Use of Cholinesterase Activity in Environmental Monitoring †Research Paper

Use of Cholinesterase Activity in Environmental Monitoring – Research Paper Free Online Research Papers Use of Cholinesterase Activity in Environmental Monitoring: Importance of Kinetic Parameters Determination In Estaurine Fish Research Paper Abstract The aim of the present work was to determine the kinetic parameters and cholinesterase (ChE) activity from two teleost fishes: the croacker Micropogonias furnieri (Scianidae) and sea catfish Cathorops spixii (Ariidae), to verify their suitability as sentinels of aquatic pollution by anticholinesterasic compounds. Fish were collected in a reference and in a polluted site in Southern Brazil and brain ChE was used as enzyme source. Inhibition kinetic parameters employing ChE from C. spixii showed that fish collected in the reference site presented more affinity (Ka) for eserine than those collected in the polluted site and the contrary was observed for the carbamylation constants (Kc), overall resulting in similar inhibition constants (Ki). Considering the extremely low sensitivity to in vitro inhibition by eserine, M. furnieri seems to be an unsuitable species to be employed as an environmental sentinel for pollution of anticholinesterasic compounds. Results obtained in the present study point to the importance of kinetic studies when cholinesterasic activity is employed as a biomarker in environmental quality monitoring programs. Keywords: biomarkers, cholinesterase, eserine, fish, kinetic parameters, estuarine environments 1. Introduction Some pesticides, including organophosphorus and carbamates, are known to selectively inhibit cholinesterase (ChE) activity (Valbonesi et al., 2003). When directly released into the environment, these molecules can reach rivers and sometimes the sea, leading to the contamination of various aquatic ecosystems (Mora et al., 1999). The relationship between the presence of these kind of compounds and ChE activity has been widely studied and employed as a biomarker in aquatic invertebrate and vertebrate species (Bocquenà © et al., 1997; Sturm et al., 1999; Rodriguez-Fuentes Gold-Bouchot, 2000; De la Torre et al., 2002). The use of biochemical measurements in organisms as an indicator of pollution can complement chemical analysis, giving information about the adaptive or deleterious responses in organisms exposed to a certain amount of chemicals. Moreover, among biological effects of pollutants, biochemical ones occur more quickly, thus providing earlier warning signal before other toxicological end points, including death, are evident. (Livingstone, 1998). Since organophosphorus and carbamates have a relatively short half-life, the assessment of cholinesterase (ChE) inhibition is a useful tool to evaluate their environmental impact on aquatic biota, even when they are not longer detectable in the environment (Valbonesi et al., 2003) and, as mentioned above, considerable efforts have been made in the last two decades to develop and validate measurements of biological parameters to complement the information given by the chemical analysis of contamination. The main advantage of using biomarkers at low levels of biological organization is the possibility to detect deleterious effect pollutants before being evidenced at higher levels of biological organization. Among biochemical markers, the measurement of fish cholinesterase activities has become a classical tool for biomonitoring pollution in marine (Bocquenà © et al., 1990) and continental waters (Sturm et al., 1999). However, before employment of ChE as a biomarker of anticholinestera sic compounds in monitoring programms, is important to analyze the sensitivity to this kind of molecules (Varà ² et al., 2003), also for the fact that potential effects of organophosphorus and carbamate pesticides are widely variable for different fish species (Ferrari et al., 2004; Silva Filho et al., 2004). The dynamics of the interaction of ChE with organophosphate and carbamate compounds has been shown to depend largely upon the affinity of a particular insecticide for the enzyme, commonly represented as the enzyme affinity for a particular insecticide, which is commonly represented as the affinity constant Ka (Wang Murphy, 1982). Silva Filho, et al. (2004) showed extremely great differences in the inhibition kinetic parameters between several fish species, an important point to be considered in the selection of a sentinel organism in biomonitoring programs. In this context, the concentration of eserine that inhibits 50% of cholinesterase activity (IC50) and inhibition kinetic parameters are important characteristics for the selection of sensitive ChEs to be employed as biomarkers. Considering the facts previously described, the objectives of the present study were to determine the kinetic parameters and eserine sensitivity of brain ChE from two estuarine fish species, Micropogonias furnieri (Teleostei: Scianidae) and Cathorops spixii (Teleostei: Ariidae) collected in polluted and non-polluted sites in Southern Brazil. The white mouth croaker Micropogonias furnieri (Desmarest, 1823), is a subtropical fish found in muddy and sandy bottoms in coastal waters. Its feeding habit varies along the ontogenic development and season: juveniles feed on benthic migratory crustaceans and sessile mollusks, while adults are benthic feeders, occasionally preying on fish (Isaac, 1998). Sea catfish Cathorops spixii (Spix and Agassiz, 1829) is a demersal tropical cat fish found in shallow coastal marine waters and brackish estuaries, lagoons and river mouths, as well as in hypersaline waters. In South America, its distribution includes Atlantic and Caribbean rivers and estuaries from Colombia to Brazil. Adults feed mainly on invertebrates and small fishes, while juveniles feed on amphipods, isopods and copepods (Cervigà ³n et al., 1992). This study is part of a research project developed along the Brazilian coast, the RECOS (â€Å"Uso e Apropriaà §o de Recursos Costeiros†) project in the scope of the Millenium Institute (Brazilian Ministry of Science and Technology). One of the objectives of RECOS project is the standardization of sampling protocols, quantitative and qualitative evaluations of biochemical, physiological and histological biomarkers in different animal species collected from polluted and non-polluted sites. In the present study biochemical biomarker responses were analyzed in fish collected in different seasons (winter and summer), to evaluate the natural variability of ChE activity and its sensitivity to eserine inhibition. 2. Materials and methods 2.1. Chemicals Acetylthiocholine iodide, eserine (physostigmine), 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB) were obtained from Sigma (St. Louis, MO). The protein content was determined using a commercial kit from Doles Reagentes (Belo Horizonte, Brazil), based on Biuret method. 2.2. Organisms Micropogonias furnieri was collected in summer and winter seasons in reference (unpolluted) site, â€Å"Ilha dos Marinheiros† (32 °02’005† S and 52 °12’151† W) and in a polluted site, â€Å"Saco da Mangueira† (32 °04’369† S and 52 °06’473† W). Cathorops spixii was collected only in summer in a reference site, â€Å"Baà ­a das Laranjeiras† (25 °31’271† S, 48 °29’690† W) and in a polluted one, â€Å"Baà ­a de Parangua (25 °21’050† S, 48 °25’97† W) (Figure 1). In every case, ten fish were collected in each season and site. Immediately after collection, fish were anesthetized with benzocaine (200 ppm), measured (total length and weight) and head isolated and stored at -20 oC until arrival at the laboratory, where they were kept at -80 oC before biochemical determinations. It should be mention that up to date no chemical characterization was perfo rmed in the locals referred as polluted and unpolluted. However, the local â€Å"Ilha dos Marinheiros† is far from any obvious pollution source, whereas â€Å"Saco da Mangueira† is located near to fertilizer industries. ELTON/ADALTO/VANESSA: Uma frase equivalente para os locais de amostragem no Paranagu seria importante a meu ver. 2.3. Enzyme extraction Fish whole brain was dissected and then homogenized (1:20) in cold phosphate buffer (0.05 M) containing 20% glycerol at pH 7.40. The homogenate was then centrifuged at 850 xg (4 °C) for 15 min. The supernatant was again centrifuged at 12,800 xg (4 °C) during 15 min. The supernatant of this last centrifugation was used as enzyme source. 2.4. Enzyme assay Cholinesterase activity was determined using the method described by Ellman et al., (1961). Phosphate buffer (0.05 M, pH 7.40) was placed at least for 15 min in a water bath at 25 °C. Aliquots of homogenate, DTNB and substrate (acetylthiocholine iodide- ATch) were then added and the absorbance (412 nm) was immediately determined, during 90 s, in an ELISA reader (Victor 2, Perkin Elmer). To determine substrate affinity (Km) and maximum cholinesterase activity (Vmax), different ATch concentrations ranging from 0.025 to 9 mM were assayed, being the cholinesterase activity expressed as nmol/min/mg proteins. In each experiment, a first blank without substrate was assayed to evaluate the reaction of protein thiol groups with DTNB, and a second blank without sample was used to estimate the rate of spontaneous substrate hydrolysis. 2.5. In vitro enzyme inhibition by eserine The sensitivity of brain ChE to inhibition by eserine was investigated. ChE activity was measured on extracts after 5 min of incubation at 25 °C with several eserine concentrations, ranging from 110-4 to 1 mM. Enzyme activity was measured as described above. Inhibition was expressed as a percentage of ChE activity after eserine exposure respect control enzymatic activity. Kinetic parameters of enzyme inhibition were also estimated employing the carbamate eserine. The inhibition of an enzyme (E) with an inhibitor (I) can be summarized as follow (Main, 1964): where (EI)R represents a reversible enzyme-inhibitor complex and (EI)I an irreversible one. The affinity equilibrium constant is defined as Ka= K-1/K1 and Kc represents the carbamylation constant (Hastings et al., 1970). The bimolecular inhibition constant, Ki is defined as Ki= Kc/Ka. The constants Ka and Ki can be estimated according to the following equation: 1/i = ?t/(2.303*?log10 ?)*Ki – 1/Ka, where i represents the inhibitor concentration and ?t/(2.303*?log10 ?) is the reciprocal of the pseudo-first-order rate of enzyme inhibition at a fixed concentration (i) of the inhibitor (Monserrat et al., 2002). Six concentrations ranging from 0.3 to 10 mM were tested, at least in duplicate and after four or five different times of incubation (range: 30-360 s). 2.6. Data analysis Enzyme kinetic parameters (Vmax and Km) were estimated by fitting experimental data to Michaelis-Menten equation. IC50 values were obtained through probit analysis (Monserrat Bianchini, 1998). Linear regression and ANCOVA was employed to estimate and compare inhibition kinetic parameters (Ki and Ka). Statistical analysis of enzyme activity was performed using ANOVA followed by a posteriori comparisons using the Newman-Keuls test. A significance level of 5% was employed in all cases. 3. Results Fish from of both species were homogeneous (P>0.05) in length and weight at the different sampling sites and seasons analyzed, and for this reason only the general mean is reported. For M. furnieri, the mean weight and total length of fish collected were 25.78  ± 7.72 g and 14.41 ±1.83 cm, respectively (n= 40). For C. spixii, mean weight and total length of sampled fish were estimated in 35.77 ±11.45 g and 16.14g ±1.56 cm, respectively (n= 40). The Michaelis-Menten constants (Km and Vmax) for brain ChE of M. furnieri showed different patterns. Km values were statistically similar (P>0.05) in all seasons and sampling sites. On the other hand, Vmax showed a complex response, since fish collected in the reference site showed higher values (P0.05) in the Km values was observed in summer, the only season analyzed. However, higher (P