Jayme Cunha Bastos

Ghrelin and leptin modulate the feeding behaviour of the hawksbill turtle Eretmochelys imbricata during nesting season

Female sea turtles rarely have been observed foraging during the nesting season. We investigated the levels of ghrelin, leptin and other physiological and nutritional parameters in nesting hawksbill sea turtles in Brazil. We found that levels of serum leptin (appetite-suppressing protein) decreased over the nesting season, while an increasing trend was observed in ghrelin (hunger-stimulating peptide). Both findings are consistent with the prediction that post-nesting females will begin to forage after the nesting season, , either during or just after their post-nesting migration.

The interaction of methyl-parathion with serum and albumin of the neo-tropical fish Piaractus mesopotamicus

The interaction of methyl-parathion with serum and albumin of pacu, Piaractus mesopotamicus, was studied, using the fluorescence quenching technique. Pacu is a neo-tropical fish specie inhabitant of rivers from western regions of Brazil. Methyl-parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) is an organophosphorous pesticide still used in agriculture and fish farming in many countries. The quenching of fluorescence can be mathematically expressed by the Stern-Volmer equation to calculate quenching constants. Stern-Volmer curves analysis is able to give important information about the pesticide-albumin interaction. Our results showed that the serum quenching reached 10% when the molar ratio of pesticide/albumin was about 7:1 for the three temperatures of the experiment. For the pure albumin quenching of 10%, methyl-parathion concentrations were 6, 7 and 9 times higher than albumin at 20, 25 and 30 degrees C, respectively. The calculated Stern-Volmer constants at 25 degrees C were 9.73x10(3)(+/-4.9x10(2))M(-1) for serum and 9.20x10(3)(+/-2.0x10(2))M(-1) for albumin. It was observed that albumin quenching is the phenomenon contributing to the quenching of the pacu serum fluorescence for methyl-parathion concentration lower than 10microM, suggesting that the protein is the most important carrier for the pesticide in serum.

Cholinesterase activity of muscle tissue from freshwater fishes: Characterization and sensitivity analysis to the organophosphate methyl‐paraoxon

The biochemical characterization of cholinesterases (ChE) from different teleost species has been a critical step in ensuring the proper use of ChE activity levels as biomarkers in environmental monitoring programs. In the present study, ChE from Oreochromis niloticus, Piaractus mesopotamicus, Leporinus macrocephalus, and Prochilodus lineatus was biochemically characterized by specific substrates and inhibitors. Moreover, muscle tissue ChE sensitivity to the organophosphate pesticide methyl-paraoxon was evaluated by determining the inhibition kinetic constants for its progressive irreversible inhibition by methyl-paraoxon as well as the 50% inhibitory concentration (IC50) for 30 min for each species. The present results indicate that acetylcholinesterase (AChE) must be present in the muscle from P. mesopotamicus, L. macrocephalus, and P. lineatus and that O. niloticus possesses an atypical cholinesterase or AChE and butyrylcholinesterase (BChE). Furthermore, there is a large difference regarding the sensitivity of these enzymes to methyl-paraoxon. The determined IC50 values for 30 min were 70 nM (O. niloticus), 258 nM (P. lineatus), 319 nM (L. macrocephalus), and 1578 nM (P. mesopotamicus). The results of the present study also indicate that the use of efficient methods for extracting these enzymes, their kinetic characterization, and determination of sensitivity differences between AChE and BChE to organophosphate compounds are essential for the determination of accurate ChE activity levels for environmental monitoring programs.

Influence of norbixin on plasma cholesterol-associated lipoproteins, plasma arylesterase/paraoxonase activity and hepatic lipid peroxidation of Swiss mice on a high fat diet

Effects of the carotenoid norbixin (7.7 and 92.8 mg/kg) added to drinking water were studied in male Swiss mice fed with a high fat diet for 4 weeks. High fat diet decreased, by 55.7%, the levels of high-density lipoprotein (HDL)-cholesterol (P < 0.05) increased, by 167%, the levels of very low-density lipoprotein (VLDL)-cholesterol/low-density lipoprotein (LDL)-cholesterol (P < 0.0001). When the high fat diet was ingested with norbixin, HDL-cholesterol and VLDL/LDL-cholesterol remained near control levels while plasma triglycerides were not altered. High fat diet decreased the activity of plasma arylesterase/paraoxonase (EC3.1.8.1) by 46% (P < 0.0001), a reduction that was also counteracted by norbixin. Basal levels of thiobarbituric acid-reactive substances (TBARS), measured in liver homogenates, were not increased by high fat diet. However, there was a significant increase in TBARS levels (P < 0.001) when liver homogenate from mice submitted to high fat diet was incubated with Fe2+ and H2O2. Under the same oxidative conditions, liver homogenate was much less responsive to peroxidation (P < 0.0001) when high fat diet was associated with norbixin. Thus, norbixin, a common carotenoid food additive, alters plasma lipid levels and paraoxonase activities in Swiss mice fed with an atherogenic diet. Further studies should be performed in order to determine the lowest effective norbixin concentration as this carotenoid is ingested with industrialized food in very tiny amounts.

A high density lipoprotein from Piaractus mesopotamicus, pacu, (Osteichthyes, Characidae), is associated with paraoxonase activity.

We have characterized the serum lipoprotein profile and localized the serum paraoxonase activity of pacu, Piaractus mesopotamicus, a tropical fish species. The total lipoprotein profile of pacu serum obtained after KBr density ultracentrifugation shows the predominance of HDL (1.1267 g/mL). SDS-PAGE electrophoresis revealed a negligible amount of LDL. Pacu HDL was purified by gel filtration column on HPLC, and its molecular mass was estimated to be 246 kDa. Protein composition was 35%, and comprised four protein components with molecular masses of 45, 38, 25 and 12.5 kDa. Lipids represent 58% of total HDL, comprising 40% neutral lipids and 18% phospholipids by weight. The HDL contains 7% of carbohydrates, and mannose was the only sugar detected by paper chromatography in HDL hydrolysates. HDL-containing fractions showed the major paraoxonase activity. Purification of HDL resulted in a 23-fold enrichment of this activity. This is the first experimental evidence demonstrating the association of paraoxonase activity with a HDL in fish.

Bioconcentration and Acute Intoxication of Brazilian Freshwater Fishes by the Methyl Parathion Organophosphate Pesticide

Three species of freshwater Brazilian fishes (pacu, Piaractus mesopotamicus; piavussu, Leporinus macrocephalus, and curimbatá, Prochilodus lineatus) were exposed to an acute dose of 5 ppm methyl parathion organophosphate pesticide. Three to five individuals per species were exposed, one at a time, to 40 liters tap water spiked with Folidol 600. Pesticide concentrations and cholinesterase (ChE) activities were evaluated in serum, liver, brain, heart, and muscle. The bioconcentration of methyl parathion was similar for all studied fishes. Brain tissue showed the highest pesticide concentration, reaching 80 ppm after exposure for 30 min to methyl parathion. Three to 5 hours of 5 ppm methyl parathion exposure provoked the death of all P. lineatus at 92% brain AChE inhibition, whereas fish from the other two species survived for up to 78 hours with less than 80% brain AChE inhibition. Our results indicate that acute toxic effects of methyl parathion to fish are correlated with brain AChE sensitivity to methyl paraoxon.

Paraoxonase activity in liver of Pacu, Piaractus mesopotamicus Holmberg (Characidae)

Enzymatic production of p-nitrophenol in liver of Piaractus mesopotamicus Holmberg, 1887 was consistently assayed at pH 8.5 using 7.5 mM paraoxon as substrate. This pacu liver paraoxonase activity was activated by NaCl. Apparent values of KM were 2.42 x 10-3 M in the presence of 0.5 M NaCl and 8.99 x 10-3 M without NaCl. Apparent maximum velocity values calculated in the absence and presence of 0.5 M NaCl were 1.09 x 10-3 µmoles/min/mg of proteins and 1.29 x 100-3 µmoles/min/mg of proteins, respectively. These Vmax values are fifty-fold the value described for trout (Salmo trutta Linnaeus, 1758) liver paraoxonase. Paraoxonase activity of pacu liver homogenates was recovered as much in cytosolic as in particulate cellular subtractions, but the particulate subtractions showed higher specific activities. The data presented here indicate that hepatic hydrolysis of organophosphorous pesticides may not be an important detoxification process in pacu.

Paraoxonase activity in sera from Piaractus mesopotamicus Holmberg (Characidae) and Hypostomus punctatus Valenciennes (Siluridae)

A paraoxonase activity present in serum of two Brazilian fish species was consistently assayed at pH 8.5 using 7.5 mM paraoxon final concentration. The paraoxonase activity was more activated by 0.5 M NaCl in serum of Piaractus mesopotanricus Holmberg, 1887 (pacu) than in serum of Hypostomus punctatus Valenciennes, 1840 (cascudo). Apparent values of KM were 3.3 x 10-3 M for cascudo and pacu paraoxonase activity in the presence of 0.5 M NaCl. Apparent maximum velocity values calculated in the presence of 0.5 M NaCl were 6.1 and 6.5 nmole/min/mL of serum for cascudo and pacu, respectively. Vmax/KM ratio values of determinations in the presence and absence of 0.5 M NaCl showed that NaCl had a more evident effect on increasing the affinity of serum paraoxonase for paraoxon in pacu serum. Young specimens of pacu showed a marked decreased paraoxonase serum activity when kept in tanks treated with 0.25 ppm methyl-parathion.

The importance of an efficient extraction protocol for the use of fish muscle cholinesterases as biomarkers

Esterase activity found in muscle extracts is useful to evaluate harmful effects of anticholinesterase pollutants. Yet, most procedures applied in the extraction of fish muscle esterases in order to investigate their activity as a biomarker of environmental exposure comprise the homogenization of muscle tissue in low-salt solutions, followed by centrifugation to separate the supernatant as the enzyme source. However, acetylcholinesterase (AChE), the main target in these monitoring efforts, is a membrane-bound protein and is only present in muscle extracts if homogenization is carried out using chaotropic high-salt solutions. In this context, four extraction procedures using muscle tissue from six fish species were evaluated in order to establish a reproducible and reliable AChE assay for the determination of this biomarker. Results indicate that over 80% of AChE activity might be lacking in low-salt supernatants, and that the highest activities are obtained after extraction with solutions containing either 1molL-1 NaCl or 1molL-1 NaCl plus 3% Triton X-100, preserving almost 100% esterase activity over acetylthiocholine as substrate after centrifugation. Thus, many studies in the literature suffer from theoretical flaws and report erroneous AChE activity, since typical muscle AChE activity, the end-point biomarker for anticholinesterase pollutants, may have not been consistently assayed.

Toxinas de cianobactérias e microalgas marinhas: um desafio para a ecotoxicologia aquática

The purpose of this review is to draw attention to the growing number of secondary metabolites produced by microorganisms in aquatic environments, specially cyanobacteria and marine microalgae. The first group is a producer of major hepatotoxins, neurotoxins and dermatotoxins. Cyanobacteria are responsible for most poisoning events in epicontinental aquatic environments. Eukaryotic microalgae, in marine environments such as diatoms and dinoflagellates, are great producers of a variety of phycotoxins. These syndromes are caused by consumption of contaminated mussels or ciguatera intoxication by consumption of fish (CFP). The current situation demands attention because many events occur and are not notified for important historical studies on seasonal flower blooms. Methodological issues and skilled labor also hinder more precise diagnosis of the blooms. The cooperation between different professionals with different backgrounds as taxonomists, chemists, biologists and environmental engineers is essential for ecotoxicological studies on the risk assessment of these blooms and suggest changes in environmental legislation.