FUNDAMENTAL

AND

APPLIED

A Microassay

TOXICOLOGY

16, 110-l 16 (1991)

Method for Neurotoxic

Esterase Determinations’

LINDA~ORRELLAND MARION EHRICH' Virginia-Maryland

Regional

Received

College

March

of Veterinary

Medicine,

12. 1990; accepted August

Blacksburg,

Virginia

24061

13, 1990

A Microassay Method for Neurotoxic Esterase Determinations. CORRELL, L., AND EHRICH, M. (1991). Fundam. Appl. Toxicol. 16, 110-l 16. A microtiter plate reader with an associated computer to average triplicate samples and subtract blanks was used for reading and calculating neurotoxic esterase (NTE, also known as neuropathy target esterase) activities in spinal cord regions of hens 4 hr after administration of diisopropylphosphorofluoridate (DFP, 0.5 m&kg SC). Although NTE inhibition is an early indicator of organophosphorus ester-induced delayed neuropathy, DFP-induced inhibition was not greater in regions of the spinal cord where pathological changes are most notable. Acetylcholinesterase (AChE) activities and protein determinations were also done on these tissues using microassay methods. DFP-induced AChE inhibition was similar to NTE inhibition. In addition to the capability to be used for small regional esterase activity measurements, the microassay was advantageous because the number of samples incorporated into a single assaywas increased and the time needed for the NTE assay was reduced by 50%. Total volume of incubate in each well was 0.3 ml; the incubate contained l/20 quantities of sample and reagents necessary in more conventional assays.Validation of the microassay was performed by comparison with more conventional assayswhen measuring inhibition of NTE and AChE in brains of control and experimental hens of two different genetic strains (B 13B I3 and B21B21 white leghorns). Experimental birds were given DFP, 0.5 mgjkg SC,24 hr before samples were collected. NTE activities in brains of control hens were similar using both types of NTE analytical procedures. Percentage inhibition of NTE caused by DFP was within 4% using both assay procedures in both strains of hens. The microassay was sensitive enough to detect NTE activity in 42 rug of hen brain after 15 min of incubation. Hen lymphocytes could also be examined for effectsof organophosphorus esters on NTE activity, with 14. I + 2.2 and 8.3 + 2.2 pmol/l5 min/mg protein in 1 X IO6 cells measured in samples taken before and 4 hr after administration of 0.5 mg/kg SCDFP. o 1991 society ofToxicology.

Information on the capability of organophosphorus esters to inhibit neurotoxic esterase (NTE, also known as neuropathy target esterase) is used to indicate the potential of these chemicals to induce a progressive, delayed neuropathy before the lesions occur (Johnson, 1982). NTE activity in neural tissue is inhibited within hours after exposure of man and animals to certain organophosphorus esters, ’ Presented in part at the 29th Annual Meeting of the Society of Toxicology, February 12-16, 1990, Miami Beach, FL. * To whom all correspondence should be addressed.

0272-0590/91 $3.00 Copyright 0 I99 I by the society of Toxicology. All rights of reproduction in any form rewwd.

yet neuropathy is not evident for several weeks. NTE activity is usually measured in homogenates of whole brain or spinal cord, yet lesions are most notable in relatively restricted regions. In the spinal cord, the distal portions of the longer tracts are affected, such as the rostral cervical regions of ascending tracts (the spinocerebellar and gracilis tracts) and the lumbosacral regions of decending tracts. Lesions in brain are considered continuations of affected ascending spinal pathways (Davis and Richardson, 1980; Jortner, 1984; Tanaka and Bursian, 1989). To study NTE inhibition in

110

MICROASSAY

these regions, a microassay method was developed that required less than 0.5 mg of spinal cord as the enzyme source. This procedure allowed incubations for NTE studies to be done in microtiter wells and results to be read on a microplate spectrophotometer. As the same tissues used for measurement of NTE activities can be used for measurement of acetylcholinesterase (AChE) activities, a microtiter version of the assay of Ellman et al. ( 196 1) was used as well. For both NTE and AChE determinations, microassay allowed for inclusion of larger numbers of samples per assay, decreased the quantities of enzyme source and reagents, and reduced the time necessary for esterase activity measurements. MATERIALS

AND

METHODS

The microassay for NTE in brain and spinal cord. The NTE assay was based on the method described for hen brain by Sprague et al. (198 I). In this conventional assay, I part tissue was homogenized in 6.5 parts 50 mM Tris buffer containing 0.2 mM EDTA, pH 8.0, using a PotterElvehjen glasshomogenizer. One milliliter ofhomogenate diluted with this buffer (containing 6.7 mg brain or 9.26 mg spinal cord) was incubated with and without 0.0116 mg mipafox and/or 0.1376 mg paraoxon (prepared as 100X concentrates in acetone and 10 pl added to the incubation mixture) in 2 ml of 50 mM Tris buffer. Phenyl valerate was used as substrate for the enzyme ( 1.064 mg added in 1 ml 0.03% Triton-X in saline). Hydrolysis of phenyl valerate in the 4-ml incubate was determined after a I5-min incubation period. Hydrolysis of phenyl valerate by NTE, a membrane-bound enzyme, was stopped with 2 ml of a solution containing the detergent sodium dodecyl sulfate (5% SDS, 0.02% 4-aminoantipyrine in 0.5 M Tris buffer, pH 9.6). One milliliter ofan aqueous solution containing 4 mg potassium ferricyanide was then added to provide the oxidant for condensation of phenol with 4aminoantipyrine. The product of this reaction is orange, and absorbance was read at 5 10 nm (Gottlieb and Marsh, 1946). Final concentrations of reagents in the 7-ml volume on which absorbances were determined were 9.2 pM mipafox, 7 1 PM paraoxon, 0.85 mM phenyl valerate, 1.4% SDS, 0.0057% 4-aminoantipyrine, and 1.7 mM potassium ferricyanide. Protein concentrations were determined spectrophotometrically at 595 nm after binding to Coomassie brilliant blue G-250 (Bradford, 1976), using a commercially available kit (Bio-Rad, Richmond, CA). Bovine serum albumin provided in the kit was used to prepare the protein standard curve. NTE activity, expressed as nanomoles of phenol formed/ 15 min/milligram of pro-

FOR NTE

111

tein, was determined as the difference in enzyme activity between incubates containing paraoxon and incubates containing paraoxon and mipafox (Johnson, 1977). In the microassay, quantities of brain, spinal cord, and reagents were l/20 of those used in the conventional assay, but concentrations were equivalent. A volume of 50 pl of 1:150 brain homogenate (0.33 mg) or 50 r.11of 1:110 spinal cord homogenate (0.45 mg), prepared in 50 mM Tris, 0.2 mM EDTA, pH 8.0, was incubated with and without 25 pl containing 0.58 pg mipafox and/or 6.88 pg paraoxon. These organophosphates were prepared as solutions of 1.16 and 13.76 mg/ml acetone, respectively, and diluted 1:50 in Tris-EDTA buffer before being added to the microtiter well. The plate was mixed after the addition of each reagent using the automix on the microtiter plate reader (Molecular Devices,Menlo Park, CA) and then placed in a 37°C water bath for 20 min. Fifty microliters containing 53 pg phenyl valerate dissolved in 0.03% T&on-X in saline was then added to the microtiter wells and the plate mixed twice more. Following a 15-min incubation at 37°C 50 r.dof a solution of 10% SDS and 0.04% 4-aminoantipyrine in 0.5 M Tris was added to the microtiter wells, and the plates were mixed twice. This was followed by addition of 50 pl of 0.4% potassium ferricyanide solution, mixing three times, a 15-min incubation at room temperature, and reading of the plate at 5 10 nm. Color was stable between 10 and 45 min after the fenicyanide solution was added. Substrate and tissue blanks were included in each assay. The SOFTmax program (Molecular Devices, Menlo Park, CA) used with the spectrophotometer could be programmed to subtract the blanks (reagents but no enzyme), average the triplicate readings, and print the results. Protein was determined by microassay using the Bio-Rad kit described above (Brogden and Dickinson, 1983; Simpson and Sonne, 1982). In the microassay procedure, 250 ~1 Coomassie brilliant blue G-250, diluted in distilled water, was mixed at room temperature with 10 rl sample or protein standard (bovine serum albumin in water, 0.01870.8 mg/ml), providing a 0.26-ml volume that was l/10 of that recommended for assayin test tubes. The SOFTmax program for protein determinations was used to calculate milligrams of protein included in the wells. The microassay for NTE in other tissues. The procedure described above was used with 1.25 mg hen sciatic nerve and 50,COOhen lymphocytes as sources of NTE. Both were provided in 50-&l volumes added to the microtiter wells. Hen sciatic nerve homogenates were prepared using a I: 40 dilution of nerve in 50 mM Tris buffer containing 0.2 mM EDTA, pH 8.0. The nerve was minced and then dispersed, on ice, using a Polytron apparatus followed by mixing in a Potter-Elvehjen glass homogenizer to prepare a homogenous suspension. When hen lymphocytes were used as the enzyme source, blood was collected in heparinized syringes, diluted 1:1 with phosphate-buffered saline, layered above 4 ml Ficoll, and then centrifuged at 2500 rpm for 20 min to separate the lymphocytes. After cen-

112

CORRELL AND EHRICH

trifugation, lymphocytes were removed from above the Ficoll, resuspended in 2 vol of Hanks’ buffer (Sigma Chemical Co., St. Louis, MO), and recentrifuged at 1000 rpm for 10 min. The supernatant was removed, and the pellet was gently resuspended in 2 vol Hanks’ buffer and centrifuged again. After removal of the supematant, the pellet was suspended in 1 ml Hanks’ buffer, the cells/milliliter were determined using a Coulter counter, and concentration was adjusted to 1 X lo6 cells/ml using Hanks’ buffer. The microassayfor AChE. For the conventional AChE assay,in which the total volume of the incubate was 5 ml, homogenates containing 0.8 mg brain or 5.9 mg spinal cord in 0.1 M Na phosphate buffer (pH 8.0) were incubated with 2 mg dithionitrobenzoic acid (DTNB), 0.75 mg Na bicarbonate, and 1.084 mg acetylthiocholine iodide for a period of 30 min. This time period was used to provide an absorbance at 4 12 nm that was high enough in control tissues (0.50-2.00) that residual activity could easily be detected in samples from organophosphate-treated animals (Ellman et al., 196 1). Substrate was present in sufficient excessto allow color development over this period of time. The microassay method for AChE was derived from the methods of Ellman et al. (196 I) and Brogdon and Dickinson (1983). Contents of microtiter wells contained 0.048 mg brain, 0.35 mg spinal cord, or 1.25 mg sciatic nerve, which had been added as 50 ~1 of tissue homogenates in 0. I M Na phosphate buffer, pH 8.0. Also preincubated in the microtiter wells was 150 ~1 buffer and 50 /II of a solution containing 0.12 mg DTNB and 0.045 mg Na bicarbonate. The automix function on the microplate reader was used for mixing after the addition (buffer, followed by tissue and DTNB-bicarbonate). Fifty microliters containing 0.06504 mg acetylthiocholine iodide was used to start the reaction. After mixing, the initial absorbance was determined. The plate was then placed in a 27°C water bath for 30 min. At the end of this time. the plate was mixed again and a second absorbance obtained. Activity was expressed as micromoles of acetylthiocholine hydrolyzed/minute/gram of tissue using 1.36 X IO4 as the extinction coefficient of the yellow anion produced by combination of thiocholine with DTNB. The report option of the SOFTmax system associated with the microliter plate reader was programmed to average the triplicate readings and subtract the blanks (reagents but no enzyme: enzyme but no substrate). In vivo studies. Tissue samples (brain, spinal cord, sciatic nerve) and lymphocytes for NTE and AChE measurements were obtained from 1Cmonth White Leghorn hens with known haplotypes of the B system, B 13B 13 or B2 lB2 1. The haplotype designations B 13 and B2 1 are symbols for genetically similar birds that differ by a particular set of alloantigens determined by loci within the major histocompatibility complex of the chicken (Briles et al., 1983; Ehrich et al., 1986). These hens were hatched and raised in the Department of Poultry Science, Virginia Tech, being

reared on littered floors until 18 weeks of age, when they were transferred to individual wire cages 30 X 50 X 50 cm. Feed and water were provided ad libitum. Diisopropylphosphorofluoridate (DFP, 100% pure, Aldrich Chemical Co., Milwaukee, WI) was administered subcutaneously at a dosage of 0.5 mg/kg to groups of four or five hens. The dosage was provided in a solution of 0.25 ml/kg in corn oil. Controls were untreated. Hens were pretreated with 8 mg/kg atropine administered by intramuscular injection 20 min before administration of DFP. Atropine (4 mg/kg) was readministered 0.5, 3, 6, and 1I hr after DFP. Euthanasia for tissue collection was done by cervical dislocation 4 or 24 hr after administration of DFP. Samples collected at 4 hr included lymphocytes, cerebrum, cerebellum, brain stem, cervical spinal cord, thoracic spinal cord, lumbosacral spinal cord, and sciatic nerve. Whole brain and spinal cord were collected at 24 hr. The areas at which sections are cut for best visualization of organophosphate-induced delayed neuropathy (OPIDN) (Jortner, 1984) were dissected from the rostra1 cervical cord and the lumbosacral spinal cord. Brain and spinal cord samples were also obtained from 70-day-old, male Long-Evans rats (Charles River, Raleigh, NC). Groups of four were pretreated with 20 mg/kg atropine im before being given either saline or mipafox, 30 mg/kg ip (Lark Enterprises, Webster, MA), 4 hr prior to tissue collection. Results are presented as means + SE, with a minimum of four different animals used to provide samples for each determination. Comparisons between enzyme activities measured on the same samples using conventional and microassay procedures were made using the Student t test, with p < 0.05 considered a statistically significant difference. Variability among duplicate or triplicate incubates of the same sample was expressed as a percentage of the difference of the extremes over the mean.

RESULTS NTE and AChE inhibitions determined by microassay in sections of spinal cord, brain regions, and sciatic nerve 4 hr after administration of DFP to B 13B 13 hens are presented in Table 1. Activities of NTE and AChE in sciatic nerve and in all spinal cord regions of control hens were less than activities in the brain stem, cerebellum, and cerebrum. Inhibition of esterases after DFP administration was not greater in the cervical or lumbar regions of the spinal cord than in the region between. The average percentage variabilities among sample replicates of cervical spinal cord and of lumbar spinal cord used for NTE de-

MICROASSAY

113

FOR NTE

TABLE 1 REGIONAL ESTERASEACTIVITY AFTER DFP ADMINISTRATION TO Bl3Bl3 Activity in untreated hens”

HENS Activity after DFP b (% inhibited)

Enzyme

Region

NTE

Cerebrum Cerebellum Brain stem Cervical spinal cord Thoracic cord Lumbar cord Sciatic nerve

399 t468 f 380 It 107 + 93 * 87 f 21 +

13 13 29 9 3 I 3

252 311 206 54 33 42 9.5

AChE

Cerebrum Cerebellum Brain stem Cervical spinal cord Thoracic cord Lumbar cord Sciatic nerve

8.8 + 21.4 f 11.2 + 3.1 + 1.4 * 2.2 & 0.29 +

1.4 1.5 1.2 0.6 0.1 0.2 0.05

5.0 * 11.3 + 7.2 + 1.8 f 0.4 k 1.1 + 0.20 +

rt + f + + k +

21 62 35 9 3 9 0.6

(37) (34) (46) (50) (65) (52) (64)

1.2 2.1 1.8 0.3 0.1 0.2 0.04

(43) (47) (36) (40) (70) (51) (32)

DNTE expressed as nmol phenyl valerate hydrolyzed/l 5 min/mg protein; AChE as pmol acetylthiccholine hydrolyzed/ min/g tissue; mean f SE, n = 5. Percentage inhibition after DF’P administration is included in parentheses. b DFP, 0.5 mg/kg SC,4 hr before sample collection.

terminations were 4.3 (range, 0.8-7.9; 12= 10) and 2.6 (range, 0.9-4.4; IZ = lo), respectively. The average percentage variabilities among sample replicates of cervical spinal cord and of lumbar spinal cord used for AChE determinations were 2.6 (range, O-6.0) and 4.2 (range, 0.6-9. l), respectively.

Results from comparison of conventional and microassay methods are presented in Table 2. NTE activities in brains from control and mipafox-treated rats were, respectively, 15 1 + 4 and 15.2 + 4.3 nmol/ 15 min/mg protein using the conventional assay and 173 + 11 and 24 & 4 nmol/l5 min/mg protein

TABLE 2 COMPARISONOF ESTERASEACTIVITIES DETERMINED BY Two PROCEDURES Tissue source

Treatment’

NTE (Con)b

MicroNTE

AChE (Con)b

MicroAChE

Bl3Bl3 hen brain

0 DFP

417 * 43 50 zk 2 (88%)

414 + 44 58 + 1 (86%)

11.8 f 0.5 4.5 + 0.2 (62%)

13.6 f 0.4 5.2 f 0.3 (62%)

B2lB21 hen brain

0 DFP

403 + 20 81 + 11 (80%)

316 f 21 16 + 4 (76%)

12.7 -c 0.8 4.3 f 0.2 (66%)

16.0 f 0.8 4.8 f 0.1 (70%)

Long-Evans rat spinal cord

0 Mipafox

143 * 14 25 + 4 (83%)

124 + 3 12 + 3 (90%)

3.2 f 0.1 0.8 + 0.1 (75%)

3.9 + 0.4 1.5 f 0.1 (62%)

a DFP, 0.5 mg/kg sc. 24 hr before collection of hen brains; mipafox, 30 mg/kg ip, 4 hr before collection of rat spinal cords. b Con. conventional assay.Sprague et nl. ( 198 1) for NTE and Ellman et al. ( 196 1) for AChE. Values are expressed as nmol phenyl valerate hydrolyzed/l 5 min/mg protein for NTE and as rmol acethylthiocholine hydrolyzed/min/g brain for AChE, mean f SE, n = 4-5. Percentage inhibition after DFP administration is included in parentheses.

114

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AND EHRICH

using the microassay. The only significant difference between control values for NTE and AChE using the two assay procedures was in brains from B2 1B2 1 hens. Variability of both NTE and AChE activities within the groups of control and test animals was similar when either the microassay or the conventional assays for NTE and AChE were used. Strain of hen did not appear to contribute to sensitivity to OP-induced NTE inhibition. Activities of brain NTE and AChE after DFP administration to hens were similar when either the conventional or the microassay methods were used for enzyme determinations; the largest difference in percentage inhibition induced by organophosphates measured by the two assay procedures was rat spinal cord AChE. Using the microassay procedures, the average percentage variabilities of sample replicates were 3.9 for NTE (range, 1.0-4.2; n = 18) and 3.5 for AChE (range, 0.2-5.0; n = 18). Using the conventional assays, the average percentage variabilities of sample replicates were 5.6 for NTE (range, 0.8-9; y1= 18) and 4.3 for AChE (range, O-8.2; n = 18). Serial dilutions of hen whole brain homogenate were used to determine the minimal quantity of enzyme needed to provide at least a 2.5-fold difference between activity measured in microtiter incubates containing paraoxon + mipafox and those containing paraoxon alone. For a 15-min incubation period, enzyme activity was linear to 42 /*g of hen brain. The average percentage variability of sample replicates was 5.9 (range, 0.6-15.8; n = 12). The microtiter assay procedure was also useful for determination of NTE activity and inhibition by DFP (0.5 mg/kg SC,4 hr earlier) in hen lymphocytes. Activities of 14.1 + 2.2 and 8.3 + 2.2 pmol/l5 min/mg protein in 1 X lo6 lymphocytes were determined in samples from control and experimental hens, respectively (mean + SE, 12= 5). DISCUSSION The present experiments demonstrated that neither quantity of NTE nor sensitivity of NTE

to DFP-induced inhibition could identify regions of the spinal cord most susceptible to lesions characteristic of OPIDN. As reported previously (Johnson, 1982; Dudek and Richardson, 1982) activity of NTE was greater in brain, although lesions of OPIDN are less notable there (Jortner, 1984; Tanaka and BurSian, 1989). The present experiments also demonstrated that the method for NTE activity determinations could be modified so that incubations of small amounts of tissue could be done in 0.3ml microtiter wells. Results obtained were similar to NTE activities measured when 20fold-greater quantities of enzyme and reagents were used. Proportional activities of NTE determined by microassay in whole brain and brain regions, spinal cord, sciatic nerve, and lymphocytes of untreated hens were demonstrated to correspond to those previously reported (Johnson, 1982; Dudek and Richardson, 1982; Olajos and Rosenblum, 198 1; Lotti et al., 1986). Time necessary for an NTE assay that included 24 samples and associated blanks, including weighing and homogenization of tissue samples, required 4 hr when incubations were done in duplicate test tubes using the conventional method of Sprague et al. ( 198 1). When this assay was repeated using the microassay procedure, total time required was 2 hr and incubations were done in triplicate. The AChE determinations using the conventional assay procedure (Ellman et al., 1961) and the microassay procedure took about the same amount of time, but the microassay increased the potential to analyze more samples at once. Other investigators have noted that less time is needed for microassay methods for AChE activities (Ray and Clark, 1988; Hammond and Forster, 1989; Brogdon and Dickinson, 1983) but none included tissue homogenates as enzyme sources in their procedures. Although the microassay procedures are economical, caution must be taken when they are used. Accuracy of pipetting is necessary when such small volumes are used; tiny bub-

MICROASSAY

FOR NTE

115

bles, for example, will produce large errors. Dickinson, 1983; Simpson and Sonne, 1982), With care, however, variability among repliand collection of kinetic and concentrationcate incubates of the same sample can be response data in vitro (Ray and Clark, 1988). equivalent to the variability that occurs among Microassay procedures have also been used for replicate incubates of the same samples using the rapid determination of activities of enthe conventional assays. Safety is another item zymes such as acid and alkaline phosphatases, to take into consideration when organophos,&glucuronidase, and succinate dehydrogenase phates are used for NTE assays. Even though (Young et al., 1983). The potential for inquantities needed in the microassay are small, creased use of microassay procedures for bioopen containers of paraoxon and mipafox are chemical studies is likely to continue to inused to provide solutions for multiwell pipet- crease as investigators attempt to economize ters. both time and resources. The present results indicate that microassay methods are useful for determination of NTE ACKNOWLEDGMENTS and AChE activities after exposure of animals to organophosphorus esters. As demonstrated The authors acknowledge the technical assistance of here, microassay methods may also be useful Joanne Huntington, JeffMusser, Candace Remcho, Kristel for studies of anatomical relationships between Furhman, and Amy Nostrandt. Dr. Bernard Jortner disNTE activity and sites for axonal degradation sected the hen spinal cord to localize regions of neuropathy in the central nervous system of hens with for study of NTE activity. Hens of the B 138 13 and B2 I B2 1 OPIDN. Other research efforts that may ben- strains were provided by Drs. Paul Siegel and Ann Dunnington of the Department of Poultry Science. The work efit from the capability to determine NTE ac- was partially supported by USDA-Hatch funds. tivity in the small samples used in microassay are those investigating OPIDN in rodent tissues and those in which NTE is being purified REFERENCES and characterized. In addition, microassay for NTE activity could be more useful than con- Anonymous (1985). Neurotoxicity. Federal Register 50, 39,458-39,470. ventional assays when serial samples of lymphocytes are used to monitor potential for BRADFORD, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utiOPIDN in humans and experimental animals lizing the principle of protein-dye binding. Anal. (Johnson, 1982; Dudek and Richardson, 1982; Biochem. 72,248-254. Lotti et al., 1986). Furthermore, the microasBRILES, W. E., BRILES, R. W., TAFFS, R. E., AND STONE, H. W. (1983). Resistance to a malignant lymphoma in say procedure for NTE could be useful and chickens is mapped to subregion of major histocomeconomical for routine screening of a large patibility (B) complex. Science 219, 977-979. number of samples, as this test is required for BROGDON, W. G., AND DICKINSON, C. M. (1983). A miorganophosphorus esters that are proposed to croassay systemfor measuring esteraseactivity and proenter the market classified as insecticides tein concentration in small samples and in high-pressure liquid chromatography eluate fractions. Anal. Biochem. (Anonymous, 1985) because significant in131,499-503. hibition (>70%) of NTE is considered predicDAVIS, C. S., AND RICHARDSON, R. J. (1980). Organotive of subsequent delayed neuropathy (Johnphosphorus compounds. In Experimental and Clinical son, 1982; Davis and Richardson, 1980). Neurotoxicify (P. S. Spencer and H. H. Schaumburg, eds.), pp. 527-544. Williams & Wilkins, Baltimore. Uses of microassay procedures for AChE have been previously reported and include de- DUDEK, B. R., AND RICHARDSON,R. J. (1982). Evidence for the existence of neurotoxic esterase in neural and tection of low levels of organophosphates lymphatic tissue ofthe adult hen. Biochem. Pharmacol. (Hammond and Forster, 1989), determination 31, 1117-1121. of enzyme activity in chromatographic sam- EHRICH,M., BRILES,T. W., BRILES,W. E., DUNNINGTON, ples in purification studies (Brogdon and E. A., MARTIN, A., SIEGEL, P. B., AND GROSS, W. B.

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(1986). Neurotoxicity of triorthotolyl phosphate in chickens of different genotypes in the presence and absence of deoxycorticosterone. Pot&. Sci. 65, 375-379. ELLMAN, G., COURTNEY, K. D., ANDRES, V., JR., AND FEATHERSTONE, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. I, 88-95. GOTTLIEB, S., AND MARSH, P. B. (1946). Quantitative determination of phenolic fungicides. Ind. Eng. Chem. 18, 16-19. HAMMOND, P. S., AND FORSTER,J. S. (1989). A microassay-based procedure for measuring low levels of toxic organophosphorus compounds through acetylcholinesterase inhibition. Anal. Biochem. 180, 380-383. JOHNSON, M. K. (1977). Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol. 37, 113- 115. JOHNSON,M. K. (1982). The target for initiation of delayed neurotoxicity by organophosphorus compounds. In Reviews in Biochemical Toxicology (E. Hodgson, J. R. Bend, and R. M. Philpot, eds.), Vol. 4, pp. 141-212. Elsevier, New York. JORTNER, B. S. (1984). Pathology of organophosphorusinduced delayed neurotoxicity. In Proceedings of the Fourteenth Conference on Environmental Toxicology (J. D. MacEwan and E. H. Vemot, eds.), p. 117. National Technical Information Service, Springfield, VA. LQTTI, M., MORETTO, A., ZOPPELLARI, R., DAINISE, R., RIZZUTO, N., AND BARUSCO,G. (1986). Inhibition of

lymphocytic neuropathy target esterase predicts the development of organophosphate-induced delayed polyneuropathy. Arch. Toxicol. 59, 176- 179. OLAJOS, E. J., AND ROSENBLUM, I. (1981). Membranebound and soluble esteraseactivities in various hen brain regions after diisopropyl phosphorofluoridate and trichlorfon treatment. Neurotoxicology 2,463-470. RAY, R., AND CLARK, 0. E. (1988). A computer-assisted automated calorimetric microassay system for determination of enzyme activity, kinetics, and concentration-response relationship. FASEB J. 2, A 154 1. SIMPSON, I. A., AND SONNE, 0. (1982). A simple, rapid, and sensitive method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation. Anal. B&hem. 119, 424-427.

SPRAGUE, G., SANDVIK, L. L., BROOKINS-HENDRICKS, M. J., AND BICKFORD, A. A. (1981). Neurotoxicity of two organophosphorus ester flame retardants in hens. J. Toxicol. Environ. Health 8, 507-5 18. TANAKA, D., JR., AND BURSIAN,S. J. (1989). Degeneration patterns in the chicken central nervous system induced by ingestion of the organophosphorus delayed neurotoxin tri-ortho-tolyl phosphate: A silver impregnation study. Brain Res. 484, 240-256. YOUNG, J. D., LIU, C. C., LEONG, L. E.. DAMIANO, A., PERSECHINI,P. M., AND COHN, Z. A. (1983). An automated calorimetric microassay system for rapid and quantitative determination of soluble enzymatic activities. BioTechniques 5, 572-576.

A microassay method for neurotoxic esterase determinations.

A microtiter plate reader with an associated computer to average triplicate samples and subtract blanks was used for reading and calculating neurotoxi...
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