4»EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-60Q/1-80-016
February 1980
Research and Development
Development of an
in vitro Model for
Screening
Organophosphates
for Neurotoxicity
(Pilot Study)
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-80-016
February 1980
DEVELOPMENT OF AN IN VITRO MODEL FOR SCREENING
ORGANOPHOSPHATES FOR NEUROTOXICITY (PILOT STUDY)
by
Doyle G. Graham
Department of Pathology
Duke University
Durham, NC 27710
Interagency Contract No.
68-02-2953
Project Officer
August Curley
Health Effects Research Laboratory, EPA
Research Triangle Par*, N.C. 27711
HEALfH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report was prepared by the Department of Pathology, Duke
University and has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of Duke University or the U.S. Environmental Protection
Agency, nor does mention of trade names of commercial products constitute
endorsement or recommendation for use.
11
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance the
quality of our environment in order to promote the public health and welfare
and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park, conducts
a coordinated environmental health research program in toxicology, epidemio-
logy, and clinical studies using human volunteer subjects. These studies
address problems in air pollution, non-ionizing radiation, environmental
carcinogenesis and the toxicology of pesticides as well as other chemical
pollutants. The Laboratory participates in the development and revision
of air quality criteria documents on pollutants for which national ambient
air quality standards exist or are proposed, provides the data for registra-
tion of new pesticides or proposed suspension of those already in use,
conducts research on hazardous and toxic materials, and is primarily respon-
sible for providing the health basis for non-ionizing radiation standards.
Direct support to the regulatory function of the Agency is provided in the
form of expert testimony and preparation of affidavits as well as expert
advice to the Administrator to assure the adequacy of health care and
surveillance of persons having suffered imminent and substantial endanger-
ment of their health.
The pilot study was designed to test the feasibility of using murine
or human neuroblastoma cells in tissue culture for the assessment of neuro-
toxicity of organophosphorus insecticides. The following organophosphorus
compounds were evaluated: DFP, TOCP, parathion, leptophos, EPN, DEF, and
merphos.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
111
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ABSTRACT
An in vitro model utilizing neuroblastoma cell lines was developed
for the differentiation of organophosphoras compounds which have the
potential for induction of delayed degeneration of peripheral nerves and
the spinal cord. IMR-32 neuroblastoma cells, derived from a human tumor,
showed specific (SH)-norepinephrine uptakes. The greater effect of
alkyl over aryl OP compounds suggests a relationship with water solubility,
thus equilibrium between adsorption to agar and the surrounding medium
or lysosomal contents. The effect of parathion was intermediate, less
that that for merphos, DEF and EPN but greater than that for leptophos
and TOCP. Electron microscopic observations suggest further studies
to define a morphologic lesion peculiar to the clinically delayed
neurotoxic OP compounds.
iv
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Final Report
EPA Contract 68-02-2953
DEVELOPMENT OF AN IN VITRO MODEL FOR SCREENING
ORGANOPHOSPHATES FOR NEUROTOXICITY (PILOT STUDY)
Introduction
One to two weeks after exposure to certain organophosphorus compounds,
man and many animals develop degeneration of peripheral nerve and of the long
tracts of the spinal cord (1-12). The differentiation of this lesion as neuronal
rather than of myelin (1,3) is widely accepted. Whether the focus of injury is
within the cell body .(13) or in the axon (14,15) has not been settled, but current
literature suggests that the concept of a dying-back polyneuropathy, long-postula-
ted to explain toxic, nutritional, and hereditary neuropathies (16-22), does not
adequately characterize -the sequences of events in organophosphorus-induced
delayed neurotoxicity. Rather, it would appear that the initial site of injury is
within the axon and that this is then followed by Wallerian degeneration peripheral
to the lesion (15,22).
Within the axon, however, the molecular or subcellular target for organophos-
phorus-induced injury is poorly defined. Aldridge, Johnson, and others have
described "neurotoxic esterase", an enzyme activity defined by its capacity to
hydrolyze phenyl phenyl-acetate or phenyl valerate and by its inhibition by
neurotoxic organophosphorus compounds (4,5,22-25).
Johnson has proposed using inhibition of "neurotoxic esterase" as a screening
method for detecting the potential of organophosphorus compounds to induce
delayed neurotoxicity and has postulated that inhibition of this enzyme is related
to the mechanism by which these agents cause this disorder. While inhibition of
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this activity correlates with neurotoxicity potential with rare exceptions, the
intracellular location of "neurotoxic esterase", the properties of the enzyme as a
protein, its role within the cell, and the relationship between phosphorylation of
this enzyme and axonal degeneration, all remain undefined (k). Further, a time-
dependent process, termed "aging" is required between phosphorylation and inhibi-
tion_of this enzyme. This phenomenon is likewise poorly understood and difficult to
dissect in whole brain homogenates where, besides "neurotoxic esterase" there are
at least 4 additional esterases with similar substrate specificities (4,5).
During the past three years Dr. M.B. Abou-Donia and I have evaluated a
series of phenylphosphonothioates for the capacity to induce delayed neurotoxicity
in vivo using hens and ducklings (7-12,28). In these studies we have developed a
grading system for assassing clinical ataxia and have evolved much-needed
standardized treatment protocols in which histopathologic verification of clinical
neurotoxicity can be observed as lesions in peripheral nerve and spinal cord. These
protocols include daily oral or topical applications of small doses and single large
dose studies. In the latter protocols we have been able to test compounds at levels
50 to 100 times the LD_fl dose through the vigorous prophylactic administration of
atropine sulfate to counter the cholinergic manifestations of acute organophos-
phorus toxicity. Most previous in vivo studies have not included histopathologic
study, so that differentiation of the clinical state of acute toxicity from delayed
neurotoxicity has been uncertain. It is for this reason that in the development of
an in vitro model for differentiating neurotoxic from non-neurotoxic organophos-
phorus compounds we have chosen to use those which we have characterized in
vivo.
In the course of our in vivo studies the need for an in vitro model which could
be used for screening became obvious. The neurobiastoma lines we have used as a
tool for exploring the pathogenesis of Parkinson's disease (26,27) have now become
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a model system for organophosphorus-induced delayed neurotoxicity. The advan-
>
tages of neuroblastoma cells in tissue culture as an m vitro system are many.
These cells differentiate in tissue culture, form neurites, demonstrate electrical
activity, and synthesize and store specific neurotransmitters (28-32). Thus these
cell lines provide neurobiologists with the heretofore unavailable opportunity to
study neuronal metabolism and pathology divorced from blood-tissue barriers and
the interposition of myelin and glial cells. Further, using cloned lines from either
human or murine sources, statements can be made in reference to one type of
neuron as compared with another (e.g., adrenergic vs cholinergic), a distinction not
readily achieved in vivo. In addition, cloned neuroblastoma lines in tissue culture
present a pure population of neurons in an optimal setting for morphological
observations (phase and electron microscopy, autoradiography) and are available in
sufficient quantities for chemical studies.
This pilot study was designed to test the feasability of using murine or human
neuroblastoma cells in tissue culture for the assessment of neurotoxicity of
organophosphorus insecticides. By testing a variety of agents known to result in
delayed neurotoxicity in chickens and, in some cases, in man, it was hoped that
morphologic and/or biochemical end points could be defined which distinguished
these insecticides from those not associated with delayed neurotoxicity. The tissue
culture lines utilized were the Ml 15 clone of the C1300 murine neuroblastoma and
the human IMR-32 neuroblastoma line. The following organophosphorus compounds
were evaluated: DFP, TOCP, parathion, leptophos, EPN, DEF, and merphos.
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Materials and Methods
Tissue culture lines: The N115 adrenergic clone of the murine C1300
neuroblastoma, developed in the Nirenberg laboratory at NIH was obtained from
Dr. David Duch, Burroughs Wellcome Co. and was maintained in serial passage in
Dulbecco's modification of Eagle's MEM medium with 20% fetal calf serum,
penicillin, (250 i.u/ml), streptomycin (100 mg/ml) and glutamine. IMR-32 adrener-
gic neuroblastoma was started from frozen stock in the laboratory of Dr, Darell
Bigner at Duke. It, too, was grown in Dulbecco's medium.
Organophosphorus compounds: DFP (diisopropyl phosphofluoridate) was ob-
tained from Aldrich Chemical Co., Milwaukee, WI, EPN from E.I. duPont
deNemours and Co., Inc., Wilmington, Del., leptophos from Velsicol Chemical Co.,
Chicago, 111., DEF and merphos from Chemagro Corp., Kansas City, Mo., TOCP
from Eastman Kodak Co., Rochester, N.Y., parathion from Pfaltz and Bauer.
Exposure of neuroblastoma cells to organophosphorus compounds: In experi-
ments with DFP, ampoules were chilled on dry ice and opened in a fume hood,
immediately diluting in isopropanol in order to reduce hydrolysis. Isopropanol
solutions were then added to medium without fetal calf serum, mixed and rapidly
added to the tissue culture flasks. Control cells were exposed to medium to which
an equal volume of isopropanol had been added. Exposure was for 3 hours, after
which the cells were evaluated for the capacity for ( H)-norepinephrine uptake or
washed and covered with tissue culture medium containing fetal calf serum in the
multiple-exposure experiments. In the latter, on successive days neuroblastoma
cells in Falcon flasks were washed with medium deficient in fetal calf serum prior
to exposure to DFP for 3 hour periods at 37°C.
Exposure to the remaining organophosphorus compounds required that
methodology be developed for the uniform exposure of tissue culture cells to
agents poorly soluble in aqueous media. Each compound was dissolved in ether,
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then layered over a rapidly stirring sterile suspension of agar particles (0.25%).
Stirring continued until the ether had evaporated and the organophosphorus
compound had been adsorbed by the agar. Then aliquots were pipetted into medium
to yield a final concentration of organophosphorus compound of 1 mg/ml. The
uniform suspension was then pipetted into flasks containing neuroblastoma cells,
with incubation for 3 hours at 37°C. Control cultures were treated with medium
containing an equal amount of agar. At the end of the exposure the cells were
Q
examined by phase contrast microscopy, then separate flasks were exposed to ( H)-
norepinephrine or prepared for electron microscopy as described below.
Electron microscopy: Cells were fixed m situ with a) glutaraldehyde (4% in
0.1 M sodium cacodylate buffer pH 7.*), b) 4% aqueous KMnCK, or c) 4% aqueous
KMnO^ after 30 minutes exposure to 10 p M 5-hydroxydopamine (3,^,5-trihydroxy-
phenylethylamine). Glutaraldehyde-fixed cells were post-fixed in 1% OsCX. All
preparations were then dehydrated and embedded in Epon. After solidification the
plastic flasks were broken away from the sheet of epon and sites chosen for thin
sections. Grids were examined and photographed on a Hitachi HS 11 transmission
electron microscope.
3 3
( H) norepinephrine uptake: ( H) norepinephrine (25-30 Ci/m mole) was added
to complete medium to a final concentration of 10" M and pipetted to the bottom
of a vertical Falcon flask. Flasks were then laid flat to allow contact of medium
with neuroblastoma cells for 10 min at 37°C in a 5% CO2 atmosphere. At the end
of this period the flasks were placed vertically in ice and ( H) norepinephrine
medium withdrawn. The cells were then dissociated with trypsin, washed with
centrifugation using phosphate-buffered saline, then the ( H)-norepinephrine ex-
tracted with O.iM perchloric acid for scintillation counting. The perchloric acid
pellets were then digested with 1.0 M NaOH for protein determinations.
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Protein concentrations: Protein content was quantified by the Pulley and
Grieve modification of the Lowry method in which protein is precipitated in cold
10% TCA, then dissolved in IN NaOH after which buffered sodium dodecylsulfate
is added to prevent precipitation upon addition of the Folin-Phenol Reagent (33).
Scintillation counting: Aliquots of perchloric acid extracts of neuroblastoma
cells exposed to ( H)-norepinephrine were added to 10 ml. volumes of aquasol II
(New England Nuclear Corp., Boston, MA) and counted in a Beckman LS-I50
scintillation spectrometer.
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Results
Initial studies with the N115 adrenergic clone of the murine C1300 neuroblas-
toma showed a 21% reduction in ( H)-norepinephrine uptake after 3 hours exposure
to DFP at 1 mg/ml in the absence of fetal calf serum. Two observations gave us
reason to doubt the adrenergic differentiation of these cells, which would diminish
the usefulness of ( H)-norepinephrine uptake as a sensitive index of neurite injury:
norepinephrine could not be detected in perchloric acid extracts of these cells, and
( H)-norepinephrine uptake was only slightly inhibited by 10~ M cocaine.
Accordingly, we focused our efforts on another cell line, the human IMR-32
neuroblastoma. In medium containing ImM ascorbate and 2 uM iproniazid these
cells demonstrated active ( H)-norepinephrine uptake, linear for at least 10
minutes, which was inhibited >_ 90% by 10 M cocaine. Perchloric acid extracts of
the IMR-32 cultures however, have not been demonstrated to contain norepine-
phrine. Our working hypothesis is that these cells are dopaminergic rather than
noradrenergic; this will be established through the. demonstration of dopamine and
tyrosine hydroxylase and the concommitant absence of norepinephrine and dopa-
mine 3-hydroxylase.
In order to study the effects of DFP on the IMR-32 cells we had to deal with
two properties of this organophosphorus compound, its rapid hydrolysis in aqueous
solutions and its reactivity with nucleophiles in serum proteins. Accordingly, DFP
dilutions were made in isopropanol, and exposure to neuroblastoma cells was
effected in medium without fetal calf serum as soon after addition of DFP to
medium as was possible. Under these conditions exposure to DFP at 1 mg/ml for 3
hours resulted in a reduction of ( H)-norepinephrine uptake from (mean + SEM)
35,245 + 1,087 cpm/mg protein to 19,680 + 1,507 cpm/mg protein (W.2% reduction,
p < 0.005). Reducing the concentration of DFP to 0.5 or 0.25 mg/ml and daily
exposure up to 8 days at 0.25 mg/ml yielded minor to no differences in ( H)-norepi-
nephrine uptake.
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Exposure of neuroblastoma cells to the remaining organophosphorus com-
pounds required that water-insoluble compounds be dispersed in a form that would
allow for uniform exposure to the cells in tissue culture. Adsorption to agar
particles gave excellent agreement among triplicate assays for ( H)-norepinephrine
uptake, and electron microscopy disclosed evidence for phagocytosis of the agar
particles by the neuroblastoma cells, as will be discussed below.
In Table I are presented the results of experiments in which IMR-32 cells
were exposed to medium containing agar particles, with and without adsorbed
organophosphorus compounds. In each instance the final concentration was 1
mg/ml. The greatest inhibition of ( H)-norepinephrine uptake was observed with
the alkyl organophosphorus compounds, merphos, DEF, and parathion, while lesser
degrees of inhibition were found with the aryl compounds TOCP, EPN, and
leptophos. In all instances the reductions were statistically significant.
Only a limited amount of electron microscopy was completed in this study.
Cells were examined from each exposure group reported in Table I. In these
studies we were able to ascertain that adsorped organophosphorus compounds at a
final concentration were not cytotoxic, that neurofibrils, neurotubules, and dense
core vesicles typical of catecholamine neurons could be observed, that the agar
particles were taken into the neuroblastoma cells by phagocytosis, and that on two
occasions, after exposure to leptophos and DEF, tightly packed bundles of
micro filaments were observed within the neuroblastoma cells.
Representative control cells are illustrated in Figs. 1-4. The neuroblastoma
cultures are a replicating population of cells with continuous cell division (Fig. 2)
and cell death. Dead cells lose their adsorption to plastic and would thus be
washed off prior to ( H)-norepinephrine exposure; relating ( H)-norepinephrine
uptake to protein concentration, therefore, served as a reliable method for
correcting for the flask to flask variation in cell density and cell death. The close
8
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apposition between cells and neurite processes from other cells was readily
observed (Figs. 3,4). Vacuoles with debris suggesting agar phagocytosis were
observed (Fig. 1-4) and are more clearly seen in later figures.
Neuroblastoma cells which had been exposed to parathion are seen in Figs. 5
and 6. The large vacuoles containing agar are most dearly seen in Fig. 5. In Fig. 6
the two adjacent parathion exposed cells are seen to be well-preserved without
changes of cell injury. Again the agar particles can be seen in phagocytic vacuoles.
Similarly after exposure to TOCP there was no increase in the proportion of
dead cells, and those remaining showed no evidence for cell injury. Continuation of
replication side-by-side with cell death can be seen in Fig. 8 in which cells had
been exposed to EPN/agar for 3 hours. Among the EPN-exposed cells dense core
vesicles typical of catecholamine-storage vacuoles, were observed in cell bodies
and neuritic processes along with neurotubules (Fig. 9).
After leptophos exposure, dividing cells and dead cells were also observed
(Fig. 10). Dense core granules could be seen in neurites (Fig. 11) and cell bodies
(Fig. 14). Cytoplasmic extensions or neurites were noted to contain closely-packed
bundles of microfilaments (actin) (Figs. 12,13) which were not observed in control
or parathion-exposed cells but were observed aftei1 DEF exposure (Fig. 15).
The marked reductions in ( H)-norepinephrine uptake effected by DEF and
merphos were not accompanied by excess cell death (Figs. 15-17). Neurosecretory
granules (dense core vesicles) were readily observed in cell bodies (Fig. 16).
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DISCUSSION
This study has shown that hydrophobia or gano phosphor us compounds can be
exposed to neuroblastoma cells in tissue culture through adsorption to agar
particles. By electron microscopy numerous phagocytic vacuoles could be obser-
ved, and in many, debris probably representing agar was readily seen. Close
agreement was seen between triplicate flasks with regard to the capacity for ( H)-
norepinephrine uptake, suggesting that cell exposure was relatively uniform.
IMR-32 neuroblastoma cells, derived from a human tumor, showed specific
( H)-norepinephrine uptake (>90% inhibition by 10 M cocaine). The uptake of
( H)-norepinephrine was reduced after a 3 hour exposure to all organophosphorus
compounds tested. The greater effect of alkyl over aryl organophosphorus
compounds suggests a relationship with solubility in water, thus the equilibrium
between adsorption to agar and the surrounding medium or lysosomal contents.
The effect of par at hi on, which has not been demonstrated to cause delayed
neurotoxicity in vivo, was intermediate, less than that for merphos, DEF, and EPN,
but greater than that for leptophos and TOCP.
Two alternative explanations can be proposed for these results. One is that
there is no relationship between reduced ( H)-norepinephrine uptake and the
capacity of a given organophosphorus compound to cause delayed neurotoxicity.
The other is that parathion might be capable of causing delayed neurotoxicity but
cannot be demonstrated to do so in vivo because of the severe cholinergic poisoning
caused by this compound.
Electron microscopic observations suggest that further study may define a
morphologic lesion peculiar to the clinically delayed neurotoxic organophosphorus
compounds. Cells exposed to leptophos and DEF were found to contain tightly
packed bundles of microfilaments (actin). This finding was not present in control
or parathion-exposed cells, yet should be viewed with caution until a more
complete electron microscopic study can be completed.
10
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Publication: Graham, D.G., Lee, J.S., and Abou-Donia, M.B., "The use of human
neuroblastoma cells in tissue culture in the study of organophosphorus-indured
delayed neurotoxicity," To be presented at the annual meeting of the Society of
Toxicology, Washington, D.C., March 10,1980.
Doyle G. Graham
Associate Clinical Professor of Pathology
Principal Investigator
December 12, 1979
11
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TABLE I
The Effect of Agat-Adsorbed Organophosphorus
Compounds on ( H) Norepinephrine Uptake
by IMR-32 Neuroblastoma
Compound
( H) norepinephrine
uptake
(cpm/mg protein + SEM)
% Inhibition
1. Control
Leptophos
EPN
2. Control
DEF
Merphos
3. Control
TOCP
Parathion
76,271 +1,565
63,629+3,890
47,5*8+1,286
203,627 +24,081
110,625 +4,580
76,357 +9,415
120,555+1,566
84,942 +4,391
75,360 + 1,896
16.6
37.7
45.7
62.5
29.5
37.5
0.05
0.001
0.01
0.01
0.005
0.001
12
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LEGENDS TO FIGURES
Fig. 1. Control cells. The IMR-32 human neuroblastoma line grows well in
tissue culture. This figure shows the cytoplasmic vacuolization which is seen when
these cells are exposed to medium containing agar particles (left side of figure).
On the right are portions of 3 dead cells in among well-preserved cells, illustrating
that in tissue culture there is continuous cell turnover and cell death. X12,500.
Fig. 2. Control cells. Along with occasional dead cells, evidence that these
cells are replicating is regularly observed as here where a cell is found in mitosis
(telophase). Vacuoles resulting from exposure to agar particles are seen in the
lower right corner. X21,000.
Fig. 3. Control cells. By electron microscopy one can see what is readily
apparent by phase contrast microscopy, that these cells send out cytoplasmic
processes which come into close apposition with each other. One in the bottom
center contains neurotubule-like structures. Two in the upper portion of the figure
contain vacuoles in which the debris may represent agar particles. X42,500.
Fig. 4. Control cells. The ribosome-rich cell below contains cytoplasmic
vacuoles. Processes from neighboring neuroblastoma cells come into close contact
with the cell body and each other. The larger process above contains neurotubule-
iike structures and a less well preserved dense core vacuole. X*2,500.
Fig. 5. Parathion-exposed cells. The vacuoles in the cytoplasm of these cells
contain lacy ingested agar particles. X 12,500.
Fig. 6. Parathion-exposed cells. Two neuroblastoma cells with intact nuclear
arid cytoplasmic structures are seen here. In the cell on the right, neurotubule-like
structures run roughly parallel to the plasma membrane. The cell on the left
contains agar particles in cytoplasmic vacuoles. X42,500.
Fig. 7. TOCP-exposed cells. As in control cultures scattered dead cells are
observed among cells with no evidence of cell injury.
13
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Fig. 8. EPN-exposed cells. A dead cell (upper center) is seen among well-
preserved neuroblastoma cells, one of which is dividing (left center). An agar
particle, to which £PN was adsorbed in this experiment, can be seen in the lower
right corner. X 12,500.
Fig. 9. EPN-exposed cells. One of the cytoplasmic processes (neurites) in the
upper portion of this figure contains the dense core vacuoles typical of catechola
mine-storage vesicles. Additional dense core vesicles are seen in the lower right
along with neurotubule-like structures, ribosomes and mitochondria. X42,500.
Fig. 10. Leptophos-exposed cells. Cytoplasmic vacuoles reflecting phagocytosis
of leptophos - containing agar particles can be seen in most of the cells. The cell
on the left with islands of chromatin is dividing, while on the right a cell is seen
with the pyknotic nucleus characteristic of cell death. X 12,500.
Fig. 11. Leptophos-exposed cells. The cell in the lower right contains numerous
cytoplasrnic vacuoles. A cytoplasmic process (left center) contains many dense
core vesicles. X21,000.
Fig. 12. Leptophos-exposed ceils. The cell in the lower half contains numerous
cytoplasmic vacuoles. In the upper right the neuroblastoma cell contains a densely
packed bundle of micro filaments. X42,500.
Fig. 13. Leptophos-exposed cells. The cell on the right contains cytoplasmic
vacuoles plus filaments and neurotubule-like structures. A portion of a cell
containing bundles of microfilaments is seen in left center. X42,500.
Fig. 14. Leptophos-exposed cells. High magnification detail of dense core
granules reveals the morphology of catecho'lamine-coritaining vesicles. X70,000
Fig. 15. DEF-exposed cells. A dividing cell is observed in the lower left corner.
The cells contain cytoplasrnic vacuoles. Two cells contain bundles of tightly
packed neurofilaments. X12,500.
Fig. 16. DEF-exposed cells. Cells have debris-containing cytoplasmic vacuoles,
dense core granules, and neurotubule-like structures. X42,500.
14
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Fig. 17. Merphos-exposed cells. A mixture of vacuole-containing living cells and
dead cells is seen. X 12,500.
15
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References
1 Cavanagh, 3.B. The toxic effects of tri-ortho-cr^syl phosphate nn the
nervous system. An experimental study in hens. J. Neurol. Neurosurg,
Psychiat. 17:163-172, 1954.
2. Durham, F.W., Gaines, T.B., and Hayes, W.3. Paralytic and related effects of
certain organic phosphorous compounds. Arch. Ind. Health 13:326-330, 1956.
3. Cavanagh, 3.B. Peripheral nerve changes in ortho-cresyl phosphate poisoning
in the cat. 3. Path. Bact. 87:365-383, 1964.
4. 3ohnson, M.K. The delayed neuropathology caused by some organophosphor-
ous esters: mechanism and challenge. CRC Crit. Rev. Toxicol. 3:289-316,
1975.
5. 3ohnson, M.K. Organophosphorus esters causing delayed neurotoxic effects.
Mechanism of action and structure/activity studies. Arch. Toxicol. 34:259-
288, 1975.
6. Cavanagh, 3.B. Peripheral neuropathology caused by chemical agents. CRC
Crit. Rev. Toxicol. 2:365-417, 1973.
7. Abou-Donia, M.B. and Preissig, S.H. Delayed neurotoxicity of leptophos:
Toxic effects on the nervous system of hens. Toxicol. Appl. Pharmacol.
35:269-282, 1976.
8. Abou-Donia, M.B., and Preissig, S.H. Delayed neurotoxicity from continuous
low-dose oral administration of leptophos to liens. Toxicol. Appl. Pharmacol.
38:595-608, 1976.
9. Preissig, S.H. and Abou-Donia, M.B. The neuropathology of leptophos in the
hen: a chronologic study. Env. Res. 17:242-250, 1978.
10. Herin, R.A., Komeil, A.A., Graham, D.G., Curley, A., and Abou-Donia, M.B.
Delayed neurotoxicity induced by organophosphorus compounds in the wild
Mallard duckling: effect of leptophos. 3. Env. Path. Toxicol. 1:233-240,
1978.
11. Abou-Donia, M.B. and Graham, D.G. Delayed neurotoxicity of O-ethyl
O-4-nitrophenyl phenylphosphonothioate: subchronic (90-day) oral admini-
stration in hens. Toxicol. Appl. Pharmacol. 45:685-700, 1978.
12. Abou-Donia, M.B., and Graham, D.G. Delayed neurotoxicity from long-term
low-level topical administration of leptophos to the comb of hens. Toxicol.
Appl. Pharmacol. 46:199-213, 1978.
13. Majno, G. and Karnovsky, M.L. A biochemical and morphologic study of
myelination and demyelination-HI. Effect of an organophosphorus compound
(Mipafox) on the biosynthesis of lipid by nervous tissue of rats and hens. 3.
Neurochem. 8:1-16, 1961.
14. Prineas, 3.P. The pathogenesis of dying-back neuropathies. Part I. An
ultrastructural study of experimental triothocyresyl phosphate intoxication in
the cat. 3. Neuropath. Exp. Neurol. 28:571-597, 1969.
33
-------
15. Bouldin, T.W. and Cavanaugh, 3.B. Organophosphorus neuropathy. I. A
teased-fiber study of the spatio-temporal spread of axonal degeneration. II.
A fine-structural study of the early stages of axonal degeneration. Am. 3.
Pathol. 94:241-252, 253-270, 1979.
16. Prineas, 3.P. The pathogenesis of dying-back neuropathies. Part II. An
ultrastructural study of experimental acrylamide intoxication in the cat. 3.
Neuropath. Exp. Neurol. 28:598-621, 1969.
17. Schaumberg, H.H., Wisniewski, H.M., and Spencer, P.S. Ultrastructural
studies of the dying-back process. I. Peripheral nerve terminal degeneration
in systemic acrylamide intoxication. 3. Neuropath. Exp. Neurol. 33:260-284,
1974.
18. Spencer, P.S. and Schaumberg, H.H. Ultrastructural studies of the dying-
back process. III. The evolution of peripheral, giant axonal degeneration. 3.
Neuropath. Exp. Neurol. 36:276-299, 1977.
19. Spencer, P.S. and Schaumberg, H.H. Ultrastructural studies of the dying-
back process. IV. Differential vulnerability of PNS and CNS fibers in
experimental central-peripheral distal axonopathies. 3. Neuropath. Exp.
Neurol. 36:300-320, 1977.
*
20. Koch, T., Schultz, P., Williams, R.f and Lampert, P. Giant axonal neuro-
pathy: A childhood disorder of microfilaments. Ann. Neurol. 1:438-451,
1977.
21. Towfighi, 3., Gonatas, N.K., Pleasure, D., Cooper, H.M., and McCree, L.
Glue sniffer's neuropathy. Neurol. 26:238-243, 1976.
22. Spencer, P.S. and Schaumberg, H.H.: Industrial neuropathies. Neupotoxicol.
1:427-430, 1977.
23. Earl, C.3. and Thompson, R.H.S. Cholinesterase levels in the nervous system
in tri-ortho-cresyl phosphate poisoning. Br. 3. Pharmacol. 7:685-694, 1952.
24. Davison, A.N. Some observations on the cholinesterases of the central
nervous system after the administration of organophosphorus compounds. Br.
3. Pharmacol. 8:212-220, 1953.
25. Paulsen, E. and Aldridge, W.N. Studies on esterases in the chicken nervous
system. Biochem. 3. 90:182-187, 1964.
26. Graham, D.G. Oxidative pathways for catecholamines in the genesis of
neuromelanin and cytotoxic quinones. Mol. Pharmacol. 14:633-643, 1978.
27. Graham, D.G., Tiffany, S.M., Bell, W.R., 3r., and Gutknecht, W.F. Autooxi-
dation vs. covalent binding of quinones as the mechanism of toxicity of
dopamine, 6-hydroxydopamine and related compounds for C1300 neuroblast-
oma cells in vitro. Mol. Pharmacol. 14:644-653, 1978.
28. Blume, A., Gilbert, F., Wilson, S., Farber, 3., Rosenberg, R., and Nirenberg,
M. Regulation of acetylcholi nest erase in neuroblastoma cells. Proc. Nat.
Acad. Sci. USA 67:786-792, 1970.
34
-------
29. Tumilowicz, 3.J., Nichols, W.W., Cholon, J.J., and Greene, A.E. Definition of
a continuous human cell line derived from neuroblastoma. Cancer Res.
30:2110-2118, 1970.
30. Biedler, J.L., Helson, L., and Spengler, B.A. Morphology and growth,
tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous
culture. Cancer Res. 33:2643-2652, 1973.
31. West, G.3., Uki, J., Hershman, H.R., and Seeger, R.C. Adrenergic, choliner-
gic, and inactive human neuroblastoma cell lines with the action-potential
Na++ ionophore. Cancer Res. 37:1372-1376, 1977..
32. Seeger, R.C., Rayner, S.A., Banerjee, A., Chung, H., Lang, W.E., Neustein,
H.B., and Benedict, W.F. Morphology, growth, chromosomal pattern, and
fibrinolytic activity of two new human neuroblastoma cell lines. Cancer Res.
37:1364-1371, 1977.
33. Pulley, J.D., and Grieve, P.A. A simple technique for eliminating interfer-
ence by detergents in the Lowry method of protein determination. Anal.
Biochem. 64:136-140, 1975.
35
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Development of an iji_ vitro Model for Screening
Organophosphates for Neurotoxicity (Pilot Study)
5. REPORT DATE
February 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Doyle R.Graham
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Pathology
Duke University Medical Center
Durham, NC
'10. PROGRAM ELEMENT NO.
! 1EA615
|11. CONTRACT/GRANT NO.
! 68-02-2953
12. SPONSORING AGENCY NAME AND ADDRESS
Toxic Effects Branch, ETD (MD-67)
HERL, USEPA
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final 10/1/78 - 9/3079
,14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. A3STHACT
An in vitro model utilizing neuroblastoma cell lines was developed for the
differentiation of organophosphorus compounds which have the potential for induction
of delayed degeneration of peripheral nerves and the spinal cord. IMR-32 neuroblastoma
cells, derived from a human tumor, showed specific (3H)-norepinephrine uptake. The
greater effect of alkyl over aryl OP compounds suggests a relationship with water
solubility, thus equilibrium between adsorption to agar and the surrounding medium
or lysosomal contents. The effect of parathion was intermediate, less than that
for merphos, DEF and EPN but greater than that for leptophos and TOCP. Electron
microscopic observations suggest further studies to define a morphologic lesion
peculiar to the clinically delayed neurotoxic OP compounds.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Delayed neurotoxicity
Organophosphorus compounds
Neuroblastoma cell lines
Norepinephrine
06FJ
18. O'STRISUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY C!~A£3 , Thu eieport)
Unclassified
21. "^O. OF ?A£
40
120. SECURITY.CL^SS .Thupagt/
I Unclassified
22. S
cPA Form 2220-1 !R«v. 4-771 =>o6Viou3-
36
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