Interference of goethite in the effects of glyphosate and Roundup® on ZFL cell line.

Goethite (α-FeOOH) brings important perspectives in environmental remediation, as, due to its physicochemical properties, this iron oxide can adsorb a wide variety of compounds, including glyphosate. This study aimed to evaluate the effects of goethite nanoparticles (NPs), glyphosate (Gly), Roundup® (Rd), and co-exposures (Gly + NPs and Rd + NPs) on zebrafish liver cell line (ZFL). ZFL cells were exposed to NPs (1, 10, and 100 mg L-1), Gly (3.6 mg L-1), Rd (10 mg L-1), and co-exposures (Gly + NPs and Rd + NPs), or only to saline for 1, 6, and 12 h. Cell viability was assessed by Trypan blue, MTT, and neutral red assays. The generation of reactive oxygen species and total antioxidant capacity were also determined, while genotoxicity was quantified by the comet assay. Both NPs and Rd in isolation produced cytotoxic effects at 6 h and genotoxic effects at 1 and 6 h. Rd + NPs resulted in synergistic effects, intensifying the toxicity. Cells exposed to Gly did not present toxic effects and Gly + NPs resulted in the suppression of toxic effects observed for NPs. The presence of other components in Roundup® seems to favor its toxicity compared to the active ingredient. In conclusion, according to the in vitro model, the concentrations used were not safe for the ZFL lineage.

Overview of the toxic effects of titanium dioxide nanoparticles in blood, liver, muscles, and brain of a Neotropical detritivorous fish.

The toxicity of titanium dioxide nanoparticles (TiO2 -NP) in the blood, liver, muscle, and brain of a Neotropical detritivorous fish, Prochilodus lineatus, was tested. Juvenile fish were exposed to 0, 1, 5, 10, and 50 mg L-1 of TiO2 -NP for 48 hours (acute exposure) or 14 days (subchronic exposure) to evaluate changes in hematology, red blood cell (RBC) genotoxicity/mutagenicity, liver function (reactive oxygen species (ROS) production, antioxidant responses, detoxification, and histopathology), acetylcholinesterase (AChE) activity in muscles and brain, and Ti bioaccumulation. TiO2 -NP did not cause genetic damage to RBC, but acutely decreased white blood cells (WBC) and increased monocytes. Subchronically, RBC decreased, mean cell volume and hemoglobin increased, and WBC and lymphocytes decreased. Therefore, NP has the potential to affect immune system and increase energy expenditure, reducing the fish's ability to avoid predator and to resist pathogens. In the liver, acute exposure decreased ROS and increased glutathione (GSH) content, while subchronic exposure decreased superoxide dismutase activity and increased glutathione-S-transferase (GST) activity and GSH content. GSH and GST seem to play an essential role in metabolizing NP and ROS, likely increasing hepatocytes' metabolic rate, which may be the cause of observed cell hypertrophy, disarrangement of hepatic cords and degenerative morphological alterations. Although most studies indicate that the kidney is responsible for metabolizing and/or eliminating TiO2 -NP, this study shows that the liver also has a main role in these processes. Nevertheless, Ti still accumulated in the liver, muscle, and brain and decreased muscular AChE activity after acute exposure, showing neurotoxic potential. More studies are needed to better understand the biochemical pathways TiO2 -NP are metabolized and how its bioaccumulation may affect fish homeostasis and survival in the environment.

Mitochondria-rich cells adjustments and ionic balance in the Neotropical fish Prochilodus lineatus exposed to titanium dioxide nanoparticles.

Manufactured titanium dioxide nanoparticles (TiO2-NP) have been intensely applied in numerous industrial products and may be a risk for aquatic systems as they are not completely removed from domestic and industrial wastes after water treatment. This study evaluated the osmo- and ionic balance, Na+/K+-ATPase, H+-ATPase and carbonic anhydrase activities and the mitochondria-rich cells (MRC) in the gills and kidney of the Neotropical fish Prochilodus lineatus after 2 (acute) and 14 (subchronic) days of exposure to nominal 0, 1, 5, 10 and 50 mg L-1 TiO2-NP. The nominal concentrations corresponded to 0.0, 0.6, 1.6, 2.7 and 18.1 mg L-1 suspended TiO2-NP, respectively, in the water column one hour after NP introduction and were maintained for at least 24 h. Acute exposure to TiO2-NP decreased plasma osmolality and Ca2+ levels. Na+/K+-ATPase, H+-ATPase and carbonic anhydrase activities were inhibited in the gills, but not in the kidney. Total MRC density did not change in gills and kidneys. At gill surface, total MRC density decreased in fish exposed to 50 mg L-1 TiO2-NP and the total MRC fractional surface area unchanged although, there were some changes in the fractional area of MRC with apical microvilli (MRCm) and MRC with apical sponge-like structure (MRCs). MRCm was more abundant than MRCs. After subchronic exposure, there was no change in plasma osmolality, ionic balance and enzyme activities. Total gill MRC density increased in the filament epithelium and renal tubules. In the gills, MRC contacting water exhibited some adjustments. Total MRC and fractional surface area unchanged, but there was an increase of MRCs contacting water at gill surface after exposure to10 and 50 mg L-1 TiO2-NP. MRC proliferation in filament epithelium and in renal tubules as well as the increasing MRCs at gill surface may have contributed to avoid change in plasma osmolality, ionic balance and enzyme activities and suggested a cellular physiological and morphological response to restore and maintain osmotic and ionic homeostasis after subchronic exposure.

Chromium accumulation and biomarker responses in the Neotropical fish Prochilodus lineatus caged in a river under the influence of tannery activities.

In order to assess the effects of tannery effluents (TE) in organism health, juveniles of Prochilodus lineatus were submitted to in situ tests at four different river locations: site A - upstream of the tannery; site B - next to the tannery; and sites C and D - downstream of the tannery. After 96 h exposure in the river, samples of fish tissue, river water and sediment were collected in order to quantify chromium (Cr) concentrations. Tissue samples were used to assess the activity of ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the content of glutathione (GSH) and metallothionein (MT) and the occurrence of lipid peroxidation (LPO) and DNA damage. Higher Cr concentrations were detected in the water and sediments from site B and in the liver of fish confined at site B, compared to the other sites. Fish caged at site B demonstrated higher levels of liver MT and hepatic EROD activity in relation to fish caged at the other sites. Moreover, fish from site B presented increased liver and branchial GST activities, as well as more GSH in the liver, than fish from site A. There were no significant variations in the occurrence of LPO and DNA damage among fish caged at the different sites. Thus, TE increased Cr levels in the water, sediments, and fish livers and stimulated the synthesis of MT and GSH and the activities of EROD and GST. In conclusion, TE affect the quality of the river and promote changes in biochemical biomarkers and Cr accumulation in P. lineatus.

Evaluation of caffeine effects on biochemical and genotoxic biomarkers in the neotropical freshwater teleost Prochilodus lineatus.

Caffeine is often found in aquatic environments, leading to concerns regarding its adverse consequences for aquatic biota. Biochemical and genotoxic biomarkers were analysed in juveniles of Prochilodus lineatus to evaluate the effects of caffeine. Fish were exposed to caffeine (0.3, 3 and 30 µg L-1) for either 24 h or 168 h. Longer exposure to caffeine resulted in a significant reduction in the activity of the phase I biotransformation enzyme ethoxyresorufin-O-deethylase (EROD) in the brain but a significant increase in the liver. Changes in glutathione content (GSH), glutathione S-transferase (GST) activity, and lipid peroxidation were not found in the liver and brain of fish exposed to caffeine. DNA damage in erythrocytes were also not found. These results show that caffeine may interfere with the biotransformation mechanism of P. lineatus after 168 h exposure, but it does not generate sufficient changes to trigger a state of oxidative stress.

Biomarkers of waterborne copper exposure in the Neotropical fish Prochilodus lineatus.

The main goal of the present study was to investigate the effects of acute exposure to copper (Cu) using a Neotropical freshwater fish as sentinel species through multi biomarkers analysis at different biological levels. Juveniles of Prochilodus lineatus were kept under control condition (no Cu addition in the water) or exposed to environmentally relevant concentrations of waterborne Cu (5, 9 and 20µgL(-1)) for 96h. These concentrations were selected to bracket the current Brazilian water quality criteria for Cu in fresh water (9 and 13µgL(-1) dissolved copper). Endpoints analyzed included ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, reduced glutathione (GSH) and metallothionein-like protein (MT) concentration, lipid peroxidation (LPO) level, tissue damage index, and incidence of free melano-macrophages (FMM) and melano-macrophage centers (MMC) in the liver. They also included DNA damage (frequency of nucleoids per comet class, number of damaged nucleoids per fish and DNA damage score) in erythrocytes, as well as muscle and brain acetylcholinesterase (AChE) activity and behavioral parameters (swimming distance and velocity, time spent swimming and swimming activity in the upper and lower layers of the water column). Fish exposed to any of the Cu concentrations tested showed increased liver MT concentration and LPO level, higher number of damaged nucleoids in erythrocytes per fish, and inhibited muscle AChE activity. Also, increased liver SOD activity was observed in fish exposed to 9 and 20µgL(-1) Cu. Fish exposed to 5 and 9µgL(-1) Cu spent lower amount of time swimming. Fish exposed to 9µgL(-1) Cu showed increased swimming distance and velocity while those exposed to 20µgL(-1) Cu had lower swimming distance and velocity, as well as, spent less time swimming in the lower layer of the water column when compared to those kept under control condition. These findings indicate that Cu exposure at environmentally relevant concentrations (below or close to the current Brazilian water quality criteria) induced significant biological (histological, biochemical and genetic) and ecological (swimming and exploratory abilities) damages in the Neotropical fish P. lineatus. They also suggest that MT concentration, DNA damage (comet assay), LPO (TBARS method), SOD and AChE activity, together with swimming behavior analyses are potential biomarkers to assess and monitor areas impacted by Cu in fresh water.

Effects of the water soluble fraction of gasoline on ZFL cell line: Cytotoxicity, genotoxicity and oxidative stress.

This work aimed to evaluate the effects of different dilutions of gasoline water-soluble fraction (GSF) on Danio rerio hepatocyte cell line (ZFL). Two tests were used to assess cell viability, MTT reduction assay (MTT) and the Trypan blue (TB) exclusion test. Oxidative stress was evaluated through the quantification of reactive oxygen species (ROS) and the assessment of the total antioxidant capacity against peroxyl radicals (ACAP) and the comet assay was employed to assess DNA damage. ZFL cells were exposed to 5, 10, 25 and 50% GSF or only to saline for 1, 3 and 6h. The GSF exhibited concentration-dependent cytotoxicity, and longer exposure times resulted in lower cell viability as indicated by both MTT and TB assays. The establishment of oxidative stress in cells exposed to GSF was not observed at any exposure period and the lower ROS levels could be related to the increased antioxidant capacity after 6-hour exposure. DNA damage was significantly increased after exposure to GSF at the three experimental times. Taking together these results show that GSF has a genotoxic potential at the lower concentrations and becomes cytotoxic at higher concentrations and that ZFL can be considered a good biological model for in vitro toxicological studies.

Evaluation of the side effects of poly(epsilon-caprolactone) nanocapsules containing atrazine toward maize plants.

Poly(epsilon-caprolactone) (PCL) nanocapsules have been used as a carrier system for the herbicide atrazine, which is commonly applied to maize. We demonstrated previously that these atrazine containing polymeric nanocapsules were 10-fold more effective in the control of mustard plants (a target species), as compared to a commercial atrazine formulation. Since atrazine can have adverse effects on non-target crops, here we analyzed the effect of encapsulated atrazine on growth, physiological and oxidative stress parameters of soil-grown maize plants (Zea mays L.). One day after the post-emergence treatment with PCL nanocapsules containing atrazine (1 mg mL(-1)), maize plants presented 15 and 21% decreases in maximum quantum yield of photosystem II (PSII) and in net CO2 assimilation rate, respectively, as compared to water-sprayed plants. The same treatment led to a 1.8-fold increase in leaf lipid peroxidation in comparison with control plants. However, all of these parameters were unaffected 4 and 8 days after the application of encapsulated atrazine. These results suggested that the negative effects of atrazine were transient, probably due to the ability of maize plants to detoxify the herbicide. When encapsulated atrazine was applied at a 10-fold lower concentration (0.1 mg mL(-1)), a dosage that is still effective for weed control, no effects were detected even shortly after application. Regardless of the herbicide concentration, neither pre- nor post-emergence treatment with the PCL nanocapsules carrying atrazine resulted in the development of any macroscopic symptoms in maize leaves, and there were no impacts on shoot growth. Additionally, no effects were observed when plants were sprayed with PCL nanocapsules without atrazine. Overall, these results suggested that the use of PCL nanocapsules containing atrazine did not lead to persistent side effects in maize plants, and that the technique could offer a safe tool for weed control without affecting crop growth.

Bioaccumulation of nickel and its biochemical and genotoxic effects on juveniles of the neotropical fish Prochilodus lineatus.

Juveniles of the freshwater fish Prochilodus lineatus were exposed to three concentrations of nickel (Ni): 25, 250 and 2500 µg L(-1) or water only for periods of 24 and 96 h to test for Ni bioaccumulation, its effects on antioxidant defenses and metallothioneins, and the occurrence of DNA damage. After exposure, the fish were sampled and tissue removed from the gills, liver, kidney and muscle to test for Ni accumulation and conduct biochemical (gills and liver) and genotoxic (blood cells and gills) analyses. The results showed that Ni accumulates in the organs in different proportions (kidney>liver>gills>muscle) and accumulation varied according to exposure time. Metallothionein (MT) levels increased in the liver and gills after exposure to Ni, implying that the presence of Ni in these tissues could induce MT synthesis. We also observed that Ni exposure affected antioxidant defenses, increasing lipid peroxidation in the liver of fish exposed to Ni for 96 h at the highest concentration tested. DNA damage increased in both blood cells and gills of fish exposed to all Ni concentrations, indicating the genotoxic potential of Ni on fish. We therefore concluded that Ni accumulates in various tissues and results in oxidative and DNA damage in P. lineatus, and that the maximum permitted Ni concentration set in Brazilian legislation (25 µg L(-1)) for freshwaters is not safe for this species.

Acute effects of cadmium on osmoregulation of the freshwater teleost Prochilodus lineatus: enzymes activity and plasma ions.

Cadmium (Cd) is a trace element that is very toxic to fish. It is commonly found in surface waters contaminated with industrial effluents. When dissolved in water, Cd can rapidly cause physiological changes in the gills and kidneys of freshwater fish. The objective of this study was to evaluate the acute effects of Cd on the osmoregulation of the Neotropical fish Prochilodus lineatus. Juvenile fish were exposed to Cd at two concentrations [1 (Cd1) and 10 (Cd10) µgL(-1)] for 24 and 96h. The effects of Cd were evaluated through the analysis of ions (Na(+), K(+), Ca(2+), and Cl(-)) and plasma osmolality, and by measuring the activities of enzymes involved in osmoregulation obtained from the gills and kidney. Fish exposed to Cd for 24 and 96h showed a decrease in Na(+)/K(+)-ATPase activity in the gills and kidney. The activity of carbonic anhydrase decreased in the gills after 24h and in both tissues after 96h of Cd exposure. The gill Ca(2+)-ATPase activity also decreased with Cd exposure, with a concomitant drop in the plasma concentration of Ca(2+). Despite the hypocalcemia, there were no changes in the concentration of the ions Na(+), K(+), and Cl(-) or in plasma osmolality. Among the enzymes involved in ion transport, H(+)-ATPase was the only enzyme that showed increased activity in gills, whereas its activity in kidney remained unchanged. The results of this study demonstrate that waterborne Cd at the maximum concentrations set by Brazilian guidelines for freshwater affects the gills and kidney functions of P. lineatus. Acute exposure to Cd resulted in the decrease of the activity of enzymes, which culminated with the loss of the fish's ability to regulate the levels of calcium in the blood, leading to hypocalcemia.

Effects of the surfactant polyoxyethylene amine (POEA) on genotoxic, biochemical and physiological parameters of the freshwater teleost Prochilodus lineatus.

The surfactant polyoxyethylene amine (POEA) is added to several formulations of glyphosate herbicides that are widely used in agriculture and can contaminate aquatic ecosystems. In the present study, an integrated approach examining genotoxic, biochemical and physiological parameters was employed to evaluate acute effects of POEA on the Neotropical fish Prochilodus lineatus. Juvenile fish were exposed to 0.15 mg·L(-1) (POEA 1), 0.75 mg·L(-1) (POEA 2) and 1.5 mg·L(-1) (POEA 3) of POEA or only water (CTR), and after 24h exposure samples of blood and liver were taken. Compared with CTR, liver of fish exposed to POEA 2 and POEA 3 showed increased activity of 7 ethoxyresorufin-O-deethylase and increased content of glutathione, whereas the activity of glutathione-S-transferase was diminished. On the other hand, fish of the group POEA 1 showed an increase in the activity of superoxide dismutase and in the occurrence of lipid peroxidation. Fish exposed to POEA 3 presented increased hepatic activity of glutathione peroxidase and reduced plasma cortisol. The exposure to POEA at all concentrations tested caused an increase in plasma lactate and a decrease in the hepatic activity of catalase, in the number of red blood cells and in hemoglobin content. The comet assay used for analyzing DNA damage in blood cells indicated the genotoxicity of the surfactant at all concentrations tested. Taken together these results show that POEA can cause effects at various levels, such as hemolysis, DNA damage and lipid peroxidation, which are directly related to an imbalance in the redox state of the fish.

Cytotoxic, biochemical and genotoxic effects of biodiesel produced by different routes on ZFL cell line.

Transesterification has proved to be the best option for obtaining biodiesel and, depending on the type of alcohol used in the reaction, the type of biodiesel may be methyl ester or ethyl ester. Leaking biodiesel can reach water bodies, contaminating aquatic organisms, particularly fish. The objective of this study was to determine whether the soluble fraction of biodiesel (Bd), produced by both the ethylic (BdEt) and methylic (BdMt) routes, can cause cytotoxic, biochemical and genotoxic alterations in the hepatocyte cell line of Danio rerio (ZFL). The metabolic activity of the cell was quantified by the MTT reduction method, while genotoxic damage was analyzed by the comet assay with the addition of specific endonucleases. The production of reactive oxygen species (ROS) and antioxidant/biotransformation enzymes activity also were determined. The results indicate that both Bd increased ROS production, glutathione S-transferase activity and the occurrence of DNA damage. BdMt showed higher cytotoxicity than BdEt, and also caused oxidative damage to the DNA. In general, both Bd appear to be stressors for the cells, causing cytotoxic, biochemical and genetic alterations in ZFL cells, but the type and intensity of the changes found appear to be dependent on the biodiesel production route.

Assessment of domestic landfill leachate toxicity to the Asian clam Corbicula fluminea via biomarkers.

In order to evaluate the effects of domestic landfill leachate to bivalves Corbicula fluminea, clams were exposed to different leachate concentrations (v/v): 2, 3, 6 and 10 percent, corresponding to dilutions observed along a stream that receives this effluent, or only to clean water for comparisons. After 5 and 15 days of exposure the activity of the biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the multixenobiotic resistance mechanism (MXR) and lipid peroxidation (LPO) in gills and digestive gland and metallothionein (MT) content in gills were evaluated. Differences in biomarkers responses were observed between gills and digestive gland, except for MXR that decreased in both tissues of clams exposed to 6 percent for 5 days. EROD activity in gills was reduced in all leachate concentrations after 5 days and only in 2 percent after 15 days exposure, while an EROD increase was observed in digestive gland after 15 days exposure to 6 percent. GST activity increased only in the gills of clams exposed to 10 percent for 5 days. LPO varied between tissues and different conditions. A significant increase in LPO was observed in the gills, after 5 days exposure to 2 and 6 percent, and in digestive gland after 5 and 15 days exposure to 2 and 3 percent. MT content in the gills increased after 15 days exposure to 2 percent. In conclusion, different leachate concentrations tested here caused biochemical changes in C. fluminea, but due to the observed variability in biomarkers responses among leachate concentrations, it was difficult to determine patterns or thresholds concentrations.

Genotoxic effects of the herbicide Roundup Transorb and its active ingredient glyphosate on the fish Prochilodus lineatus.

Roundup Transorb (RT) is a glyphosate-based herbicide and despite its wide use around the world there are few studies comparing the effects of the active ingredient with the formulated product. In this context the purpose of this study was to compare the genotoxicity of the active ingredient glyphosate with the formulated product RT in order to clarify whether the active ingredient and the surfactant of the RT formula may exert toxic effects on the DNA molecule in juveniles of fish Prochilodus lineatus. Erythrocytes and gill cells of fish exposed to glyphosate and to RT showed DNA damage scores significantly higher than control animals. These results revealed that both glyphosate itself and RT were genotoxic to gill cells and erythrocytes of P. lineatus, suggesting that their use should be carefully monitored considering their potential impact on tropical aquatic biota.

Genotoxic and biochemical effects of atrazine and Roundup(®), alone and in combination, on the Asian clam Corbicula fluminea.

The present study aimed to evaluate biochemical and genotoxic effects of the herbicides atrazine (ATZ) and Roundup(®) (RD) separately, as well as their mixture, on the freshwater clam Corbicula fluminea after 96 h exposure. Animals were exposed to 2 and 10 ppb of ATZ (ATZ2 and ATZ10), 2 and 10 ppm of RD (RD2 and RD10) and the following mixtures: 2 ppb ATZ+2ppm RD (AR2) and 10 ppb ATZ+10 ppm RD (AR10). Activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the multixenobiotic resistance mechanism (MXR), reduced glutathione concentrations (GSH) and lipid peroxidation (LPO) were measured in gills and digestive gland. DNA damage was determined in clams hemocytes through the comet assay. The gills were more susceptible to the action of the herbicides and the results showed that ATZ2 and ATZ10 caused a significant reduction in EROD and the mixture leads to a significant decrease in EROD and MXR. No significant change in the biotransformation parameters was observed in the digestive gland. Regarding the primary antioxidant defenses, SOD activity increased in the gills of clams exposed to ATZ10 and RD10 and in the digestive gland of animals exposed to RD2 and RD10, CAT activity was significantly reduced only in digestive gland of clams exposed RD10 while GPX increased in the gills after exposure to ATZ2 and RD10. The exposure to RD10 caused a significant increase in LPO in both gills and digestive gland. While the exposure to ATZ and RD separately did not increase DNA damage, the exposure to AR2 and AR10 caused a significant increase in the occurrence of DNA damage. In conclusion, this study showed that both herbicides applied alone caused effects on C. fluminea; ATZ interfered mostly in biotransformation while RD interfered mainly in antioxidant defenses leading to lipid peroxidation. The herbicides mixture showed antagonistic effects on the gills EROD and on lipid peroxidation in gills and digestive gland and synergistic effects on the gills MXR and on DNA damage in the hemocytes.

Lead accumulation and its effects on the branchial physiology of Prochilodus lineatus.

The purpose of this work was to determine the tissue accumulation of lead (Pb) and its effects on osmoregulatory processes of the freshwater fish Prochilodus lineatus. Juvenile fish were exposed to Pb (from 1.7 to 0.7 mg of dissolved Pb L(-1)) for 6, 24 and 96 h and Pb accumulation was analyzed in the gills, liver, kidneys, blood cells and muscle. The following parameters were also analyzed: hematologic (hemoglobin content, hematocrit and number of red blood cells), metabolic (blood glucose), endocrine (blood cortisol), osmo ionic (plasma osmolality and Na(+), K(+), Cl(-) and Ca(+2) concentrations), gill enzymes (Na(+)/K(+)-ATPase and carbonic anhydrase), chloride cell (CC) density and CC location in the gills. Pb accumulated in all the analyzed tissues, with the kidneys showing the highest concentration, followed by the gills and liver. The lowest Pb concentrations were found in blood cells and muscle. Pb promoted an increase in blood glucose after 6 and 24 h exposure. Gill Na(+)/K(+)-ATPase was inhibited after 24 h of exposure, but its activity was restored at 96 h, probably due to the increase in CC in gill lamellae. Plasma Na(+) was reduced after 6 and 96 h, while K(+) concentrations increased at all the experimental times. Fish exposed to Pb showed reduced plasma Ca(+2) at all experimental periods. Hematologic parameters remained unchanged. Overall, this study demonstrated that Pb interferes in osmoregulatory processes of P. lineatus and the proliferation of CC in the gills is a response in order to reestablish adequate ion concentrations.

Physiological effects of gasoline on the freshwater fish prochilodus lineatus (characiformes: prochilodontidae)

The purpose of this work was to evaluate the effects of the water-soluble fraction of gasoline (WSFG) on the Neotropical freshwater fish Prochilodus lineatus. The WSFG was prepared by mixing gasoline in water (1:4) and animals were exposed for 6, 24 and 96h to 5% diluted WSFG or only to water. After exposure, blood was collected from the caudal vein and the gills were removed. The following parameters were analyzed: hematological (hemoglobin, hematocrit, number of red blood cells), osmo-ionic (plasma Na+, Cl- and K+ and plasma osmolarity), metabolic (total plasma proteins and glucose), endocrine (cortisol), density and distribution of chloride cells [CC] in the gills (immunohistochemistry), and branchial Na+/K+-ATPase (NKA) activity. Hemolysis was found to occur after 96h exposure to WSFG, as indicated by the decrease in the hematological parameters analyzed, followed by an increase in plasma K+. Secondary stress response was revealed by the occurrence of hyperglycemia in the three periods of exposure, despite the absence of significant increases in the plasma cortisol. The exposure to WSFG also caused an increase in the quantity of CC and in plasma Na+, after 24h, as well as in the enzymatic activity of NKA and plasma osmolarity, after 24h and 96h. These results indicate that fish exposed to the WSFG showed physiological adjusts to maintain their osmotic balance. However, the increase in the quantity of CC in the lamellae may interfere in the gas exchange impairing respiration.

O objetivo deste trabalho foi avaliar os efeitos da fração solúvel da gasolina (FSG) em Prochilodus lineatus. A FSG foi preparada misturando-se a gasolina à água (1:4) e os animais foram expostos por 6, 24 e 96h à FSG diluída 5% ou apenas à água. Após a exposição, o sangue foi coletado pela veia caudal e as brânquias foram retiradas. Os seguintes parâmetros foram analisados: hematológicos (hemoglobina, hematócrito, quantidade de células vermelhas), osmo-iônicos (concentrações plasmáticas de Na+, Cl-, K+ e osmolaridade), metabólicos (concentrações plasmáticas de proteínas totais e glicose), endócrino (cortisol plasmático), densidade e distribuição de células-cloreto [CC] nas brânquias (imunohistoquímica) e a atividade da enzima Na+/K+-ATPase (NKA) branquial. Hemólise foi observada após 96h de exposição à FSG, indicada pela diminuição dos parâmetros hematológicos analisados, seguido pelo aumento do K+ plasmático. Houve resposta secundária de estresse visualizada pela ocorrência da hiperglicemia nos três tempos de exposição, apesar da ausência de diferenças significativas na concentração plasmática do cortisol. A exposição à FSG também provocou aumento na quantidade das CC e na concentração de Na+, após 24h, e na atividade enzimática da NKA e osmolaridade, após 24h e 96h. Esses resultados indicam que peixes expostos à FSG apresentaram ajustes fisiológicos para manter o equilíbrio osmótico. Entretanto, o aumento na quantidade de CC nas lamelas pode ter interferido nas trocas gasosas prejudicando a respiração.

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.

Biochemical and genetic alterations in the freshwater neotropical fish Prochilodus lineatus after acute exposure to Microcystis aeruginosa

Microcystins are secondary metabolites produced by different species of cyanobacteria, such as Microcystis aeruginosa (MA). In this study, the biochemical and genetic effects of lyophilized MA were evaluated in the neotropical fish Prochilodus lineatus exposed to 1 or 2 mg L-1 lyophilized MA (treated group) or only water (control group) in static toxicity tests for 24 and 96 h. The gills and liver were used in the analysis of biotransformation enzymes and antioxidant defenses, blood and gill cells in genetic analysis and in brain and muscle it was determined the activity of acetylcholinesterase (AChE). The results showed the biotransformation pathway activation due to the increase in hepatic CYP1A and in branchial and hepatic glutathione S-transferase (GST). The antioxidant defense proved to be greatly affected by MA exposure leading to changes, both in gills and liver, in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and in the content of tripeptide glutathione (GSH). Lipid peroxidation was not detected, but damage to DNA molecule was observed in blood cells. In conclusion, it can be state the lyophilized MA is able to promote changes in the biochemical and genetic parameters of P. lineatus.

As microcistinas são metabólitos secundários produzidos por diferentes espécies de cianobactérias, como a Microcystis aeruginosa (MA). Neste estudo, os efeitos bioquímicos e genéticos de liofilizado de MA foram avaliados para juvenis da espécie de peixe Neotropical Prochilodus lineatus expostos a 1 ou 2 mg L-1 de liofilizado de MA (grupo tratado) ou apenas à água (grupo controle), em testes de toxicidade estáticos, durante 24 e 96 h. As brânquias e o fígado foram usados para as análises das enzimas de biotransformação e defesas antioxidantes, células do sangue e das brânquias para análises genéticas e no cérebro e músculo foi determinada a atividade da acetilcolinesterase (AChE). Os resultados mostraram ativação da via de biotransformação devido ao aumento da atividade da CYP1A hepática e da atividade da glutationa S-transferase (GST) hepática e branquial. As defesas antioxidantes foram muito afetadas pela exposição a MA levando a alterações, tanto no fígado como nas brânquias, na atividade da superóxido dismutase (SOD), da catalase (CAT), da glutationa peroxidase (GPx), glutationa redutase (GR) e no conteúdo do tripeptídeo glutationa (GSH). Apesar dessas alterações a peroxidação lipídica não foi detectada em nenhum dos tecidos, mas danos na molécula de DNA foram observados nas células do sangue. Em conclusão, pode-se afirmar que o liofilizado de MA é capaz de promover alterações em parâmetros bioquímicos e genéticos de P. lineatus.

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.