EPA    Cholinesterase
          Methodologies
          Workshop
          Abstracts
          Sheraton Crystal City
          Arlington, Virginia
          December 4-6,1991

                               PAGE  01 OF  34

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                                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

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                        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
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               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

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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)
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                 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.
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                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
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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.
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                    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.
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            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.
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               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.
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                            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.
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                     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.
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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.
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             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
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   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.
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          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

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                           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

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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

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     PRESENTERS' OUTLINES

Cholinesterase Methodologies Workshop
        December 3-5,1991
                               PAGE     20 OF    34

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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

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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

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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

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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

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    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

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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

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             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

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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

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   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

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                      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

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                   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

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          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

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          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

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                                                         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.

-------