Zebrafish (Danio rerio) ability to activate ABCC transporters after exposure to glyphosate and its formulation Roundup Transorb®.

The emergent demand for food production has increased the widespread use of pesticides, especially glyphosate-based herbicides as they can protect different types of crops, especially transgenic ones. Molecules of glyphosate have been found in water bodies around the world, and its presence can cause negative effects on non-target organisms, such as fish. Glyphosate toxicity appears to be systemic in fish but does not affect their organs equally. Also, its formulations can be more toxic than pure glyphosate. In this sense, we investigated if these variations in toxicity could be related to ATP binding cassette subfamily C (ABCC) transporters and the cellular detoxification capacity, following exposure to herbicides. Thus, adults of Danio rerio were exposed (24 and 96 h) to glyphosate and Roundup Transorb® (RT) at an environmental concentration of 0.1 mg/L, and the activity of ABCC proteins and gene expression of five isoforms of ABCC were analyzed. Glyphosate and RT exposure increased ABCC protein activity and gene expression up to 3-fold when compared to controls, indicating the activation of detoxification mechanisms. Only in the brain of D. rerio, the exposure to RT did not stimulate the activity of ABCC proteins, neither the expression of genes abcc1 and abcc4 that responded to the exposure to pure glyphosate. These results may suggest that the brain is more sensitive to RT than the other target-tissues since the mechanism of detoxification via ABCC transporters were not activated in this tissue as it was in the other.

Effects of learning on mTOR pathway gene expression in the brain of zebrafish (Danio rerio) of different ages.

Target of rapamycin (TOR) is a protein kinase involved in the modulation of mRNA translation and, therefore, in the regulation of protein synthesis. In neurons, the role of TOR is particularly important in the consolidation of long-term memory (LTM). One of the modulators of TOR is brain-derived neurotrophic factor (BDNF), which activates the TOR signaling pathway to promote protein synthesis, synapse strengthening, and the creation of new neural networks. We investigated the gene expression pattern of this pathway during memory consolidation in zebrafish of different ages. Our findings demonstrate that TOR activation in old animals occurs in the early phase of consolidation, and follows a pattern identical to that of BDNF expression. In younger animals, this increase in activation did not occur, and changes in BDNF expression were also not so remarkable. Furthermore, the expression of the main proteins regulated by the synthesis of TOR (i.e., 4EBP and p70S6K) remained identical to that of TOR in all age groups.

NPY and sbGnRH gene expression in juvenile and adult male Brazilian flounder Paralichthys orbignyanus / Expressão gênica do NPY e do sbGnRH em machos juvenis e adultos de linguado Paralichthys orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.

O objetivo deste estudo foi avaliar a expressão gênica do neuropeptídeo Y (NPY) e da variante sea bream do hormônio liberador de gonadotrofinas (sbGnRH) em linguados machos juvenis e adultos. O hipotálamo foi isolado para a extração de RNA total. Após a síntese de cDNA, a PCR em tempo real foi usada para avaliar a expressão gênica. Foi observado um aumento de aproximadamente duas vezes nos níveis de NPY e de aproximadamente três vezes nos níveis de sbGnRH nos peixes adultos. Esses resultados demonstram que estes peptídeos podem estar envolvidos na regulação, via hipotálamo, da maturação sexual no linguado.

mRNA Expression and activity of ion-transporting proteins in gills of the blue crab Callinectes sapidus: effects of waterborne copper.

Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2‰) and full (30‰) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 µM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodium-potassium adenosine triphosphatase [Na(+)/K(+)-ATPase], hydrogen adenosine triphosphatase [H(+)-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Na(+)/K(+)/2Cl⁻) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl⁻ cotransporter. However, Cu did not affect Na(+)/K(+)-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30‰). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30‰) than in diluted seawater (2‰).

Reactive oxygen species generation and expression of DNA repair-related genes after copper exposure in zebrafish (Danio rerio) ZFL cells.

Copper is an essential metal to aquatic animals, but it can be toxic when in elevated concentrations in water. The objective of the present study was to analyze copper effects in zebrafish hepatocytes (ZFL cell-line). The number of viable cells and copper accumulation were determined in hepatocytes exposed in vitro to different copper concentrations (5-30mgCu/L). Intracellular reactive oxygen species (ROS) formation, total antioxidant capacity against peroxyl radicals, and expression of genes related do DNA repair system were also measured in hepatocytes exposed to 5 and 20mgCu/L. After 24h of exposure, hepatocytes showed an exponential kinetics of copper accumulation. Copper exposure (24 and 48h) significantly reduced hepatocyte number in all concentrations tested, except at the lowest one (5mgCu/L). Exposure to 20mgCu/L for 6, 12 and 24h significantly increased intracellular ROS formation. However, no significant change in total antioxidant capacity was observed. After 12 and 24h of exposure to 20mgCu/L, a significant decrease in expression of p53 and CDKI genes was observed. Conversely, expression of Gadd45alpha, CyclinG1 and Bax genes was significantly induced after 24h of exposure to 20mgCu/L. In hepatocytes exposed to 5mgCu/L, any significant alteration in expression of these genes was observed. In a broad view, most of genes encoding for DNA repair proteins were inhibited after copper exposure, especially in hepatocytes exposed to 20mgCu/L. Taken all together, results obtained suggest that the increased intracellular ROS formation induced by copper exposure would be responsible for the alteration in gene expression pattern observed.

Short-term responses to cadmium exposure in the estuarine polychaete Laeonereis acuta (polychaeta, Nereididae): subcellular distribution and oxidative stress generation.

Some effects of cadmium exposure (100 microg/L for 4, 8, 12, and 24 h) on the estuarine polychaete Laeonereis acuta (Nereididae) were evaluated. This polychaete was able to accumulate cadmium in the body, with the metal stored mainly in the cytosolic fraction (>10 kDa). Activity of the antioxidant enzymes superoxide dismutase, glutathione S-transferase, and glutathione reductase (GR) as well as the total oxyradical scavenger capacity, the glutamate cysteine ligase catalytic subunit gene expression, and the metallothionein-like proteins content were not affected by cadmium at any exposure time tested. Catalase (CAT) activity, however, was significantly lower (p < 0.05) in worms treated with cadmium compared with that in controls after 8 h of exposure. At the same exposure time, lipid peroxide levels were increased (p < 0.05) in worms exposed to cadmium compared with those in control worms. Interestingly, CAT and GR activities decreased over time (p < 0.05) independent of cadmium treatment, which is a result that could be attributed to starvation. The effects caused by cadmium in the present study were observed only after 8 h of exposure, demonstrating that cadmium can generate oxidative stress.