Marta M. Souza

Copper and the herbicide atrazine impair the stress response of the freshwater fish Prochilodus lineatus

In order to evaluate the effects of copper and atrazine on the stress response of the freshwater fish Prochilodus lineatus, juvenile fish were pre-exposed to copper (20 μg L(-1)) or atrazine (10 μg L(-1)) for 24 h and then submitted to air exposure for 3 min. Simultaneously fish kept in dechlorinated water for 24 h were subjected to air exposure and a non-stress group was not subjected to air stress or any contaminants. Animals were sampled immediately (t0) and after 1, 3 and 6 h of air exposure (t1, t3 and t6 respectively) for the analysis of plasma cortisol, glucose and Na(+), hepatic glycogen, branchial Na(+)/K(+)-ATPase (NKA), number of red blood cells per cubic millimeter of blood (RBC), hematocrit (Hct) and hemoglobin content (Hb). In fish pre-exposed to copper the stress response was inhibited, and at t1 and t3 both cortisol and glucose remained significantly lower compared to fish subjected to air stress only. In fish pre-exposed to atrazine there was no rise in cortisol, but there was an increase in plasma glucose, RBC, Hct and Hb at t0 and a return of these parameters to basal levels at t1, as they did not differ significantly in relation to non-stressed fish. Animals pre-exposed to either Cu or atrazine showed a significant reduction in NKA activity at t1 and t3, in relation to air stressed fish. These results clearly indicate that copper and atrazine impair cortisol stress response of P. lineatus and that fish subjected to a contaminant-induced stress, either by copper or atrazine, may not be able to respond to any additional stressors.

Effect of copper on ion content in isolated mantle cells of the marine clam Mesodesma mactroides

The effect of copper on ion content (Na(+), K(+), Ca(2+), and Cl(-)) was evaluated in isolated mantle gills of the marine clam Mesodesma mactroides. Clams were collected at the Mar Grosso Beach (São José do Norte, Rio Grande do Sul [RS], southern Brazil), cryoanesthetized, and had their mantles dissected. Mantle cells were isolated and incubated in a calcium-free phosphate solution without (control) or with Cu (CuCl(2)). Cells were exposed to Cu for 1 h (5 µM) or 3 h (2.5 and 5 µM). In cells incubated with 2.5 µM Cu, a significant decrease in intracellular Cl(-) content was observed. However, in cells incubated with 5.0 µM Cu, significant reductions in Na(+), K(+), and Cl(-) intracellular content were observed. Given the mechanisms involved in ion transport in mantle cells of the marine clam M. mactroides, the findings described here suggest that Cu exposure inhibits carbonic anhydrase and Na(+)/K(+) -ATPase activity. Also, it can be suggested that Cu is competing with Na(+) for the same mechanisms of ion transport in the cell membrane, such as the Na(+) channels and the Na(+)/K(+)/2Cl(-) cotransporter. Results from the present study also clearly indicate that processes involved in cellular anion regulation are more sensitive to Cu exposure than those associated with the cellular cation regulation. Characterization of sites for Cu accumulation and toxicity in aquatic animals is important for derivation of metal binding constants at the biotic ligand. Also, identification of the mechanism of metal toxicity is needed for modeling metal accumulation in the biotic ligand and its consequent toxicity. Therefore, the findings reported here are extremely valuable for the development of a biotic ligand model version for marine and estuarine waters.

Regulation of muscle hydration upon hypo- or hyper-osmotic shocks: differences related to invasion of the freshwater habitat by decapod crustaceans.

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages.

Cytotoxicity of the association of pesticides Roundup Transorb® and Furadan 350 SC® on the zebrafish cell line, ZF-L.

The present study aimed to ascertain the cytotoxicity of pesticides commonly used in rice cultivation, through in vitro assays employing the ZF-L cell line. The in vitro analyses investigated three cellular targets (cell membrane integrity, mitochondrial activity and lysosomal stability) in cells exposed to concentrations of Roundup Transorb® (67.7 μg L(-1), 135.4 μg L(-1) and 270.8 μg L(-1)), Furadan 350 SC® (0.1 μg L(-1), 0.05 μg L(-1) and 0.02 μg L(-1)). We also tested these products in combination. We analyzed the defensive capacity of the cells by measuring the activity of xenobiotic extruder proteins, as well as the expression of these same proteins. Cytotoxic effects of both pesticides were observed individually, as well as with the mixture of both products; including an inhibitory effect on the activity of xenobiotic extrusion. When exposed to the insecticide Furadan, and also the mixture of Furadan and Roundup, there was an increase in the expression of P glycoproteins (P-gps). There was also a negative correlation with cytotoxicity, mainly exhibited by mitochondrial activity and lysosomal integrity, but also with respect to the activity of P-gps. We observed that concentrations below those allowed by law were toxic regarding all parameters tested in this study, with the exception of mitochondrial function. Taken together, our results suggest that toxicity may be due to the surfactants present in the commercial formulations.

Cell damage induced by copper: an explant model to study anemone cells.

Sea anemones are benthic organisms, of low mobility and can be directly affected by water pollution. This work studied the defense mechanisms and DNA damage caused by copper toxicity in cells from the anemone Bunodosoma cangicum. For this, exposure of anemones cells were held, kept in primary culture through explant of podal disk to copper (7.8 and 15.6 μg/L), and the control group, for 6 and 24h. Cytotoxicity was seen through the viability and cell number, MXR phenotype through the accumulation of rhodamine-B, ROS generation by H2DCF-DA and DNA damage by comet assay. The results obtained show that there is a drop in viability and number of cells, especially after exposure of 24h in 15.6 μg/L. There is an induction of the MXR activity only at 7.8 μg/L for 24h. As for ROS, there is an increase in the generation of reactive species in greatest concentration of copper for 6h, and in both for 24h, which leads to oxidative stress, which culminates with a DNA damage. What was evidenced by the increase of the tail size, % DNA presented and moment of tail. Therefore, the copper represents an adversity to the anemones cells, being cytotoxic and genotoxic.

Efficiency of Neutral Red, Evans Blue and MTT to assess viability of the freshwater microalgae Desmodesmus communis and Pediastrum boryanum

This study assessed the efficacy of three cell viability assays – Methyl‐thiazolyl‐tetrazolium (MTT), Evans Blue, and Neutral Red – for two freshwater microalgae species, Desmodesmus communis and Pediastrum boryanum (Chlorophyceae), in order to find suitable techniques to detect the levels of pollution in water ecosystems. Following exposure to a glyphosate‐based herbicide, our results showed that Evans Blue did not adequately measure cell viability in either species, while MTT and Neutral Red were able to detect decreased cellular viability for both algae in response to herbicide exposure. Overall, however, Neutral Red proved to be more sensitive than MTT for these algae.

Mesodesma mactroides Gill Cells Exposed to Copper: Does Hyposmotic Saline Increase Cytotoxicity or Cellular Defenses?

Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions.

Toxicity induced by glyphosate and glyphosate-based herbicides in the zebrafish hepatocyte cell line (ZF-L)

Glyphosate is the active component of many commonly used herbicides; it can reach bodies of water through irrigated rice plantations. In the present study, we evaluated the effect of glyphosate and Roundup® (a glyphosate-based herbicide) in established culture of the zebrafish hepatocyte cell line ZF-L after 24 and 48 h of exposure to concentrations of 650 and 3250 µg/L. We observed a reduction in metabolic activity and lysosomal integrity, and an increase in cell number after 24 h of Roundup® exposure at the highest concentration. An increase in active mitochondria and apoptotic cells was observed following 24 h exposure to glyphosate and Roundup®, while only exposure to Roundup® induced an increase in necrotic cells. Rhodamine B accumulation decreased after 48 h exposure to 650 µg/L of Roundup®; this reduction is indicative of increased activity of ABC pumps. Overall, the present findings highlighted the hazard of glyphosate exposure not only in the commercial formulation but also glyphosate alone, since both can induce damage in the ZF-L cell line primarily through the induction of apoptosis.

Characterization of MXR activity in the sea anemone Bunodosoma cangicum exposed to copper

Transmembrane proteins of the ABC family contribute to a multiple xenobiotic resistance (MXR) phenotype in cells, driving the extrusion of toxic substances. This phenotype promotes a high degree of protection against xenobiotics. The present study provides a better understanding of the MXR activity in the podal disk cells of Bunodosoma cangicum exposed to copper, and further establishes the relationship between protein activity (measured by accumulation of rhodamine-B) and bioaccumulation of copper in these cells. Sea anemone cells were exposed for 24h to copper (0, 7.8 and 15.6μg/L) in presence and absence of MXR blocker (verapamil 50μM). Results indicate that copper exposure increases intracellular metal content when ABC proteins were blocked, causing an increase in cellular death. The present study also verified the relationship between MXR activity, ATP depletion, and general metabolic activity (by MTT). MXR activity decreased in treatment groups exposed to copper concentrations of 15.6μg/L and 10mM energy depleting potassium cyanide. Metabolic activity increased in cells exposed to 7.8μgCu/L, but 15.6μgCu/L was similar to 0 and 7.8μg/L. The presence of copper decreased the ABC proteins expression. The present study improves the knowledge of MXR in anemone cells and shows that this activity is closely associated with copper extrusion. Also, the copper exposure is able to modify the metabolic state and to lead to cytotoxicity when cells cannot defend themselves.

Aquaporin in different moult stages of a freshwater decapod crustacean: expression and participation in muscle hydration control.

Crustaceans, during their moult cycle, at the stages of both pre-moult and post-moult, need water uptake. This movement of water creates a challenge for the regulation of cell volume. The cells of freshwater decapods require a high regulatory capacity to deal with hyposmotic stresses, given the need to face dilution of the haemolymph during their moult cycles. This study investigated the variation in the expression of water channels (aquaporins) along the moult cycle of a freshwater palaemonid shrimp, focusing on their role in cell volume regulation. Moults in Palaemonetes argentinus have been investigated along three stages of its moult cycle: intermoult, late pre-moult and recent post-moult. For the evaluation of tissue volume regulation, the weight of isolatedmuscle, subjected to isosmotic and hyposmotic salines, was followed for 60min. The expression of AQP during the different moult stages was evaluated by immunocytochemistry. Muscle from the three moult stages in isosmotic conditions showed the same pattern of tissue volume regulation. When muscle from animals in pre-moult and intermoult were submitted to hyposmotic stress they swell, followed by volume regulation, while in post-moult the regulation is compromised. The difference in volume regulatory control between pre-moult and post-moult may be related to a possible regulation of water channels, as AQP expression was equal at these stages. This study presents novel findings for crustaceans in general, in the demonstration that AQP expression changes during the moult cycle of a decapod crustacean, together with the regulation of cell volume with the participation of AQPs.