A method to measure total antioxidant capacity against peroxyl radicals in aquatic organisms: application to evaluate microcystins toxicity.

Determination of total antioxidant capacity, instead of the measurements of limited number of antioxidants, is very important for the understanding of how antioxidants interact with reactive oxygen species (ROS). Several techniques already exist with this propose, although some of them are extremely time-consuming. A new methodology is proposed, based on the detection of ROS by fluorometry (ex/em: 485/520 nm) employing 2',7'-dichlorofluorescein diacetate (H(2)DCF-DA) as substrate. Supernatant of homogenized samples from different organs (gill, muscle, liver, and brain) of the teleost fish Jenynsia multidentata (Anaplebidae) were exposed to peroxyl radicals generated by thermal (35 degrees C) decomposition of 2,2'-azobis (2 methylpropionamidine) dihydrochloride (ABAP, 4 mM). Different protein concentrations (0.5, 1, 2 and 8 mg/ml) were assayed to get the best signal and curve fitting of fluorescence data over time (30 min). Total antioxidant capacity against peroxyl radicals was estimated as the difference in ROS area with and without ABAP, relative to the fluorescence registered without ABAP. For application of this methodology, J. multidentata specimens were exposed for 24 h to microcystins, cyanotoxins known to induce oxidative stress. Almost all organs showed a lower antioxidant capacity (p<0.05) in samples with 8 mg proteins/ml, when compared to protein content of 1-2 mg/ml. In liver samples, higher (p<0.05) free iron content was determined in samples with 8 mg proteins/ml. Sensitivity test employing GSH spiked in homogenized samples showed the protocol efficiency in detecting total antioxidant capacity. In the test with microcystins a dose-dependent decrease (p<0.05) of antioxidant competence in gills and brain and an inverse result with liver samples were observed. The use of antioxidant defenses was efficient in avoiding oxidative damage, as the content of oxidized proteins was not altered. Data obtained show the potential of this new methodology to be used in ecotoxicological studies.

Modulation of antioxidant and detoxification responses mediated by lipoic acid in the fish Corydoras paleatus (Callychthyidae).

Lipoic acid (LA) has been reported as a potential therapeutic agent due its antioxidants proprieties. It was considered its effect in different organs (gills, brain, muscle and liver) of the fish Corydoras paleatus (Callychthyidae). LA (70 mg/kg of body mass) was added to a commercial fish diet, organisms being fed daily (1% body weight). Sixty animals (mean mass: 2.37+/-0.09 g) were placed randomly in aquariums and received (+LA) or not (-LA) lipoic acid enriched diet during four weeks. After, fish were killed and the brain, muscle, gills and liver were dissected. LA treatment reduced significantly (p<0.05) reactive oxygen species concentration in brain and increased (p<0.05) glutamate-cysteine ligase activity in brain and liver of the same experimental group. LA fed organisms showed higher (p<0.05) brain glutathione-S-transferase activity, indicating that LA improves the detoxification and antioxidant capacity face components that waste glutathione in phase II reactions. A conspicuous reduction of protein oxidation was observed in muscle and liver of +LA organisms, indicating that the treatment was also effective in reducing oxidative stress parameters.

Antioxidant responses and reactive oxygen species generation in different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae).

The aim of this study was to analyze the total antioxidant capacity (TOSC), generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) in the different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae) sampled at non-polluted (NOPOL) and polluted (POL) sites from Lagoa dos Patos (Southern Brazil). Organisms collected at POL during summer showed similar (p>0.05) TOSC values along the body, but worms collected at NOPOL presented higher (p<0.05) TOSC values in the posterior (P) region in respect of anterior (A) region and middle (M) region. TOSC in the P region at NOPOL was higher (p<0.05) compared with the same body region of worms at POL. In summer, ROS concentration was higher in A and M regions of worms at POL in respect of the organisms at NOPOL. During winter all the regions showed higher ROS in worms sampled at POL. It was registered absence of season influence on LPO content, but in the P region at NOPOL in summer there were lower LPO levels compared with the others regions (p<0.05). In vitro assays showed that P region, despite a higher basal ROS, presented a higher competence to cope with pro-oxidants compared with A and M regions (p<0.05), corroborating the field results. A lower proteic sulfhydril content was observed in P in respect of the other regions (p<0.05) supporting the idea of a highest oxidant condition in this region. The results indicate that worms collected at the POL site are confronted to higher ROS concentrations, affecting its antioxidant capacity, a result that depends of body regions.