Red Blood Cell Cholinesterase Research Articles

Health surveillance and biological monitoring of pesticide formulators in India

Results of health survey and biological monitoring in pesticide formulators exposed to a combination of pesticides, an organophosphorus (OP) insecticide (phorate) and a persistent chlorinated insecticide (technical hexachlorocyclohexane; HCH; BHC) are reported. (I) Exposure of 160 workers to a combination of pesticides (malathion, parathion, DDT and HCH) resulted in 73% of the workers showing toxic signs and symptoms. Formulators showed marked inhibition of whole blood, plasma and red blood cell cholinesterase (ChE) activity and slightly higher concentrations of DDT and HCH in serum. An interesting observation was that over 25% of the formulators showed ECG aberrations. The ECG changes were not related to whole blood ChE activity. (II) Exposure to the chlorinated insecticide HCH in 19 workers engaged in the manufacture of technical HCH resulted in toxic signs and symptoms in over 90% of the subjects. The HCH concentrations in serum showed a ten-fold increase. Changes in the liver enzymes ornithine carbamyl transferase (OCT), 7-glutamyl transpeptidase (GGTP), leucine aminopeptidase (LAP) and in immunoglobulin M(IgM) showed possible effects on liver and humoral immunity. ECG monitoring showed evidence of cardiac effects. (III) Exposure of 40 formulators to a highly toxic OP insecticide (phorate) showed that over 60% of the workers suffered from toxic effects in spite of using a complete set of protective clothing. A marked and progressive inhibition in whole blood and plasma ChE activity was found during the two weeks of exposure to phorate. An appreciable recovery in ChE activity was observed 10 days after cessation of exposure. These surveys have established the need to practice and develop biological monitoring techniques to assess exposure and predict health risks in workers occupationally exposed to pesticides.

Paradoxical Elevation of Plasma Cholinesterase

Biological monitoring of workers exposed to hazardous or toxic substances is common in industry. Plasma and red blood cell (RBC) cholinesterase activity level monitoring is used to detect early toxic effects of exposure to organophosphate and carbamate insecticides. However, the validity of plasma cholinesterase level monitoring alone in such exposures is of questionable value since depressed, normal, and elevated levels of activity in test results lead to risk assessment dilemmas. The finding of an elevated plasma cholinesterase level in an exposed plant pathologist led to referral for evaluation of test results. We reviewed the prevalence and significance of elevated plasma cholinesterase levels. The prevalence of elevated plasma cholinesterase values ranges from 1.0 percent to 12.9 percent, depending on the kind of testing and populations surveyed. The benefits and/or risks of elevated levels over time remain to be elucidated.

Clinical confirmation of organophosphate poisoning of agricultural workers.

A group of 31 lettuce harvesters exposed to the organophosphate pesticide mevinphos presented to a local emergency room with moderate cholinergic symptoms and eye and skin irritation, with 22 of the subjects (76%) reporting three or more symptoms. None had baseline cholinesterase values, and plasma cholinesterase activity for all but two workers was above the lower limit of the laboratory normal range. None of the workers received antidotes and all were released for return to work. Twenty-nine workers sought additional care when symptoms persisted, and were followed by the investigators until 12 weeks after exposure. Plasma and red blood cell (RBC) cholinesterase increased until 14 days after exposure. Plasma cholinesterase was estimated to have been inhibited by an average of 15.6% (p less than 0.01), and RBC cholinesterase by 5.6% (p less than 0.01). These findings support the utility of sequential postexposure plasma cholinesterase analyses in confirmation of suspect organophosphate-induced illness when baseline values are not available.

Farm worker exposure to terbufos [phosphorodithioic acid, S-(tert-butylthio) methyl O,O-diethyl ester] during planting operations of corn.

Eleven farmers were monitored for dermal and respiratory exposure to terbufos [phosphorodithioic acid, S-(tert-butylthio) methyl O,O-diethyl ester] during a typical working day while planting corn and applying COUNTER® 15-G systemic insecticide-nematicide. The average estimated dermal exposure was 72 Μg/hr while the estimated respiratory exposure was 11 Μg/hr. The results of urinary alkyl phosphate analyses were all negative showing no detectable absorption of terbufos. Plasma and red blood cell cholinesterase values of exposed farmers showed no significant difference in activity when compared to either preexposure or control values indicating no adverse physiological effects from the exposure. Based on these results, the use of COUNTER 15-G does not present a significant hazard, in terms of acute toxicity, to farmers using this product for the control of corn insects.

Two-step immunoaffinity purification of acetylcholinesterase from rabbit brain.

Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X-100 from rabbit brain was purified 2,000-fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium-Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver-staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogeneous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km = 0.16 +/- 0.01 mM) were close to those of the native enzyme (Km = 0.12 +/- 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two-step immunopurification procedure presented in this communication offers a convenient route to homogeneous neural AChE in quantities useful for detailed biochemical and immunochemical study.

Aldicarb sulfoxide/aldicarb sulfone mixture in drinking water of rats: effects on growth and acetylcholinesterase activity.

A 1:1 mixture of aldicarb sulfoxide/aldicarb sulfone was administered to young adult Wistar rats via the drinking water at nominal concentrations of 19.2, 4.8, 1.2., 0.3, 0.075, or 0 ppm for 29 d. Blood was collected after 8, 15, and 29 d of treatment for plasma and erythrocyte cholinesterase determinations, and brain cholinesterase was determined at sacrifice. Body weight, food intake, and water consumption were measured weekly. Body weight gain and water consumption were reduced at 7, 14, 21, and 29 d in male and female rats at 19.2 ppm. Food consumption was reduced in males at 7, 14, 21, and 29 d but was reduced in females only on d 7. Both plasma and erythrocyte cholinesterase activity were reduced after 8, 15, and 29 d in male and female rats at 19.2 ppm. Males at 4.8 ppm showed reductions in plasma activity only after d 8 and in erythrocyte activity only after d 29. Female rats at 19.2 ppm also displayed depressions in brain cholinesterase activity not observed in similarly treated males. Since the only effects noted at 4.8 ppm were reductions in plasma and red blood cell cholinesterase activity in males only and at only one of three sampling periods, these two instances are not believed to be of any biological significance. The data suggest that 4.8 ppm in drinking water is a no observable ill-effect level for exposure of rats to aldicarb residues based on the parameters measured in this study.

Effect of high-fat and high-protein diets on toxicity of parathion and dichlorvos.

Growing male rats that weighed 120 +/- 5 g were kept for 30 days on the following synthetic diets: high protein diet (HPD), 59% casein; high fat diet (HFD), 50% saturated fat; and normal diet (ND), 19% casein, 10% saturated fat, and 60% sucrose. Other essential dietary ingredients were included in all the diets. All animals were injected at the end of the 30-day period with parathion [10 mg/kg intraperitoneal (ip) injection as a single dose] or dichlorvos (30 mg/kg ip as a single dose) to compare the effect of dietary pretreatments on mortality from parathion and dichlorvos. A lower dose of parathion (7.5 mg/kg) and dichlorvos (20 mg/kg) was employed in another set of experiments to compare the spontaneous regeneration of plasma and red blood cell (RBC) cholinesterase (ChE) activity at 2 hr, 1 day, 3 days, and 5 days after administration of parathion or dichlorvos. The effect of these diets on hepatic microsomal oxidases was also determined. Results showed that diets per se did not affect initial plasma and RBC ChE activity. The HPD and HFD significantly protected against mortality from parathion but not from dichlorvos. Hepatic microsomal cytochrome P-450 and aminopyrine demethylase activity were unchanged, but aniline hydroxylase activity was increased significantly by HPD and HFD. Parathion oxidase in hepatic microsomes was significantly increased in rats fed HFD only. For the HPD, spontaneous regeneration of ChE diminished in RBCs in parathion-intoxicated rats and in plasma and RBCs of dichlorvos-intoxicated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of a dermal self-exposure model to worker reentry.

The dermal toxicity of five commonly used organophosphate insecticides was investigated with a mouse intermittent self-exposure model. Blood cholinesterases were monitored on d -3 and -1 before exposure and for 4--6 d during exposure to foliar residues. Responses were much greater in unmuzzled than in muzzled animals due to oral contamination. After two 10-h exposures, muzzled mice showed log-linear cholinesterase responses across a wide range of foliar pesticide concentrations. Foliar pesticide levels that caused 50% depression in plasma or red blood cell cholinesterase were determined with log-probit dose-response analysis. The greatest cholinesterase responses for both emulsifiable concentrate and encapsulated formulations were found with diazinon, followed by parathion and methyl parathion. Azinphos-methyl and mevinphos produced no significant responses in muzzled mice at maximal foliar concentrations. Symptomatology, food consumption, and body weight provided less sensitive indicators of response than cholinesterases. No consistent relation existed between the mouse intermittent self-exposure toxicities and mouse dermal LD50 values. Use of data from acutely exposed animals to predict the hazard of intermittent foliar exposure appears inadvisable.

Acute dermal toxicities of various organophosphate insecticides in mice.

Dermal LD50 values for five organophosphate insecticides were determined in mice by application of solutions to hind feet. Values were simultaneously generated for the ED50 (milligrams per kilogram) for both cholinesterase and acetylcholinesterase. Lethality was greatest with mevinphos, followed by parathion, methyl parathion, diazinon, and azinphosmethyl. LD50 values were higher than reported values for mice treated on shaved back skin. Cholinesterase ED50 values roughly agreed with LD50 values for mevinphos, parathion, methyl parathion, and azinphos-methyl, but diazinon appeared much more inhibitory of blood than neuronal cholinesterase. Red blood cell and plasma cholinesterase activities were equally sensitive for all but mevinphos and diazinon.

Levels of metrifonate and dichlorvos in plasma and erythrocytes during treatment of schistosomiasis with Bilarcil.

A method for the simultaneous quantitation of metrifonate (0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate) and dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) in human blood has been worked out. It is based upon multiple labelling of the compounds with deuterium and gas phase analysis using the mass spectrometer as a selective detector. The amount of DDVP in plasma is about 1% of the amount of metrifonate. In erythrocytes the corresponding amount of DDVP is 0.5% or less of metrifonate. Both compounds reach peak levels in blood within two hours after oral dosing and are detectable for at least eight hours. Cholinesterase activity in plasma reaches zero levels within 15 min. and remains inhibited for more than eight hours. Red blood cell cholinesterase is inhibited only 60-80%. According to kinetic calculations, clearance of metrifonate occurs primarily via dichlorvos. If dichlorvos is the only active component, which in all likelihood it is, it's slow release may be important in the schistosomicidal effect. Clinical data in seven metrifonate treated patients revealed that mild vertigo subsiding in a few hours was the most common side effect.

Exposure of field workers to organophosphorus insecticides: cotton.

The red blood cell and plasma cholinesterase (ChE) levels of cotton scouts inspecting foliage for insect infestation and damage were monitored. Group means for ChE activity were significantly depressed at one or more time(s) during four of the eight growing seasons studied. Although no symptoms of organophosphate poisoning were confirmed, several scouts exhibited ChE depressions over 50% of pre-exposure levels.

Cut flowers: a potential pesticide hazard.

Following reports of ten cases of possible organophosphate pesticide poisoning in florists exposed to pesticide residues on cut flowers, we conducted a prospective random-sample survey to determine residual pesticide levels on flowers imported into the United States via Miami, Florida. A sample of all flowers imported into Miami on three days in January 1977 showed that 18 (17.7 per cent) of 105 lots contained pesticide residue levels greater than 5 ppm, and that three lots had levels greater than 400 ppm. Azodrin (monocrotophos) was the most important contaminant with levels of 7.7--4,750 ppm detected in nine lots. We examined 20 quarantine workers in Miami and 12 commercial florists exposed to contaminated flowers. Occasional nonspecific symptoms compatible with possible organophosphate exposure were noted, but we found no abnormalities in plasma or red blood cell cholinesterase levels. This study documents a previously unrecognized potential source of occupational pesticide exposure and suggests that safety standards should be set for residue levels on cut flowers.

Exposure of field workers to organophosphorus insecticides: sweet corn and peaches.

Plasma and red blood cell cholinesterase levels of professional agricultural workers engaged in packing sweet corn and thinning peaches were monitored. Workers with extensive contact with mechanically harvested sweet corn (the corn had been treated one or two days before harvest with a combination of ethyl and methyl parathion) exhibited significant depression of cholinesterase. Gloves, worn by 40% of the workers, provided some protection from absorption of pesticide residues. No significant cholinesterase depression was found in workers thinning peaches which had been previously treated with parathion.

Worker reentry study involving phosalone application to citrus groves

This study was conducted to provide information for the establishment of a worker reentry period after applying phosalone to citrus groves in California. Fourteen healthy male agricultural workers (volunteers) ranging in age from 18 to 72 years were given physical examinations, including cholinesterase (ChE) determinations, prior to their entry into the phosalone-treated grove; four individuals treated in like manner served as controls. Blood and urine were sampled at the end of each work day. Two separate areas of a citrus grove were treated (6 lb per acre), so that the workers would enter the respective areas 14 or 21 days after the application. Cloth patches were attached to clothing at four sites (chest, shoulder, thigh and back) to collect dislodgeable residues for analysis. Leaf samples were taken to determine the dislodgeable residue of phosalone and its oxon at Day 0 and intervals thereafter. No adverse effects were observed with dislodgeable residues as high as 3.6 μg/cm 2 and no phosalone or its metabolites (phosalone-oxon and 2-amino-5-chlorophenol) were detected in urine. The red blood cell ChE activity of the workers was not significantly different from controls according to the t test on deviations, while the deviations in plasma ChE activity of the workers and controls were significantly different in both the 14- and 21-day studies. Dislodgeable residues on cloth patches were lower in the 14-day study and reflected the differences in dislodgeable residues on leaves for the two reentry periods. On the basis of this study, health scientists recommended a 21-day reentry interval.

Quantitative structure activity study of a series of substituted 0,0-dimethyl 0-(p-nitrophenyl) phosphorothioates and 0-analogs

The lethality of seven 0,0-dimethyl 0-(p-nitrophenyl) phosphorothioates and their corresponding oxons has been determined in adult, male mice. The anticholinesterase potency of the oxons for bovine, erythrocyte acetylcholinesterase has also been determined. The LD50 values in mg/kg were: methylparathion, 33.1; fenitrothion, 988; chlorothion, 794; decapthon, 331, 0,0-dimethyl 0-(3 ethyl-4-nitrophenyl) phosphorothioate, 1396; 0,0-dimethyl 0-(3-isopropyl-4-nitrophenyl) phosphorothioate, 1379, and 0,0-dimethyl 0-(3,5-dimethyl-4-nitropenyl) phosphorothioate, 630. The lethality values for the 0-analogs were: methyl paraoxon, 21.8; fenitrooxon, 120; chloroxon, 280; dicapthoxon, 102; dimethyl-3-ethyl-4-nitrophenyl phosphate, 191; dimethyl-3-isopropyl-4-nitrophenyl phosphate, 321; dimethyl-3,5-dimethyl-4-nitrophenyl phosphate, 630. The pI50 values for inhibition of bovine red blood cell cholinesterase by the oxons were: methyl paraoxon, 7.2; fenitrooxon, 6.2; chlorooxon, 7.0; dicapthoxon, 7.1; dimethyl-3-ethyl-4-nitrophenyl phosphate, 6.0; dimethyl-3-isopropyl-4-nitrophenyl phosphate, 6.0; and dimethyl-3,5-dimethyl-4-nitrophenyl phosphate, 5.4. All the substitutions on the 4-nitrophenyl nucleus decreased toxicity whereas conversion of the phosphorothioates to oxons increased toxicity. The substituted oxons were all less toxic than methyl paraoxon while the substituted phosphorothioates were all less toxic than methyl parathion.

Effect of glucocorticoids on liver and blood cholinesterases

The effects of oral prednisone on plasma and red blood cell cholinesterases have been studied in human volunteers. While the activity of plasma pseudocholinesterase is depressed by glucocorticoid administration, the activity of red blood cell true cholinesterase is not affected. Cortisol administration to rats, intraperitoneal or subcutaneous, inhibited liver pseudocholinesterase activity, indicating a possible depression of the enzyme protein synthesis.

Effects on nervous function in volunteers ingesting mevinphos for one month

To investigate reported nervous system effects of chronic exposure to organophosphorus pesticides, mevinphos 25 μg/kg, was administered daily to eight male subjects for 28 days; results were compared to those in a control group. At the end of exposure, a 7% decrease in slow fiber motor nerve conduction velocity and a 38% increase in Achilles tendon reflex force were found. No effect on neuromuscular transmission was observed. Red blood cell cholinesterase depression was 19%. Results confirm similar deviations found in pesticide workers.

Environmental Temperature and The Percutaneous Absorption of a Cholinesterase Inhibitor, Vx

S-(2-diisoproplaminoethyl) 0-ethyl methylphosphonothioate (VX), an anticholinesterase liquid of low volatility, was applied to the skin of 139 men at environmental temperatures of -18 degrees, 2 degrees, 18 degrees, or 46 degrees C. The skin was decontaminated after 3 hr and the men spent the next 21 hr at about 27 degrees C. The amount of VX penetrating the skin was estimated from the inhibition of red blood cell cholinesterase. The decimal fraction of the dose that penetrated in 3 hr ranged from 0.04 at -18 degrees C to 0.32 at 46 degrees C for the cheek and from 0.004 at +18 degrees C to 0.029 at 46 degrees C for the forearm. Further increase in cholinesterase inhibition after decontamination was evidence of a deposit of VX in the skin. The amount of VX remaining in the skin after decontamination was greater in the forearm and less in the cheek at higher temperatures.

Human subject experiments to estimate reentry periods for monocrotophos-treated tobacco.

Thirteen to fifteen human volunteers worked for eight-hr periods in tobacco fields treated 96, 72, and 48 hr previously with monocrotophos (3-hydroxy-N-methyl-cis-crotonamide dimethyl phosphate). At the 48-hr reentry period, during which time no rainfall occurred, both post-exposure plasma and red blood cell cholinesterase levels of the worker group were depressed from pre-exposure levels although the decline was less than 9% in both cases. Rainfall in excess of one inch fell during the 96- and 72-hr reentry intervals, and no important change in cholinesterase levels was noted. Experiments with mice were in general agreement with the human tests. Dimethyl phosphoric acid in urine of the worker group collected prior to, and three hr after, exposure was unchanged in all cases. Rain appeared to have markedly reduced dislodgable residues.

Interaction of reversible inhibitors with catalytic centers and allosteric sites of cholinesterases

The kinetics of inhibition of the human red blood cell cholinesterase with galanthamine tacrine and oxazyl (ambenonium) and the effect of these drugs on chick, mouse, cat and rat blood plasma enzyme activity was studied. Galanthamine proved to bind with acetylcholinesterase in the anionic areas of the catalytic centres, oxazyl interacted in the area of the allosteric anionic site, and tacrin interacted with the hydrophobic areas of the enzyme.