Fungicides from rice cultivation (tebuconazole and azoxystrobin) alters biochemical and histological markers of hammertoad tadpoles (Boanafaber).

Tebuconazole (TBZ) and azoxystrobin (AZX) are fungicides frequently used in rice cultivation. Despite protecting crops against fungal diseases, these compounds can contaminate the natural environments close to the crops, exerting negative effects on non-target organisms, the present study aimed to characterize the contamination by fungicides of a river that flows in an area dominated by rice cultivation in the north of the state of Santa Catarina, SC, Brazil. Concentrations of TBZ and AZX found in the field were used to evaluate their negative effects on development, biochemical biomarkers and histopatology of the liver of a native tadpole species, the hammerfrog (Boana faber). Tadpoles were exposed for 16 days to the lowest (1.20 µg/L) and highest (2.60 µg/L) concentration of TBZ, lowest (0.70 µg/L) and highest (1.60 µg/L) concentration of AZX, and the mix of both fungicides at lowest and highest concentration of each found in field analyses. Exposure to the lower TBZ concentration and both concentrations of the Mix accelerated the development of tadpoles. AZX caused an increase in the activities of glutathione S-transferase (GST), carboxylesterase (CbE) and glucose-6-phosphate dehydrogenase (G6PDH) in the liver, an increase in the levels of protein carbonyls (PC) in the liver and an increase in the activity of acetylcholinesterase (AChE) in muscle of tadpoles. TBZ, on the other hand, generated an increase in GST, G6PDH, PC and histopathological severity scores in liver and in muscle AChE activity. The effects were more intense in the groups exposed to the Mix of contaminants. No treatment altered brain AChE. The data showed that the fungicides from in rice cultivation found in natural aquatic environments around the crops pose risks to the health of the animals, compromising their metabolism and development.

Effects of the herbicide ametryn on development and thyroidogenesis of bullfrog tadpoles (Aquarana catesbeiana) under different temperatures.

Thyroid hormones (TH) are essential for the metamorphosis of amphibians and their production can be influenced by environmental stressors, such as temperature fluctuations, and exposure to aquatic pollutants, such as herbicides. In the present study we evaluated the influence of different temperatures (25 and 32 °C) on the effects of the herbicide ametryn (AMT, 0 - control, 10, 50 and 200 ng.L-1) for 16 days on thyroidogenesis of bullfrog tadpoles. Higher temperature and AMT exposure caused a delay in the development of tadpoles, despite no differences were noted in weight gain and total length of the animals. Levels of triiodothyronine (T3) and thyroxine (T4) were not altered neither by AMT nor by temperature, but the highest temperature caused a decrease in total area and number of follicles in the thyroid gland. Transcript levels of thyroid hormone receptors alpha and beta (TRα and TRß) and iodothyronine deiodinase 3 (DIO3) were lower at 32 °C, which is consistent with developmental delay at the higher temperature. Tadpoles exposed to 200 ng.L-1 of AMT at 25 °C also presented delayed development, which was consistent with lower TRα and DIO3 transcript levels. Lower levels of estradiol were noted in tadpoles exposed to AMT at the higher temperature, being also possibly related to a developmental delay. This study demonstrates that higher temperature and AMT exposure impair thyroidgenesis in bullfrog tadpoles, disrupting metamorphosis.

Fullerene and omega-3 and omega-6 fatty acids on fish brain antioxidant status.

The carbon nanomaterial fullerene (C(60)) can act as anti or pro-oxidant. The aim of this study was to evaluate, in cell suspensions of carp brains (Cyprinus carpio, Cyprinidae), the effect of C(60) after a pre-treatment with polyunsaturated fatty acid (PUFAs) such as omega-3 (docosahexaenoic acid, DHA) and omega-6 (linoleic acid, LA). Assays consisted of a pre-treatment with PUFA (48 h) and then exposure to C(60) (2 h). Cell viability and total anti-oxidant capacity did not differ (p > 0.05). A reduction (p < 0.05) was observed in reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) concentration in fish brain cells pre-exposed with PUFA groups and then exposed or not with C(60). An antioxidant effect of C(60) was evident since in control group (cells not pre-exposed to PUFA), a significant (p < 0.05) reduction of intracellular ROS concentration was observed, although this reduction was not enough to reduce the TBARS levels. Cysteine levels presented a reduction (p < 0.05) in all groups exposed to C(60). For glutathione (GSH), an increase (p < 0.05) was registered in cells exposed to C(60) without PUFAs pre-treatment and in the C(60) group pre-treated with DHA. Overall C(60) appears to play an antioxidant role that is modulated by PUFA, taking into account its effects on intracellular ROS concentration and MDA levels. Results also suggest that C(60) influences GSH synthesis, as showed for the augmented levels of this antioxidant and also for the lowering of the intracellular cysteine concentration.

Influence of temperature on biomarker responses of bullfrog tadpoles (Lithobates catesbeianus) exposed to the herbicide ametryn.

The S-triazine herbicide ametryn (AMT) is relatively low adsorbed in soils and has high solubility in water, thus believed to affect non-target aquatic organisms such as amphibians. Temperature increases can intensify the effects of herbicides, possibly increasing the susceptibility of amphibians to these compounds. The aim of this study was to evaluate the influence of temperature (25 and 32 °C) on the responses of biochemical biomarkers in bullfrog tadpoles (Lithobates catesbeianus) exposed to different concentrations of AMT (0, 10, 50 and 200 ng.L-1) for a period of 16 days. The antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) and the biotransformation enzyme glutathione S-transferase (GST) had their activity decreased at the highest temperature (32 °C). SOD activity was reduced at 200 ng.L-1 and 32 °C compared to the control at the same temperature. AMT exposure also decreased the activities of alanine aminotransferase and gamma glutamyl transferase. On the other hand, the activities of acetylcholinesterase, carboxylesterase, alkaline phosphatase, levels of lipid peroxidation and protein carbonyl, as well genotoxic markers (micronucleus and nuclear abnormalities frequencies) were unchanged. The evaluation of integrated biomarker response index (IBR) indicated highest variations at the concentration of 200 ng.L-1 at 32 °C, suggesting that the combination of high AMT concentrations and temperatures generate more pronounced negative effects to tadpoles.

Hepatic and blood alterations in Lithobates catesbeianus tadpoles exposed to sulfamethoxazole and oxytetracycline.

In this study the effects of environmentally realistic concentrations of the antibiotics sulfamethoxazole (SMX) and oxytetracyclyne (OTC) on Lithobates catesbeianus tadpoles were evaluated, through the analyzes of the frequencies of micronucleus and nuclear abnormalities in erythrocytes, alterations in leucocytes, liver histopathology, and changes in hepatic esterase activities and oxidative stress biomarkers. The animals were exposed for 16 days at concentrations of 0 (control), 20, 90 and 460 ng L-1. No significant difference was found in the frequencies of micronucleus and nuclear abnormalities. The two highest concentrations of SMX and all concentrations of OTC caused a significant increase in the number of lymphocytes. A significant decrease in the number of neutrophils compared to the control group was observed for all concentrations tested of both antibiotics. Also, decrease in the activity of glutathione S-transferase and high histopathological severity scores, indicating liver damage, were found in tadpoles exposed to the two highest concentrations of SMX and all concentrations of OTC. The main changes in the liver histopathology were the presence of inflammatory infiltrate, melanomacrophages, vascular congestion, blood cells and eosinophils. Esterase activities were unchanged. Indeed, the two highest concentrations of OTC caused a reduction in the activities of superoxide dismutase and glucose 6-phosphate dehydrogenase, while the highest concentration inhibited the activity of glutathione peroxidase and increased protein carbonyl levels. These results evidences that environmentally realistic concentrations of SMX and OTC in aquatic environments are capable to significantly disrupt tadpoles' physiology, possibly affecting negatively their survival rate in natural environments.

Biochemical biomarkers and metals in Perna perna mussels from mariculture zones of Santa Catarina, Brazil.

The activity of cholinesterase (ChE), glutathione-S transferase (GST), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH) and catalase (CAT) was evaluated in the gill and digestive glands of the Perna perna mussel transplanted to three non-contaminated mariculture zones under the influence of distinct physical-chemical characteristics. Differences among sites for ChE, GST and CAT activities in gill, as well as ChE, GST and G6PDH activity in digestive gland of mussels, were found and possibly related to differences in physicochemical characteristics of the sites and/or biological status of the mussels. Mussels that were transplanted to another, more urbanized site (Ponta do Lessa) with similar physicochemical characteristics to one of the farming sites (Sambaqui), was also chosen to evaluate biomarker responses to pollution. Activities of ChE, GST and GR in the digestive glands and CAT in the gills were higher in the polluted site. GR was the only biomarker to be unaltered in different farming sites, but induced in the pollution site. The trace metal concentrations in the mussels were low and unlikely to cause the changes observed in the biomarker levels. The present study strongly suggests that monitoring programs should compare sites with similar physicochemical characteristics when using a complementary biomarker approach. In addition, the baselines for the biomarkers and metal used in the present study can serve as a reference for the monitoring of these mariculture zones in future monitoring programs employing P. perna.

Assessment of Doñana National Park contamination in Procambarus clarkii: integration of conventional biomarkers and proteomic approaches.

Aquatic ecosystems of Doñana National Park (DNP) were monitored using Procambarus clarkii as bioindicator in four campaigns carried out between 2003 and 2004 to assess environmental quality possibly threatened by agrochemicals used in nearby areas. An integrated approach was carried out, by combining the responses of well-established biomarkers and the massive analysis of biological effects at the proteomic level. In sites potentially polluted, lower catalase, glucose-6-P dehydrogenase, and esterase activities, and higher malondialdehyde, metallothionein and glutathione levels were found. Two-dimensional gel electrophoresis resolved >2500 gill spots, and image analysis detected that 35 showed significant intensity differences between the reference site and the other seven sites studied. The superiority of proteomic approaches was clearly recognized in our study since four different protein expression patterns were established based in the fold-number of up-/down-regulation of the 35 differentially expressed proteins. Sites located within Doñana Biological Reserve were essentially free of contaminants and those near the DNP limits were only slightly polluted. The higher proteomic responses found at the upper "Rocina" and "Partido" courses indicate that non-persistent agrochemicals are mainly used in Doñana surroundings. The highest responses corresponded to rice growing areas placed between the Guadiamar stream and the Guadalquivir River, according to the extended and intensive use of agrochemicals in such areas.

Combined effects of temperature and clomazone (Gamit®) on oxidative stress responses and B-esterase activity of Physalaemus nattereri (Leiuperidae) and Rhinella schneideri (Bufonidae) tadpoles.

Temperature is an important factor influencing the toxicity of chemicals in aquatic environments. Neotropical tadpoles experience large temperature fluctuations in their habitats and many species are distributed in areas impacted by agriculture. This study evaluated the effects caused by the exposure to clomazone (Gamit®) at different temperatures (28, 32 and 36 °C) on biochemical stress responses and esterase activities in Physalaemus nattereri and Rhinella schneideri tadpoles. Results evidenced that temperature modulates the effects of clomazone on biochemical response of tadpoles. Antioxidant enzymes, including catalase, superoxide dismutase (SOD), and glucose-6-phosphate dehydrogenase had their activities increased by clomazone in P. nattereri treated at higher temperatures. The biotransformation enzyme glutathione-S-transferase (GST) was also induced by clomazone at 32 and 36 °C. In R. schneideri, clomazone failed to alter antioxidant enzymes at 28 °C, but SOD and GST were increased by clomazone at higher temperatures after three days. All enzymes had their activities returned to the control levels after eight days in R. schneideri. Lipid peroxidation was induced in both species exposed to clomazone at 32 and 36 °C, but not at 28 °C. Acetylcholinesterase was not sensitive to clomazone and temperature, while most treatments impaired carboxylesterase activity. Integrated biomarker response (IBR) was notably induced by temperature in both species, and a synergic effect of temperature and clomazone was mostly observed after three days of exposure. These findings imply that tadpoles from tropical areas may present differential responses in their physiological mechanism linked to antioxidant defense to deal with temperature fluctuations and agrochemicals presence in their habitats.

Impact of genetic polymorphisms in key enzymes of homocysteine metabolism on the pathophysiology of sickle cell anemia.

This work aimed at studying a possible influence of methylenetetrahydrofolate reductase (MTHFR; c. 677C>T) and cystathionine ß-synthase (CBS; 844ins68) polymorphisms on overall oxidative status of sickle cell anemia (SCA) patients and on routine markers, correlating them with hydroxycarbamide (HC) treatment. We evaluated 95 unrelated and diagnosed SCA patients. All patients received a prophylactic treatment with folic acid of 5mg/day, while 41 (43.2%) of them were under hydroxycarbamide (HC) treatment (average dose: 22mg/kg/day). MTHFR and CBS polymorphisms were identified by Polymerase Chain Reaction. Biochemical parameters were measured using spectrophotometric and chromatographic methods. Routine markers were developed by specialized laboratory. We did not find any effect of 677T and "I" allele combination on the biomarkers evaluated. On the other hand, MTHFR 677T mutation was related to a depletion of antioxidant capacity, according to the decreased catalase activity and a reduction about 30% of glutathione levels. Moreover, the presence of the insertion was related to about 23% less biomolecule oxidation levels and lower monocytes count, but about 14% higher lactate dehydrogenase activity. These findings may contribute to highlight that the MTHFR and CBS polymorphisms involvement in SCA pathophysiology is likely to be far more complex than it was explored to date.

Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: antioxidants, lipid peroxidation and DNA damage.

Oxidative stress can take place in marine bivalves under a series of environmental adverse conditions. The study of different systems related to oxidative stress in these organisms can give important information about their physiological status and also about environmental health. Bivalves have been proposed as good sentinel organisms in pollution monitoring studies through the analysis of biochemical biomarkers, and most of the biomarkers analyzed are those related to oxidative stress. However, it is very important to know how other environmental factors not associated to the presence of pollutants might affect these parameters. We have studied a series of mechanisms related to oxidative stress in mussels which inhabit the Brazilian coast, especially in Perna perna species, subjected to different stress conditions, such as the exposure to different contaminants in the laboratory and in the field, the exposure of mussels to air and re-submersion, simulating the tidal oscillations, and in mussels collected at different seasons. Both oxidative damage levels and antioxidant defense systems were strongly affected by the different environmental stress. This review summarizes the data obtained in some studies carried out in bivalves from the Brazilian coast.