O uso da esponja marinha Hymeniacidon heliophila (Wilson, 1911) (Filo Porifera) como modelo experimental para a avaliação dos efeitos do hormônio sintético 17α-etinilestradiol

Abstract

A key environmental problem faced in recent years is that of emerging micropollutants termed endocrine disrupters, which are dumped into the oceans. Among these substances, synthetic estrogen 17α-ethinylestradiol has been identified as the main compound responsible for the endocrine changes in aquatic organisms. The main objectives of this research were: standardization of exposure bioassays of sponge Hymeniacidon heliophila (Wilson, 1911) to synthetic estrogen contraceptive 17α-ethinyl estradiol, and establishment of the most appropriate technique for extraction of total messenger RNA from this marine sponge. For this purpose, during three campaigns, marine sponges of the species H. heliophila were collected in the region of the rocky shore of Itaipu Beach. The individuals were removed alive and intact from the rocky shore, always submerged in marine water, and packed in transparent plastic bags containing water from the collection site; where they remained until acclimatized in a marine aquarium of 200 liters, previously arranged in the Laboratory of Marine Genetics (LGMar), at the State University of Rio de Janeiro (UERJ), Maracanã Campus. Two types of bioassays were performed with marine sponges exposed to the synthetic hormone 17α-ethinyl estradiol: A) Exposure bioassays using " beta fish" type aquaria with different concentrations of the hormone: control; 40; 200; 1000 and 5000 ng / L-1, and different exposure times: 10; 40; 120 minutes; six and 24 hours; B) Bioassays in 50 mL "Falcon " tubes, with the concentration of the hormone 5000 ng / L-1 and times of 40 and 120 minutes. In addition, two methodologies were chosen for RNA extraction: A) after the recommended exposure times, the sponge samples were contained in RNAlater and stored in the freezer at -80ºC until RNA extraction; B) RNA extraction from the sponge samples were performed immediately after the bioassays. The H. heliophila species presented a good performance for laboratory maintenance, and the two bioassay conditions analyzed were quite satisfactory, since no signs of stress were recorded in the organisms during their handling for the toxicity tests. The standardization of the laboratory maintenance conditions of the H. heliophila marine sponge and the determination of a positive control of effect in the two types of bioassays applied, contributes to the establishment of this organism as a promising animal model to be used in ecotoxicological trials. The RNA obtained through the storage of the sponge samples in freezer -80°C did not present a fragmentation pattern that allowed the identification of bands in the gel. The total RNA extracted immediately after the bioassay provided the expected pattern, that is, the presence of two bands, when analyzed on 1% agarose gel, corresponding to ribosomal RNA 28S and 18S. As a main result of this study we have the model of bioassay of exposure to micropollutants standardized for the model marine sponge species, Hymeniacidon heliophila, and for pollutant 17α-ethinyl estradiol