United States
                                   Environmental Protection
                                   Agency
                                   Health Effects Research
                                   Laboratory
                                   Research Triangle Park NC 27711
AEPA
                                   Research and Development
                                   EPA-600/S1-81-043  July 1981
Project  Summary
                                   Development  of  an In   Vitro
                                   Neurotoxicity  Assay
                                   Mary L. Barth and Rudy J. Richardson
                                     The aim of this project was to inves-
                                   tigate the development of a totally in
                                   vitro neurotoxicity assay system using
                                   the enzyme neurotoxic esterase. Such
                                   a system was sought to allow rapid
                                   assessment of a large number of com-
                                   pounds for neurotoxicity.
                                     In addition to a thorough biochemical
                                   characterization of several tissues
                                   containing neurotoxic esterase,  a
                                   phylogenetic study of NTE occurrence
                                   was conducted. This search was made
                                   to identify sources of NTE for study.
                                     Purified or partially purified neuro-
                                   toxic esterase was to be immobilized
                                   on a solid matrix for eventual use in a
                                   continuous flow reactor.
                                     This Project Summary was  devel-
                                   oped by EPA's Health Effects Research
                                   Laboratory, Research Triangle Park,
                                   NC, to announce key findings  of the
                                   research project that is fully  docu-
                                   mented in a separate report of the
                                   same title (see Project Report ordering
                                   information at back).

                                   Introduction
                                     One of the major concerns of the U.S.
                                   Environmental Protection Agency's
                                   Health Effects Research Laboratory is
                                   the testing and assessment of chemical
                                   pesticides for toxicity. Of particular
                                   concern are widely used pesticide com-
                                   pounds that may adversely affect human
                                   health.
                                     Many organophosphorus (OP) com-
                                   pounds are well suited for use as pesti-
                                   cides because of their natural anti-
                                   cholinesterase activity. However, these
                                   compounds, as well as industrially used
                                   OP compounds without anticholinester-
                                   ase  activity, can produce a delayed
                                   neurotoxic effect resulting in the distal
                                   degeneration of nerve axons. This often
                                   irreversible effect, characterized by
                                   concomitant ataxia and paralysis,  is
                                   called organophosphorus distal axon-
                                   opathy (OPDA).
                                     Neurotoxic esterase (NTE) has been
                                   identified as the enzymatic site of the
                                   primary biochemical lesion observed  in
                                   OPDA. This membrane-bound phenyl
                                   valerate hydrolase is found in the neural
                                   tissue of the adult hen. The neurotoxic
                                   potential of organophosphorous com-
                                   pounds is thus often predicted by dosing
                                   adult hens  in vivo and assaying brain
                                   NTE activity after a suitable interval
                                   (usually 24  hours).
                                     This study was undertaken by the
                                   University of Michigan  in support of the
                                   Health Effects Research Laboratory's
                                   research goal of developing reliable, yet
                                   rapid and inexpensive, in vitro methods
                                   for assaying chemicals. Specifically, the
                                   study was  initiated to determine the
                                   feasibility of developing an in vitro assay
                                   system employing isolated NTE as an
                                   indicator of neurotoxicity.  This system
                                   was based on the differential sensitivity
                                   of phenyl valerate hydrolyzing esterase
                                   to neurotoxic and non-neurotoxic in-
                                   hibitors. Using the assay, a compound
                                   (i.e., inhibitor) was designated "neuro-
                                   toxic" strictly on the basis of its ability to
                                   produce OPDA. That is, even if it pro-
                                   duced acute neurotoxicity, it was called
                                   "non-neurotoxic" unless  it produced
                                   the specific  syndrome of OPDA.
                                     The development  of an  effective in
                                   vitro system required that a thorough
                                   characterization of NTE be made and
                                   that potential sources of NTE be identi-
                                   fied. Previously, the presence of NTE

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had been established only for hen brain.
To locate and characterize other NTE
sources, several tissue types from hens,
chicks, rats, and humans suspected of
having NTE activity were assayed. In
addition, a phylogenetic search for NTE
occurrence was conducted. (This search
was not conducted to find sources of
NTE for this study, but more for general
informational purposes.)
  For the tissues chosen, biochemical
characterization of NTE activity  was
achieved by generating and comparing
inhibitor titration curves and by deter-
mining  inhibitor lso values, substrate
hydrolysis ratios, thermal inactivation
curves, and  pH profiles. These param-
eters were used to assess the similarity
of NTE activity from different sources or
after  different treatments, such as
solubilization  and immobilization. To
assess the relative merit of different
NTE preparations for eventual use as a
component in the in vitro test system,
additional measurements of stability
were made under various conditions of
medium composition and temperature.
  Complete neurotoxic and non-neuro-
toxic inhibitor titration  curves were
done for the following tissues of the
hen: brain, spinal cord, peripheral
nerve, red muscle, white muscle, heart,
liver, kidney, spleen, and lymphocytes
isolated from spleen and blood. Titration
curves were also generated for chick,
rat, and human brain lymphocytes and
platelets.
  For hen and chick brain tissue charac-
terizations, homogenates were prepared
from the brain tissue of White Leghorn
"spent" hens and White Leghorn chicks.
Paired  samples of tissue homogenate
were preincubated in buffer with either
a non-neurotoxic inhibitor (paraoxon) or
paraoxon and a neurotoxic inhibitor
(mipafox). After preincubation, a phenyl
valerate dispersion was added to the
solution and vortexed. Following an
antipyrene dye reaction, the NTE activity
was determined by  measuring  the dif-
ference in absorbance  between the two
conditions (i.e., paraoxon and paraoxon
+ mipafox). This assay was modified as
required for individual experiments or
for use of tissues other than brain (e.g.,
lymphocytes and platelets).
  Paraoxon titration curves were gener-
ated by varying the paraoxon concen-
tration  in the preincubation mixture
over a concentration range. For tissues
that showed very high phenyl valerate
esterase activity at low inhibitor con-
centrations,  an aliquot of the preincu-
bation mixture was used for incubation
with phenyl valerate. For each tissue
studied, inspection of the paraoxon
titration curve, together with consider-
ation of the absolute activity remaining
at the higher paraoxon concentration,
yielded an optimum paraoxon concen-
tration for use in the mipafox titration.
  Mipafox titration curves were gener-
ated by including paraoxon in the pre-
incubation medium (at the selected
optimum concentration) and  varying
mipafox over a concentration range.
  When working with lymphocytes
isolated from  spleen  and  blood, the
assay and reagent volumes were reduced
by a factor of  20 to accommodate the
small  amounts of tissue available. For
peripheral nerve and small quantities of
brain tissue, assay and reagent volumes
were reduced by a factor of 2.
  Results of the phylogenetic search for
NTE activity showed NTE to be present
in the neural tissue  of birds, fish,
reptiles, amphibians, and mammals
whose ancestors evolved in the last 400
million years. Sheep, steer, lizard, newt,
skate, shark, and sea lamprey were
examined. Apparent NTE activity was
found in all of these species,  with the
possible exceptions of skate and shark.
No evolutionary pattern was discernible
from the species examined.
  Paraoxon and  mipafox  preincubated
in hen brain homogenate produced
titration curves  which confirmed the
presence of  NTE in hen brain. The
presence of NTE in spinal cord and
peripheral nerve tissue was also con-
firmed.
  No  NTE activity was found for red
muscle, white muscle,  liver, or kidney
tissue in the hen. For heart muscle, NTE
activity was tentatively identified at 14%
that of brain on a per gram wet weight
basis. Spleen and blood lymphocyte
curves were very similar to brain curves,
and lso values were also similar to brain
Iso values using paraoxon.
  Differential titration curves for human
lymphocytes and platelets were very
similar to those obtained from hen brain
and lymphocytes, and showed the same
plso for  mipafox. Rat brain tissue was
shown to have only about half the NTE
activity of hen brain. NTE was relatively
plentiful in both the lymphocytes and
platelets of the human.
  In order to compare the properties of
the NTE obtained from different sources,
the following enzymological character-
istics were used: heatdenaturation, pH-
activity relationship, substrate specificity.
and inhibitor specificity. Lymphocyte,
platelet, and brain phenyl valerate
hydrolases from hens, rats, and humans
were  compared with respect to these
characteristics.
  Preliminary heat denaturation experi-
ments showed 55°C to  be optimal for
studies using hen NTE. For rat enzyme,
50°C  was used. Brain and lymphocyte
homogenates were preincubated at
these temperatures, then diluted with
buffer. Aliquots of these dilutions were
assayed for NTE activity; brain homoge-
nate was also assayed for anticholines-
terase.
  Hen lymphocyte and brain NTE dena-
tured  at similar rates. However, acetyl-
cholinesterase denatured at a different
rate, indicating that NTE denaturation is
not simply due to general denaturation
of proteins at this temperature. Rat NTE
denatured very rapidly at 55°C, differing
significantly from the hen enzyme. A
similar increased  heat sensitivity was
not observed in the chick. Thus, either
this characteristic is not related to both
species'  resistance to OPDA, or the
species and age resistance are due to
distinct mechanisms.
  Hydrolysis ratios were used to infer
that the activity in two tissue homoge-
nates was attributable to the same
enzyme(s). The hydrolysis  ratio,  at a
specific inhibitor  concentration,  was
calculated by dividing the specific activity
obtained for phenyl valerate by that
obtained for phenyl phenylacetate. This
ratio  was plotted against mipafox or
diisopropyl fluorophosphate (DFP) con-
centration. The ratios for each tissue at
each inhibitor concentration were quite
similar, indicating that  the hydrolase
activity in these tissues was attributable
to the same enzyme. A striking feature
of the data is that these enzymes behave
similarly in the two tissues with respect
to the two inhibitors, and moreover, the
ratio of substrate hydrolysis changed in
the same way for both tissues.
  Since the tissue distribution portion
of the study revealed the presence of
NTE in peripheral lymphocytes, and this
enzyme had biochemical characteristics
very similar to NTE in hen brain, the
possibility of  using  lymphocyte  NTE
activity as an indicator of central nervous
system NTE activity was explored. Dose-
response curves were generated frorr
brain and lymphocyte tissues dosec
orally with tri-orr/?o-cresyt phosphate
(TOCP). The correlation coefficient for i
plot of percent NTE inhibition in  braii
versus lymphocytes was 0.92. Thit
suggested that lymphocyte  NTE couli

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 be used to monitor brain NTE activity
 and detect neurotoxic exposure in the
 hen.
   This correlation was expanded to
 include a variety of other neurotoxic and
 non-neurotoxic compounds. After initial
 experiments, 4 h post-dose was adopted
 as the time before assay. The response
 of brain and lymphocyte NTE activity at 4
 h  post dose to the other  neurotoxic
 compounds chosen to expand the cor-
 relation gave  a correlation coefficient
 for a plot of percent inhibition of  lym-
 phocyte NTE versus percent inhibition
 of brain NTE at 0.85. This result showed
 that lymphocyte NTE was a good monitor
 of brain  NTE  and could  be  used  as a
 biomonitor of delayed neurotoxicity
 exposure and  potential in hens.
   However, the data obtained did not
 demonstrate  conclusively that  hen
 brain NTE and the enzyme operationally
 defined as NTE in lymphocytes were
 identical. Dosing with non-neurotoxic
 compounds caused a stimulation of NTE
 activity, i.e., there was a dichotomy of
 response in lymphocyte NTE to neuro-
 toxic and non-neurotoxic compounds.
 Proof that  the enzymes are the same
 must await further work on isolation of
 the enzyme from both  tissue sources.
 However, the similarities in response to
 heat, pH, substrates, and inhibitors in
 vitro, as well  as a similar response to
 neurotoxic OP compounds 4 h after
 dosing, support the idea that NTE exists
 in lymphocytes as well as in neural
 tissue and can be used as an indicator of
 neurotoxicity.
   In order to obtain NTE for solubiliza-
 tion, and ultimately'for immobilization,
 subcellular fractionation of hen brain
 was carried out. Up to 70% of the NTE in
 brain was recovered in  the microsomal
 fraction (Pa). The objective of the solubili-
 zation experiments was to maximize the
 amount of NTE solubilized, while mini-
 mizing NTE.activity lost from detergent
 inactivation.
  Once optimal solubilization conditions
were determined through stability stu-
dies, microsomal NTE was obtained for
 use in solubilization experiments. All
fractions—microsomes,  microsomes
plus detergent, a supernatant obtained
by microsome-detergent centrif uging,
and a microsomal pellet—were assayed
for NTE activity. Inhibitor characteristics,
pH activity, and pH denaturation were
investigated. Studies were conducted in
a systematic manner, maintaining strict
attention to detergent/protein ratios.
  Ovine and bovine brain were investi-
gated as starting material for the prepa-
 ration of large quantities of soluble NTE.
 Brain homogenates from these species
 had approximately one-half the specific
 activity of hen brain. Activity was solu-
 bilized from both sources with unbuf-
 fered Triton X-100 according to the
 method used for hen brain.  The ovine
 preparation was found to be very un-
 stable, with half-life of 4-8 days at 4°C.
 The solubilized bovine preparation had a
 half-life of about 12 days at 4°C. An
 attempt was made  to expedite the
 isolation of bovine brain microsomes by
 isoelectric precipitation with citrate.
 Using this technique, 60-80% of the
 activity could be recovered, but the
 activity'could no longer be solubilized
 with detergent.
   Immobilization of NTE was attempted
 on a variety of supports using several
 different binding methods.  The most
 extensive studies were carried out on
 ion-exchange resins, using ionic bind-
 ing. Immobilization was also  attempted
 by means of hydrophobic binding to a
 phenoxyacetyl cellulose support and by
 three different  methods of covalent
 bonding.
   NTE was successfully immobilized
 with retention of catalytic activity by
 both covalent and ionic bonding. How-
 ever, neither of these preparations
 equaled the stability of the free enzyme
 stored under comparable conditions.
 Both forms of immobilized NTE were ob-
 tained with sufficient activity on the
 matrix to be useful as an enzyme prep-
 aration. The stability of the ionically
 bound form is adequate for storage over
 about one week. This form reacts nor-
 mally in the NTE assay and retains the
 inhibitor characteristics of the native
 enzyme.

 Conclusions
  Brain tissue from  a wide  variety of
 primitive and advanced species was
 found to possess apparent NTE activity,
 with the possible exception of shark and
 skate, where mipafox \so values were
 shifted to values approximately 10-fold
 higher than found in hen brain. Chick
 and hen brain were found to have
 comparable activity and were identical
 by all characterization parameters used.
  A major new finding was the existence
 of NTE in hen, rat, and human lympho-
cytes and in rat and human platelets. By
 all criteria used to characterize enzymatic
activity, hen lymphocyte and brain NTE
were identical. Moreover, the activity
responded similarly after dosing with
neurotoxic OP compounds, if  measure-
ments  were carried out at  4 h post
 dosing. At 24 h, some compounds did
 not  produce similar responses on the
 brain and lymphocyte enzymes. Lack of
 correlation at longer times may be due
 to the dynamics of the lymphocyte pool
 compared with the relatively stable
 situation in brain.
  The best solubilization preparations
 proved to be from  hen or chick brain
 using Triton X-100 detergent. Incor-
 poration of a lipid mixture,  asolectin,
 into the solubilization step was found to
 prevent loss of activity, indicating that
 NTE is a lipid-dependent enzyme. Solu-
 bilized  NTE retained all the  character-
 istics of  native enzyme. This is an
 encouraging result  in terms of further
 work on the isolation and use of NTE in
 an in vitro test system. Brain from larger
 species (ovine; bovine) conferred no
 advantage over chicken brain as a
 starting material for  solubilization.
 These species  gave activity that was
 relatively less stable or that  differed in
 inhibitor characteristics from native
 chicken brain NTE.
  A considerable quantity of detailed
 information on stability of NTE under
 various conditions was compiled in this
 study. In terms of the most  promising
 preparation for  further development of
 purification and an in vitro test system,
 the best conditions for storage appeared
 to be Triton X-100 solubilized NTE from
 chicken brain microsomes, stored un-
 buffered at-18°C.
  During immobilization studies, cova-
 lent attachment to matrices resulted in
 extensive inactivation of the enzyme.
 The  optimum immobilization  system to
 date  is binding to Triton X-100 solu-
 bilized NTE from chicken brain to DEAE-
 Sephacel™. The bound enzyme retains
 the inhibitor characteristics of native
 enzyme. However,  the  immobilized
 enzyme was not as stable as the soluble
 form under identical storage conditions.

 Recommendations
  Since chick and hen brain were found
to have comparable activity,  and to be
 identical by all characterization param-
eters used, it is recommended that chick
brain be used as a tissue source for
future work on the isolation of pure NTE.
  Further research is also needed to
determine the reason why at 24 h some
compounds do  not produce  similar
responses on brain and lymphocyte
NTE.
  Overall, it is recommended that efforts
be continued to develop an in vitro assay
system  based on NTE. Because of the
lack of stability of immobilized forms of
                                                                                      > US GOVERNMENT PRINTING OFFICE 1981 -757-012/7205

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    the enzyme, it may still be necessary to
    perform assays  in the  batch mode.
    Emphasis should be placed on obtaining
    pure NTE in order to obviate the dif-
    ferential character of the assay. Also,
    further attention should be directed
    toward  convenient ways  to assess the
    aging reaction,  which is the second
    element, in addition to simple inhibition,
    required to have neurotoxicity.
           Mary L. Barth and Rudy J. Richardson are with the Toxicology Research Labora-
             tory. University of Michigan, Ann Arbor, Ml 48109.
           Ronald L. Baron is the EPA Project Officer (see below):
           The complete report, entitled "Development of an In Vitro Neurotoxicity Assay,"
             (Order No. PB 81-208 159; Cost: $11.00, subject to change) will be available
             only from:
                   National Technical Information Service
                   5285 Port Royal Road
                   Springfield, VA 22161
                   Telephone: 703-487-4650
           The EPA Project Officer can be contacted at:
                   Health Effects Research Laboratory
                   U.S.  Environmental Protection Agency
                   Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
        CMlCA<

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