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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