Anti-Inflammatory, Anti-Oxidative and Anti-Apoptotic Effects of Thymol and 24-Epibrassinolide in Zebrafish Larvae.

Thymol (THY) and 24-epibrassinolide (24-EPI) are two examples of plant-based products with promising therapeutic effects. In this study, we investigated the anti-inflammatory, antioxidant and anti-apoptotic effects of the THY and 24-EPI. We used zebrafish (Danio rerio) larvae transgenic line (Tg(mpxGFP)i114) to evaluate the recruitment of neutrophils as an inflammatory marker to the site of injury after tail fin amputation. In another experiment, wild-type AB larvae were exposed to a well known pro-inflammatory substance, copper (CuSO4), and then exposed for 4 h to THY, 24-EPI or diclofenac (DIC), a known anti-inflammatory drug. In this model, the antioxidant (levels of reactive oxygen species-ROS) and anti-apoptotic (cell death) effects were evaluated in vivo, as well as biochemical parameters such as the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), the biotransformation activity of glutathione-S-transferase, the levels of glutathione reduced and oxidated, lipid peroxidation, acetylcholinesterase activity, lactate dehydrogenase activity, and levels of nitric acid (NO). Both compounds decreased the recruitment of neutrophils in Tg(mpxGFP)i114, as well as showed in vivo antioxidant effects by reducing ROS production and anti-apoptotic effects in addition to a decrease in NO compared to CuSO4. The observed data substantiate the potential of the natural compounds THY and 24-EPI as anti-inflammatory and antioxidant agents in this species. These results support the need for further research to understand the molecular pathways involved, particularly their effect on NO.

Evaluation of the developmental effects of a glyphosate-based herbicide complexed with copper, zinc, and manganese metals in zebrafish.

The use of glyphosate-based herbicides (GBH) has increased dramatically, being currently the most used herbicides worldwide. Glyphosate acts as a chelating agent, capable of chelate metals. The synergistic effects of metals and agrochemicals may pose an environmental problem as they have been shown to induce neurological abnormalities and behavioural changes in aquatic species. However, as their ecotoxicity effects are poorly understood, evaluating the impacts of GBH complexed with metals is an ecological priority. The main objective of the study was to evaluate the potentially toxic effects caused by exposure to a GBH (1 µg a.i. mL-1), alone or complexed with metals (Copper, Manganese, and Zinc (100 µg L-1)), at environmentally relevant concentrations, during the early period of zebrafish (Danio rerio) embryo development (96 h post-fertilization), a promising model for in vivo developmental studies. To clarify the mechanisms of toxicity involved, lethal and sublethal development endpoints were assessed. At the end of the exposure, biochemical and cell death parameters were evaluated and, 24 h later, different behavioural responses were assessed. The results showed that metals induced higher levels of toxicity. Copper caused high mortality, low hatching, malformations, and changes in biochemical parameters, such as decreased Catalase (CAT) activity, increased Glutathione Peroxidase (GPx), Glutathione S-Transferase (GST), reduced Glutathione (GSH) and decreased Acetylcholinesterase (AChE) activity, also inducing apoptosis and changes in larval behaviour. Manganese increased the activity of SODs enzymes. Zinc increased mortality, reactive oxygen species (ROS) levels, superoxide dismutase activity (SODs) and caused a decrease in AChE activity. Embryos/larvae exposed to the combination of GBH/Metal also showed teratogenic effects during their development but in smaller proportions than the metal alone. Although more studies are needed, the results suggest that GBH may interfere with the mechanisms of metal toxicity at the biochemical, physiological, and behavioural levels of zebrafish.

Behavioural toxicity of environmental relevant concentrations of a glyphosate commercial formulation - RoundUp® UltraMax - During zebrafish embryogenesis.

The use of herbicides with glyphosate as an active ingredient (a.i.) has increased dramatically in recent years, with its residues often being found in either soil or water. Nevertheless, concerns have arisen about its harmful side effects for both ecosystems and wildlife health. Therefore, the objective of this work was to assess the effects of a commercial formulation of glyphosate (RoundUp® UltraMax), at environmentally relevant concentrations on zebrafish embryos through a set of behavioural patterns. Zebrafish embryos were exposed to 0, 1, 2 and 5 µg a.i. mL-1 concentrations of the glyphosate formulation for 72 h (from 2.5 to 75 h post-fertilization (hpf)). After exposure, larvae were washed and allowed to develop until 144 hpf. At this point, the larvae behaviour was evaluated using a battery of tests to assess the general exploratory motility, escape-like responses, anxiety-related behaviours and social interactions. In addition, cortisol levels were assessed. No significant changes were observed relative to the exploratory behaviour in the standard open field. The anxiety-related behaviours were similar among groups, and no social interference was observed following exposure to these glyphosate concentrations. Likewise, cortisol levels remained similar among treatments. Still, the larvae exposed to 5 µg a.i. mL-1 did not react to the presence of an aversive stimulus, supporting glyphosate-induced changes in the sensory-motor coordination during development. In general, these results indicate a possible neurotoxic effect of this glyphosate-based formulation that should be further evaluated. In addition, the results obtained could impose a risk for wildlife sensitive species that should not be neglected.

Dose-dependent effects of a glyphosate commercial formulation - Roundup® UltraMax - on the early zebrafish embryogenesis.

The use of herbicides with glyphosate as an active ingredient, the so-called glyphosate-based herbicides (GBH), has increased dramatically in recent years currently being the most widely used in the world. Therefore, glyphosate residues have been detected in water and soils near the application sites. Recent studies indicate that GBH may cause adverse effects on vertebrates although these have been attributed to the presence of adjuvants in the commercial formulations rather than to the sole compound. Accordingly, the objective of this work was to investigate the lethal and sub-lethal developmental effects, neurotoxic potential and oxidative stress responses of zebrafish embryos to Roundup® Ultramax (RU) exposure. Embryos were exposed during 72 h to 0, 2, 5, 8.5 µg a.i. mL-1 of RU. Increased mortality was observed in embryos exposed to concentrations above 8.5 µg a.i. mL-1 as well as increased number of malformations. Decreased heart rate and hatchability were also observed. By contrast, exposure to concentrations that do not evoke teratogenic outcomes induced a dose-dependent decrease of heart rate although not inducing significant developmental changes. However, histological changes were not observed in the larvae exposed to these concentrations. Moreover, the generation of reactive oxygen species, the antioxidant enzymes activities (SOD and CAT), the GST biotransformation activity, the glutathione levels (GSH and GSSG), the oxidative damage (MDA) and the acetylcholinesterase and lactate dehydrogenase were similar among groups following exposure. Overall, available evidence suggests a dose-dependent toxicological effect of this formulation at concentrations that are not routinely detected in the environment. However, additional studies should be performed to better understand the underlying molecular mechanisms in favor of this formulation.