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.

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.

A novel butyrylcholinesterase from serum of Leporinus macrocephalus, a Neotropical fish.

We show here that serum of piaussu, a Neotropical characin fish, has the highest butyrylcholinesterase activity so far described for humans and fish. To clarify whether this cholinesterase could protect piaussu against anticholinesterase pesticides by scavenging organophosphates, we purified it 1700-fold, with a yield of 80%. Augmenting concentrations (from 0.01 to 20 mM) of butyrylthiocholine activated it. The pure enzyme was highly inhibited by chlorpyriphos-oxon (ki=10,434x10(6) M-1 min-1) and by the specific butyrylcholinesterase inhibitor, isoOMPA (ki=45.7x10(6) M-1 min-1). Electrophoresis of total serum and 2-D electrophoresis of the purified cholinesterase showed that some enzyme molecules could circulate in piaussu serum as heterogeneously glycosylated dimers. The enzyme's N-terminal sequence was similar to sequences found for butyrylcholinesterase from sera of other vertebrates. Altogether, our data present a novel butyrylcholinesterase with the potential of protecting a fish from poisoning by organophosphates.