Cláudia B.R. Martinez

Histopathology of gills, kidney and liver of a Neotropical fish caged in an urban stream

Histological changes in gills, kidney and liver were used to evaluate the health of the Neotropical fish species Prochilodus lineatus, subjected to in situ tests for 7 days in a disturbed urban stream and in a reference site, during winter and summer. In fish caged in the urban stream the most common lesions were epithelial lifting, hyperplasia and hypertrophy of the respiratory epithelium, lamellar fusion, and aneurysms in the gills; enlargement of the glomerulus, reduction of Bowmans space, occlusion of the tubular lumen, cloudy swelling and hyaline droplet degeneration in the kidneys; hepatocytes with hypertrophy, cytoplasmic and nuclear degeneration, melanomacrophage aggregates, bile stagnation and one case of focal necrosis in the liver. The lesions were comparatively most severe in the liver. Histopathology showed to be a very suitable biomarker for use in conjugation with the in situ test, because the seasonal variation did not interfere in the results and it was possible to differentiate the sites in the urban stream from the reference site.

Roundup causes oxidative stress in liver and inhibits acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus

This work aimed to evaluate Roundup effects on biochemical biomarkers of the neotropical fish Prochilodus lineatus. Fish were acutely exposed (6, 24 and 96 h) to 10 mg L(-1) of Roundup (RD) or only water (control) and samples of liver, for antioxidants analysis, and brain and muscle, for acetylcholinesterase (AChE) determination, were collected. Fish exposed to RD for 24h showed reduction on superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, and increased glutathione (GSH) content. After 24 and 96h, fish of RD group showed increased glutathione-S-transferase (GST) activity and lipid peroxidation. AChE activity was inhibited in brain after 96h and in muscle after 24 and 96h of exposure. Thus, acute exposure to RD stimulated the biotransformation pathway, with increased GST, but interfered on the antioxidant defenses, with reduction of SOD and GPx activity, leading to the occurrence of lipid peroxidation. Inhibition of AChE showed that RD acts as a contaminant with anti-AChE action.

Genotoxic effects of Roundup on the fish Prochilodus lineatus.

Glyphosate-based herbicides, such as Roundup, represent the most extensively used herbicides worldwide, including Brazil. Despite its extensive use, the genotoxic effects of this herbicide are not completely understood and studies with Roundup show conflicting results with regard to the effects of this product on the genetic material. Thus, the aim of this study was to evaluate the genotoxic effects of acute exposures (6, 24 and 96 h) to 10 mg L(-1) of Roundup on the neotropical fish Prochilodus lineatus. Accordingly, fish erythrocytes were used in the comet assay, micronucleus test and for the analysis of the occurrence of nuclear abnormalities and the comet assay was adjusted for branchial cells. The results showed that Roundup produces genotoxic damage in erythrocytes and gill cells of P. lineatus. The comet scores obtained for P. lineatus erythrocytes after 6 and 96 h of exposure to Roundup were significantly higher than respective negative controls. For branchial cells comet scores were significantly higher than negative controls after 6 and 24 h exposures. The frequencies of micronucleus and other erythrocyte nuclear abnormalities (ENAs) were not significantly different between Roundup exposed fish and their respective negative controls, for all exposure periods. In conclusion, the results of this work showed that Roundup produced genotoxic effects on the fish species P. lineatus. The comet assay with gill cells showed to be an important complementary tool for detecting genotoxicity, given that it revealed DNA damage in periods of exposure that erythrocytes did not. ENAs frequency was not a good indicator of genotoxicity, but further studies are needed to better understand the origin of these abnormalities.

Effects of Roundup Transorb on fish: Hematology, antioxidant defenses and acetylcholinesterase activity

Roundup Transorb(RDT) is a glyphosate-based herbicide containing a mixture of surfactants. The objective of this work was to evaluate the effects of this herbicide on the Neotropical fish Prochilodus lineatus. Juvenile fish were acutely exposed (6, 24 and 96 h) to 1 mg L(-1) of RDT (RDT 1), 5 mg L(-1) of RDT (RDT 5) or only water (control) and blood samples for hematological analysis, liver for antioxidants analysis, and brain and muscle for acetylcholinesterase (AChE) determination, were collected. RDT effects were more evident in fish exposed to the higher concentration of the herbicide. Hematologic alterations appeared only after 96 h exposure, when fish showed an increase in the hematocrit and in the number of both red and white blood cells. After 6h exposure fish showed a transient reduction in superoxide dismutase and catalase activity. RDT also inhibited glutathione-S-transferase, after 6 and 24h of exposure. The reduction in these enzymes is probably related to the occurrence of lipid peroxidation (LPO) in fish exposed to the herbicide for 6h. LPO returned to control levels after 24 and 96 h exposure to RDT, when fish showed an increased activity of glutathione peroxidase. The content of reduced glutathione also increased after 96 h exposure. Thus, after 24 and 96 h the antioxidant defenses were apparently enough to combat ROS, preventing the occurrence of oxidative damage. The exposure to RDT for 96 h led to an inhibition of AChE in brain and muscle at rates which may not be considered a life-threatening situation.

Biochemical and physiological biomarkers in Prochilodus lineatus submitted to in situ tests in an urban stream in southern Brazil.

The aims of the present study were to verify the suitability of short-term in situ test with the neotropical fish Prochilodus lineatus and to evaluate from a set of biochemical and physiological biomarkers the ones which could work as sensitivity tools for the environmental quality assessment. In situ tests were carried out for 1 week in winter and summer, at three sites along an urban stream heavily contaminated by anthropogenic activities and at a reference site. The variables analyzed were: hemoglobin content (Hb), plasma concentrations of cortisol, glucose, total protein, Na(+) and Cl(-), plasma osmolarity, liver activities of glutathione-S-transferase (GST) and catalase and interrenal cells area. Results showed that glycemia, interrenal cell size and GST activity, which were significantly higher in fish caged in the urban stream, were best able to distinguish between the most disturbed sites and the reference and caged P. lineatus showed to be a promising tool for the assessment and monitoring of tropical aquatic ecosystems.

Acute effects of nitrite on ion regulation in two neotropical fish species

To broaden the understanding of physiological responses of tropical fish to environmental stressors, the effects of nitrite on haematological parameters and plasma and red blood cell ion regulation were studied in two neotropical fish species, Astyanax altiparanae and Prochilodus lineatus. Both fish species were exposed to NaNO2 (30 mg l(-1)) over a 96-h period and blood samples were taken for ion and haematological analyses. The results revealed that nitrite leads to a decrease in P. lineatus blood haematocrit and haemoglobin content and an increase in blood methaemoglobin. A. altiparanae did not exhibit any significant difference in these haematological parameters. During the exposure to NO2- both fish species had significantly reduced plasma Na+ concentration and red blood cell (RBC) K+ concentration, but only P. lineatus showed an increase in extracellular K+ concentration. When RBC volume was analyzed in vitro, after 2 min of exposure to NaNO2, a 36% shrinkage was observed in P. lineatus cells, while only a 10% shrinkage was observed in A. altiparanae cells. These results suggest that for P. lineatus, nitrite entrance into the cell leads to methaemoglobin formation and K+ efflux, causing red cell shrinkage and increased plasma K+. However, A. altiparanae proved to be a species more resistant to nitrite, exhibiting fewer responses to this compound.

Atrazine promotes biochemical changes and DNA damage in a Neotropical fish species.

The effects of Atrazine, an herbicide used worldwide and considered as a potential contaminant in aquatic environments, were assessed on the Neotropical fish Prochilodus lineatus acutely (24 and 48 h) exposed to 2 or 10 μg L(-1) of atrazine by using a set of biochemical and genetic biomarkers. The following parameters were measured in the liver: activity of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and glutathione S transferase (GST), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), content of reduced glutathione (GSH), generation of reactive oxygen species (ROS) and occurrence of lipid peroxidation (LPO); in brain and muscle the activity of acetylcholinesterase (AChE) and DNA damage (comet assay) on erythrocytes, gills and liver cells. A general decreasing trend on the biotransformation and antioxidant enzymes was observed in the liver of P. lineatus exposed to atrazine; except for GR, all the other antioxidant enzymes (SOD, CAT and GPx) and biotransformation enzymes (EROD and GST) showed inhibited activity. Changes in muscle or brain AChE were not detected. DNA damage was observed in the different cell types of fish exposed to the herbicide, and it was probably not from oxidative origin, since no increase in ROS generation and LPO was detected in the liver. These results show that atrazine behaves as enzyme inhibitor, impairing hepatic metabolism, and produces genotoxic damage to different cell types of P. lineatus.

How aluminium exposure promotes osmoregulatory disturbances in the neotropical freshwater fish Prochilus lineatus

The aim of this study was to understand the effects of the interaction between aluminium and low pH in a native fish species Prochilodus lineatus. Thus, juveniles of this neotropical fish species were exposed to 196 microg L(-1) of dissolved aluminium in acid water (Al group), only to acid water (pH group) or to water with neutral pH (CTR group) for 6, 24 and 96 h. Al effects were evaluated with regard to hematological parameters (hemoglobin, hematocrit and red blood cell number), plasma ions and osmolarity, density and distribution of chloride cells (CC), Na(+)/K(+)-ATPase activity in the gills, metabolic (protein and glucose) and endocrine (cortisol) parameters. The fish exposed to Al had increased hematological and metabolic parameters in relation to the CTR group after all periods of exposure. In fish exposed to Al for 24 and 96 h plasma ions and osmolarity were significantly lower and the identification of the enzyme Na(+)/K(+)-ATPase by immunohistochemistry indicated a reduction in the number of CC in the gills. Enzyme activity was 50% lower in fish exposed to Al in all experimental times. Taken together these results showed that acute exposure to Al causes an ionic unbalance, probably related to the effects of Al on Na(+)/K(+)-ATPase activity, on the distribution and number of chloride cells in the gills as well as the effects associated with the stress response caused by the presence of the metal.

In vivo and in vitro exposures for the evaluation of the genotoxic effects of lead on the Neotropical freshwater fish Prochilodus lineatus

In the present study, in vivo and in vitro exposures were used to assess the genotoxicity of lead (Pb) to the freshwater fish Prochilodus lineatus. The comet assay using blood, liver and gill cells, and the occurrence of micronuclei (MN) and other erythrocytic nuclear abnormalities (ENA) were used to assess the genotoxic potential of lead in vivo. Metallothionein content (MT) was measured in fish liver in order to evaluate the protection of fish against Pb toxicity. Fish erythrocytes were exposed to Pb in vitro (1, 3 and 6 h) and the number of viable cells, DNA integrity, using the comet assay, and lysosomal membrane stability, measured by the neutral red retention assay (NRRA) were analyzed. The results of the comet assay after in vivo toxicity tests (6, 24 and 96 h) showed that Pb was genotoxic for all the three tissues analyzed after 96 h exposure. A significant increase in liver MT content was observed after 6 and 24 h of Pb exposure. MN frequency did not increase after Pb exposures, but the frequency of the other ENA, such as kidney-shaped nuclei, segmented nuclei and lobed nuclei, showed a significant increase after 24 and 96 h, indicating that ENA is a better biomarker for Pb exposure than MN alone after short-term exposures. The results of the comet assay performed with erythrocytes in vitro exposed to lead confirmed its genotoxic effect and showed that DNA damage increased with increasing exposure time. Moreover, the NRRA clearly indicated that Pb induces a destabilization of the lysosomal membrane. These results demonstrate the potential genotoxicity and cytotoxicity of lead after acute exposures.

Hematological and biochemical alterations in the fish Prochilodus lineatus caused by the herbicide clomazone

The indiscriminate use of herbicides has led to the contamination of water bodies, possibly affecting the health of aquatic biota. Therefore, to evaluate the possible effects of the clomazone-based herbicide (Gamit(®) 500) on the fish Prochilodus lineatus, juveniles were exposed for 96h to three concentrations (1, 5 and 10mgL(-1)) of clomazone, and an analysis was made of their hematological parameters: hemoglobin (Hb); hematocrit (Hct); red blood cell (RBC) count; mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC) and biochemical parameters: glutathione S-transferase (GST); catalase (CAT); glutathione peroxidase (GPx) and acetylcholinesterase (AChE). Hct presented a significant decrease at the concentration of 10mgL(-1), while the parameters Hb, HCM and MCHC presented a significant decrease at the two higher concentrations, indicating an anemic condition. The RBC increased significantly at the lowest concentration, possibly due to the release of new red blood cells into the bloodstream in response to splenic contraction, which may occur as an adaptive response to the stressor agent. P. lineatus presented activation of the biotransformation pathway, indicated by augmented hepatic activity of the enzyme GST and hepatic activation of the antioxidant enzyme CAT at the higher concentrations. Liver GPx was significantly inhibited at the higher concentrations, which may indicate the efficient action of CAT in the elimination of H2O2 or its competition with GST for the same substrate (GSH). AChE activity in brain and muscle was inhibited at the higher concentrations, indicating the neurotoxic effects of the herbicide in the fish. The hematological and biochemical alterations led to the conclusion that the herbicide clomazone has toxic effects on the species P. lineatus, and that its presence in the environment may jeopardize the health of these animals.