EPA Cholinesterase
Methodologies
Workshop
Abstracts
Sheraton Crystal City
Arlington, Virginia
December 4-6,1991
PAGE 01 OF 34
-------
TABLE OF CONTENTS
WORKSHOP AGENDA
ABSTRACTS
Characteristics of Esterases: Enzymology and
Biology of Cholinesterases, Stephen Brimijoin
Factors Affecting Cholinesterase Inhibition,
David E. Lenz
Microtiter Assay for Cholinesterases, B.P. Doctor .
Determination of Cholinesterase Activity in Plasma,
Erythrocytes, and Brains of Experimental Animals
Based on EUman's Method, Byong Han (Paul) Chin
Cholinesterase Measurements in Tissues from
Carbamate-Treated Animals, Stephanie Padilla ...
Cholinesterase Data Obtained from 'Historical
Control Animals, Jeffrey M. Charles
Cholinesterase Data Collected from Bioassays Performed
for Hazard Evaluations, Karen Hamemik
Strawman Protocol, Brian Dementi
Existing Guidelines for Assessing Cholinesterase Inhibition
Caused by OPs and Carbamates, Bruce Jaeger
Cholinesterase Inhibitors: Ongoing and Proposed Research
Needs, Hugh A. Tilson
New Opportunities in Cholinesterase Testing Paradigm,
Dan W. Hanke
PARTICIPANT LIST
PAGE 02 OF 34
-------
CHOLINESTERASE METHODOLOGIES WORKSHOP
Sheraton Crystal City
Arlington, Virginia
December 4-5,1991
AGENDA
WEDNESDAY. DECEMBER 4. 1991
7:30AM-4:30PM
8:30AM-8:40AM
8:40AM-9:10AM
9:10AM-9:20AM
9:20AM-9:45AM
9:45AM-10:OOAM
10:00 AM-10:15AM
10:15AM-10:35AM
10:3SAM-10:550AM
10:55AM-11:25AM
11:25AM-11:45AM
11:45AM-12:15PM
Participant and Observer Registration
Opening Remarks
Barry Wilson
Session I - Chemistry/Biology of Esterases
Session Chairperson - David Lenz
Characteristics of Esterases: Enzymology and Biology of Cholinesterases
Stephen-Brimijoin •
Discussion
Factors Affecting Cholinesterase Inhibition
David Lenz
Discussion
Break
Session II- Methods for Measuring ChEI
Session Chairperson • B. P. Doctor
Overview/Microtiter Assay for Cholinesterase
B.P. Doctor
Determination of Cholinesterase Activity in Plasma, Erythrocytes, and
Brains of Experimental Animals Based on EUman's Method
Paul Chin "
Discussion
Radiometric Method: Cholinesterase Measurements in Tissues from
Carbamate-Treated Animals
Stephanie Padilla
Discussion
PAGE 03 OF 34
-------
Session III - Cholinesterase Measurements: Variabilities Observed in Results
Facilitator - Barry Wilson
2-OOPM-2:45PM Cholinesterase Data Collected from Bioassays Performed for Hazard
Evaluations
Karen Hamemik
2:45PM-2:55PM Questions
2'55PM-3:40PM Cholinesterase Data Obtained from Historical Control Animals
Jeffrey Charles
3:40PM-4:OOPM Break
4:OOPM-5:OOPM Discussion
5-.OOPM Wrap-Up
Barry Wilson
TfflWSnAV DEPVMBF.R S. 1991
7:30AM-8:30AM Observer Registration
Recapitulation of Day I
8:30AM-8:40AM Chemistry/Biology Session
David Lenz
8:40AM-8:50AM Analytical Methods
B.P. Doctor
8:50AM-9:OOAM Historical Control and Toricity Testing Bioassay Data
Barry Wilson
Session IV - Protocol/Guidelines for ChE Determinations: Future Research Needs
Session Chairperson - Stephanie PadUla
9:OOAM-9:10AM Introduction
Stephanie PadiOa (not confirmed)
9:10AM-930AM Cholinesterase Assay Strawrnan Protocol
Brian Dementi
9:30AM-10KX)AM Discussion
10:OOAM-10:15AM New Developments in Laboratory Testing
Juergen Thyssen
10:15AM-10:30AM Existing Guidelines for Assessing Cholinesterase Inhibition Caused By
OPs and Carbamates
Bruce Jaeger
10:30 AM-10:45 AM Discussion
10:45AM-11:OOAM Break PAGE 0 4 OF 04
-------
11:OOAM-11:20AM
11:20-11:40AM •
11:40AM-12:OOPM
12:OOPM-12:20PM
12:20PM-1:30PM
1:30PM-4:OOPM
Session V - Future Directions
Session Chairperson - Hugh Tilson
Cholinesterase Inhibitors: Ongoing and Proposed Research Needs
Hugh Tilson
New Opportunities in Cholinesterase Testing Paradigm
Dan Hanke
Discussion
Summary of Workshop
Barry Wilson
Formal Workshop Adjourns/Lunch
Executive Session (Drafting of Summary Reports)
PAGE 05 OF 34
-------
CHARACTERISTICS OF ESTERASES: ENZYMOLOGY
AND BIOLOGY OF CHOLINESTERASES
W. Stephen Brimijoin
Mayo Clinic
Rochester, Minnesota 55905
Among the many classes of esterases, the most important from a physiologic and
toricologic standpoint are the B-esterases, acetyl-cholinesterase (AChE) and
butyrylcholinesterase (BuChE). AChE. concentrated in synapses, is necessary for cholinergic
neurotransmission, but there is a margin of safety and a significant fraction of the AChE activity
must be inhibited before signs of toricity are evident. There is no known function for AChE in
non-synaptic sites like the erythrocyte or for BuChE in plasma and elsewhere, but the activities
of these enzymes can be used as indicators of exposure to environmental anticholinesterases.
To predict and interpret the effects of anticholinesterases on AChE and BaChE. understanding—
of the tissue distribution of the enzymes and their molecular forms is required. Tissue
distribution has major implications for the accessibility of enzyme to toxicants, especially in view
of blood-brain and blood-nerve barriers that block the free diffusion of molecules with low lipid-
solubility. The occurrence of multiple molecular forms of AChE and BuChE is also of crucial
importance. These molecular forms are best understood as assemblies of identical, or nearly
identical, catalytic subunits, with or without accessory molecules that promote attachment to the
cell surface membrane or the extracellular synaptic basal lamina. The functional significance
and regulation of the molecular forms of AChE and BuChE are still being elucidated, but it is
already clear that the enzyme forms have characteristic cellular locations and rates of renewal.
These factors affect both the extent of inhibition and the rate of recovery after
anticholinesterase exposure. Such considerations underscore the need for appropriately
designed tests to determine (a) potential anticholinesterase activity of target compounds in vivo;
(b) biological risks associated with environmental use of these compounds; and (c) actual
adverse effects in individuals and populations at risk. Conventional activity-based assays of
AChE and BuChE are suitable tests for most purposes, while more elaborate immunoassays can
be used in order to define the levels of cholinesterase protein.
P/toE
06 OF 34
-------
FACTORS AFFECTING CHOLINESTERASE INHIBITION
David E. Lervz
Biochemical Pharmacology Branch
US Army Medical Research Institute of Chemical Defense
Aberdeen Proving Ground, MD 21010
Cholinesterases (ChEs) have been classified by their preference to catalyze a particular
substrate. Acetylcholinesterase (AChE), which is found in red cells, the central nervous system, and
in nerve endings, displays a marked preference for the neurotransmitter acetylcholine.
Butyrylcholinesterase (BuChE), which is most prevalent in the plasma as well as a variety of tissues
such as heart, exhibits less substrate specificity but has maximal catalytic activity for the hydrolysis
of butyrylcholine. Both enzymes have been described as having an esteratic site and an anionic site.
With the recent elucidation by Sussman and coworkers of the 3-dimensional structure of torpedo
AChE the traditional model of the active site has been revised. While an-esteratic-site-aas—
confirmed, the anionic site was found to be comprised of aromatic amino acids rather than any
amino acid with a formal positive charge. This finding nay require some reexamination of the large
body of inhibition data that exists for AChE with respect to structure/activity relationships.
However, at this time it does not seem likely that the classical inhibition kinetic model based oh
Michaelis-Menton kinetics will have to be abandoned.
ChEs are inhibited by compounds which can phosphorylate or carbamylate the active site.
Many of the studies were carried out in vitro. Earlier studies that were carried out jij yjy£ were
often incomplete with respect to a detailed analysis of the pharmacokinetics of the inhibition of
ChE in a particular tissue. Within the last few years however there have been a series of
pharmacokinetic studies of the inhibition of ChE (usually AChE) which have examined the effect
of the route of administration of the inhibitor, e.g., intramuscular, inhalation, or intravenous, in
several animal species. The effect of naturally occurring target enzymes other than ChE, such as
carboxylesterase (CaE) on the pharmacokinetics of AChE inhibition has been determined as have
the pharmacokinetics of diastereomeric ChE inhibitors. In all studies, the detoxication of
organophosphorus ChE inhibitors has been markedly affected by the relative tissue concentrations
of CaE. It was also determined that ChE inhibitors can affect blood flow to different organ systems
and that this correlated with the relative extent of ChE inhibition in these organ systems.
<
Often the extent of ChE inhibition has been determined by measuring ChE activity in a
blood sample, either whole blood or plasma, and then those results extrapolated to the whole
organism. Recent studies by Shih, Jimmerson, and Maxwell have revealed that there is a poor
correlation between blood AChE activity and plasma BuChE activity with respect to each other or
to central nervous system (whole brain) AChE activity. These initial findings were confirmed in
subsequent studies and expanded upon to reveal a poor correlation between blood AChE or BuChE
and regional brain AChE activity, suggesting that measurement of blood ChE activity after exposure
to a ChE inhibitor is a poor indicator of central toxicity. These investigators did report a good
correlation between brain AChE activity and toxic signs. They further noted that while blood ChE
activity did not correlate well with brain regional AChE the former was the initial site of ChE
inhibition and as such provided a very sensitive indicator of exposure to a ChE inhibitor.
Studies of ChE inhibition and hence the toxicity of various ChE inhibitors in vivo have
on "F 34
-------
revealed a large variability between animal models. This has often been attributed to 'species
variation* with little additional explanation. Initial studies by Fonnum and Stern identified a role
for CaE in affecting inhibitor toxicokinetics. This was expanded upon by Maxwell who
demonstrated that the concentration of CaE in circulation could explain the difference in toxicity
of ChE inhibitors in different species. The enzyme CaE is particularly prevalent in rodent blood
but almost absent in human and non-human primate blood. By selectively inhibiting the activity
of CaE, the differences in species toxicity could be eliminated. These data must be interpreted
carefully however, because too high of dose of the CaE inhibitor can also affect ChE activity.
Despite the numerous variables that affect the inhibition of ChE, e.g., form of ChE affected,
route of administration of inhibitor, concentration of endogenous CaE, alterations in blood flow,
stereochemistry of the inhibitor, correlation between blood ChE activity and toxicity, inhibitor
phanr.acokinetics, and animal species, there have been attempts to define some unifying models
whereby inhibitor concentration and extent of ChE inhibition in a particular tissue can be
determined. Most of these efforts have centered around the use of physiological pharmacokinetic
modeling. The difficulty with this approach is that much of the data needed to solve the large set
of simultaneous differential equations that describe-ihfi-jhvsiologicjrocess are not available^-
However in one of the few cases where the data were available, Maxwell and coworkers were able
to use this approach to correlate inhibitor concentration with the extent of ChE inhibition in a
variety of tissues. Their preliminary success suggests that this approach may offer a method of
extrapolating toxicity data across species up to and including humans.
References
1. V.R. Jimmerson, T.-M. Shih and R.B. Mailman (1989) Toxicology 57 241-254.
2. V.R. Jiramerson, T.-M. Shih, D.M. Maxwell, A. Kaminskis and R.B. Mailman (1989) Fund.
Appl. Tox. 13 568-575.
3. V.R. Jiramerson, T.-M. Shih, D.M. Maxwell and R.B. Mailman (1989) Toxicol. Lett. 48 93-
KB.
4. D.M. Maxwell and K.M. Brecht (1991) Neurosci. Biobehav. Reviews 15 135-139.
5. D.M. Maxwell, K.M. Brecht, D.E. Lenz and B.L. O'Neill (1988) J. Pharrn. Exep. Ther. 246
986-991.
6. D.M. Maxwell, D.E. Lenz, W.A. Croft A. Kaminskis and H.L. Frdehlich (1987) Tox. Appl.
Pharm. 88 66-76. *
7. D.M. Maxwell, K.M. Brecht and B.L Q-Niell (1987) Toxicol. Lett 39 35-42.
8. D.M. Maxwell, C.P. Vlahacos nd D.E. Lenz (1988) Toxicol. Lett 43 175-188.
08 OF 34
-------
MICROTTTER ASSAY FOR CHOLINESTERASES
B. P. Doctor
Division of Biochemistry
Walter Reed Army Institute of Research
Washington, DC 20307-5100
A microtiter plate adaptation of the Ellman colorimetric procedure for measurement of
acetyl- or butyrylcholinesterase activity is described (Doctor et al. (1987) AnaL gjficjiejn., 166:399-
403). This method uses ELISA plate reader for rapid analysis of multiple samples and is particularly
suitable for analysis of cholinesterase activity on sucrose gradients and column chroraatography
fractions. This procedure is rapid, sensitive, and does not require radioactive material.
This procedure has been used for determination of ChEs in whole blood and plasma
obtained from mice, rats, monkeys, and humans. It is also used in the simultaneous determmarion_
-of-protenrand-AChE of the same sample and to determine tneTinhibition of CBE activity by
^1 LS4 WhMAA* •***»• M • ^^•AAtf **• **»v WMB^W • • • f —
monoclonal antibodies. It is further modified to measure trace amounts of organophosphates.
PAGE 09 OF 34
-------
DETERMINATION OF CHOUNESTERASE ACTIVITY IN PLASMA,
ERYTHROCYTES, AND BRAINS OF EXPERIMENTAL ANIMALS
BASED ON ELLMAN'S METHOD
Byong Han (Paul) Chin
Health Effects Division, Office of Pesticide Programs
U.S. Environmental Protection Agency
Washington, DC 20460
The method described by Ellraan and coworkers (1961) is the principal method used in U.S.
laboratories for determining plasma, RBC, and/or brain cholinesterase (ChE) activity in both human
and laboratory animals. Among the numerous methods, this method has been widely used because
this method is easily automated and the automated method is reproducible, precise, and more easily
performed than the manual method. The method uses acetylthiocholine iodide as substrate which
Is hydrolyzed to thiocholine iodide and acetic acid by the cholinesterase activity in biological tissues.
The liberated thiocholine reduces color reagent, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) to 5-
thio-2-nitrobenzoic acid which is measured spectrophotometrically. This presentation will cover the
following subjects related to the Ellman's method: basis of reactions; optimal concentrations for
substrate, DTNB, and buffer; optimal pH; and spectrophotometric measurement of the end product.
In addition, two most commonly used automated procedures, which are based on a continuous flow
system (e.g., Technicon AutoAnalyzer) and centrifugal system (e.g., CentrifiChem), will be reviewed
in terms of their instrumentation (e.g., reaction time, reaction temperature, and an absorbance
reading); sample output (no. of samples analyzed/hour); and reproducibility of analysis.
10 OF 34
-------
CHOLINESTERASE MEASUREMENTS IN TISSUES FROM
CARBAMATE-TREATED ANIMALS
Stephanie Padilla
Neurotoricology Division (MD-74B)
U. S. Environmental Protection Agency
Research Triangle Park, NC 27711
Between 1964 and 1975, many different investigators, using diverse approaches, attempted
to develop a sensitive, quick, and safe assay for cholinesterase activity using radiolabeled substrate.
The culmination of these efforts was a publication in 1975 by Johnson and Russell describing the
radiometric method which is stilt used today. Briefly, the entire assay takes place in a scintillation
vial with the tissue and substrate (pH]acetylcholine) combined in a total volume of 100 jiL at the
bottom of the vial, acidic buffer is added to stop the reaction, and the scintillant (approximately 5
ml) is then added to extract the_jadlQlab.eledjimducL_Whffn placed in.a-scintfllarion-counter.-only-
the substrate that has been hydrolyzed is counted.
A spectrophotometric assay, by its nature, requires extensive dilution of the tissue in order
for proper passage of the beam of light; therefore, because of this necessary dilution step, the
spectrophotoraetric method may underestimate the amount of cholinesterase inhibition in tissues
from carbaraate-treated animals. On the other hand, because the radiometric method may be
conducted on undiluted or minimally diluted tissues, it would superior for determining
cholinesterase activity in tissues from carbaraate-treated animals. In our laboratory we routinely
use both type of assays to measure cholinesterase activity in tissues collected from animals: the
spectrophotoraetric assay (i.e., Ellman assay [Ellraan et al., Biochem. gharm., 7:88-95]); and a
radiometric assay (Johnson and Russell. Anal. Biochenu 64:229-239,1975). Using these two assays
to determine cholinesterase activity in the same tissue samples, we have determined that the Ellraan
method consistently underestimates cholinesterase inhibition in tissues from carbaraate-treated
animals. We have, however, as have other laboratories, developed strategies for conducting Ellman
assays on carbamate-treated tissues and will make recommendations for storage, preparation, and
analysis of tissues from carbamate-treated animals.
PAGE 11 OF 34
-------
ABSTRACT
CHOLINESTERASE DATA COLLECTED FROM BIOASSAYS
PERFORMED FOR HAZARD EVALUATIONS
Karen L. Hamernik, Ph.D.
HED Cholinesterase Workgroup Representative
Office of Pesticide Programs
United States Environmental Protection Agency
Members of the Health Effects Division (HED) Cholinesterase
Workgroup have prepared a document entitled "Measurement of
Cholinesterase Activity in Toxicological Studies Submitted to the
Environmental Protection Agency in Support of Pesticide
Registration: Analysis of Data Variability. A Summary Report."
The document represents the results of a first effort by the
Workgroup to examine the reliability of the methods being used to
measure Cholinesterase activity in studies submitted to^HED/DPP as
part of the regulatory process. Major objectives were to identify
the methods, protocols, standard operating procedures, and study
designs used by laboratories performing plasma, erythrocyte, and
brain Cholinesterase bioassays for submitted studies, to analyze.
the variability in the data, and to attempt to identify factors
contributing to the variability observed.
Twenty-eight (28) studies from HED toxicology files, 16 rat and 12
dog, were finally chosen for analysis after certain selection
criteria had been applied. Test materials were organophosphorus or
carbamate pesticides. Most of the studies were 1 year (dog) and 2
year (rat) feeding studies although some shorter term studies were
included. In all studies, the Ellman method or a modification
thereof, had been used to measure Cholinesterase activity. The
availability of a Standard Operating Procedure (SOP) from the
testing laboratory was an important, although not absolute, factor
in determining which studies were analyzed.
An overview of the modifications in the Ellman method used in the
studies was prepared from information contained in the SOPs and
hardcopies of the study reports. *
Summarization and comparison of the Cholinesterase assay data and
data variability within and among studies was facilitated by the
use of statistical measures such as the mean and standard
deviation, coefficient of variation, median, and range.
Highlights of the modification overview and data evalutions will be
presented during the talk.
«« 12 OF 34
-------
CHOLINESTERASE DATA OBTAINED FROM
HISTORICAL CONTROL ANIMALS
Jeffrey M. Charles
NACA Representative
Jay Mark Consulting
Chapel Hill, NC 27514
Working with EPA, ihe National Agricultural Chemicals Association (NACA) provided
historical control data on plasma, red blood cell (RBC), and brain cholinesterase (ChE) from six
laboratories chosen by EPA. The laboratories included: Bush Run Research Center, Export,
Pennsylvania; CIBA-Geigy, Summit, New Jersey, Haskell Laboratories, Newark, Delaware; Hazleton
Washington, Vienna, Virginia; Hazleton Wisconsin, Madison, Wisconsin; andMobay Corporation,
Stilwell, Kansas. The laboratories provided the data from all rat and dog studies (that included
iiniiE)JnitMttluUun^^
.
the individual animal data obtained at all time points in the study. Written methodologies,
Standard Operating Procedures (SOPs), and/or protocols documenting the clinical methodologies
employed in performing the ChE assays on plasma, RBC, and brain were also provided. All
modifications of the procedures used over this 5-year period were provided.
A total of 78 studies were provided by the NACA member laboratories. Of the 56 rat
studies provided, 28 were subchronic studies of 2 to 18 weeks in duration, 16 were reproduction
studies of 9 to 22 months of duration, and 12 were 2-year chronic studies. Data from 22 dog studies
were provided. Of these, 10 were subchronic studies of 2 to 13 weeks in duration and 12 were 1-
year chronic studies.
In all the studies examined, ChE activity was assayed according to the modified method of
Ellman et al. Parameters used to evaluate ChE variation included mean +. S.D. and the coefficient
of variation (CV)- CV is a statistical index that has been used in the analysis of non-uniform
parameters such as plasma ChE in animal studies.
Data will be presented describing the variability in the methods used in the six laboratories
in determining plasma, RBC, and brain cholinesterase activity in rats and dogs. Comparison will
be made within each laboratory and within given studies.
PAGE 13 OF 34
-------
ABSTRACT
CHOLINESTERASE ASSAY STRAWMAN PROTOCOL
Brian Dementi, Ph.D.
U.S.E.P.A.
A presentation based upon the Eliraan, et al. cholinesterase
methodology, which examines the various parameters in the
assay in light of contemporary literature and industrial
procedures with the objective of providing guidance in
drafting a more definitive assay procedure for routine
cholinesterase assays. This involves an examination of
issues pertaining to many of the parameters in the assay
ag *"" thoae_ha«iiiq. to-do-wi-th— the-
preparation and storage of tissue samples for assay of
cholinesterase activity.
Examples of topics under discussion include acetylthiocholine
substrate concentration, non-enzymatic hydrolysis of substrate,
dithionitrobenzoate indicator and its stability under various
conditions, alternative indicators, spectrophotometer wavelength
selection, hemoglobin absorption interference, assay temperature,
enzyme unitology, factors of concern in assaying blood cholinesterases
including in particular the handling of ery throcy tes , use of
anticoagulants, non-ionic detergent, distinguishing between
butyrylcholinesterase and acetylcholinesterase in blood
and other tissues such as brain, etc.
14
-------
EXISTING GUIDELINES FOR ASSESSING CHOLINESTERASE
INHIBITION CAUSED BY OPS AND CARBAMATES
Brace Jaeger
EPA Office of Pesticide Programs
Crystal Mall #2
1921 Jefferson Davis Highway
Crystal City, VA 22202
Guidance for conducting cholinesterase assays in laboratory rodents and dogs was outlined
in 1959 by FDA in their booklet: "Appraisal of the Safety of Chemicals in Foods, Drugs and
Cosmetics (edited 1975)". In 1972, EPA outlined, for the first time, its own proposed guidelines
for conducting cholinesterase assays. These were expanded in 1978 to make cholinesterase
measurements a requirement for ragfctrarinn nf f>P< and rartnmarre Tn 1
-------
CHOUNESTERASE INHIBITORS: ONGOING AND PROPOSED RESEARCH NEEDS
Hugh A. Tilson
Neurotoxicology Division
Health Effects Research Laboratory
United States Environmental Protection Agency
Research Triangle Park, NC 27711
Cholinesterase inhibitors are used in many countries to control a variety of pests. These
agents are designed to produce neurotoxicity in the target organism and humans may manifest
neurotoric signs and symptoms following accidental exposure. During the last four years, various
workshops, colloquia, and briefings have been sponsored by EPA to address scientific issues
concerning regulation of these chemicals. During the course of these meetings, several research
needs were identified.
One of the most difficult questions concerns the definition of an adverse effect. Although
there is some consensus that inhibition of brain Cholinesterase may be adverse, more research is
needed to determine the neurotoxicity associated with inhibition of blood Cholinesterase. There
is consensus that inhibition of blood Cholinesterase is a valid indicator of exposure. In addition,*
research is needed to determine the relationship between blood and brain Cholinesterase levels, the
role of various esterases, and possible regional differences in sensitivity to the Cholinesterase
inhibitors. Other endpoints of neurotoxicity, particularly cognitive dysfunction, need to be
developed and validated. A number of studies have indicated that developing organisms may be
differentially sensitive to the effects of Cholinesterase inhibition. In addition, there is some evidence
that repeated exposure to Cholinesterase inhibitors may accelerate the aging process. Other
research is addressing why tolerance develops to some, but not all, effects following repeated
exposure and if residual neurotoxicity exists following cessation of exposure. Research also is
needed to determine the relationship between noncholinergjc effects of these agents with the
manifestation of neurotoxicity. Recent research has indicated that in vitro methodologies might
have utility in the screening of Cholinesterase inhibitors for neurotoxicity, as well as in investigations
of structure-activity relationships and chemicals mixtures.
PAGE 16 OF 34
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDE PROGRAMS
HEALTH EFFECTS DIVISION
CHOLINESTERASE METHODOLOGIES WORKSHOP DECEMBER 3-5 1991
SHERATON CRYSTAL .CITY HOTEL, ARLINGTON, VIRGINIA
NEW OPPORTUNITIES IN CHOLINESTERASE TESTING PARADIGMS
DAN WOLF HANKE, PH. D.
ABSTRACT
With advancing technologies come new research opportunities for
testing and development within the general field of the
cholineraic synapse. Among those opportunities are__the_U5.e-jpf_
cloned and purified cholinesterases (ChEs) as well as cloned
nicotinic and muscarinic receptors and a synthetic receptor.
There are also opportunities that lie within the purview of
intra-neuronal factors affecting developmental aggregation of
acetylcholine receptors (AChRs) and exocytosis of acetylcholine
(ACh) at nerve terminals attendant to nerve impulse transmission.
My presentation today, however, focuses on the development of
three ChEs and their possible use in an in vitro screening
paradigm with all the attendant advantages and disadvantages
associated with in vitro approaches. Two of the ChEs are human
recombinant acetylcholinesterase (AChE) and human recombinant
butyrylcholinesterase (BChE) cloned from brain and liver cDNA
libraries respectively by Mona Soreq et al. at the Hebrew Un of
Jerusalem in Jerusalem, Israel. The third ChE is AChE purified
from fetal bovine serum via affinity chromatography on a
procainamide-sepharose matrix by BP Doctor e^ al. at the Walter
Reed Army Institute of Research (WRAIR) in Washington, DC. The
use of these ChEs in an in vitro testing paradigm could rapidly
and inexpensively generate reproducible, comparative toxicity
data on pesticides. The pesticide inhibition data would be
evaluated relative to AChE or BChE as well as relative to
standard reference organophosphorus (OP) and carbamate compounds.
Determination of the kinetic constants for ChE inhibition could
provide information on acute toxicity. The availability of pure
human and bovine ChEs may assist in directing end-use pesticide
products towards appropriate applications peculiar to human or
animal exposures respectively. The use of these three ChEs in an
in vitro pesticide testing paradigm could help to standardize
laboratory procedures, to generate reliable historical reference
data within and across laboratories, and to assist in
prioritizing pesticides for more cost-effective running the
gauntlet of requisite animal studies.
17 of 34
-------
CHOLINESTERASE METHODOLOGIES WORKSHOP
Sheraton Crystal City
Arlington, VA
December 3-5, 1991
PARTICIPANT LIST
Karl Baetcke
Office of Pesticide Programs (H7509C)
U.S. Environmental Protection Agency
Crystal Mall #2
1921 Jefferson Davis Highway
Crystal City, VA 22202
703-557-7397
FAX 703-557-2147
_SlepheiLBnmijoin_
Professor, Department of Pharmacology
Mayo Clinic
200 1st Street, SW
Rochester, MN 55905
507-284-8165
FAX 507-284-9111
Jeffrey Charles
NACA Representative
JayMark Consulting
207 Longwood Drive
Chapel Hill, NC 27514
919-489-7753
FAX 919-493-3837
Paul Chin
Office of Pesticide Programs (H7509C)
U.S. Environmental Protection Agency
Crystal Mall #2
1921 Jefferson Davis Highway
Crystal City, VA 22202
703-557-4376
FAX 703-557-2147
Brian Dementi
Office of Pesticide Programs (H7509C)
U.S. Environmental Protection Agency
Crystal Mall #2
1921 Jefferson Davis Highway
Crystal City, VA 22202
703-557-7403
FAX 703-557-2147
B.P. Doctor
Director
Division of Biochemistry
Walter Reed Army Institute of Research
Building 40C, Room B041
Walter Reed Army Medical Center
Washington, DC 20307-5100
202-576-3001
FAX 202-576-1304
Karen Hamernik
Office of Pesticide Programs (H7509C)
U.S. Environmental Protection Agency
Crystal Mall #2
1921 Jefferson Davis Highway
Crystal City, VA 22202
703-557-5467
FAX 703-557-2147
Daniel Hanke
Office of Pesticide Programs (H7509C)
U.S. Environmental Protection Agency
Crystal Mall #2
1921 Jefferson Davis Highway
Crystal City, VA 22202
703-557-0357
FAX 703-557-2147
Bruce Jaeger *
Office of Pesticide Programs (H7509C)
U.S. Environmental Protection Agency
Crystal Mall #1
1921 Jefferson Davis Highway
Crystal City, VA 22202
703-557-4369
FAX 703-557-2147
PAGE
18 OF 34
-------
Page Two
David E. Lenz Pam DiBona
Chief, Biochemical Pharmacology Branch Eastern Research Group, Inc. (ERG)
Department of the Army 6 Whitteraore Street
Medical Research Institute of Chemical Arlington, MA 02174
Defense, Building E 3100 617-641-5324
Aberdeen Proving Ground, MD 21010-5425 FAX 617-648-3638
301-671-2372
FAX 301-676-7045
Stephanie Padilla
Neurotoxicology Division (MD-74B)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
919-541-3956
FAX 919-541-4849
Hugh Tilson
Neurotoxicology Division (MD-74B)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
919-541-2671
FAX 919-541-4849
Juergen Thyssen
Mobay Corp.
Corporate Toxicology
17745 S. Metcalf
Stilwell, KS 66085
913-897-9272
FAX 913-897-9125
Barry W. Wilson
Professor
Department of Avian Sciences
3202 Meyer Hall
University of California
Davis, CA 95616
916-752-1300/752-3519
FAX 916-752-8960
Contractor Support
Kate Schalk
Eastern Research Group, Inc. (ERG)
6 Whittemore Street
Arlington, MA 02174
617-641-5324
FAX 617-648-3638
PAGE 19 op 34
-------
PRESENTERS' OUTLINES
Cholinesterase Methodologies Workshop
December 3-5,1991
PAGE 20 OF 34
-------
EPA Workshop on Cholinesterase
CHARACTERISTICS OF ESTERASES - ENZYMOLOGY AND BIOLOGY OF
CHOLINESTERASES by W. S. Briroijoin, PhD.
Professor of Pharmacology
Mayo Clinic
Rochester, MN
I. INTRODUCTION
A. Overview of Esterases
1. Enzyraology of Esterase Classes
a) A-Esterases (Arylesterase)
b) B-Esterases (AChE, BuChE, Carboxylesterase)
c) C-Esterases (Acetylesterase)
U. GENERAL CONSIDERATION OF CHOLINESTERASE
ArMotecutar-Fbrras
1. Structural Organization
2. Dynamic Interrelations
3. Molecular Biology
B. Cellular and Subcellular Distribution
1. Intracellular Sites
2. Membrane Anchoring
3. Extracellular Sites
C. Functional Roles
1. Synaptic
2. Other
D. Brief Introduction to Assay Methods
m. ACUTE AND CHRONIC INHIBITION
A. Accessibility'to Inhibitors
1. Routes of Inhibitor Access
2. Blood vs. Peripheral Tissues
3. Central Nervous System
B. Turnover and Replacement
C Signs of Inhibition
1. Overt Signs
2. Biochemical Determinations on Tissues and Fluids
3. Combined Assays for Enzyme Activity and Immunoreacnvity
IV. CONCLUSION
PAGE 21 OF
-------
EPA Workshop on Cholinesterase Methodology
Session I
FACTORS AFFECTING ChEI by David E. Lenz
I. INTRODUCTION
A. Three-Dimensional Structure
1. Active Site
2. Channel
B. Classic Kinetics of Inhibition
C. Issues of Importance
1. Metabolism/Pharraacokinetics
2. Blood ChE Activity vs CNS
3. CaE Activity vs Species Toxcity
H. METABOUSM/PHARMACOK1NETICS
'A. Classic Studies of Where Organophosphorus (O) Compounds Bind
B. Recent OP/AChE Pharmacokinetic Studies
1. Billy Martin
2. David Lenz
a) CaE Inhibitor Absent
b) CaE Inhibitor Present
3. Hendrick Benschop '
a) Stereoisomer Considerations
b) Species Differences
C Blood Flow and Affect on ChE Inhibition in Tissue
m. ACTIVITY OF ChE IN BLOOD vs CNS
A. Source of Enzyme vs Activity After Inhibition
1. Blood AChE/BuChE
2. Brain ChE
B. Activity vs Behavioral Effects
1. Blood ChE
2. Regional Brain ChE
3. Behavioral Measurements
IV. CaE ACTIVITY vs TOXICTTY
A. Effect of Dose of Inhibitor on CaE vs ChE Activity
B. Effect of CaE Activity on Species Toxicity
V. SUMMARY
A. Recapitulation of Problem in Light of Current Information
B. The Potential of Physiological Pharmacokinetic Modeling to Extrapolate from
Extent of ChE Inhibition to Toxicity
22 * 34
-------
METHODS FOR MEASURING CHOLINESTERASE INHIBITION AN OVERVIEW
B. P. Doctor, Director, Division of Biochemistry
Walter Reed Army Institute of Research
I. INTRODUCTION
A. Methods: Ellman, Titermetric, Radionetric, (Johnson
and Russell), Others
1. Principles of each method
2. Areas of applicability for each method
3. Cost, Ease, reliability, and reproducibility
4. Data collection, interpretation and time factor
B. Factors: (Effecting Ellman Method)
1. Temperature of reaction
2. Reactants, substrates
3. Reaction volume, buffer, pH
-4-^Sene-artei-vitiy to light •
5. Wavelength
6. Interference by presence of contaminants
7. Enzyme standard
8. Hydrolysis (non-enzymematic) of substrates
C. Sample Preparation
1. Blood, plasma, erythrocytes
2. Dilution, how much in what media
3. Haemoglobin interference
4. Acquisition, transportation, storage, shelf-life
5. Periodicity (more than one sample)
6. Brain, muscle other
D. Additional Considerations
l. Rate constants of carbamates
2. Rate constants of organophosphates
3. Use of inhibitors to distinguish various ChEs
4. Distribution, clearance and Circulation rate of
carbamates and OP's
5. One enzyme source for standard
E. Alternate Methodology.
1. Modified Ellman method/Micro-titer plate assay
2. Biosensor techniques
3. Other
PAGE 23 OF 34
-------
CHE WORKSHOP (Dec. 3-5, 1991)
OUTLINE
DETERMINATION OF CHOLINESTEPASE ACTIVITY IN PLASMA, ERYTHROCYTES ,
AND BRAINS OF EXPERIMENTAL ANIMALS BASED ON ELLMAN'S METHOD
Byong Han (Paul) Chin, Health Effects Division, Office of Pesticide
Programs
U.S. Environmental Protection Agency, Washington, DC 20460
I . INTRODUCTION
A. Commonly used methods for determining cholinesterase
activity in both human and laboratory animals
spectrophotometric method of Eiiman and co-workers
(1961)
2. Electrometric method of Michel (1949)
B. Other methods
1. Titrimetric method
2. Radiometric method
3. Fluorimetric method
II. ELLMAN'S METHOD
A. Basis of reactions
B. Conditions of assay
1. pH
2. Color reagent, 5,5-dithio-bis-2-nitrobenzoic acid
(DTNB)
3. Substrate concentration
4. Buffer concentration
III. INSTRUMENTAL ANALYSIS BASED ON ELLMAN'S METHOD
•r
A. Based on continuous flow system (e.g., Technicon
AutoAnalyzer)
B. Based on centrifugal system (e.g., CentrifiChem)
1. Instrumental settings (e.g., reaction time,
reaction temperature, and an absorbance reading)
2. Sample output (No. of samples analyzed/hour)
3. Reproducibility of analysis
PAGE 24 OF 34
-------
Use of the Radlometric Method for Chollnesterase Measurements:
Advantages and Disadvantages
by
Stephanie Padllla, Ph.D.
Neurotoxioology Division
U.S. Environmental Protection Agency
Research Triangle Park, NO 27711
I. BRIEF HISTORY OF THE DEVELOPMENT OF THE METHOD
II. OUTLINE OF THE PRESENTMETHOD__
— •
III. DISADVANTAGES OF THE RADIOMETRIC METHOD
A. Endpoint Reaction
B. Employs a radioactive Isotope
C. Requires a scintillation counter
D. Generates hazardous waste
IV. ADVANTAGES OF THE RADIOMETRIC METHOD
A. Extremely sensitive; wide range of linearity
B. Quick ;
C. Can use undiluted tissue samples >
1. Radlometric method limits dilution-Induced dephosphorylatlon
anddecarbamylation. •
2. An example comparison of the spectrophotometrle vs.
racfiometric method for measuring AChE in carbamate-
treated tissue. :
V. CONCLUSIONS AND RECOMMENDATIONS
PAGE 25 OF 34
-------
CIIOLINE3TERASE DATA COLLECTED FROM BIOASSAYS PERFORMED FOR HAZARD
EVALUATIONS
Speaker: Karen L. Haraernik, Ph.D.
Office of Pesticide Programs
United States Environmental Protection Agency
I. Introduction
A. Project purpose, scope, and participants
B. Project goals
1. Examine choBnestsrase (ChE) method reliability
a. Identify methods/protocols TLsed
b. Analyze ChE data variability
c. Attempt to identify factors contributing to
-variabflityobserved"
2. Build ChE historical control database
n, How Studies Containing ChE Data Were Selected For Analysis
HL How Studies Were Analyzed
A. Summarization and comparison of methods, modifications,
protocols, and procedures used
5, Data collection, statistical treatment, and summarization
TV. Parameters Examined For Potential Contribution to Data Variability
A. Results in control animals
L Species/strain
2. Sex
3. Age
4. Type ChE measured (plasma, erylhrocyte, brain)
5. Method related-
a. Protocol, laboratory Standard Operating Procedure (SOP),
method modifications
b. Instrumentation
c Sample collection
d. Sample processing
B. Variability in data from animals treated with an
organophosphonis agent or a carbamaie
C. Other considerations
(Intra- and interlaboratory differences in absolute
ChE activity measurements)
PAGE
26 OF 34
-------
c^clinesterase Assay Strawman Protocol
by Brian Dementi
U.S.E.P.A. Office of Pesticide Programs
I. pTTRODUCTTON
II. gpOTjINSSTSRASE ASSAY PROTOCOL
a. Scope
Methodology for cholinesterase activity in plasma,
erythrocytes, brain and other tissue, as derived
from Ellaan, additional journal publications and
industrial SOPs
b. Assay Procedure
1. Laboratory equipnent, reagents, etc.
2. Present draft procedure with item by_Jt.fm rpview-o£-
psraffieters for consideration by workgroup
c. Seaeiraen Collection/Preoqra'fclon
Review technical aspects of the
obtaining, preparation, star age, etc. of biological
samples
d. Reporting of Results
e. Statistical Analysis of,Data
r."T 31 OF
-------
EXISTING GUIDELINES FOR ASSESSING CHOLINESTERASE INHIBITION CAUSED
BY OPs AND CARBAMATES
I. BRIEF HISTORY
A. 1959: FDA - Appraisal of the Safety of Chemicals in Foods,
Drugs and Cosmetics (ed. 1975)
2 species: rat and dog
blood: plasma and erythrocytes
10 males/10 females per dose
pre-dose measurements: 5 weekly determinations
dosing: levels from maximum tolerated to "no effect level"
enzyme measurements: beginning of test period at one and
two weeks and then biweekly up to 12 weeks in 5 rats
of each sex per group
post-dose measurements: same 5 M/F at 1 and 4 weeks after
..treatment.
NOTE: " when plasma and erythrocyte provide the most
sensitive index of cumulative effect, the response
in dog has approached very closely that in the
human." 4M/4F per dose; use each as their own
control; 5 weekly pre-dose levels on each dog;
during test, measure at 1 and 2 weeks then biweekly
for duration of 90 day study.
B. 1972: EPA - Proposed Toxicology Guidelines
2 species: rat and dog
tissues: plasma and rbc
brain at termination
test duration: rat - 90 days and 2 years
dog - 90 days or 6 months
number of animals: rat - 15 M/F (90 day); 25-50 M/F (2 yr)
dog - 4M/4F
dosing: 3 levels from effect to no-effect level, plus
control
enzyme measurements: 3, 6, 12, 18 and 24 months (rat)
3 and 6 months (dog)
C. 1975: EPA - Guidelines for Registering Pesticides in U.S.
(Proposed)
Neurotoxicity: "The rat or dog is appropriate for
demonstrating acetylcholinesterase inhibition."
D. 1978: EPA - Proposed Guidelines for Registering Pesticides
in the U.S.; Hazard Evaluation: Humans and Domestic
Animals
2 species: rat and dog
tissues: plasma and rbc
brain at termination
test duration: 90 days (rat); 6 months (dog)
number of animals: rat - 10 M/10F
dog - 6 M/6 F
dosing: 3 levels plus control, from no-effect to effect
PAGE 28 OF 34
-------
enzyme measurements: twice before, twice during and at
termination. In dogs, serial determinations may be
useful to provide a time course of development of
inhibition, extent of inhibition, and recovery from
inhibition (e.g. after removal from treated diet).
Dogs should be fasted 1 day prior to obtaining blood
samples.
test duration: 24 - 30 months (rat) enM/Kn-v
number of animals: at least 8/sex/group (out of 50M/50F)
dosing levels: 3 plus control .
enzyme measurements: before dosing, every 6 months during
and at termination.
E. 1982 and 1984 (Revised): EPA - Pesticide Assessment
Guidelines, Subdivision F, Hazard Evaluation: Humans
and Domestic Animals
species •. rat and_dog
tissues: not stated
duration: 90 day
number of animals: rat - 10 M/10F
dog - 4M/4F
dosing levels: 3 plus control
enzyme measurements: rats - termination
dogs - beginning, monthly intervals or midway
through the test and at termination
testing duration: rat - 12 -24 months
dog - 12 months
number of animals: rat - 10 M/10F out of 20M/20F
dog - 4M/4F
dosing levels: 3 plus control
enzyme measurements: at least 3 times; beginning,
middle and end
F. 1991: EPA - Pesticide Assessment Guidelines, Subdivision
F, Hazard Evaluation: Human and Domestic Animals
Addendum 10 - Neurotoxicity
Delayed Neurotoxicity of OP Substances Following Acute and
28 Day Exposures
Study Conduct: Biochemical Measures - (A) NTE Assay.
"Depending on the duration of acute signs as an indication
of the disposition of the test material, the time for
sacrifice for NTE and AChE assessment may be chosen at a
different time to optimize detection of effects."
(B) AChE Measures. "Assay of acetylcholinesterase in the
brains of the same birds shall also be performed."
References. Ellman, G.L. Bioehem. Pharmacol. 7:88-95
(1961).
PAGE 29 OF 34
-------
OVERVIEW OF TESTING GUIDELINES
DOCUMENT
1959
FDA
1972
EPA GL
1978
EPA GL
1982/84
PRE-DOSE PERIODIC
MEASURES
5 weekly 1, 2 wks
biweekly
for 12 wks
N.S. 3, 6, 12,
18 and 24
months (rat)
3 , 6 mos .
(6 mo. dog)
2 X before 90 day
2 X during
and term
Before 24-30 Month
every 6 mos.
and term.
N.S. 90 day
POST-DOSE
MEASURES
1 and 4
weeks
N.S.
N.S.
N.S.
N.S.
TISSUES METHOD
pi , rbc N.S.
pi , rbc , N.S.
brain
(term)
pi , rbc , N.S.
brain
(term)
pi, rbc, N.S.
brain
(term)
pi, rbc, N.S.
brain
(term)
EPA GL
1991
EPA GL
Neurotox
(Hen)
term, (rat); N.S,
begin, monthly
or midway, term
(dog)
1-2 vr
begin, midway, N.S.
term.
N.S.
Term.
N.S.
N.S.
brain
N.S,
N.S.
Ellman
(1961)
PAGE 30 OF 34
-------
RESEARCH NEEDS: ONGOING AND PROPOSED
Hugh A. Til son, Neurotoxicology Division, Health
Effects Research Laboratory, U.S. EPA
Research Triangle Park, North Carolina
I. INTRODUCTION
A. Cholinesterase Inhibitors in the Environment
B. Recent Meetings on Cholinesterase Inhibitors
1. June 1988, Risk Assessment Forum Colloquium
2. May 1989, FIFRA SAP Open Meeting
3. September 1989, Joint FIFRA SAP/SAB Open Meeting
4. May 1991, OPP/HERL Briefing on Research Needs
II. IDENTIFIED RESEARCH NEEDS
A. Definition of Adverse Effect
1. Use of Cholinesterase levels in blood, brain
2. Regional differences in brain
3. Different esterase forms
4. Use of neurobehavioral endpoints
B. Appropriate Surrogate for Human
1. Critical evaluation and analysis of species
2. Different species of rodents
3. Different strains of rats
C. Developing Organism
1. Brain acetylCholinesterase and development
2. Relative sensitivity of developing organism
D. Repeated Exposure
1. Tolerance to some but not all effects
2. Residual effects following cessation of exposure
E. New Issues
1. Accelerated aging and delayed onset neurodegeneration
2. Ocular toxicity
3. In vitro neurotoxicology
PAGE 31 OF 34
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDE PROGRAMS
HEALTH EFFECTS DIVISION
CHOLINESTERASE METHODOLOGIES WORKSHOP DECEMBER 3-5 1991
SHERATON CRYSTAL CITY HOTEL, ARLINGTON, VIRGINIA
NEW OPPORTUNITIES IN CHOLINESTERASE TESTING PARADIGMS
DAN WOLF HANKE, PH. D.
OUTLINE
I. Opening remarks.
II. Recombinant human acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE).
Generation and expression of the cDNA clones according to
Mona Soreg et al.
Use of synthetic oligodeoxynucleotide probes to search cDNA
libraries originating from fetal and adult brain to identify
o cDNA clones for AChE and to search cDNA libraries
originating from fetal and adult liver to identify cDNA
clones for BChE.
o Generation of multiple copies of the AChE and BChE cDNAs
using the polymerase chain reaction (PCR).
o in vitro transcription of the synthetic mRNAs from the AChE
and BChE cDNAs
Translation of the synthetic mRNAs encoding for AChE and
o BChE in microinjected Xenopus oocytes to generate and to
characterize the recombinant AChE and BChE. *
Mass expression of the AChE and BChE synthetic mRNAs in
o bacteria- Escherichia coli- to generate large quantities of
the recombinant AChE and BChE.
III. AChE from fetal bovine serum (FBS).
Affinity chromatography preparation of electrophoretically
pure AChE according to BP Doctor et al.
Cytotoxic serum, unusable for tissue culture, is mixed with
o sepharose attached to procainamide, which is the ligand
binding the anionic site of AChE.
IHGE T? QF
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
CHOLINESTERASE METHODOLOGIES WORKSHOP 3-5 DECEMBER 1991
NEW OPPORTUNITIES IN CHOLINESTERASE TESTING PARADIGMS
DAN WOLF HANKE, PH. D.
o pack the mixture onto a column and elute the AChE off the
column with decaasethcnium.
o The collected AChE is assayed and electrophoresed to
establish specific activity and purity respectively.
IV Suggested in vitro ChE screening paradigm using pure human
recombinant AChE and BChE as well as AChE from FBS
Set standard enzyme activity control rate of reaction (slope
o of curve fT absorption plotted aqalast-tiae) that i-s—1-i-near
for~~at~Teasiri5 min regardless of assay method.
Set standard negative enzyme control with DFP or
o physostigmine at 20 % inhibition or use discrete increments,
e.g., 20 %, 50 %, and 75 % inhibition.
Test pesticide against the appropriate negative control at
the same concentration(s). Result will be an immediate
measure of ChE toxicity relative to the negative control.
o Also will have ability to immediately differentiate between
the pesticide toxicity relative to each specific ChE, which
is comparatively more difficult to do from a blood sample
containing both ChEs thereby requiring selective inhibitors.
May decide to stop here at this point if the relative
toxicity of the pesticide is "too great" to warrant testing
in vivo. Otherwise, continue on with the paradigm and
o determine next the rate constants, Kd, k,, and kz, for
pesticide inhibition of each ChE. Inferences can be made
from the rate constants whether acute toxicity may be
involved.
If the pesticide toxicity to the ChEs is "significant" then
o test oximes as antidotes for accidental poisoning and for
use on labels.
Run animal studies after the previous step (or in parallel
o from the outset). The in vitro data can help to prioritize
an order for testing an array of pesticides in animals based
on their toxicity to the pure ChEs.
PAGE 33 OF 34
-------
PROTECTION
rufti TMr.om»«. ENVIRONMENTAL i-KOTECTI
CHOLINESTERASE METHODOLOGIES WORKSHOP 3-5
NEW OPPORTUNITIES IN CHOLINESTERASE 1
DAN WOLF HANKE, PH. D
TES1ING
v-
A. Advantages.
° relatively inexpensive.
o quick and easy to do- same day results.
AGENCY
DECEMBER 1991
PARADIGMS
*- «**
o the ChEs can be stable for at least one year when stored properly.
CO
CO
pestlciae
rate constants may be used to estimate acute toxicity.
°Xiffle"reaCtiVati°n screeni"9 «n identify
B. Disadvantages.
o availability on contract and commitment to quantity.
and food
uses in or around cattle
antidotes for labeling
higher with the additional
' down-the-lirie.
------- |