Metabolic, immunologic, and histopathologic responses on premetamorphic American bullfrog (Lithobates catesbeianus) following exposure to lithium and selenium.

The presence of chemicals and the destruction of freshwater habitats have been addressed as one of the reasons for the decline in the amphibians' populations worldwide. Considering the threat that these animals have been suffering in tropical regions, the present study tested if the Brazilian legislation, concerning the permissive levels of lithium and selenium in water bodies and effluents, warrants the protection of aquatic life. To do so, we assessed the metabolic, immunologic, and histopathologic alterations in liver samples of American bullfrog (Lithobates catesbeianus), at the premetamorphic stage, through biomarkers indicative of general energetic status, i.e., glucose, lipid, and protein metabolism using biochemical and histochemical approaches. The immunologic responses were assessed by the quantification of melanomacrophage centres (MMCs); the histopathologic evaluation of the liver sections was also performed. The assay was carried out over 21 days with two periods of sampling (after 7 and 21 days) to assess the effects of exposure over time. The animals were exposed to the considered safe levels of lithium (2.5 mg L-1) and selenium (10 µg L-1), both, isolated and mixed. The exposed animals showed alterations in glucose and lipid metabolism throughout the experiment. The intense presence of MMCs and histopathological responses are compatible with hepatotoxicity. The toxicity expressed by the employed animal model indicates that the Brazilian environmental legislation for the protection of aquatic life needs to be updated. With this study, we intend to provide data for better environmental policies and bring attention to sublethal effects triggered by the presence of contaminants in the aquatic environment.

Comet assay protocol for Bombus atratus fat body and pericardial cells (Hymenoptera, bombini) at a safe concentration of mercury.

The decline of the Bombus population is closely related to the presence of environmental pollutants. Among these pollutants, trace metals represent a major concern, which includes mercury, a known genotoxic substance. The induction of genotoxicity may be demonstrated by the comet assay (a.k.a. single-cell gel electrophoresis), a simple and sensitive method for DNA damage estimating. The current work provided, for the first time, a protocol of comet assay for Bombus atratus using mercury as a standard chemical at safe concentrations according to the Environment National Council of Brazil, and the World Health Organization. Bees were collected and divided into three groups (n = 11 each), in which the exposed groups received a 0.2 ppb or a 1 ppb of mercury solution, and the control group received water. The bioassay was performed for 48 h at controlled temperature and humidity conditions, according to the OECD guideline toxicological test method for B. terrestris. The samples were stained with different dyes to observe the efficacy of each one. Variations of parameters in methodology, such as concentration and time of exposure to lysis solution as well as the electrophoretic process, allowed the observation of comets at different levels. DAPI and acridine orange presented an unstable fluorescence, and silver nitrate dye was more effective. Therefore, the comet assay was shown to be an effective method to evaluate genotoxic effects in bees. The obtained results may be helpful for the establishment of a suitable protocol for future genotoxicity assessment in neotropical bees using different doses of xenobiotics.