. Atividade da catalase e a medida de TBARS apresentaram aumento significativo na colheita da folha de fumo em relação ao grupo não exposto e ao momento da aplicação de pesticidas. Finalmente, esta investigação sugere níveis maiores de dano ao DNA avaliados pelo ensaio Cometa e pelo teste de MN em linfócito e em mucosa oral, nos diferentes momentos da safra do fumo (aplicação de pesticides e colheita), chamando a atenção o significativo aumento de dano ao DNA no momento da entresafra. Modulação dos genes de metabolismo foi observada, onde GSTM1 nulo e PON1 Gln/Gln tiveram maiores níveis de dano ao DNA, em linfócitos e em células da mucosa oral respectivamente, no momento da aplicação de pesticidas e CYP2A6*1/*1 e CYP2A6*9/- tiveram maiores níveis de dano ao DNA (em linfócitos) e de morte celular (em células da mucosa oral) na colheita. Não houve influência dos genes envolvidos no reparo em relação aos diferentes biomarcadores no grupo exposto. Os resultados indicam que exposição crônica a pesticidas, tanto químicos sintéticos como natural, pode ativar o sistema de enzimas antioxidantes. Por fim, nosso estudo chama a atenção à necessidade de formação profissional e informação sobre práticas seguras no uso de pesticidas e, além disso, na fumicultura, na manipulação das folhas fumo.Agricultural workers engaged in tobacco cultivation are constantly exposed to large amounts of pesticides as well as to the nicotine present in raw tobacco leaves. Pesticides have been considered potential chemical mutagens. Studies have assumed that nicotine absorbed through the skin results in the characteristic green tobacco sickness (GTS), an occupational illness reported by tobacco workers. This study sought to determine genotoxic effects in farm workers occupationally exposed to agrochemicals and nicotine. The genomic instability, cell death as well as cholinesterase and cotinine levels in the blood of tobacco farmers were investigated. In order to verify relation of genetic susceptibility with biomarkers results, GSTT1, GSTM1, GSTP1, CYP2A6, PON, OGG1, RAD51, XRCC1 and XRCC4 genes polymorphisms was evaluated. Determination of oxidative stress markers and trace elements content was accomplished. In the first moment, peripheral blood samples were collected from 30 agricultural workers and 30 non-exposed subjects to investigate occupational exposure in three different crop times (off-season, during pesticides application and leaf harvest). In this study a significant increase in DNA damage, assessed by Comet assay, was observed in tobacco farmers compared to the nonexposed group, for all different crop times, and a significant increase in micronucleated cells was detected in the off-season group. No correlation was found between age and exposure time in relation to biomarker tests. The DNA damage was greater in males than in females, but with a significant difference only in off-season group. No difference, in cholinesterase activity, was seen among the group of farmers and non-exposed group. Increased level of cotinine was observed in leaf harvest group. In the second moment, peripheral blood and buccal cells samples were collected about from 111 agricultural workers, at two different crop times (during pesticides application and leaf harvest), and 56 non-exposed individuals. Results showed nuclear anomalies in the blood and buccal cells in tobacco farmers exposed to mixture of the substances with genotoxic and cytotoxic potential. Difference in blood and buccal micronucleus cytome assay results was found among two different times in the tobacco crop. Effects of PON1 and CYP2A6 genetic polymorphisms on the modulation of DNA damage induced by pesticides and cell death were observed in buccal micronucleus cytome (BMNcyt) assay. No statistically significant difference was found between genders and exposure time for all parameters analyzed. No difference was observed in hematocrit values between the groups. Correlation was observed in age and cytokinesis-blocked micronuclei (CBMN) assay results in nonexposed and pesticide application group. In relation to personal protective equipment (PPE) use, increased MN frequency in pesticide application was observed. From the nine markers of individual susceptibility studied in the CBMN assay, only GSTM1 null and CYP2A6*9, showed significant associations with results in farmers cells. Increase in cotinine level was observed in leaf harvest, when compared to non-exposed group. Cholinesterase (BChe) level was similar in the farmer and non-exposed group. No significant effect on the BChe activity and cotinine level was observed in exposed group with reference to different PON1 and CYP2A6 genotypes, respectively. In pesticide application was found increase in chromiun, magnesium, aluminum, chloride, zinc and potassium, when compared to non-exposed and leaf harvest group. Superoxide dismutase (SOD) activity presented a significant increase in exposed groups (pesticide application and leaf harvest) in relation to non-exposed group, and pesticide application presented higher values than leaf harvest group. The catalase (CAT) activity and TBARS presented a significant increase in leaf harvest than non-exposed group and pesticide application. Finally, this investigation suggests that DNA damage increases, analyzed by Comet, CBMN and BMNcyt assays, at different tobacco crop stages (pesticides application and tobacco leaf harvest), calling attention to the significant increase in the DNA damage during the off-season. Effect of individual genotype of the metabolism genes on the level of different biomarkers evaluated in exposed group demonstrated an influence on increase in damage of GSTM1 null (blood cells) and PON1 Gln/Gln (buccal cells) on pesticides application group, and a influence of CYP2A6*1/*1 (blood cells) and CYP2A6*9/- (buccal cells) on tobacco leaf harvest group. Individual genotype of the DNA repair genes of exposed groups did not show influence on the different biomarkers in this study. In pesticide application was found increase of trace elements content. The results also indicated that chronic exposure to pesticides, synthetic and natural, can influence antioxidant enzymes activity. Our study drives the attention once more to the need for occupational training on safe working practices and safe work environment for farm workers. Developing countries should use such data to establish safety occupational rules during the use of pesticides and mainly during tobacco leaf harvest

Genotoxicidade ocasionada pelas folhas do fumo (Nicotiana tabacum) - expostas ou não a agrotóxico - em Cantareus aspersus

A produção de fumo na região sul do Brasil, mais precisamente no Estado do Rio Grande do Sul (RS), exerce grande importância na atividade econômica e social. Na área econômica, o fumo é responsável pela arrecadação de grandes somas em impostos. No campo social, a atividade fumageira é grande geradora de empregos diretos e indiretos. Os agricultores que trabalham nas lavouras de fumo estão em constante contato com a planta. Consequentemente, estes trabalhadores se expõem tanto aos compostos orgânicos e inorgânicos presentes nas folhas, como aos pesticidas naturais e sintéticos relacionados. “Green tobacco sickness” (GTS) é conhecida como risco ocupacional à saúde de trabalhadores do campo no plantio de tabaco. Os sintomas têm sido atribuídos ao envenenamento agudo por nicotina seguido por contato dermal com as folhas do fumo. Contudo autores salientam que os efeitos sinérgicos da nicotina e dos pesticidas devem ser examinados. Neste trabalho utilizou-se Cantareus aspersus com o objetivo de identificar a ação genotóxica e/ou mutagênica das folhas de Nicotiana tabacum através da exposição dermal. O caracol terrestre tem sido utilizado nos últimos anos devido a sua fácil aclimatação e manipulação em laboratório e por sua sensibilidade e resistência aos testes de genotoxicidade. Nestes animais foram realizados testes de biomonitoramento de exposição: Ensaio Cometa e quantificação dos elementos químicos pela técnica PIXE; bem como, testes de biomonitoramento de efeito: Teste de Micronúcleo e quantificação do Citocromo P450. Nosso estudo dosou a quantidade de nicotina na água, exposta à superfície da folha, por cromatografia líquida de alta eficiência (HPLC) e realizou “screening” fitoquímico de N. tabacum. Trinta moluscos terrestres foram expostos a diferentes tratamentos: dez expostos a folhas de tabaco sem pesticida; dez expostos a folhas de tabaco com pesticida e dez expostos a folhas de Lactuca sativa L. (grupo controle). Células da hemolinfa foram coletadas após 0, 24, 48 e 72 horas de exposição, o dano ao DNA foi avaliado pelo Ensaio Cometa em todos os períodos e a freqüência de micronúcleos somente em 72 horas. Resultados significativos foram encontrados nos grupos expostos a folhas de fumo, através do Ensaio Cometa, no período de 24, 48 e 72 horas de exposição e em 72 horas através doTeste de Micronúcleos, quando comparados ao grupo controle. Não houve diferença significativa entre caracóis expostos a folhas de fumo com e sem pesticida, portanto, podemos afirmar que o pesticida flumetralin não contribuiu com os danos ao DNA observados. “Screening” fitoquímico revelou presença de alcalóide, cumarina, traços de saponina e flavonóides. Inibição da atividade da enzima do Citocromo P450 ocorreu somente no grupo exposto a folhas de fumo sem pesticida. Doze elementos inorgânicos diferentes foram quantificados nas folhas de tabaco e nos caracóis expostos a folhas sem pesticida e, dez nos caracóis expostos a folhas com pesticida. Por cromatografia foram dosados 0,02% de nicotina por folha. A presença de alcalóides (nicotina), cumarinas, saponinas, flavonóides e diferentes metais provavelmente influenciaram o efeito mutagênico e genotóxico em C. aspersus expostos ao fumo. Estes efeitos possivelmente tenham sido causados pela complexa mistura presente nas folhas que podem interagir produzindo efeitos sinérgicos, antagônicos ou influenciando a absorção de um dado composto. As enzimas do sistema Citocromo P450 em C. aspersus expostos a folhas de fumo sem veneno podem ter sido inibidas pela nicotina, pelos flavonóides e pelo cobre. Por fim, nosso estudo providencia dados biológicos e químicos sobre C. aspersus expostos à folha de fumo e confirma a sensibilidade do Ensaio Cometa e do Teste de Micronúcleos na avaliação de misturas complexas, indicando associação entre nicotina, cumarina e interação flavonóide/metal (principalmente cobre e ferro) como principais causadores de dano ao DNA em células de hemolinfa do molusco terrestre C. aspersus por folhas de N. tabacum.Tobacco production in southern Brazil, precisely in Rio Grande do Sul state (RS) play a major role in economic and social activity. Tobacco farmers are routinely exposed to complex mixtures present in tobacco leaves including organic and inorganic compounds. Green tobacco sickness (GTS) is a form of nicotine poisoning that affects workers who have direct dermal contact with tobacco plants during cultivation and harvesting. However, some authors suggest that the synergistic effects of nicotine and pesticide should be examined. The purpose of this study was to evaluate the genotoxic and mutagenic effect of tobacco leaves, with and without exposure to flumetralin in Cantareus aspersus through dermal exposure. The land mollusk was utilized because of its easy acclimatization and manipulation in the laboratory and for its sensibility and resistance in genotoxicity tests. Biomonitoring exposure test was performed on these animals: Comet Assay and chemical elements quantification by PIXE; biomonitoring tests for effects were also carried out: Micronuclei Test and cytochrome P450 quantification. Nicotine dosage was performed in water exposed to the surface of tobacco leaves, by High Performance Liquid Chromatography (HPLC) and screening phytochemistry in Nicotiana tabacum. Thirty land mollusks were exposed to different treatments; ten snails to tobacco leaves with pesticide; ten to tobacco leaves without pesticide and ten snails exposed to lettuce leaves (control group). Hemolymph cells were collected after 0, 24, 48 and 72 hours, the DNA damage was evaluated by single cell gel assay for all these periods, and the frequency of micronuclei only in 72h. Significant results were found in groups exposed to tobacco leaves during 24, 48 and 72 hours of exposure by Comet Assay and by Micronucleus Test when compared to the control group. No difference was observed between different periods or between exposure to leaves with and without pesticide. This result shows that no genotoxic effect can be attributed to pesticide. Chemical results found alkaloid, coumarin, saponin traces, flavonoids and different metals. Inhibition of cytochrome P450 enzymes occurs only in the group without pesticide. Twelve inorganic elements were found in tobacco leaves and in snails exposed to tobacco leaves without pesticide. Ten inorganic elements were found in tobacco leaves with pesticide. Nicotine dosage found 0.02% of this alkaloid per leaf. The presence of coumarin, saponin traces, flavonoids, alkaloid (nicotine) and different metals probably causes genotoxic effects on land mollusks exposed to tobacco leaves with and without pesticide. This induction of DNA damage by dermal exposure to tobacco leaves was caused by a complex mixture present in the leaves. The enzymes of cytochrome P450 systems were inhibited by nicotine, flavonoids and copper. Our study provided chemical and biological data on C. aspersus exposed to tobacco leaves. The results of our study confirm the sensibility of the Comet Assay and Micronucleus Test for the evaluation of the complex mixture, indicating an association between nicotine, coumarin and flavonoid/metal interaction (mainly copper and iron) as the main causes of DNA damage in hemolymph cells from the land mollusk C. aspersus by N. tabacum

Avaliação dos efeitos da poluição em duas espécies de peixes nos Rios Tramandaí e Mampituba (RS) através do teste de micronúcleos

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