J. Cunha Bastos

Brain acetylcholinesterase as an in vitro detector of organophosphorus and carbamate insecticides in water

Abstract An inexpensive but accurate enzymatic method is proposed for the detection of carbamate and organophosphorus pesticides contaminating water supplies. The method uses an acetylcholinesterase preparation obtained after extraction of rat brain microsomal fraction with Triton X-100. The method is based on inhibition of acetylcholinesterase in the presence of the pesticides. Some phosphorothionate insecticides (e.g. parathion, malathion), which are not direct acetylcholinesterase inhibitors, can also be activated by preincubation with the enzyme preparation. Enzyme assay is performed by a potentiometric method based on the formation of acetic acid in the incubation mixture. Interference of any eventual buffering capacity of the sample can be easily corrected. Malathion, parathion, diazinon and deoxicarbamate inhibited the enzyme at least 20% when they were added to the medium in the limit concentration recommended for public water supplies (0.1 mg/l). The method was evaluated in samples collected from selected locations of Paraiba do Sul river, Rio de Janeiro, Brazil, and it proved to be sufficiently practical and accurate as an alarm routine test for such pesticide classes.

Activation of parathion by liver of Hypostomus punctatus, a Brazilian benthic fish (Cascudo)

Abstract 1. Organophosphorus activation ability of Hypostomus punctatus liver was demonstrated by an in vitro metabolization curve of parathion into paroxon. 2. A calibration curve of paroxon acetylcholinesterase inhibition indicated that about 6.0% of parathion was biotransformed after 2 hr of incubation when the medium contained 0.5 mg of parathion per litre and 0.15 mg of protein. 3. Identification of incubation products by thin layer and gas chromatography showed that paroxon was not the unique resulting product. 4. Microsomal liver activation of parathion was largely dependent on reduction by NADH or NADPH but was only partially inhibited by SKF-525A and CO.

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.

Drug metabolism components in liver microsomes from Hypostomus punctatus, a brazilian benthic fish (cascudo)

1. The major components of hepatic drug biotransformation system were identified in a Brazilian freshwater benthic fish. 2. Cytochrome P-450 difference spectra were obtained adding 0.02 mM phenazine ethosulphate and 2 mM ascorbate to microsomal suspensions. Basal levels of P-450 were high (0.9 nmol/mg of microsomal protein) and were not induced by 3-MC. 3. Microsomal NADPH-cytochrome C reductase activity was determined in presence of 1.3 x 10(-4) M NADPH, 3.3 x 10(-5) M cytochrome C, 1.0 x 10(-4) M EDTA, 66 micrograms of microsomal protein per ml in a 0.3 M Tris-HCl buffer, pH 8.6. Basal levels of NADPH-cytochrome C were 152.7 nmoles/min/mg of microsomal protein.

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.