Responses to ROS inducer agents in zebrafish cell line: differences between copper and UV-B radiation.

Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm(2)) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways.

Effects of anti-inflammatory diclofenac assessed by toxicity tests and biomarkers in adults and larvae of Danio rerio.

The entrance of the anti-inflammatory diclofenac in water bodies is a consequence of inappropriate use, incorrect disposal, and inefficiency of wastewater treatment plants (WWTPs) in removing this drug from sewage, among others. Effects of diclofenac on non-target aquatic organisms still need to be clarified. The objective of this work was to evaluate the toxic effects of the diclofenac on larvae and adults of Danio rerio. The LC50 values were 5.49 mg/L and 5.22 mg/L for the adult and larvae, respectively. A set of biochemical and genotoxic biomarkers were evaluated in fish exposed to an environmentally relevant concentration of 2 µg/L DCF and a no observed effect concentration (NOEC) of 3 mg/L diclofenac. At the NOEC, an increase in activities of glutathione-S-transferase (GST) enzyme and an increase in ATP binding cassette (ABC) transporters in gills of adult fish was observed; also, an increase in lipoperoxidation (LPO) was seen in the gills of adults and whole larvae. These results indicate that diclofenac activates the fish detoxification processes and may affect fish health.

Antifouling biocide dichlofluanid modulates the antioxidant defense system of the brown mussel Perna perna.

Dichlofluanid is a fungicide employed as a booster biocide in antifouling paints, but information its toxicity to aquatic organisms is scarce. This study aims to evaluate biomarker responses in the mussel Perna perna exposed to dichlofluanid. Mussels were exposed to 0 (control), 0.1 µg/L (environmental concentration), 10, and 100 µg/L of dichlofluanid for 24 and 96 h. Byssus formation, oxygen consumption, and oxidative stress response were evaluated in gills and digestive glands. The results demonstrated that even the lowest dichlofluanid concentration causes a reduction in byssus biomass and water content. The higher concentrations caused an acute increase in oxygen consumption, which only returned to control levels after 96 h of exposure. ACAP levels and antioxidant enzyme activities were affected in both tissues with a larger effect observed in gill tissues as demonstrated by the IBR index. The overall results demonstrated that environmentally relevant concentrations of dichlofluanid would be deleterious to aquatic organisms.

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.

Effects of glyphosate on cholinesterase activity of the mussel Perna perna and the fish Danio rerio and Jenynsia multidentata: in vitro studies.

Although the herbicide glyphosate [N-(phosphonomethyl)glycine] is not classified as an acethylcholinesterase inhibitor, some studies have reported reduction in the acethylcolinesterase activity after in vivo exposure to both its pure form and its commercial formulations. Considering this controversy, the objective of the present study was to investigate, in vitro, the effects of glyphosate exposure on cholinesterase activity of the brown mussel Perna perna and of two fish species: zebrafish Danio rerio and onesided livebearer Jenynsia multidentata. For this purpose, samples of different tissues (brain and muscle for fish; gills and muscle for mussel) were homogenized and pre-incubated with different glyphosate concentrations before cholinesterase activity determination. Results demonstrated that cholinesterase from different fractions of all species tested was inhibited by glyphosate. The concentrations of glyphosate that inhibits 50% of cholinesterase activity (IC50) ranged from 0.62 mM for P. perna muscle to 8.43 mM for J. multidentata brain. According to this, cholinesterase from mussel seems to be more sensitive to glyphosate exposure than those from the fish D. rerio and J. multidentata.

Molecular and biochemical biomarkers responses in the mussel Mytilus edulis collected from Southern Brazil coast.

Marine ecosystems are typically subjected to a variety of stressors containing complex xenobiotics mixtures. This study aims to evaluate the responses of molecular and biochemical biomarkers in the mussel Mytilus edulis providing data for environmental monitoring programs development in Southern Brazil. Mussels were collected at a polluted site, near Patos Lagoon outfall, and at a control site. Gills, muscle and mantle samples were used for biomarker determinations. Mussels collected at the polluted site significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities and decreased catalase (CAT) activity. Moreover, an increase in sod1, gstπ and hsp70 mRNA expression was observed. Overall, biochemical and molecular biomarkers responses were observed, but these responses varied depending on the analyzed tissue. These results indicate possible contaminants effects on organisms and the need for effective environmental monitoring programs in this ecosystem.