Lúcia Guilhermino

Inhibition of acetylcholinesterase activity as effect criterion in acute tests with juvenile Daphnia Magna

In this work we investigated the possibility of using the enzyme acetylcholinesterase (AChE) activity in Daphnia magna homogenates, both in vivo and in vitro conditions, as a specific method for rapid toxicity evaluations. The results from in vivo and in vitro AChE inhibition tests were compared with 48 hours EC50 values obtained in conventional acute bioassays. EC50 values from in vivo AChE inhibition tests were: 2.4 micrograms/l for parathion, 0.2 microgram/l for paraoxon; DCA and cadmium at the concentrations tested had no effects on enzyme activity. I50 values were 764 micrograms/l for parathion, 0.08 micrograms/l for paraoxon and 3367 micrograms/l for cadmium; DCA did not affect AChE activity measured in in vitro conditions. EC50 values from conventional acute tests were: 2.2 micrograms/l for parathion, 0.2 microgram/l for paraoxon, 163 micrograms/l for DCA and 9.5 micrograms/l for cadmium. Our results indicated that the in vivo AChE inhibition test is selective, being very sensitive to detect toxicity of the organophosphates tested. The in vitro AChE inhibition assay is less time consuming, requires less human effort and produces less toxic waste than conventional acute bioassays and the in vivo AChE inhibition test. However, it does not take into account the effect of the metabolization of the toxicants inside live organisms; since the organophosphate metabolism may be activative or degradative, the toxic potential of the parent compound may be under or over evaluated in in vitro conditions.

Should the use of inhibition of cholinesterases as a specific biomarker for organophosphate and carbamate pesticides be questioned

Recently there has been evidence that contaminants other than organophosphate and carbamate pesticides may inhibit the activity of the enzyme acetylcholinesterase (AChE) both under in vitro and in vivo conditions. In this study we investigated the in vitro effect of three detergents \[dodecyl benzyl sulphonate (DBS), sodium dodecyl sulphate (SDS) and a mixture commonly used as domestic detergent (X)] and three metals \[molybdenum, barium and chromium (VI)] on AChE activity of Mytilus galloprovincialis haemolymph. All the detergents tested significantly inhibited the activity of the enzyme. The lowest observed effect concentrations were equal to 12 5 for DBS and 50 mg l-1 for SDS and X. Among the metals, molybdenum and barium had no effect on AChE activity, whereas chromium (VI) significantly depressed the activity of the enzyme at concentrations equal to or higher than 25 mg l-1. These results suggest that the use of AChE as a specific biomarker for organophosphate and carbamate pesticides should be quest...Recently there has been evidence that contaminants other than organophosphate and carbamate pesticides may inhibit the activity of the enzyme acetylcholinesterase (AChE) both under in vitro and in vivo conditions. In this study we investigated the in vitro effect of three detergents \[dodecyl benzyl sulphonate (DBS), sodium dodecyl sulphate (SDS) and a mixture commonly used as domestic detergent (X)] and three metals \[molybdenum, barium and chromium (VI)] on AChE activity of Mytilus galloprovincialis haemolymph. All the detergents tested significantly inhibited the activity of the enzyme. The lowest observed effect concentrations were equal to 12 5 for DBS and 50 mg l-1 for SDS and X. Among the metals, molybdenum and barium had no effect on AChE activity, whereas chromium (VI) significantly depressed the activity of the enzyme at concentrations equal to or higher than 25 mg l-1. These results suggest that the use of AChE as a specific biomarker for organophosphate and carbamate pesticides should be questioned and that the use of this enzyme as a biomarker could be extended.

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: Linking biomarkers to behaviour ☆

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC(50)s of copper and mercury (dissolved throughout metal concentrations) at 96h were 568 microg L(-1), and 62 microg L(-1), respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 microg L(-1) for copper and 3 microg L(-1) for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 microg L(-1), for copper and from 3 to 25 microg L(-1) for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.

Lactate dehydrogenase activity as an effect criterion in toxicity tests with Daphnia magna straus

Activity of lactate dehydrogenase (LDH) was used as an effect criterion in toxicity tests with Daphnia magna. In the first part of the work, the conditions for the use of LDH activity in toxicity tests with juveniles and adults of D. magna, were optimized. The influence of parameters such as the number of animals per sample, nutritional status, age and the presence of eggs in the brood chamber were investigated. In the second part of the study, both in vivo and in vitro tests based on the alteration of LDH activity of D. magna were developed and tested using zinc chloride as test substance. The results obtained indicate that LDH activity of D. magna may be used as an indicative parameter in aquatic toxicity tests.

Do metals inhibit acetylcholinesterase (AChE)?: Implementation of assay conditions for the use of AChE activity as a biomarker of metal toxicity

Abstract The enzymatic activity of acetylcholinesterase (AChE) has been shown to be altered by environmental contaminants such as metals. However, the available literature illustrates a background of contradictory results regarding these effects. Therefore, the main purpose of this study was to investigate the potential of five metal ions (nickel, copper, zinc, cadmium and mercury) to inhibit AChE activity in vitro. First, to accomplish this objective, the possible interference of metals as test toxicants in the Ellmans assay, which is widely used to assess AChE activity, was studied. The potential influence of two different reaction buffers (phosphate and Tris) was also determined. The results suggest that the selected metals react with the products of this photometric technique. It is impossible to ascertain the artefactual contribution of the interaction of the metals with the technique when measuring AChE inhibition. This constitutes a major obstacle in obtaining accurate data. The presence of phosphate ions also makes enzymatic inhibition difficult to analyse. Attending to this evidence, an assay using the substrate o-nitrophenyl acetate and Tris buffer was used to investigate the effects of metals on AChE activity. O-nitrophenyl acetate is also a substrate for esterases other than cholinesterases. It is therefore only possible to use it for the measurement of cholinesterase activity with purified enzymes or after a previous verification of the absence of other esterases in the sample tissue. Under these conditions, the results indicate that with the exception of nickel, all tested metals significantly inhibit AChE activity.

In vitro and in vivo inhibition of Daphnia magna acetylcholinesterase by surfactant agents: possible implications for contamination biomonitoring

This study was designed to investigate the effect of two surfactants, dodecyl benzyl sulfonate (DBS), sodium dodecyl sulfate (SDS), and of a domestic detergent (Y) on the AChE activity of the crustacean cladoceran Daphnia magna. All the chemicals significantly inhibit the activity of the enzyme, both in vitro and in vivo conditions. In vitro lowest observed effect concentration (LOEC) values ranged from 12.5 to 100 mg/l and correspondent IC50 (50% inhibition concentration) values ranged from 6.6 to 58.5 mg/l. In vivo LOEC values ranged from 2 to 11.9 mg/l, while EC50 (50% effect concentration) values ranged from 11.4 to 56.7 mg/l. AChE inhibition by environmental contaminants such as surfactants, detergents and metals may lead to false diagnostics and even wrong conclusions in biomonitoring studies based on the use of AChE as a specific biomarker for organophosphorous and carbamate compounds.

Acute effects of Benzo[a]pyrene, anthracene and a fuel oil on biomarkers of the common goby Pomatoschistus microps (Teleostei, Gobiidae).

The objective of this study was to investigate the effects of two different PAHs and a complex petrochemical mixture on the common goby, Pomatoschistus microps, using selected biomarkers as effect criteria. Benzo[a]pyrene (BaP) and anthracene were used as reference substances, while the water accommodated fraction of #4 fuel-oil (#4 WAF) was used as an example of a petrochemical mixture. P. microps was used since it is both a suitable bioindicator and a good test organism. Groups of fish were exposed to different concentrations of each of the test substances for 96 h and the activities of several enzymes commonly used as biomarkers were determined at the end of the bioassays. All the substances inhibited P. microps acetylcholinesterase (AChE) indicating that they have at least one mechanism of neurotoxicity in common: the disruption of cholinergic transmission by inhibition of AChE. An induction of lactate dehydrogenase (LDH) activity was found in fish exposed to BaP or to anthracene, suggesting an increase of the anaerobic pathway of energy production. On the contrary, inhibition of LDH was found in fish exposed to #4 WAF, suggesting a distinct effect of the mixture. An induction of P. microps glutathione S-transferase (GST) activity was found in fish exposed to BaP or to #4 WAF, while an inhibition was observed after exposure to anthracene. These results suggest that GST is involved in the detoxification of BaP and #4 WAF, but not of anthracene. All the substances increased catalase activity and isolated PAHs also increased superoxide dismutase, glutathione reductase and glutathione peroxidase activities, while #4 WAF did not cause significant alterations on these enzymes. These results suggest that all the substances may induce oxidative stress on P. microps, with BaP and anthracene apparently having more oxidative stress potential than #4 WAF.

Effect of dichlorvos on cholinesterase activity of the European sea bass (Dicentrarchus labrax)

In this study, the acute toxicity of the organophosphorous pesticide (OP) dichlorvos and both in vitro and in vivo effects of dichlorvos on cholinesterase (ChE) activity of the European sea bass (Dicentrarchus labrax) were investigated. The characterisation of ChE and the “normal” range of activity in brain and muscle of non-exposed fish were determined in a first phase of the study. Acetylthiocholine was the substrate preferred of both brain and muscle ChE. Eserine sulphate and BW284C51 significantly inhibited the brain and muscle enzyme activity at low concentrations (μM range). Iso-OMPA had a significant effect in muscle, but not in brain tissue. These results suggest that acetylcholinesterase (AChE) is the predominant ChE form in brain tissue. In contrast, both acetylcholinesterase and butyrylcholinesterase seem to exist in muscle. Using acetylthiocholine as substrate, the “normal” range of fingerling head and muscle ChE were 58.05±2.11 and 118.03±8.67 U/mg protein, respectively. Corresponding values for juveniles were 43.32±4.42 and 19.44±2.44 U/mg protein for brain and muscle, respectively. Dichlorvos significantly inhibited the activity of ChE in the selected tissues, both in vitro and in vivo conditions. Differences in ChE sensitivity were found in relation to the age of the fish and the tissue analysed. The present study also showed that fingerlings of the European sea bass are relatively resistant to in vivo acute (96 h) dichlorvos exposure to concentrations between 0.125 and 1 mg/L, being able to tolerate high percentages of head ChE inhibition (37% and 76%) without lethal effects.

Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica

In this study, the acute toxicity of the organophosphorous pesticides dichlorvos and chlorpyrifos to two different species of Artemia (A. salina and A. parthenogenetica) was evaluated. In addition, the in vivo effect of these two pesticides on cholinesterase (ChE) activity of both A. salina and A. parthenogenetica was also determined. The characterisation of the ChE, using different substrates and specific inhibitors, and the normal range of activity in non-exposed individuals were previously investigated for both species. The results obtained indicate that the ChE of A. salina is different from that of A. parthenogenetica and that both enzymes cannot be classified neither as acetylcholinesterase nor as butyrylcholinesterase since they show intermediary characteristics between the two vertebrate forms. The range of normal ChE activity was 2.65+/-0.15 U/mg protein for A. salina, and 3.69+/-0.17 U/mg protein for A. parthenogenetica. Significant in vivo effects of both pesticides on Artemia ChE activity were found, at concentrations between 5.38 and 9.30 mg/l for dichlorvos and between 1.85 and 3.19 mg/l for chlorpyrifos. Both Artemia species are resistant to these pesticides and they are able to survive with more than 80% ChE inhibition. However, A. parthenogenetica is more resistant than A. salina, with about a 95% reduction in its ChE activity respect to the control for nauplii exposed to the median lethal concentrations (LC50), without lethal effects after 24 h of exposure.

Mechanisms of cholinesterase inhibition by inorganic mercury

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X‐ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo‐first‐order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury‐induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.