Abstract: As nanopartículas de ouro (AuNPs) com diferentes tamanhos e formas têm sido amplamente estudadas em todo o mundo. A síntese de AuNPs geralmente envolve agentes de redução e solventes que apresentam problemas relacionados à toxicidade. A fim de resolver esta questão, metabólitos presentes em diversos extratos de plantas tem sido explorados para a preparação de diferentes nanopartículas. Pesquisadores mostraram que alguns fitoquímicos, como a mangiferina (MGF) e resveratrol (RESV), além de reduzirem e estabilizarem as AuNPs, são capazes de funcionalizá-las. O aumento na produção e utilização das nanopartículas tem provocado grande preocupação quanto aos impactos e riscos potenciais que estas podem causar ao meio ambiente e à saúde humana. Desta forma, o estudo teve como objetivo avaliar o nível de toxicidade das AuNPs, reduzidas e estabilizadas com MGF e RESV in vitro e in vivo, comparando-as com o método clássico de síntese de AuNPs, descrito por Turkvich, no qual o citrato de sódio é o agente redutor e estabilizante (CITR-AuNPs). Foi realizado o ensaio de citotoxicidade de acordo com a International Standard Organization (ISO 10993-5, 2009) e o ensaio de toxicidade aguda em embriões de Zebrafish (FET TEST), de acordo com o protocolo da OECD nº 236. Zebrafish é um importante modelo animal utilizado nas áreas de biologia do desenvolvimento, genética e biomedicina e é utilizado em ensaios ecotoxicológicos. Como o zebrafish apresenta 70% de similaridade genética com humanos, assim como similaridades fisiológicas e anatômicas, este modelo pode ser usado para prever efeitos de toxicidade em humanos. Além de alta taxa reprodutiva e rápido desenvolvimento embrionário, a espécie apresenta grande sensibilidade quando exposta a produtos químicos, sendo capaz de absorver rapidamente os compostos que são diretamente adicionados à água e acumulá-los em vários tecidos. O organismo é de fácil obtenção, gerenciamento e de baixo custo. Tanto as nanopartículas reduzidas e estabilizadas com MGF e RESV, quanto os redutores, não apresentaram citotoxicidade, porém as CITR-AuNPs apresentaram IC50 de 180 μg.mL-1. Em relação ao FET TEST, a taxa de letalidade dos organismos expostos a MGF e MGF-AuNPs, ambas na concentração de 350 μg.mL-1, foi de 12% e 5% respectivamente. Já a taxa de letalidade dos organismos expostos ao RESV na concentração de 165 μg.mL-1, foi de 17.5%. A CL50 obtida das RESV-AuNPs foi de 14.55 μg.mL-1, nas concentrações de 82.5 e 165 μg.mL- 1, observou-se atraso estatísticamente significativo da eclosão dos organismos, no período de 72 horas pós fecundação. A taxa de letalidade dos organismos expostos ao citrato de sódio e CITR-AuNPs, ambas na concentração de 250 μg.mL-1, foi de 3.75% e 16.25% respectivamente. Não foram encontrados valores de CL50 nas concentrações testadas das nanopartículas, exceto RESV-AuNPS. Nanopartículas com maiores concentrações do que as testadas não apresentam estabilidade, tendendo a aglomeração e precipitação.

CAVALCANTE, A.K.; BATISTA, J.G.S.; MAZIERO, J.S.; ROGERO, S.; ROGERO, J.R.; VIVEIROS, W.; KATTI, K.; LUGAO, A.B.; ALAVARSE, R.D. Avaliação da toxicidade in vitro e in vivo das nanopartículas de ouro reduzidas e estabilizadas com mangiferina e resveratrol. In: WORKSHOP DE BIOMATERIAIS, ENGENHARIA DE TECIDOS E ORGÃOS ARTIFICIAIS, 6th, October 29-31, 2019, São Paulo, SP. Abstract... 2019. p. 35-35. Disponível em: http://repositorio.ipen.br/handle/123456789/31548. Acesso em: $DATA.

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Abstract: Surface water samples from São Paulo state were collected to perform Bioluminescent Yeast Estrogen Screen (BLYES) and chemical analyses (LC-MS/MS). Results showed environmental concentrations of atrazine from 2 to 43 ng L-1 on chemical analyses. Some studies have been performed to evaluate toxic effects on non-target organisms (fish) using herbicides such as Atrazine, a moderately toxic compound classified as an endocrine disrupting chemical (EDC) that can affect reproduction of several aquatic organisms with a compromise of vitellogenin production. To determine toxicity on embryonic stages of fish to different environmental chemicals and waste water, Fish Embryo Toxicity Test (FET) was designed using Danio rerio as model specie on this test, according to OECD 236 or ISO 15088 protocols, however these protocols observe only acute toxicity based on endpoints such as coagulated eggs, nondetachment of the tail, lack of heart beating and lack of somite formation. Some abnormalities can be recorded after the exposure on FET test but they are not considered as endpoint, neither any other compromised biomarker by EDC action. In order to evaluate the possibility of using these chronic endpoints and to verify if those environmental concentrations of atrazine are ecologically relevant, compromising reproductive aspects, FET test using Danio rerio were performed to assess lethal concentrations, sublethal concentrations and vitellogenin quantification after atrazine exposure. Occurrence of morphological abnormalities (microcephaly, spine curvature, edema, reduced size) and mortality of the embryos were determined exposing 20 fertilized eggs to atrazine concentrations from 2 to 64 mg L-1. The LC50 and EC50 were obtained after 96 hours of exposure. Organisms that survived each concentration were frozen to further vitellogenin quantification. Preliminary average concentrations obtained (LC 50; 96h= 48.15 mg L-1 and EC 50; 96h= 27 mg L-1) were considerably higher than concentrations observed on environmental samples. Therefore, surface water concentrations would not cause mortality or deformity in fish emphasizing the necessity to observe possible effect on vitellogenin concentration. Data will be analyzed and compared with the environmental concentration of atrazine to stablish the potential application of vitellogenin as endpoint on FET test.

MARTINI, G.; VIVEIROS, W.; FRANÇA, D.D.; QUINAGLIA, G.; RAIMUNDO, C.C.; LOPES-FERREIRA, M.V.; ROGERO, S.O.; ROGERO, J.R. Fish Embryo Toxicity Test (FET) to evaluate atrazine effects. In: SETAC LATIN AMERICA BIENNIAL MEETING, 12th, September 7-10, 2017, Santos, SP. Abstract... 2017. p. 83-83. Disponível em: http://repositorio.ipen.br/handle/123456789/31261. Acesso em: $DATA.

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Abstract: Gold nanoparticles (AuNPs) are among the most widely studied metal nanoparticles for biomedical applications. AuNPs can be synthesized by chemical reduction. Several methods for this type of synthesis are described in the literature, one of them is the Turkevich method, which uses sodium citrate (CITR) as a reducing agent. Other methods were developed based on the use of solvent systems during nanoparticle production. Although they are efficient production methods, they are environmentally unviable. In order to address this issue, metabolites present in various plant extracts have been explored for the preparation of different AuNPs. Green nanotechnology is the nanotechnology aspect that aims to develop protocols to generate sustainable products and production processes, in order to minimize the use of toxic compounds. In this study, the phytochemicals chosen as reducing agents and stabilizers for AuNPs synthesis were mangiferin (MGF) and resveratrol (RESV). Due to their ability to interact with biological systems, along with various applications of AuNPs, their toxicity has become one of the most important concerns. Due to the increased production and use of AuNPs, their risk of reaching different environmental compartments and becoming available increases the importance of determining toxicity in various species of biological interest, such as microcrustaceans and fish. Zebrafish is an important animal model used in the areas of developmental biology, genetics, biomedicine, nanotoxicology and is also used in ecotoxicological assays. The aim of the study is to evaluate the toxicity level of MGF (MGF-AuNPs) and RESV (RESV-AuNPs) reduced and stabilized AuNPs in vitro and in vivo by comparing them with the classical AuNPs synthesis method described by Turkvich (CITR-AuNPs). The study has two strands, one of which is to evaluate the level of nanomaterial toxicity using the zebrafish animal model as a preclinical study, and the other to evaluate the level of nanomaterial toxicity using the zebrafish animal model as a study of environmental toxicity. Cytotoxicity assay according to ISO 10993-5, Zebrafish FET TEST according to OECD 236 and Microinjection Assay in Zebrafish embryos. Green nanotechnology has proven to be a valuable tool in the synthesis of AuNPs for toxicity, not requiring the use of solvents and potentially toxic substances. The toxicity of AuNPs varied according to the assay. In the cytotoxicity assay, IC50 was obtained from CITR-AuNPs, whose IC50 was about 72%, which corresponds to the Au concentration of 74.16 µg.mL-1. In FET, RESV-AuNPs caused delays in the hatching process of zebrafish embryos, and it was the only sample that could obtain the LC50 (4.41%, corresponding to the Au concentration of 6,57 µg.mL -1). In the assay in which AuNPs were microinjected into embryos, the concentration causing toxic effect was not found in 50% of the organisms.

CAVALCANTE, A.K.; BATISTA, J.G.; MAZIERO, J.d.; FERNANDES, B.V.; VIVEIROS, W.; ROGERO, S.O.; ROGERO, J.R.; LUGAO, A.B. In vitro and In vivo toxicity of Gold nanoparticles synthesized and stabilized with phytochemicals. In: PAN-AMERICAN NANOTECHNOLOGY CONFERENCE, 2nd, March 4-7, 2020, Águas de Lindoia, SP. Abstract... 2020. Disponível em: http://repositorio.ipen.br/handle/123456789/31899. Acesso em: $DATA.

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