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.

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.

Enzymatic GST levels and overall health of mullets from contaminated Brazilian Lagoons.

Glutathione S-transferase (GST) assays in non-mammalian organisms are usually conducted inappropriately, since no previous standardization of the optimal concentrations of proteins and substrates and adequate pH is conducted. Standardization is a key task to adjust enzyme assays at their kinetically correct maximal initial velocities, if one wants these velocities to indicate the amount of enzyme in a sample. In this paper GST assays were standardized in liver cytosol to compare seasonal GST levels in liver of mullet from two contaminated lagoons in the Rio de Janeiro to those from a reference bay. GST potential as a biomarker of sublethal intoxication in this species was also evaluated. Mullet liver GST levels assayed with substrates that corresponded to three different GST isoenzymes varied throughout the year. The differences indicated that mullets are suffering from sublethal intoxication from contaminants in these lagoons. Seasonal variations of activity were relevant, since these could indicate differences in xenobiotic input into the areas. An analysis of overall mullet health condition using a morphological index (the Fulton Condition Factor) and macroscopic abnormalities corroborated the differences in GST levels, with fish from one of the sites in worse overall health condition showing lower and significantly different FCF when compared to the reference site. Therefore, GST standardized activity levels are useful biomarkers of environmental contamination for mullet.

Alterations in morphometric and organosomatic indices and histopathological analyses indicative of environmental contamination in Mullet, Mugil liza, from Southeastern Brazil.

Mullet (Mugil liza) were sampled in five different areas along the Guanabara Bay, southeastern Brazil, classified as non-contaminated, moderately contaminated and contaminated. Morphometric (Fulton condition factor, relative condition factor and weight to length scaling coefficient) and organosomatic (hepatosomatic index) indices of environmental stress were analysed. Fish from the differentially contaminated areas show statistically different Fulton and relative condition factors and hepatosomatic indices, but not the weight to length scaling coefficient. The Kn and the FCF followed the same trend, with fish from São Gonçalo (1.07 ± 0.04 and 0.89 ± 0.03), Itaipu (0.84 ± 0.01 and 0.86 ± 0.01) and the Rodrigo de Freitas Lagoon (1.03 ± 0.01 and 0.87 ± 0.20) showing higher FCFs than fish from Magé (0.96 ± 0.01 and 0.81 ± 0.01). Fish from Itaipu showed significantly higher HSI values than the other sampling sites (1.68 ± 0.07), with fish from Olaria and Ipiranga showing the lowest (1.56 ± 0.12 and 1.60 ± 0.07, respectively).

In vitro and in vivo toxicological evaluation of extract and fractions from Baccharis trimera with anti-inflammatory activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Baccharis trimera (Less) DC. (Asteraceae), popularly known in Brazil as "carqueja", have been used in folk medicine to treat gastrointestinal, hepatic and renal diseases, and inflammatory processes as rheumatism. AIM OF THE STUDY: To evaluate the in vitro and in vivo toxicological effects of anti-inflammatory Baccharis trimera aqueous extract and fractions. MATERIALS AND METHODS: Aqueous extract of Baccharis trimera (AEBt) was produced by infusion in boiling water. After lyophylization AEBt was extracted with 80% ethanol, originating the ethanolic supernatant fraction (EFBt) and the aqueous sediment fraction (AFBt). Anti-inflammatory properties of AEBt, EFBt or AFBt (3, 30 or 300 µg/kg b.w.) were evaluated by the carrageenan-induced mouse paw edema using indomethacin (10mg/kg) as positive control. The growth of rat hepatoma cells (HTC) and human embryo kidney epithelial cells (HEK) was determined by protein staining assay. Cytotoxicity was assayed by the tetrazolium salt (MTT) reduction. Cyclosporin was used as reference cytotoxic drug for spleen cells and doxorubicin for HTC and HEK cells. For in vivo toxicological evaluation SW male mice were daily and oral (gavage) treated with extract/fractions at 4.2mg/kg or 42 mg/kg during 15 days. After treatment liver or kidney cells were submitted to comet assay to determine the DNA damage index, and the glutathione S-transferase activity was assayed towards ETHA (class Pi) and CDNB (several classes). Mutagenicity was evaluated by the Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102. RESULTS: The anti-inflammatory effects of EFBt were higher than those of AEBt or AFBt. Mice treatment (3-300 µg/kg) with AFBt reduced the paw edema (3h) at lower levels (29.2-37.3%; P<0.01), than those observed for AEBt (44.7-54.2%; P<0.001), EFBt (49.3-58.2%; P<0.001) or indomethacin (64.6%, P<0.001, 10mg/kg). The growth of kidney cells (HEK) was inhibited by AEBt (IC(50) 182.6 µg/ml), EFBt (IC(50) 78.1 µg/ml) and AFBt (IC(50) 86.2 µg/ml), with lower effects on HTC hepatic cell (IC(50) 308.8 µg/ml, 396.5 µg/ml and 167.9 µg/ml, respectively). As evaluated by MTT test, AFBt exhibited cytotoxicity for HEK cells (IC(50) 372.5 µg/ml), but none for HTC ones; by the way, AFBt stimulated spleen cells (EC(50) 2.2 µg/ml) while cyclosporine, a cytotoxic reference drug inhibited them with IC(50) of 0.42 µg/ml; the IC(50) for doxorubicin for HEK and HTC cells was 0.28 µg/ml and 14.4 µg/ml, respectively, at 96h. No mutagenic potential was observed. Mice treatment with AEBt or AFBt at 42 mg/kg for 15 days altered the kidney relative weight, but not at 4.2mg/kg. Baccharis trimera did not change liver, spleen or popliteal lymph node relative weight. DNA damage index of kidney cells was observed on mice treated with AEBt/AFBt, but not on animals treated with EFBt, while DNA lesions were detected on liver cells only after AFBt treatment. The general activities of hepatic GST and Pi GST were reduced by EFBt and AFBt treatment, respectively. CONCLUSIONS: Baccharis trimera did not show mutagenicity, inhibited the GST activity, a hepatic detoxification enzyme, and induced in vivo (genotoxicity) and in vitro toxicological effects to kidney cells.

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.