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.



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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.
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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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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

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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-
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     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

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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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