Caracterização de acetilcolinesterase e sua aplicação como biomarcador de nanopartícula de prata ao usar Zebrafish e Tilápia do Nilo como espécie sentinela

Abstract

The increasing commercialization of silver nanoparticle (AgNP), due wide antimicrobial activity, makes its exposure in the environment of concern, as its potential risk is largely unknown. This nanoparticle has several pathways of exposure to a living organism and can interact with macromolecules. The use of enzymatic biomarkers has been highlighted because its reflects the toxic effects of xenobiotics in natural environments. Among these, cholinesterases (ChEs) have taken place in ecotoxicological and pharmacological research, as it acts on the central and peripheral nervous system. Therefore, the aim of this study is to purify and characterize acetylcholinesterase (AChE) of Zebrafish and to evaluate its effect, together with catalase, as an AgNP biomarker, through sublethal assays using as target organism, Nile Tilapia (Oreochromis niloticus) and Zebrafish (Danio rerio). The characterization of brain and muscle ChEs Zebrafish showed similar physicochemical (basic pH and temperature ~ 30°C) and kinetic parameters ( of 0.191 e 0.230 mM, respectively), except for inhibitor specificity, in which it identified that only brain tissue has AChE, while muscle has both cholinesterases, AChE and BuChE (butyrylcholinesterase). Purification of brain AChE of Zebrafish showed a recovery factor of 3.86 and a representative subunit of 76 kDa. The ecotoxicological studies showed changes in ChEs and catalase (CAT) activity of Nile Tilapia and Zebrafish, after 96h exposure of different AgNP concentrations and morphological changes Zebrafish gills were also observed. Muscle AChE was more sensitive to AgNP than brain AChE, showing a 37.3% reduction in activity for Zebrafish e 52% for Tilapia, after exposure to higher AgNP concentration. And gill CAT was also more sensitive to AgNP than hepatic CAT, showing a 67.4% reduction in activity for Zebrafish and 50% for Tilapia. These results show that these species can be used as an efficient bioindicator to investigate the toxicity of AgNP in molecular and histopathology level. This work can clarify and guide studies of purification and application of AChE as nanoparticle biomarker.