Use of Cholinesterase Activity in Environmental Monitoring – Research Paper

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 (Bocquené 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 (Bocquené et al., 1990) and continental waters (Sturm et al., 1999). However, before employment of ChE as a biomarker of anticholinesterasic compounds in monitoring programms, is important to analyze the sensitivity to this kind of molecules (Varò 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 (Cervigón et al., 1992).

This study is part of a research project developed along the Brazilian coast, the RECOS (“Uso e Apropriação 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, “Ilha dos Marinheiros” (32°02’005” S and 52°12’151” W) and in a polluted site, “Saco da Mangueira” (32°04’369” S and 52°06’473” W). Cathorops spixii was collected only in summer in a reference site, “Baía das Laranjeiras” (25°31’271” S, 48°29’690” W) and in a polluted one, “Baía de Paranágua (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 performed in the locals referred as polluted and unpolluted. However, the local “Ilha dos Marinheiros” is far from any obvious pollution source, whereas “Saco da Mangueira” is located near to fertilizer industries. ELTON/ADALTO/VANESSA: Uma frase equivalente para os locais de amostragem no Paranaguá 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 1×10-4 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 (P<0.05) in summer and the opposite was observed in winter (Table 1). Regarding C. spixii, no significant difference (P>0.05) in the Km values was observed in summer, the only season analyzed. However, higher (P<0.05) Vmax values was registered in fish collected at the polluted site (Table 1). ChE from M. furnieri showed an extremely lower sensitivity to eserine when compare that observed for the ChE from C. spixii. The IC50 value (4,472 ?M) in the M. furnieri was significantly (P< 0.05) higher than that inC. spixii (0.077 ?M). Taking into account the extremely low reactivity of ChE of M. furnieri to eserine, only ChE from C. spixii was assayed for determination of inhibition kinetic parameters. Results shows that the affinity constants (Ka) were significantly different (P<0.05) between fish collected at the reference and polluted site (Ka =17.18 and 3.27 mM, respectively). Also differences in carbamylation constants were observed between ChE from fish collected in reference (Kc= 7.5 min-1) and polluted site (Kc= 1.24 min-1). No significant difference (P>0.05) was observed between Ki values: 0.44 and 0.38 mM-1.min-1 for fish collected at the reference and the polluted site, respectively (Figure 3).

4. Discussion
Results obtained in the present study showed lower Vmax values of ChE activity in Micropogonias furnieri collected in summer at in the polluted site and higher Vmax values in fish collected in winter at the polluted site. A similar result was observed for Cathorops spixii collected in the summer and authors like Flammarion et al. (2002) observed that differences in ChE activity in the fish Leuciscus cephalus collected in several areas were correlated to fish length and other natural factors. In fact, a negative correlation between ChE activity and fish length has been documented, including a decrease in brain ChE activity during ontogeny (Chuiko et al., 1997). However, no difference in length of fish collected at the different sampling sites and seasons was observed in the present study, indicating that the influence of this factor can be ruled out.

The increased Vmax values registered in fish collected at the polluted sites could be related to higher ChE concentrations in homogenates. Previous studies reported several potential adaptations to ChE inhibition, including increase synthesis of ChE (Kaufer et al., 1999), a response triggered by several factors, including pollutants, lowering of dissolved oxygen content in water, temperature or salinity variations. Between the abiotic factors, water temperature has shown to exert a significant effect on ChE activity (Bocquené et al., 1990; Chuiko et al., 1997). However, this was not the case in the present study, at least for M. furnieri, since water temperature varied on a seasonal basis (winter: 11.3?1.9°C, summer: 24.4?0.97°C), but not between sampling sites. Other factors like pollutants and stress can alter ChE activity, as showed in the study of Kaufer et al. (1999), where an 8-fold increase in AChE mRNA levels under exposure to anticholinesterasic compounds and a 2-fold increase by psychological stress was reported in brain mouse. In fish species, Pavlov et al. (1994) verified an augmented ChE activity after an increase adrenaline level in fish brain. Another possibility that can not be ruled out is the presence of anticholinesterasic pollutants in the sampling sites a priori considered as non-polluted. The RECOS project aimed to make a prospecting study along the Brazilian coast and results like the obtained in the present study might be used to re-evaluate the sampling site classification.

In fish species, it has been shown that the recovery period of ChE after inhibition with carbamate pesticides is shorter than with organophosphorus. Ferrari et al. (2004) verified in goldfish exposed to organophosphorus pesticides that the recovery of enzyme activity is substantial only after 35 days of transfer to clean water. However, enzyme activity recovery after inhibition with carbamate pesticides was much quicker (after 96 h). Whether occasional discharges of anticholinesterasic pesticides occurred in the reference sites during the sampling period is a fact that remains to be studied.

For both species analyzed in present study Km values were higher than those reported other aquatic species, including mollusks and crustaceans, although De La Torre et al. (2002) showed similar kinetic parameters in Cyprinus carpio and Cnesterodon decemmaculatus. However, it is remarkable the fact that both species studied here showed lower Vmax values than other fish species (Table 2). Previous studies showed that ChE specific activities, kinetic parameters, and sensitivity to anticolinesterasic compounds varied among species (Li & Fan, 1996; Chuiko, 2000). Furthermore, differences among individuals of the same species were also observed (Chuiko et al., 1997). Differences of 15-folds in brain ChE activity was reported by Chuiko et al. (2003) between different fish species, being observed that fish from Cyprinidae family posses higher enzyme activity. Although a clear explanation to these differences are still lacking, it seems that species with higher motor activities and number of nerves cells in brain tissue posses higher ChE activity (Lindeman, 1945, Leibson, 1963 APUD Chuiko, 2003).

Results from the present study do not indicate conspicuous differences in the kinetic (Km and Vmax) parameters of the two species, but a markedly difference in ChE sensitivity to eserine was observed. Table 3 summarizes the IC50 values for different aquatic species. It can be observed that some fish species are particularly sensitive to eserine (Odontesthes bonariensis and Odontesthes argentinensis, for example), whereas others including mollusks and some crustaceans, like Chasmagnathus granulata, show high resistance to eserine inhibition (Table 4). However, it can be observed that ChE from M. furnieri showed the lowest sensitivity, whereas ChE from C. spixii showed similar values to other moderately sensitive to eserine species (IC50: 0.077 ?M). Preliminary data from another studies performed in our laboratory with Ariidae species Hexanemathics hezbergii showed a similar IC50 value for eserine (0.075 ?M) in brain ChE, while Lutjanus synagris (Teleostei, Lutjanidae) showed intermediary values (0.294 ?M) between Ariidae species and M. furnieri. In fish, other authors showed low in vitro ChE sensitivity with carbamates and organophosphorus pesticides. Silva Filho et al. (2004) found different levels of ChE inhibition in neotropical fishes, using methyl para-oxon, registering IC50 values ranging from 3.34 ?M in Paralonchurus brasiliensis to 0.123 ?M in Prochilodus lineatus. These authors registered a negative relationship between Km values and IC50, an unexpected result if we consider that carbamate and organophosphates pesticides are substrate analogues that inhibit cholinesterase. In this way, it can be expected that ChE with lower substrate affinity should present lower sensitivity to anticholinesterasic agents. In fact, some authors like Monserrat & Bianchini (2001) observed a positive correlation between Km and IC50 values for several aquatic species. However, as previously mentioned, Silva Filho et al. (2004) found inverse results in neotropical fishes, fish brain ChE more sensitive to methyl-paraoxon (lower IC50), showed higher Km values.

The estimated inhibition constants were quite different in C. spixii collected at the reference and the polluted site (Fig. 3). The inhibition kinetic parameters indicate that the generation of the reversible enzyme-inhibitor complex is more easily formed in ChE from fish collected at the polluted site (lower Ka value). On the other hand, the generation of the irreversible enzyme-inhibitor complex is more likely to occur in ChE from fish collected at the reference site (higher Kc value). Although general sensitivity to eserine, as measured by Ki, seems to be similar in fish collected at the reference and polluted sites, the generation rate of the reversible and irreversible complex are not the same, indicating that the different environments are differentially influencing in some way the kinetic characteristics of C. spixii brain ChE.

Results from in vitro inhibition assays with eserine showed a low sensitivity of brain ChE from M. furnieri. However, fish collected in summer at the polluted site showed lower enzyme activity than those from the reference site, suggesting the existence of biochemical and physiological changes acting under a seasonal basis. The higher ChE activity registered in fish collected at the polluted site suggests other factors that influence cholinesterase activity, such as stress responses mediated by adrenaline, as previously reported by Pavlov et al. (1994). Overall, results obtained in the present study indicate that kinetic are necessary before studies using cholinesterase activity as a biomarker of aquatic pollution. Therefore, ChE activity measurements in fish collected at reference and polluted regions should be analyzed in a broader context, taking into account not only the enzymatic activity but also other physiological parameters affecting it or subtle differences, such as responses in terms of inhibition kinetics to standard anticholinesterasic compounds.

Acknowledgements. This research project was supported by “Projeto RECOS- Instituto do Milênio” (Brazilian Ministry of Science and Technology – V. Tortelli was a graduate recipient of a CNPq fellowship. A. Bianchini and J.M. Monserrat are research fellows from Brazilian CNPq.


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