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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.

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.

Algal blooms are phenomena produced by anthropogenic activities, despite the possible natural causes. In Araruama Lagoon, blooms occurred in 2005 and in subsequent years, causing profound changes in phytoplankton communities. These episodes triggered events of extensive fish mortality associated with low levels of dissolved oxygen. Another adverse effect associated with blooms is the production of harmful toxins such as phycotoxins produced by eukaryotic microalgae and cyanotoxins produced by cyanobacteria. Samples of fish (mullet and menhaden) and seston showed levels of microcystin by enzyme-linked immunosorbent assay (ELISA), also a seston sample (São Pedro d'Aldeia on 08/22/2007), in a period not related to fish mortality, showed cells which contained genes encoding microcystin synthetase, an enzyme responsible for the synthesis of microcystin. The succession of microalgae with the concomitant presence of potentially toxic cyanobacteria draws attention to the risk of chronic exposure by the population that uses fish as their main protein source.

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 Boletim do Observatorio Ambiental Alberto Ribeiro Lamego, Campos dos Goytacazes/RJ, v. 4 n. 1, p. 57-80, jan. / jun. 2010 58 Manildo Marciao de Oliveira et al. Boletim do Observatorio Ambiental Alberto Ribeiro Lamego, Campos dos Goytacazes/RJ, v. 4 n. 1, p. 57-80, jan. / jun. 2010 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.

Background Chronic administration of furosemide may induce thiamine deficiency and cause or aggravate myocardial dysfunction. Methods and Results Wistar rats were divided into four groups according to food and treatment: (1) thiamine standard chow with intraperitoneal furosemide administration; (2) thiamine standard chow with intraperitoneal saline administration; (3) thiamine-deficient chow with intraperitoneal furosemide administration; and (4) thiamine-deficient chow with intraperitoneal saline administration. Thiamine status was evaluated by high-performance liquid chromatography determination in plasma, erythrocytes, and myocardium, and by erythrocyte transketolase activity and the thiamine pyrophosphate effect to recover transketolase activity. Left ventricular mass index, intramyocardial arteries-to-cardiomyocyte ratio, cardiomyocyte cross-sectional area, and cardiomyocyte nuclei number were estimated. Myocardial structure was also studied by transmission electronic microscopy. Group 3 showed significantly lower blood and myocardial thiamine levels, which was not observed in group 1. Left ventricular mass index, cardiomyocyte cross-sectional area, and intramyocardial arteries-to-cardiomyocyte ratio were smaller in thiamine-deficient and furosemide-treated rats. However, no significant variation was found in the number of cardiomyocyte nuclei among the groups. Transmission electronic microscopy showed mitochondrial alterations in the thiamine-deficient groups. Conclusion The present results indicate that furosemide administration is not the primary cause of thiamine deficiency in rats with adequate thiamine intake. Furosemide aggravates thiamine deficiency only in situations associated with insufficient thiamine intake, causing cardiac structural alterations, such as myocardial fiber hypotrophy, poor microvascularization, and mitochondrial degeneration.

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 allP. lineatusat 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.

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

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 K M 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/K M 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 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.

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., Female sea turtles have rarely been observed foraging during the nesting season. This suggests that prior to their migration to nesting beaches the females must store sufficient energy and nutrients at their foraging grounds and must be physiologically capable of undergoing months without feeding. Leptin (an appetite-suppressing protein) and ghrelin (a hunger-stimulating peptide) affect body weight by influencing energy intake in all vertebrates. We investigated the levels of these hormones and other physiological and nutritional parameters in nesting hawksbill sea turtles in Rio Grande do Norte State, Brazil, by collecting consecutive blood samples from 41 turtles during the 2010–2011 and 2011–2012 reproductive seasons. We found that levels of serum leptin decreased over the nesting season, which potentially relaxed suppression of food intake and stimulated females to begin foraging either during or after the post-nesting migration. Concurrently, we recorded an increasing trend in ghrelin, which may have stimulated food intake towards the end of the nesting season. Both findings are consistent with the prediction that post-nesting females will begin to forage, either during or immediately after their post-nesting migration. We observed no seasonal trend for other physiological parameters (values of packed cell volume and serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, low-density lipoprotein, and high-density lipoprotein). The observed downward trends in general serum biochemistry levels were probably due to the physiological challenge of vitellogenesis and nesting in addition to limited energy resources and probable fasting.

The interaction of methyl-parathion with the albumin of Piaractus mesopotamicus (Holmberg 1887) (= pacu), a fish species typical of Brazilian rivers, was studied and the results compared with known values for human and bovine albumin obtained in an earlier investigation. Methyl-parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) is an organophosphorous pesticide still used in agriculture and fish farming in many countries. The fluorescence quenching technique with tryptophan as a natural probe was used to detect for the presence of methyl-parathion. Fluorescence can be mathematically expressed by the Stern-Volmer equation to calculate quenching constants, and changes in the behavior of Stern-Volmer curves at different temperatures indicate the nature of the mechanism causing the quenching. Our results indicate that methyl-parathion forms a complex with fish albumin. The estimated association constant is 9.73 x 103 (+/- 4.9 x 102) M(-1) at 25 degrees C.

Brain acetylcholinesterase (AChE) of some fishes from the coast of Rio de Janeiro State was studied as a possible pesticide biomarker in marine environmental monitoring. AChE specific activity in brain varied from 145 to 530 U/g of proteins and the Michaelis–Menten constant (KM) for acetylthiocholine varied from 104 to 291 μM among the 20 species studied. The enzyme sensitivity to methyl paraoxon, evaluated by the inhibition kinetic constants, shows that some species (Paralonchurus brasiliensis and Genidens genidens) are more sensitive (IC50-30 min = 455 and 468 nM, respectively). The less sensitive Merluccius hubbsi and Percophis brasiliensis (IC50-30 min = 3339 and 3259 nM, respectively) belong to the super-order Paracanthopterygii, which includes the more ancient species. On the other hand, more susceptible species belong to the super-order Acanthopterygii, which includes more recent species. These results suggest a possible evolutionary linkage for AChE sensitivity to methyl paraoxon. The application of inhibition kinetic constants for fish brain AChE in phylogenetic studies is still being investigated. The results have shown that a fish sentinel species should have the highest brain AChE level among the more sensitive ones.

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 °C, respectively. The calculated Stern–Volmer constants at 25 °C were 9.73×10 3 (±4.9×10 2 ) M −1 for serum and 9.20×10 3 (±2.0×10 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 10 μM, suggesting that the protein is the most important carrier for the pesticide in serum.

Acetylcholinesterase (AChE) sensitivity to the organophosphorus (OP) pesticide methyl-paraoxon was measured in fourteen species of Neotropical marine and freshwater fish found in the waters of Brazil. The rate constant for phosphorylation, k p , the dissociation constant, k d , the second order rate constant, k i , and the IC 50 value were measured at 28 °C in pH 7.5 buffer for AChE extracted from brain. In addition, the substrate affinity constant, k m , was measured with acetylthiocholine. The IC 50 for 30 min of inhibition ranged from 123 nM ( Prochilodus lineatus ) to 3340 nM ( Percophis brasiliensis ), which corresponded to k i values of 187–6.9 mM −1 min −1 . A 10-fold range in k p values from 0.21 min −1 ( Paralonchurus brasiliensis ) to 2.1 min −1 ( Dules auriga ) was associated with a 37-fold range in k d values from 4 to 150 μM. These large differences in reactivity with methyl-paraoxon were not reflected in the binding affinity for acetylthiocholine; k m values were approximately 0.1–0.3 mM for all species. These results predict that the amino acid sequence involved in AChE sensitivity differs in these fishes, and that consequently some fish species may be resistant to the toxicity of methyl-paraoxon.

Methyl parathion (MP; O,O-dimethyl O-p-nitrophenyl phosphorothioate) is an organophosphorous compound still largely used in agriculture and fish hatcheries. This pesticide is not quite selective and is potentially toxic for both vertebrates and invertebrates. Its mechanism of acute toxicity is the inhibition of the enzyme acetylcholinesterase in nervous tissue. Binding of pesticides to plasma proteins is one of many factors that influence their distribution and elimination. The free concentration available for toxic action can be effectively reduced for pesticides with high binding to plasma proteins, although the affinity of pesticides to plasma proteins is often lower than for the enzyme targets. Several different transport proteins exist in blood plasma, but albumin only is able to bind a wide diversity of xenobiotics reversibly with high affinity. It was already known that parathion (ethyl parathion) exhibits a high affinity to human and bovine serum albumins. We studied interactions of methyl parathion with these albumins by using fluorescence quenching techniques. We selectively excited the fluorescence of tryptophan residues with a 290 nm wavelength light, and observed quenching by titrating human and bovine serum albumin solutions with methyl parathion. Stern-Volmer graphs were plotted and quenching constants were estimated. Our results pointed to the formation of complexes of methyl parathion with albumins. Association constants at 25 degrees C were 3.07 x 10(4) (1.2 x 10(3))M(-1) for human serum albumin, and 1.96 x 10(4) (+/- 4.5 x 10(2))M(-1) for bovine serum albumin. At 37 degrees C, they were 1.08 x 10(4) (+/- 2.0 x 10(2))M(-1) for human serum albumin, and 8.16 x 10(3) (+/- 1.9 x 10(2))M(-1) for bovine serum albumin. Results also suggest that the primary binding site for methyl parathion on albumin is close to tryptophan residues 214 of human serum albumin and 212 of bovine serum albumin.

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.

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 K M 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.