Ecotoxicoproteômica aplicada à avaliação dos efeitos da proteína inseticida Cry1F aos estágios iniciais de peixe-zebra (Danio rerio)

Abstract

For over 50 years, the Bacillus thuringensis (Bt) soil bacterium and its Cry toxins have been widely used in agriculture as bioinsecticides. Among the Cry entomotoxins most used, we can highlight the Cry1F protein. This protein is found in transgenic cotton, maize and soybean crops to provide protection against the attack of lepidopteran insects. Although some studies indicate that Cry proteins are innocuous to non-target terrestrial organisms, very little is known about the effects of these proteins on aquatic vertebrates. In addition, studies have reported the entry of proteins into aquatic ecosystems. Thus, through the use of classic toxicological tests coupled to an ecotoxicoproteomics approach, this study had as aim to evaluate the effect of Cry1F protein, at the concentration of 1.1 mg/L (worst-case scenario of environmental contamination with Cry proteins), on the earlier life stages of the zebrafish (Danio rerio). After performing the embryotoxicity test in fish (FET Test No. 236, OECD), it was observed that, in the tested concentration, the survival ratios of the negative control (water) and Cry1F were 98.67 ± 2.36 and 91.67 ± 10.41%, with no difference between treatments (p < 0.05). In addition, embryos and larvae of zebrafish did not show any morphological or developmental changes during the 96 hours of exposure to Cry1F. However, the dosage of toxicity biomarkers showed that the enzymatic levels of acetylcholinesterase (AchE), glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) in Cry1F treated larvae were altered (p < 0.05) when compared to the negative control. Proteomic analysis showed that Cry1F treated larvae had 5 overexpressed proteins when compared to the control group: histone H4, histone H2A, cognate 71 kDa heat shock protein, Zgc: 153405 and Zgc: 163061; while 10 were underexpressed: apolipoprotein A-I, voltage-dependent anion channel 2, brain acid soluble protein homologue 1, mitochondrial ATP synthase d subunit, aldolase B fructose-biphosphate, profilin, coactasin, apolipoprotein A-Ib, Zgc: 86599 and adenylate kinase 1 isoenzyme. In addition, several metabolic pathways related to differentially expressed proteins have been altered, for example those involved in the metabolism of ATP, lipids, cholesterol and DNA. Thus, it is clear that at the concentration tested, the Cry1F protein, while do not promote any phenotypic change in zebrafish larvae, causes disorders in the proteome, as well as promotes changes in the leves of enzymatic biomarker of toxicity.