Glutathione peroxidase and glutathione S-transferase in blood and liver from a hypoxia-tolerant fish under oxygen deprivation.

Liver enzyme activities can be employed as biomarkers, but liver can only be obtained with death of the specimen. On the other hand, blood withdrawal is a non-lethal procedure. Accordingly, the hypothesis of this study is to verify if glutathione peroxidase (GPX) and glutathione S-transferase (GST) activities in blood parallel those in the liver of the hypoxia-tolerant fish, Piaractus mesopotamicus (pacu), submitted to hypoxia conditions. GPX was assayed with H2O2 in cytosols from both liver and erythrocytes and exhibited no significant variation, either in erythrocytes or in liver, when comparing pacus under normoxia with those under hypoxia (42 h). GST activity with chloro-dinitrobenzene (CDNB), an artificial substrate suitable for almost all GST isoenzymes, was compared to activity with 4-hydroxy-nonenal (4-HNE), a physiological endogenous substrate. GST activity with CDNB did not change in liver or in erythrocyte cytosols in pacus under hypoxia compared to those under normoxia. On the other hand, a significant decrease in erythrocyte activity with 4-HNE was observed after 42 h of hypoxia in both erythrocytes and liver, which may be a response to increased lipid oxidation in erythrocytes. Erythrocyte GST activity was 3-fold higher with 4-HNE than with CDNB, indicating that 4-HNE is a more appropriate substrate to determine GST activity in pacu erythrocytes.

Oestrogenicity assessment of s-triazines by-products during ozonation.

The triazines are a group of herbicides with a wide range of uses. Atrazine is, in fact, one of the most used agricultural pesticides in the world. The terbuthylazine is applied as a substitute of atrazine in some countries of Europe since 2004, when the European Union announced a ban of atrazine because of ubiquitous water contamination. In this study, both atrazine and terbuthylazine were degraded by the ozone process to estimate the efficiency on pesticide removal in water, the intermediates formed and their potential oestrogenic activity using the yeast oestrogen screen (YES) test. Both pesticides were rapidly eliminated from the medium during ozonation (applied ozone dose 0.083 and 0.02 mmol O3 L(-1), respectively). The results show that both compounds generated similar by-products from ozone degradation. Moreover, significant oestrogenic activity was detected for both atrazine and terbuthylazine intermediates, during the first minutes of ozonation. The YES assay used in this study proved to be a sensitive tool in assessing trace amounts of oestrogenic chemicals, which can represent critical issues influencing the experimental results in environmental applications.

Melatonin affects conjugation of 4-hydroxynonenal with glutathione in liver of pacu, a hypoxia-tolerant fish.

In cytosol from liver of pacu, Piaractus mesopotamicus, a hypoxia-tolerant fish that dwells in Pantanal, we found an enzyme activity capable of modulating the alkenal 4-hydroxy-2-nonenal (HNE) by conjugating it with glutathione (GST-HNE activity). HNE is a downstream metabolite from the oxidation of polyunsaturated fatty acids by reactive oxygen species arisen from mitochondria of animal cells. HNE production may increase more intensively under oxidative stress. Harmful effects to cell survival may occur when HNE increases over 10(-4) M. Pacus submitted to hypoxia in July (cold season in Pantanal) showed 40% less of this GST-HNE conjugating activity in their liver cytosol. Injecting pacus subjected to hypoxia during the cold season with a summer physiological dose of melatonin caused their liver cytosolic GST-HNE activity to increase up to the levels found in the warm season. From October to March (warm season in Pantanal), pacus are prone to oxidative stress particularly during potamodromous active oxygen-demanding swimming, when they migrate up rivers to spawn. Thus, our findings point out that the higher levels of melatonin in circulation during the summer are important to avoid the increase of 4-HNE inside liver cells of this fish species.

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal.

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.