Brain cholinesterase activity of apparently normal wild birds

Organophosphorus and carbamate pesticides are potent anticholinesterase substances that have killed large numbers of wild birds of various species. Cause of death is diagnosed by demonstration of depressed brain cholinesterase (ChE) activity in combination with chemical detection of anticholinesterase residue in the affected specimen. ChE depression is determined by comparison of the affected specimen to normal ChE activity for a sample of control specimens of the same species, but timely procurement of controls is not always possible. Therefore, a reference file of normal whole brain ChE activity is provided for 48 species of wild birds from North America representing 11 orders and 23 families for use as emergency substitutes in diagnosis of anticholinesterase poisoning. The ChE values, based on 83 sets of wild control specimens from across the United States, are reproducible provided the described procedures are duplicated. Overall, whole brain ChE activity varied nearly three-fold among the 48 species represented, but it was usually similar for closely related species. However, some species were statistically separable in most families and some species of the same genus differed as much as 50%.     

Muscular and brain cholinesterase sensitivities to azinphos methyl and carbaryl in the juvenile rainbow trout Oncorhynchus mykiss

The organophosphate azinphos methyl (AzMe) and the carbamate carbaryl are the insecticides mostly used in the irrigated valley of Río Negro and Neuquén, Patagonia, Argentina. Juvenile rainbow trout were exposed to AzMe and carbaryl and the sensitivity of skeletal muscular cholinesterase (ChE) and the time course of inhibition and recovery were evaluated. EC50 values demonstrated that AzMe was a stronger in vivo inhibitor of muscular ChE (1.05+/-0.23 microg/L) than carbaryl (270+/-62.23 microg/L). Muscular ChE was significantly less sensitive to both insecticides than brain ChE. EC50 values obtained for muscular ChE were closer than those for brain ChE to the respective pesticide lethal concentrations, pointing out the relevance of the muscular enzyme in determining acute toxicity. The recovery process of ChE activity after carbaryl exposure (500 microg/L) was fast, whereas no significant recovery was observed with AzMe (1 microg/L) after 21 days in uncontaminated media. Brain and muscular ChE were inhibited and showed a significant but not complete recovery after three consecutive 48-h exposures to AzMe (1 microg/L) followed by a recovery period of 7 days. This scheme mimics the periodical application of the insecticides in the region and suggests a certain probability of a sustained ChE inhibition under field conditions, affecting fish development and survival.     

Plasma B-esterase activities in European raptors

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and Strigidae families were characterized by a BChE contribution that dominated the total ChE activity, while in the species of the Falconidae family, AChE activity dominated. With the exception of the barn owl, CbE activity (eserine-insensitive alpha-naphthyl acetate esterase [alpha-NAE] activity) in all species was almost absent or very low. The values obtained in this study for ChE, AChE, and BChE activities and the AChE:BChE ratios for buzzard, kestrel, barn owl, and tawny owl provide a good estimate of the normal values in free-living individuals of these European species. They can be used as a baseline to evaluate the effect of anticholinesterase insecticides in the field.     

Characterization of cholinesterases in plasma of three Portuguese native bird species: application to biomonitoring

Over the last decades the inhibition of plasma cholinesterase (ChE) activity has been widely used as a biomarker to diagnose organophosphate and carbamate exposure. Plasma ChE activity is a useful and non-invasive method to monitor bird exposure to anticholinesterase compounds; nonetheless several studies had shown that the ChE form(s) present in avian plasma may vary greatly among species. In order to support further biomonitoring studies and provide reference data for wildlife risk-assessment, plasma cholinesterase of the northern gannet (Morus bassanus), the white stork (Ciconia ciconia) and the grey heron (Ardea cinerea) were characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51, and iso-OMPA). Additionally, the range of ChE activity that may be considered as basal levels for non-exposed individuals was determined. The results suggest that in the plasma of the three species studied the main cholinesterase form present is butyrylcholinesterase (BChE). Plasma BChE activity in non-exposed individuals was 0.48±0.11 SD U/ml, 0.39±0.12 SD U/ml, 0.15±0.04 SD U/ml in the northern gannet, white stork and grey heron, respectively. These results are crucial for the further use of plasma BChE activity in these bird species as a contamination bioindicator of anti-cholinesterase agents in both wetland and marine environments. Our findings also underscore the importance of plasma ChE characterization before its use as a biomarker in biomonitoring studies with birds.     

Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

Benzodiazepine-refractory status epilepticus,neuroinflammation, and interneuron neurodegeneration after acute organophosphate intoxication

Nerve agents and some pesticides such as diisopropylfluorophosphate (DFP) cause neurotoxic manifestations that include seizures and status epilepticus (SE), which are potentially lethal and carry long-term neurological morbidity. Current antidotes for organophosphate (OP) intoxication include atropine, 2-PAM and diazepam (a benzodiazepine for treating seizures and SE). There is some evidence for partial or complete loss of diazepam anticonvulsant efficacy when given 30?min or later after exposure to an OP; this condition is known as refractory SE. Effective therapies for OP-induced SE are lacking and it is unclear why current therapies do not work. In this study, we investigated the time-dependent efficacy of diazepam in the nerve agent surrogate DFP model of OP intoxication on seizure suppression and neuroprotection in rats, following an early and late therapy. Diazepam (5?mg/kg, IM) controlled seizures when given 10?min after DFP exposure (“early”), but it was completely ineffective at 60 or 120?min (“late”) after DFP. DFP-induced neuronal injury, neuroinflammation, and neurodegeneration of principal cells and GABAergic interneurons were significantly reduced by early but not late therapy. These findings demonstrate that diazepam failed to control seizures, SE and neuronal injury when given 60?min or later after DFP exposure, confirming the benzodiazepine-refractory SE and brain damage after OP intoxication. In addition, this study indicates that degeneration of inhibitory interneurons and inflammatory glial activation are potential mechanisms underlying these morbid outcomes of OP intoxication. Therefore, novel anticonvulsant and neuroprotectant antidotes, superior to benzodiazepines, are desperately needed for controlling nerve agent-induced SE and brain injury.     

Tissue-specific inhibition and recovery of esterase activities in Lumbricus terrestris experimentally exposed to chlorpyrifos

Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35 d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil.     

Using dried blood spots stored on filter paper to measure cholinesterase activity in wild avian species.

Birds of prey that are poisoned by cholinesterase inhibitors (e.g. organophosphate and carbamate insecticides) are often cared for at animal shelters, rehabilitation centres and wildlife diagnostic facilities. Plasma cholinesterase (ChE) activity is a recognized method of assessing exposure to these insecticides, but standard blood-handling protocols are difficult to follow in non-laboratory settings. The primary objective of this study was to expand upon a method for storing human blood on filter paper without the need for complicated equipment or refrigeration, and to test its utility for measurement of ChE activity in avian blood. ChE activity from whole blood, plasma, and dried blood spots was analysed from 169 wild birds and comparisons made among sample types. ChE activity measured in whole blood haemolysates and dried blood spots were significantly correlated (r = 0.74, p < 0.001), as was ChE activity measured in plasma and dried blood spots (r = 0.68, p < 0.001). This study demonstrated that monitoring pesticide exposure in birds could be conducted using elementary blood sampling, preserving and shipping techniques.     

Cholinesterase activities and sensitivity to pesticides in different tissues of silver European eel, Anguilla anguilla

Cholinesterase (ChE) activities were characterized in silver European eel, Anguilla anguilla, grown in the brackish lagoon of Comacchio (Italy). All specimens were harvested at the “lavoriero”, a traditional eel trapping weir that captures eels while leaving internal waters at the onset of reproductive migration. To our knowledge, no investigation on ChE was reported in silver eels. Therefore a first characterization of enzyme activity in muscle, brain, liver and plasma of silver eel was carried out, in the presence of different substrates, selective inhibitors, and four pesticides representative of the carbamate and organophosphate classes. Brain and white skeletal muscle showed similar ChE activities, 5- and 10-fold higher than those detected in liver and plasma, respectively. Km values of 0.31 and 0.30 mM, and Vmax values of 40.28 and 35.47 nmol min^–1 mg protein^–1 were obtained in brain and muscle ChE, respectively. Acetycholinesterase was the predominant ChE form in all tissues, as concluded by comparing the effects of BW 284c51, iso-OMPA and eserine. ChE activities in brain and muscle were significantly inhibited by in vitro treatment with pesticides, with the following order of potency: carbofuran > carbaryl > chlorpyrifos ≥ diazinon.     

MicroRNAs and extracellular vesicles in cholangiopathies

Cholangiopathies encompass a heterogeneous group of disorders affecting biliary epithelial cells (i.e. cholangiocytes). Early diagnosis, prognosis and treatment still remain clinically challenging for most of these diseases and are critical for adequate patient care. In the past decade, extensive research has emphasized microRNAs (miRs) as potential non-invasive biomarkers and tools to accurately identify, predict and treat cholangiopathies. MiRs can be released extracellularly conjugated with lipoproteins or encapsulated in extracellular vesicles (EVs). Research on EVs is also gaining attention since they are present in multiple biological fluids and may represent a relevant source of novel non-invasive biomarkers and be vehicles for new therapeutic approaches. This review highlights the most promising candidate miRs and EV-related biomarkers in cholangiopathies, as well as their relevant roles in biliary pathophysiology. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

A tryptophan in the bottleneck of the catalytic gorge of an invertebrate acetylcholinesterase confers relative resistance to carbamate and organophosphate inhibitors

Amphioxus, an invertebrate chordate, has two acetylcholinesterases (AChEs): cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2). ChE1 is up to 329-fold more resistant to a variety of carbamate and organophosphate inhibitors, including a number of insecticides, when compared with ChE2. One difference between the two enzymes is at the position homologous to Phe331 in Torpedo AChE. In Torpedo AChE, this residue is a component of the hydrophobic subsite and defines one side of the bottleneck in the catalytic gorge of the enzyme. In ChE1, the homologous residue is Trp353; in ChE2, it is Phe353. We used site-directed mutagenesis to investigate the proposal that the resistance of ChE1 to inhibition by carbamates and organophosphates was due to this difference, creating a ChE1 W353F mutant to widen the bottleneck. The mutation virtually abolishes the difference in sensitivity to the inhibitors. The ChE1 W353F mutant is only 2- to 3-fold more resistant than ChE2 to carbamates and is actually 2.5- to 10-fold more sensitive to inhibition by organophosphates. The differences in resistance are due to different affinities of the enzymes for the inhibitors, not different reactivities. Molecular modeling supports the proposal that the difference in inhibition is due to the width of the bottleneck of the gorge. Our results have implications for insecticide resistance in insects, in particular mosquitoes and aphids.     

Using dried blood spots stored on filter paper to measure cholinesterase activity in wild avian species

Birds of prey that are poisoned by cholinesterase inhibitors (e.g. organophosphate and carbamate insecticides) are often cared for at animal shelters, rehabilitation centres and wildlife diagnostic facilities. Plasma cholinesterase (ChE) activity is a recognized method of assessing exposure to these insecticides, but standard blood-handling protocols are difficult to follow in non-laboratory settings. The primary objective of this study was to expand upon a method for storing human blood on filter paper without the need for complicated equipment or refrigeration, and to test its utility for measurement of ChE activity in avian blood. ChE activity from whole blood, plasma, and dried blood spots was analysed from 169 wild birds and comparisons made among sample types. ChE activity measured in whole blood haemolysates and dried blood spots were significantly correlated (r=0.74, p<0.001), as was ChE activity measured in plasma and dried blood spots (r=0.68, p<0.001). This study demonstrated that monitoring pesticide exposure in birds could be conducted using elementary blood sampling, preserving and shipping techniques.     

Oxidative stress and cholinesterase inhibition in saliva and plasma of rats following subchronic exposure to malathion

The aim of this study was to examine whether malathion, a commonly used organophosphate (OP), might induce oxidative stress and cholinesterase (ChE) depression in saliva and plasma in rats following subchronic exposure mimicking human exposure. Malathion was administered orally at doses of 100, 500 and 1500 ppm for 4 weeks. Oxidative stress was determined by measuring the malondialdehyde concentration, the end product of lipid peroxidation, and assessing total antioxidant power. Four weeks oral administration of malathion at doses of 100 ppm, 500 ppm and 1500 ppm depressed plasma ChE activity to 45% (P<0.01), 48% (P<0.01) and 41% (P<0.01) of control, respectively. Malathion at doses of 100 ppm, 500 ppm and 1500 ppm depressed saliva ChE activity to 73% (P<0.01), 75% (P<0.01) and 78% (P<0.01) of control, respectively. Malathion at doses of 100 ppm, 500 ppm and 1500 ppm increased plasma antioxidant power by 33% (P<0.01), 59% (P<0.01) and 118% (P<0.01) of control, respectively. Malathion did not change saliva antioxidant power. Malathion at doses of 100 ppm, 500 ppm and 1500 ppm increased plasma thiobarbituric acid reactive substances (TBARS) by 61% (P<0.01), 69% (P<0.01) and 63% (P<0.01) of control, respectively. Malathion at doses of 500 ppm and 1500 ppm increased saliva TBARS by 19% (P<0.01) and 22% (P<0.01) of control, respectively. Malathion (100 ppm) did not change saliva TBARS level. We concluded that in OP subchronic exposure, depression of ChE is accompanied by induction of oxidative stress that might be beneficial in monitoring OP toxicity.     

Different sensitivity of Ca(2+)-ATPase and cholinesterase to pure and commercial pesticides in nervous ganglia of Phyllocaulis soleiformis (Mollusca)

We measured the effects in vitro of pure and commercial pesticides on Ca(2+)-activated ATPase and cholinesterase (ChE) activities in the nervous system of the slug Phyllocaulis soleiformis. The pesticides used in this study included carbamate and organophosphates, which acts as reversible and irreversible anticholinesterases, respectively. Both enzymes were insensitive to pure carbofuran (1 mM), glyphosate (1 mM) and malathion (120 microM). However, the carbamate carbofuran, in the commercial formulation Furandan 350S, inhibited ATPase and ChE activities. The organophosphate glyphosate used in the commercial preparation of Gliz 480CS inhibited ATPase activity and increased cholinesterase activity. These effects are likely due to the action of adjuvant substances of the chemical formulation. The commercial formulation (Malatol 500CE) did not alter enzymes activities. Our results suggest that cholinesterase present in the slug nervous tissue has a different behavior to those identified in vertebrate nervous tissue, since it was insensitive to pure compounds, known as anticholinesterases in vertebrates. Considering the insensitivity of the Ca(2+)-activated ATPase, we suggested that the purinergic neurotransmission and other roles of ATP might not be affected by the pure pesticides tested.     

Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.     

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.     

小峰熊蜂头部乙酰胆碱酯酶测定条件的优化及其对六种常用杀虫剂的敏感性

小峰熊蜂Bombus hypocrita是我国优势熊蜂种群之一, 因其易于饲养、 群势较强且授粉性能优良而成为我国设施农业常用优良授粉蜂种, 但常受到以乙酰胆碱酯酶(AChE)为靶标酶的有机磷和氨基甲酸酯类杀虫剂的危害。 为合理规避这两类杀虫剂对熊蜂的危害, 同时也为完善熊蜂授粉配套技术和保护野生熊蜂资源提供理论基础, 本研究利用正交试验对小峰熊蜂头部乙酰胆碱酯酶活性的测定条件进行了优化, 并明确了6种常用有机磷和氨基甲酸酯类杀虫剂对乙酰胆碱酯酶活性的影响。结果表明： 各测定因素对小峰熊蜂乙酰胆碱酯酶活性测定影响的大小顺序依次为： 酶浓度＞pH＞温度＞底物浓度＞反应时间; 小峰熊蜂头部乙酰胆碱酯酶活性的最适反应条件为： 酶浓度0.25 g 蛋白质/L, 底物浓度0.8 mmol/L, pH值7.5, 温度40℃, 反应时间5 min。毒死蜱、 三唑磷、 丙溴磷、 异丙威、 仲丁威和残杀威6种杀虫剂对小峰熊蜂头部乙酰胆碱酯酶离体抑制作用均呈现明显的剂量-效应关系, 其抑制中浓度IC₅₀分别为0.39, 1.79, 0.42, 0.04, 0.43和0.63 mmol/L。这6种杀虫剂对小峰熊蜂AChE抑制作用的强弱依次为： 异丙威＞毒死蜱＞三唑磷＞仲丁威＞残杀威＞丙溴磷, 即小峰熊蜂对异丙威最敏感, 而对丙溴磷的敏感性最弱。     

Brain cholinesterase activity of nestling great egrets, snowy egrets and black-crowned night-herons

inhibition of brain cholinesterase (ChE) activity in birds is often used to diagnose exposure or death from organophosphorus or carbamate pesticides. Brain ChE activity in the young of altricial species increases with age; however, this relationship has only been demonstrated in the European starling (Sturnus vulgaris). Brain ChE activity of nestling great egrets (Casmerodius albus) collected from a colony in Texas (USA) increased significantly with age and did not differ among individuals from different nests. Brain ChE activity of nestling snowy egrets (Egretta thula) and black-crowned night-herons (Nycticorax nycticorax) collected in one colony each from Rhode Island, Texas and California (USA) also increased significantly with age and did not differ among individuals from different nests or colonies. This study further demonstrates that age must be considered when evaluating exposure of nestling altricial birds to ChE inhibitors.     

Interactions of organophosphates with keratins in the cornified epithelium of human skin

Methods to unequivocally assess and quantify exposure to organophosphate anti-cholinesterase agents are highly valuable, either from a biomonitoring or a forensic perspective. Since for both OP pesticides and various nerve agents the skin is a predominant route of entry, we hypothesized that proteins in the skin might represent an ideal source of unequivocal and persistent biomarkers for exposure to these compounds. In this exploratory study we show that keratin proteins in human skin are relevant binding sites for organophosphates. The thick cornified epithelium of human plantar skin (callus) was exposed to a selection of relevant organophosphorus compounds and keratin proteins were subsequently extracted. After carboxymethylation of cysteine residues, enzymatic digestion of the keratins with pronase and trypsin was performed and the resulting amino acid and peptides were analyzed to assess whether covalent adducts had formed. LC-tandem MS analysis of the pronase digests demonstrated that tyrosine and to a lesser extent serine residues were selectively modified by organophosphate pesticides (both phosphorothioates and the corresponding oxon forms) under physiological conditions. In addition, modification of tyrosine with the nerve agent VX was unequivocally assessed. In order to elucidate specific binding sites, LC-tandem MS analysis of trypsin digests showed two separate tryptic keratin fragments, i.e. LASY*LDK and SLY*GLGGSK, with Y* the modified tyrosine residues, originating from keratin 1/6 and keratin 10, respectively. These preliminary findings, revealing novel binding targets for anti-cholinesterase organophosphates, will form a firm basis for the development of novel (non-invasive) methods for assessment of exposure to organophosphates. Whether this binding will also have biological implications remains an issue for further investigations.