RESUMEN
The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated that small peripheral site ligands like propidium can inhibit substrate hydrolysis simply by decreasing the substrate association and dissociation rate constants without altering the equilibrium constant for substrate binding to the acylation site. We now employ our nonequilibrium kinetic analysis to extend this model to include blockade of the dissociation of substrate hydrolysis products by bound peripheral site ligand. We also report here that acetylthiocholine can bind to the AChE peripheral site with an equilibrium dissociation constant K(S) of about 1 mM. This value was determined from the effect of the acetylthiocholine concentration on the rate at which fasciculin associates with the peripheral site. When substrate binding to the peripheral site is incorporated into our steric blockade model, hydrolysis rates at low substrate concentration appear to be accelerated while substrate inhibition of hydrolysis occurs at high substrate concentration. The model predicts that hydrolysis rates for substrates which equilibrate with the acylation site prior to the acylation step should not be inhibited by bound peripheral site ligand. Organophosphates equilibrate with AChE prior to phosphorylating the active site serine residue, and as predicted propidium had little effect on the phosphorylation rate constants for the fluorogenic organophosphate ethylmethyl-phosphonylcoumarin (EMPC). The 2nd-order phosphorylation rate constant kOP/K(OP) was decreased 3-fold by a high concentration of propidium and the 1st-order rate constant kOP increased somewhat. In contrast to propidium, when the neurotoxin fasciculin bound to the AChE peripheral site both a steric blockade and a conformational change in the acylation site appeared to occur. With saturating fasciculin, kOP/K(OP) decreased by a factor of more than 750 and kOP decreased 300-fold. These data suggest that new peripheral site ligands may be designed to have selective effects on AChE phosphorylation.
Asunto(s)
Acetilcolinesterasa/química , Inhibidores de la Colinesterasa/química , Modelos Químicos , Acetilcolina/metabolismo , Acetilcolinesterasa/sangre , Acetilcolinesterasa/metabolismo , Sitios de Unión , Inhibidores de la Colinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacocinética , Eritrocitos/enzimología , Humanos , Hidrólisis , Indicadores y Reactivos/metabolismo , Indicadores y Reactivos/farmacología , Cinética , Ligandos , Fosforilación , Propidio/metabolismo , Propidio/farmacología , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
Blood samples have been taken from chronic cannulae in the dorsal aorta of Carp under anaesthesia and at intervals of 24, 29 and 101 hours later. A few minutes after the 29 hours and 101 hours sampling was completed similar quantities (1 ml) were withdrawn from a caudal vessel. All samples were treated in the same way and determinations made of haematocrit value, total protein, glucose concentration and activity of the following plasma enzymes-acetylcholinesterase (AChE), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH). Comparisons between dorsal aorta and caudal samples at 29 hours and 101 hours showed no significant differences for any of the enzyme activities that were tested. However significant (paired "t"-test, P < 0.05) differences were observed for haematocrit and glucose concentration. It is suggested that the higher haematocrit and lower glucose of caudal samples are consistent with the expected differences between arterial and venous blood. Apart from these differences which reflect normal physiological function, values obtained for samples from the two sites were identical. It is concluded that blood sampling from caudal vessels is a satisfactory method to obtain blood for biomonitoring purposes at least for those enzymes commonly used in recent surveys.
Asunto(s)
Carpas/sangre , Acetilcolinesterasa/sangre , Alanina Transaminasa/sangre , Animales , Aorta , Aspartato Aminotransferasas/sangre , Glucemia/análisis , Proteínas Sanguíneas/análisis , Recolección de Muestras de Sangre/métodos , Cateterismo/métodos , Hematócrito , L-Lactato Deshidrogenasa/sangreRESUMEN
1. Carp with the dorsal aorta cannulated have been used to study the effects of a 5 hour exposure to methidathion at concentrations of 2 or 6 mg/litre. Blood samples taken during control, exposed and recovery periods were used to determine plasma acetylcholinesterase (AChE) activity, haematocrit value, mean cell volume, total plasma protein, and filtration time through Nucleopore filters containing pores of 8 microns. 2. A drastic inhibition of AChE activity was observed and this continued during the recovery for at least 5 days. Changes in other blood parameters were less marked and recovered soon after fish were returned to non-polluted water. The increased haematocrit and decrease in filtration time suggests some impairment of gas transfer during exposure but the depletion of AChE activity and associated muscular and neural disturbances are more serious results of pollution with this organophosphate insecticide.
Asunto(s)
Carpas/sangre , Insecticidas/toxicidad , Compuestos Organotiofosforados/toxicidad , Contaminantes Químicos del Agua/toxicidad , Acetilcolinesterasa/sangre , Animales , Proteínas Sanguíneas/análisis , Cateterismo , Deformación Eritrocítica/efectos de los fármacos , Índices de Eritrocitos , Hematócrito , Insecticidas/administración & dosificación , Dosificación Letal Mediana , Compuestos Organotiofosforados/administración & dosificación , Factores de Tiempo , Contaminantes Químicos del Agua/administración & dosificaciónRESUMEN
In the summer of 1995, 30 tonnes of eel (Anguilla anguilla) died in Lake Balaton, Hungary. An investigation was carried out to find the causes of this ecocatastrophe. During this investigation, certain biochemical parameters, i.e. the blood sugar level, the acetylcholinesterase (AChE, EC 3.1.1.7), lactate dehydrogenase (LDH, EC 1.1.2.3), glutamic-oxaloacetic transaminase (GOT, EC 2.6.1.1), and glutamic-pyruvic transaminase (GPT, EC 2.6.1.2) activities in the blood serum of the collected surviving and dying eels were examined. Deltamethrin, the active ingredient of the insecticide K-OTHRIN 1 ULV, used against mosquitoes was detected in different animal species, i.e. eel, bream (Abramis brama), pike perch (Stizostedion lucioperca), and the common gull (Larus canus) and in sediment samples from the lake. Additionally, laboratory experiments were carried out to study the effects of deltamethrin on eels. During the investigation in the field it appeared that the AChE activity was significantly lower in the blood serum of the dying eels as compared to that in living animals (P<0.05, Student's t-test). The blood glucose content exhibited a difference, too: it was 2.5 times higher in the dying eels than in the surviving ones. A huge increase in the LDH level was measured in the dying eels. The GOT activities of the serum were twice as high in the dying eels as in the living fish, while the GPT was not significantly changed. Deltamethrin was detected in different tissue samples of the dying eels: 2.70-18.1 microg/kg in the liver, 9.0-31.1 microg/kg in the gill and 3.0 microg/kg wet tissue in the muscle. Deltamethrin residues were found in tissue samples from other animals, in the following concentrations: 0.44 microg/kg in bream, 2.14 microg/kg in pike perch and 1.06 microg/kg wet tissue in dead gulls. The sediment samples collected from the sites of the devastation contained deltamethrin in a concentration of 5.50-30.00 microg/kg wet sediment at the time of the eel deaths, and in a concentration 7.00-8.75 microg/kg wet sediment a month later. Laboratory experiments with the insecticide K-OTHRIN 1 ULV revealed that 1.00 microg/l of its active ingredient, deltamethrin, caused the death of 50% of the eels after an exposure time of 96 h. During this experiments similar trends could be observed in changes of enzyme activities of the treated eels to those that were detected in filed study during the eel devastation in Lake Balaton. At the end of a one-week treatment with the insecticide at the concentration of 0.5 microg/l of its active ingredient the gills of the treated eels contained deltamethrin at 12.6-44.8 microg/kg wet tissue concentration, while at the 24th hour after the treatment (11.2-42.7 microg/kg wet tissue) deltamethrin concentration in the liver of treated eels could be detected. All the above-mentioned changes and the detected deltamethrin residue in the eels appear to demonstrate the contribution of deltamethrin to the severe eel devastation. This information on the ecological risk of pyrethroid insecticides might be useful in their further application.
Asunto(s)
Anguilas , Insecticidas/toxicidad , Piretrinas/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Glucemia/análisis , Enzimas/sangre , Femenino , Hungría , Masculino , Especificidad de la EspecieRESUMEN
1. The cholinesterases play an important role in the innervation of organs. The ratio of solubilized to membrane-bound cholinesterase and the quantitative distributions of acetylcholinesterase and butyrylcholinesterase were measured in different segments of the gut of carp (Cyprinus carpio) connected with different types of nerve-muscle synapses in different parts of the alimentary tract. 2. The inhibition of acetylcholinesterase (EC 3.1.1.7.) by the herbicide paraquat and the insecticide metidathion was measured in different parts of the gut of carp. 3. Metidathion and paraquat significantly decreased the activity of acetylcholinesterase in different segments of the alimentary tract of common carp, in a concentration-dependent manner.
Asunto(s)
Carpas/metabolismo , Inhibidores de la Colinesterasa/toxicidad , Sistema Digestivo/enzimología , Insecticidas/toxicidad , Animales , Colinesterasas/metabolismo , Sistema Digestivo/efectos de los fármacos , Herbicidas/toxicidad , Compuestos Organotiofosforados/farmacología , Paraquat/toxicidadRESUMEN
The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge with a catalytic triad characteristic of serine hydrolases, and a peripheral site at the mouth of the gorge some 10-20 A from the acylation site. Many ligands that bind exclusively to the peripheral site inhibit substrate hydrolysis at the acylation site, but the mechanistic interpretation of this inhibition has been unclear. Previous interpretations have been based on analyses of inhibition patterns obtained from steady-state kinetic models that assume equilibrium ligand binding. These analyses indicate that inhibitors bound to the peripheral site decrease acylation and deacylation rate constants and/or decrease substrate affinity at the acylation site by factors of up to 100. Conformational interactions have been proposed to account for such large inhibitory effects transmitted over the distance between the two sites, but site-specific mutagenesis has failed to reveal residues that mediate the proposed conformational linkage. Since examination of individual rate constants in the AChE catalytic pathway reveals that assumptions of equilibrium ligand binding cannot be justified, we introduce here an alternative nonequilibrium analysis of the steady-state inhibition patterns. This analysis incorporates a steric blockade hypothesis which assumes that the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Simulations based on this nonequilibrium steric blockade model were in good agreement with experimental data for inhibition by the peripheral site ligands propidium and gallamine at low concentrations of either acetylthiocholine or phenyl acetate if binding of these ligands slows substrate association and dissociation rate constants by factors of 5-70. Direct measurements with the acylation site ligands huperzine A and m-(N,N, N-trimethylammonio)trifluoroacetophenone showed that bound propidium decreased the association rate constants 49- and 380-fold and the dissociation rate constants 10- and 60-fold, respectively, relative to the rate constants for these acylation site ligands with free AChE, in reasonable agreement with the nonequilibrium steric blockade model. We conclude that this model can account for the inhibition of AChE by small peripheral site ligands such as propidium without invoking any conformational interaction between the peripheral and acylation sites.
Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Acetofenonas/metabolismo , Acetofenonas/farmacología , Acilación/efectos de los fármacos , Alcaloides , Sitios de Unión/efectos de los fármacos , Unión Competitiva/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Hidrólisis/efectos de los fármacos , Ligandos , Modelos Químicos , Propidio/metabolismo , Propidio/farmacología , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologíaRESUMEN
Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.
Asunto(s)
Acetilcolinesterasa/metabolismo , Acetiltiocolina/metabolismo , Acetilcolinesterasa/genética , Sustitución de Aminoácidos , Animales , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Catálisis , Humanos , Ligandos , Modelos Biológicos , Mutación , Fosforilación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TorpedoRESUMEN
The in vivo effects of the insecticide deltamethrin (DM) on the activity and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) were examined in different organs (brain, blood serum, heart, liver, and skeletal muscle) of carp. The chosen exposure conditions were a DM concentration of 2 micrograms/liter in the water (12 +/- 1 degrees C) for 3 days. All the treated fish survived the experiment, though the effects of the treatment were very visible: the fish always turned on their side, and the skin/scales became infected during the exposure to DM. DM did not result in a significant change in the AChE activity in any of the studied organs except the blood plasma, where the exposure resulted in an AChE activity decrease of as much as 20%. The ratio of membrane-bound to salt-soluble AChE forms was determined in the control fish. This ratio increased in the sequence heart, skeletal muscle, liver, and brain. The distribution of the AChE molecular forms was studied in the above tissues. The brain and liver contained forms G1, G4, and A12, the heart and skeletal muscle G4, A4, and A12, and the blood serum G1 and G4. The exposure to 2 micrograms/liter DM for 3 days caused hardly any changes in the pattern of the different AChE molecular forms. A small, but significant (P < 0.05) increase in the proportion of the G4 form was observed in the liver, while G1 and A12 decreased by a few percent (but insignificantly). No other tissues investigated exhibited any changes in the distribution of the AChE molecular forms.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Acetilcolinesterasa/metabolismo , Carpas/metabolismo , Insecticidas/toxicidad , Isoenzimas/metabolismo , Piretrinas/toxicidad , Acetilcolinesterasa/efectos de los fármacos , Animales , Femenino , Insecticidas/farmacocinética , Isoenzimas/efectos de los fármacos , Masculino , Nitrilos , Piretrinas/farmacocinética , Distribución TisularRESUMEN
Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 +/- 300 min-1 for DEPQ and 150 +/- 11 min-1 for EMPC, and second-order rate constants kOP/KOP were 193 +/- 13 microM-1 min-1 for DEPQ and 0.7-1.0 +/- 0.1 microM-1 min-1 for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP by factors of 2-20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216). Moreover, the binding of propidium resulted in a clear increase in kOP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of kOP/KOP were decreased by factors of 10(3) to 10(5), and kOP was decreased by factors of 300-4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin.
Asunto(s)
Acetilcolinesterasa/metabolismo , Ligandos , Compuestos Organofosforados/metabolismo , Acilación , Cumarinas/farmacología , Venenos Elapídicos/farmacología , Eritrocitos/enzimología , Humanos , Cinética , Estructura Molecular , Fosforilación , Propidio/farmacología , Unión Proteica , Quinolinas/farmacología , Proteínas Recombinantes/metabolismoRESUMEN
Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies and later confirmed by crystallography, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated how small peripheral site ligands such as propidium may inhibit substrate hydrolysis [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. In this model, the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Here, we first provide evidence that not only rate constants for substrates but also dissociation rate constants for their hydrolysis products are decreased by bound peripheral site ligand. Previous reaction schemes for substrate hydrolysis by AChE were extended to include product dissociation steps, and acetylthiocholine hydrolysis rates in the presence of propidium under nonequilibrium conditions were simulated with assigned rate constants in the program SCoP. We next showed that cationic substrates such as acetylthiocholine and 7-acetoxy-N-methylquinolinium (M7A) bind to the peripheral site as well as to the acylation site. The neurotoxin fasciculin was used to report specifically on interactions at the peripheral site. Analysis of inhibition of fasciculin association rates by these substrates revealed KS values of about 1 mM for the peripheral site binding of acetylthiocholine and 0.2 mM for the binding of M7A. The AChE reaction scheme was further extended to include substrate binding to the peripheral site as the initial step in the catalytic pathway. Simulations of the steric blockade model with this scheme were in reasonable agreement with observed substrate inhibition for acetylthiocholine and M7A and with mutual competitive inhibition in mixtures of acetylthiocholine and M7A. Substrate inhibition was explained by blockade of product dissociation when substrate is bound to the peripheral site. However, our analyses indicate that the primary physiologic role of the AChE peripheral site is to accelerate the hydrolysis of acetylcholine at low substrate concentrations.
Asunto(s)
Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/metabolismo , Acetiltiocolina/metabolismo , Unión Competitiva , Catálisis , Inhibidores de la Colinesterasa/farmacología , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Humanos , Hidrólisis , Ligandos , Modelos Químicos , Unión Proteica/efectos de los fármacos , Estereoisomerismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Thein vivo effects of deltamethrin (DM) on the blood sugar level, the acetylcholinesterase (AChE, EC 3.1.1.7) activities of the blood serum and various organs (heart, liver and intestine), the lactate dehydrogenase (LDH, EC 1.1.2.3), glutamic-oxaloacetic transaminase (GOT, EC 2.6.1.1), and glutamic-pyruvic transaminase (GPT, EC 2.6.1.2) activities of the blood serum, the adenosine triphosphatases (EC 3.6.1.3; Na(+)/K(+)-ATPase and Mg(2+)-ATPase) activities of the erythrocyte plasma membrane and the catalase (EC 1.11.1.6) activity of the liver were examined throughout 96 h in adult carp (Cyprinus carpio L.) Two sublethal concentrations, 1.0 and 1.5 µg/l of deltamethrin, were used. All fish survived the experiment except one, in an aquarium containing 1.5 ppb of DM, which died after 72 h.The AChE specific activity was significantly inhibited in the heart and intestine after 96 h at both concentrations compared to that in the control animals (P<0.05, Student'st-test), while there was no detectable difference between the two treatment. At the same time there was no detectable change in the liver. In the serum, the AChE activity almost remained unchanged; the only significant decrease could be measured after 96 h at 1.5 µg/l deltamethrin concentration. The blood glucose content exhibited interesting changes: after 24 h fish exposed at 1 µg/l DM seemed to be stressed, although this increase was not significant. When these fish became used to the new conditions (in practice this meant the presence of DM), the glucose level decreased, especially after 72 h. At the same time the control animals kept in similar circumstances showed a small insignificant decrease. Meanwhile fish in aquaria containing 1.5 µg/l DM reacted to the treatment with an increased blood glucose level after 48 h, and this did not change until the end of the treatment. The Na(+)/K(+)-ATPase activity decreased in a dose-dependant manner, while Mg(2+)-ATPase was less affected. A small increase in LDH level was observed, indicating damage of different muscle tissues. However, this phenomenon appeared only with the small dosage after 24 h (P<0.05). It has to be mentioned that the individual values varied to a large extent among of the eight fish.The GOT activities of the serum increased during the treatment. However, significant changes were only expressed after 72 and 96 h at 1 µg/l DM concentrations (P<0.01 andP<0.05), and after a similar long treatment at the high dosage (P<0.05, 72 and 96 h). The GPT did not change significantly in aquaria containing 1 µg/l DM. The only larger increase was measured after 96 h at 1.5 µg/l DM concentration (P<0.05). The catalase activity in the liver of treated carp remained practically at the same level compared to that in control fish.All these changes (concerning the primary effects of this compound) demonstrate the effect of DM on different fish enzymes, at low concentrations under laboratory conditions, which might be useful in practice for biomonitoring using fish.
RESUMEN
In the past few years, two massive eel (Anguilla anguilla L.) devastations occurred in Lake Balaton, Hungary. In 1991, 300 tons of eel perished in the western basin of the lake, while in the summer of 1995 30 tons of eel died in the eastern part of the lake. Investigations carried out to find the causes of these ecocatastrophes included measurements of certain biochemical parameters: the blood sugar level, and the acetylcholinesterase (AChE, EC 3.1.1.7), lactate dehydrogenase (LDH, EC 1.1.2.3), glutamic-oxaloacetic transaminase (GOT, EC 2.6.1.1) and glutamic-pyruvic transaminase (GPT, EC 2.6.1.2) activities in the blood serum of the collected eels. In both 1991 and 1995, deltamethrin (DM), the active ingredient of the insecticide K-OTHRIN 1 ULV used against mosquitoes, was detected in the eels; in 1995 it was demonstrated in several other animal species, i.e., bream (Abramis brama L.), pike perch (Stizostedion lucioperca L.), and the common gull (Larus canus), and in sediment samples from the lake. Additionally, laboratory experiments were carried out to study the effects of DM on eels. In 1991, eels were collected from the western (the site of the devastation) and eastern basins of the lake. The eels from the eastern basin were used as controls. At that time, the AChE activity in the blood serum of the eels from the western basin was significantly inhibited compared to that in animals from the eastern basin (P < 0.05, Student t test). Eels from the western part of the lake had GOT and GPT levels 20 and 100%, respectively, higher than those of eels from the eastern part of the lake. The blood glucose level was much higher in the eels from the affected area of the lake as compared to those from the eastern part. The brain and liver of the eels contained DM residues at 20 micrograms/kg wet tissue (Gönczy, 1992). Gönczy suspected that one of the causes of the massive eel loss in 1991 was the presence of DM in the fish. In 1995, when the eel devastation occurred in the eastern basin, moribund and surviving eels were collected from this part of the lake. The AChE activity was significantly inhibited in the blood serum of the dying eels as compared to that in surviving animals (P < 0.05, Student t test). The blood glucose content exhibited a difference too: it was 2.5 times higher in the dying eels than in the surviving ones. A huge increase in the LDH level was measured in the dying eels, indicating damage to different muscle tissues to an extent never observed previously. The GOT activities of the serum were twice as high in the dying eels as in the living fish. The GPT was not significantly changed in the serum of dying eels as compared to the surviving animals. DM was detected in different tissue samples of the dying eels: 2.7-18.5 micrograms/kg in the liver, 9.0-31.1 micrograms/kg in the gill, and 3.0 micrograms/kg wet tissue in the muscle. DM residues were found in tissue samples from other animals, in the following concentrations: 0.4 micrograms/kg in bream, 2.1 micrograms/kg in pike perch, 1.1 micrograms/kg wet tissue in dead gulls. The sediment samples collected from different places and at different times contained DM in a concentration of 5.5-30.0 micrograms/kg wet sediment at the time of the eel deaths, while the sediment samples collected from the same places a month later still contained DM at 7.0-8.8 micrograms/kg wet sediment. Laboratory experiments with the insecticide K-OTHRIN 1 ULV revealed that 1.0 microgram/liter of its active ingredient, DM, caused the death of 50% of the eels after an incubation time of 96 hr. In the liver of the dead eels, DM was detected at 2.9-20.0 micrograms/kg wet tissue. All the above-mentioned changes and the DM residue detected in the eels appear to demonstrate the contribution of DM in the severe eel devastation. This finding on the ecological risk of such types of insecticides might be useful in their further application.
Asunto(s)
Anguilla/fisiología , Enfermedades de los Peces/inducido químicamente , Insecticidas/envenenamiento , Piretrinas/envenenamiento , Contaminantes Químicos del Agua/envenenamiento , Acetilcolinesterasa/sangre , Acetilcolinesterasa/metabolismo , Alanina Transaminasa/sangre , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/sangre , Aspartato Aminotransferasas/metabolismo , Glucemia/metabolismo , Catalasa/sangre , Catalasa/metabolismo , Enfermedades de los Peces/metabolismo , Peces/metabolismo , Hungría , L-Lactato Deshidrogenasa/sangre , L-Lactato Deshidrogenasa/metabolismo , NitrilosRESUMEN
In cystic fibrosis (CF), the intense host inflammatory response to chronic infection largely accounts for the progressive pulmonary disease, and ultimately death. Neutrophils are the prominent inflammatory cells in the lungs of patients with CF, and large amounts of neutrophil elastase (NE) are released during phagocytosis. Besides having direct effects on structural elastin, NE stimulates the release of proinflammatory mediators from the respiratory epithelium and is a potent secretogogue. Therapeutic use of elastase inhibitors in CF has been complicated by difficulties in delivery to the critical site in the airway-the surface of the epithelium. We describe a unique strategy to protect the respiratory epithelial cell surface directly by capitalizing on the nondegradative transcytotic pathway of the polymeric immunoglobulin receptor (pIgR). A recombinant fusion protein was constructed consisting of an antihuman pIgR single-chain Fv (scFv) antibody linked to human alpha(1)-antitrypsin (A1AT), an inhibitor of NE. The recombinant scFv-A1AT fusion protein bound specifically to the pIgR on the basolateral surface of an epithelial cell monolayer, and was transported and released into the apical medium where the A1AT domain was capable of forming an inactivation complex with NE. Thus, A1AT linked to an antihuman pIgR scFv was delivered in receptor-specific fashion from the basolateral to apical surface and was released as an active antiprotease, indicating that it is feasible to deliver therapeutic proteins to the apical surface of epithelia by targeting the pIgR.