Ecotoxicological impacts of the Fundão dam failure in freshwater fish community: Metal bioaccumulation, biochemical, genetic and histopathological effects.

This study investigates the ecotoxicological impacts of the Fundão dam rupture, one of the major environmental disaster that occurred in Brazil and in the world mining industry history, through multi-biomarkers responses and metals bioaccumulation in the fish community of different trophic levels. Specimens of the fishes (omnivorous/herbivorous and carnivorous) were collected along the Doce River channel and its affluent Guandú River, and in different lakes and coastal lagoons adjacent to the river channel, in the Espirito Santo State, Southeast of Brazil. Four sampling collections were carried out over two years (2018 to 2020, during dry and rainy seasons). For both trophic groups the biomarkers responses indicated physiological alterations related to metals exposure and showed strong seasonal variations. The principal component analysis and integrated biomarker response index showed that DNA damage and lipid peroxidation were more associated with dry season 2 (Sep/Oct 2019) and the oxidative damage in proteins, metallothioneins concentration and the activity of superoxide dismutase in the gills showed a greater association with rainy season 2 (Jan/Feb 2020). On the other hand, the enzymes of energy metabolism, catalase and histological damage in the liver and the gills, were more associated with the dry and rainy campaigns of the first year of monitoring. The multivariate approach also suggested a temporal intensification in the bioaccumulation of metals and biological effects in the lacustrine environments. Thus, these results demonstrate that the release of mineral residues from the rupture of the Fundão mine dam affects the health status of the fish from the Doce River basin, provoking metals bioaccumulation, hepatic and branchial damage in the fish besides inducing of enzyme activity related to metal contamination, even four years after the rupture.

Impacts of tailings of Fundão dam (Brazil) rupture on marine fish: Metals bioaccumulation and physiological responses.

This study evaluated the impacts of the mining tailings after the rupture of the Fundão dam on fish communities on the Atlantic Ocean southeast coast. Four sample collections were carried out over two years (2018-2020), in seasonal periods. Omnivorous/herbivorous and carnivorous fish were collected for analysis of metal bioaccumulation, multibiomarkers of environmental contamination and histopathology. Metal bioaccumulation was stronger correlated in carnivorous fish in the dry-2018 collection, besides higher activity of antioxidant enzymes, energy metabolism and higher morphological damage; however, there was less oxidative damage and less metallothioneins induction, and these variables were strongly associated with the wet-2020 collection. In a temporal view, it was possible to observe a reduction in metal levels in fish, except in the mouth of the Doce River. These events can be explained by seasonal natural events, which tend the resuspension and boost metal levels, mainly in the mouth region during the rainy season.

Characterization of different DNA repair pathways in hepatic cells of Zebrafish (Danio rerio).

The concern about DNA damage has directed efforts toward evaluating the genotoxic potential of physical and chemical agents. Since the extent of DNA damage is also related to the capacity of the organism in repairing the DNA, the advance of toxicological studies on this area depends on the characterization of the DNA repair mechanisms in the available models. The cellular zebrafish models, for example, replace mammalian cells to answer ecologically relevant questions on aquatic toxicology. So, the aim of the present study was to characterize the nucleotide excision repair (NER) and photoreactivation (PER) in two cellular models of Danio rerio liver, primary hepatocytes and ZF-L (Zebrafish Liver) cell line. We performed kinetic studies of the DNA damage levels after exposure to 6.8 J/m2 UVC using the T4-PDG modified Comet Assay, and determined the expression levels of important genes involved in NER, PER and base excision repair using RT-qPCR. It was observed that both ZF-L cell line and primary hepatocytes exhibit similar NER and PER activity. Primary hepatocytes showed similarities in the gene expression of most of the evaluated repair genes with the original tissue. These results indicate that both primary hepatocytes and ZF-L cells are useful models for toxicological studies aiming to evaluate NER and PER in hepatic cells. Moreover, the similarities in gene expression between the cellular models suggest that the ZF-L cells retain the DNA repair characteristics of the primary hepatocytes and, thus, could serve as replacement to this primary culture, reducing the use of animals in research.

Comparison of the Base Excision Repair Activity in Liver Cell Models of Zebrafish (Danio rerio).

Fish cellular models are commonly used to study the toxic potential of environmentally relevant compounds. Several of these pollutants act on DNA and compromise its integrity. Little is known, however, about the DNA repair ability of these cellular models. Therefore, the aim of this study was to evaluate the DNA base excision repair (BER) of zebrafish Liver (ZF-L) cell line and primary hepatocytes. We performed kinetic studies of the DNA damage levels after exposure to hydrogen peroxide (H2O2, 20 µM for 10 min) using the Comet Assay. Ten minutes after H2O2 treatment, 16% and 50% of the initial damage, measured as comet tail length, were repaired in ZF-L cell line and primary hepatocytes, respectively. Primary hepatocytes repaired 50% of the damages twice as fast as ZF-L cell line and showed DNA damage levels similar to control 40 min after H2O2 treatment. The total recovery time for ZF-L model was of 180 min, which indicates the culture cells have a less efficient BER. In conclusion, both ZF-L cell line and primary hepatocytes exhibit BER activity; however, these cellular models have different repair capacity. In addition, we demonstrated that ZF-L cell line and primary hepatocytes are useful tools for ecotoxicological studies focusing on DNA single-strand breaks and BER.

Antifouling biocides: Impairment of bivalve immune system by chlorothalonil.

Marine ecosystems are subjected to a variety of contaminants. Antifouling paints, for example, have been extensively used to protect ship surfaces from marine biofouling, but their toxicity has generated great concern. Thus, we evaluated the effect of the biocide chlorothalonil on the immune system of Perna perna mussels. The mussels were exposed to 0 (control), 0.1µg/L and 10µg/L of chlorothalonil for up to 96h. After 24h and 96h of exposure, the following immune-related parameters were analyzed in the hemolymph of mussels: total hemocyte count, cell adhesion, phagocytic activity, level of reactive oxygen species, cell viability and comet assay. After 24h and 96h of chlorothalonil exposure, cellular adhesion increased and the hemocyte viability reduced. Moreover, an increase in phagocytic activity was also observed after 96h of exposure to cholorothalonil. The exposure to 10µg/L of chlorothalonil for 96h reduced the air survival capacity of mussels. Total hemocyte count, ROS generation and DNA damage were not affected by the contaminant exposure. Our results indicate that chlorothalonil affected important immune responses of the bivalves, demonstrating that this biocide has effects on non-target species. This modulation of immune system reduced the health status of mussels, which could compromise their ability to survive in the environment.

Methylene blue toxicity in zebrafish cell line is dependent on light exposure.

Methylene blue (MB) has been widely applied in the clinical area and is currently being used in aquaculture as biocide. Some recent studies have emphasized the importance of understanding the action mechanism and the MB cellular targets. In this sense, zebrafish is considered a relevant model to study the intrinsic pathway of apoptosis as well as the cellular responses involving DNA damage and repair. So, the aim of the present study was to compare MB action mechanisms in a zebrafish cell line, both in the absence (MB alone; dark toxicity) and in the presence of photosynthetically active radiation (MB+PAR; phototoxicity). There was a significant increase of the levels of reactive oxygen and nitrogen species 3 h after MB treatment, whereas this increase was only observed 12 h after treatment with MB+PAR. All treatments with MB resulted in an increase in DNA damage after 3 and 6 h. However, cell death by apoptosis was observed from 6 h after treatment with MB+PAR and 12 h after treatment with MB alone. The expression of genes related to apoptosis was altered after MB and MB+PAR treatment. Therefore, this zebrafish cell line is sensitive to the photodynamic action of MB; MB is able to generate DNA damage and induce apoptosis in this cell line both alone and in the presence of PAR. However, the pathways leading to apoptosis in this model appear to be dependent on the type of MB exposure (in the presence or absence of PAR).