Avaliação da toxicidade de nanopartículas de dióxido de titânio (TiO2) e chumbo inorgânico (Pbll) em Astyanax altiparanae (Characidae)

Orientadora : Profª Drª Marta Margarete CestariDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 22/03/2016Inclui referências : f. 70-85Resumo: O aumento das indústrias juntamente ao crescimento populacional tem elevado de forma considerável os níveis de poluição no meio ambiente e o ambiente aquático é o ecossistema mais suscetível ao despejo de substâncias. Diversas alterações estruturais e fisiológicas podem ser observadas em organismos expostos a altos níveis de contaminação, e atualmente, sabe-se que as exposições a metais, como o chumbo, causam toxicidade em diversas espécies. A utilização de nanopartículas vem crescendo em diversos campos da indústria e da medicina devido a suas características físico-químicas únicas, muito atrativas para o ramo tecnológico. Porém, pouco se sabe sobre os problemas que estes nanomateriais podem causar ao ambiente e aos seres vivos e de sua interação com outros contaminantes. O objetivo desse estudo foi avaliar a toxicidade das nanopartículas de TiO2 (NpTiO2) em baixas doses, isolada e associadas ao chumbo inorgânico (PbII) em peixes da espécie Astyanax altiparanae através de biomarcadores genéticos e bioquímicos. Os animais foram divididos em nove tratamentos: controle negativo; 5 ng/g de NpTiO2; 50 ng/g de NpTiO2; 500 ng/g de NpTiO2; 21 ?g/g de PbII; 5 ng/g de NpTiO2 + 21 ?g/g de PbII; 50 ng/g de NpTiO2 + 21 ?g/g de PbII; 500 ng/g de NpTiO2 + 21 ?g/g de PbII e controle positivo. Os bioensaios foram de 96 horas, via injeção intraperitonial, exceto o controle positivo que foi de 24 horas. Os resultados demonstraram através da análise da atividade das enzimas glutationa S-transferase e superóxido dismutase que ambos os contaminantes tanto isolados quanto em associação podem causar estresse oxidativo nas células de A. altiparanae. O PbII induziu necrose e em consequência a essa citotoxicidade ocorreu uma proliferação celular, aumentando a frequência de eritrócitos policromáticos nos grupos expostos ao PbII sozinho e associado as NpTiO2 nas três doses. O teste do micronúcleo písceo indicou que os contaminantes não tiveram efeito mutagênico, no entanto, houve um aumento no total de alterações morfológicas nucleares totais e do tipo Notched nos tratamentos de 5 ng/g+Pb e 500 ng/g+Pb. Não se observou genotoxicidade no sangue e no fígado utilizando o ensaio cometa. Entretando a maior dose dos contaminantes (500 ng/g de NPTiO2 + 21 ?g/g) causou danos ao DNA no rim, já no cérebro os tratamentos 500 ng/g de NpTiO2 e 5 ng/g + 21 ?g/g de PbII foram genotóxicas, sendo que nesta última, observou-se também o aumento na atividade da acetilcolinesterase, bem como na maior dose de NpTiO2 isolada. Em conclusão, a espécie A. altiparanae demonstrou ser um bom bioindicador, pois respondeu as baixas doses testadas nesse estudo. As NpTiO2 e o PbII demonstraram ter efeitos tóxicos e estes podem ser potencializados quando os contaminantes estão em associação. Palavras-chave: genotoxicidade; citotoxicidade; nanopartículas; dióxido de titânio; chumbo; peixes.Abstract: The increase of industries with the high population growth has considerably increased pollution levels in the environment and the aquatic ecosystem is the most susceptible to the release of substances. Several physiological and structural changes may be observed in organisms exposed to high levels of contaminants, and currently, it is known that exposure to metals such as lead, cause toxicity in several species. The use of nanoparticles is growing in various fields of industry and medicine due to its unique physical and chemical characteristics, being attractive for the technology industry. However, little is known about the problems that these nanomaterials can cause to the environment and organisms, besides its interaction with other contaminants. The aim of this study was to evaluate the toxicity of TiO2 nanoparticles (NpTiO2) in low doses, isolated and associated with inorganic lead (PbII) in fish species Astyanax altiparanae through genetic and biochemical biomarkers. The animals were separated in nine treatments: negative control; 5 ng / g NpTiO2; 50 ng / g NpTiO2; 500 ng / g NpTiO2; 21 ?g / g PbII; 5 ng / g NpTiO2 + 21 ?g / g PbII; 50 ng / g NpTiO2 + 21 ?g / g PbII; 500 ng / g NpTiO2 + 21 ?g / g PbII and positive control. Bioassays were conducted for 96 hours, with intraperitoneal injection, except for the positive control which was for 24 hours. The results demonstrated that both contaminants either alone or in association can cause oxidative stress in cells of A. altiparanae, through the analysis of the activity of the enzyme glutathione S-transferase and superoxide. The PbII induced necrosis which induced cellular proliferation, increasing the frequency of polychromatic erythrocytes in groups exposed to PbII alone and associated with the NpTiO2 in three doses. The piscine micronucleus test showed that contaminants were not mutagenic, however, there was an increase in total nuclear morphological alterations and Notched in treatments 5 ng / g + Pb and 500 ng / g + Pb. Genotoxicity in blood and liver evaluated through the comet assay was not observed. However, the higher dose of contaminants (500 ng / g NpTiO2 + 21 ?g / g) caused DNA damage in the kidney, and in the brain,treatments of 500 ng / g NpTiO2 and 5 ng / g + 21 ?g / g PbII were genotoxic, as well as presented an increase in the activity of acetylcholinesterase. This increase was also obsevedin the higher dose of NpTiO2 isolated. In conclusion, A. altiparanae proved to be a good bioindicator, since this species respond to the low doses tested in this study. The NpTiO2 and PbII have toxic effects and which were increased by the association of contaminants. Keywords: genotoxicity; cytotoxicity; nanoparticle; titanium dioxide; lead; fish

Co-exposure effects of Titanium Dioxide Nanoparticles and Metals on antioxidant systems and DNA in the fish Hoplias intermedius

Metallic nanoparticles are one of the most produced and used engineered nanomaterial and their wide applications lead to environmental contamination. The aquatic environment is the major recipient of wastes containing nanoparticles and other co-occuring contaminants. We aimed to evaluate genotoxic and biochemical effects of acute exposure to nano-TiO2 in the fish Hoplias intermedius and the interaction to metals. Besides assessing the nanoparticles’ physical-chemical properties we performed an acute exposure with 0.1; 1; 10 μg g-1 nano-TiO2, alone and with lead (21 μgg-1) and aluminium (50 μgg-1). A set of biomarkers were evaluated in the liver such as genotoxicity by comet assay and biochemical biomarkers (SOD, CAT, GPx, GSH, EROD, GST). Most of the biomarkers were altered by the metals, and the nanoparticles caused decrease in SOD (0.1 and 1μg g-1), GSH (1μg g-1), and GST (0.1 μg g-1). In co-exposure, some metal effects were attenuated. There was an increase in EROD activity for most co-exposure groups. Nano-TiO2 was not genotoxic in the experimental conditions. We did not observe any increase in DNA breaks in co-exposure, although, nanoparticles changed the response of some biochemical biomarkers

Biochemical and genotoxicity assessment of a polluted urban river using the native fish Astyanax altiparanae Garutti & Britski (Teleostei, Characidae)

Agriculture residues and domestic effluents are contaminants that reach aquatic ecosystems leading to toxic effects to environment and human health. These contaminants impacting mainly developing countries, due to precarious treatment of effluents and lack of legislation in the use of pesticides. Perequê River (South of Brazil) is impacted by pesticides (mainly glyphosate) and domestic effluents. The aims of this study were to analyze the concentration of glyphosate in water and sediments of Perequê River; and to evaluate the sublethal effects of the river contamination in Astyanax altiparanae using biomarkers. A sampling of water, sediment and fish were carried out in Perequê River (November/2016). The samples were collected in two sites, site 1 (S1) that is located on agriculture area and water catchment point; and site 2 (S2) located on the area with a domestic effluents influence. Water and sediment samples were used to glyphosate quantification. Fish were collected and tissues (brain, muscle, liver) were used to biochemical and genetic biomarkers. Glyphosate were quantified in S1, which is impacted by agriculture. Alterations in the liver biomarkers were not observed between the sites. However, the decrease in the AChE activity was observed in the S2, showing that the mixture of contaminants (urban and agriculture contaminants) can lead to anticholinesterasic effects. The sublethal effects observed can be a threat of environmental and human health