EPA-600/1-78-014
February 1978
Environmental Health Effects Research Series
SCREENING METHODS FOR TOXIC SUBSTANCES
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
-------
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.
-------
EPA-600/1-78-014
February 1978
SCREENING METHODS FOR TOXIC SUBSTANCES
by
Harish C. Sikka
Syracuse Research Corporation
Syracuse, New York 13210
Contract No. 68021786
Project Officer
Larry L. Hall
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
-------
DISCLAIMER
This report 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 the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products consitute endorsement or recommendation for use.
ii
-------
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,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing 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 endangerment of their health.
The objective of this project was to explore the feasibility of using
fluorescent probes as a technique for detecting effects produced by the
action of neurotoxic chemicals. This approach may ultimately provide a
more sensitive test for adverse biological effects needed in the hazard
evaluation of agricultural and other chemicals.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
-------
ABSTRACT
The present report deals with the application of fluorescent probe
technique to the detection of conformational changes in rat brain synaptosomes
under the influence of certain neurotoxic chemicals, chlorophenothane (DDT)
and diphenylhydantoin, which are expected to produce functional changes in
nerve membranes. Pretreatment of the rats with (1) a single dose of DDT
capable of producing neurological symptoms and (ii) repeated sub-lethal
doses of DDT did not alter the fluorescence characteristics of the hydro-
phobic probe 8-anilino-l-naphthalene sulfonic acid (ANS) bound to the
synaptosomes. DDT treatment also showed no effect on native protein
fluorescence of synaptosomes.
Exposure of synaptosomes to diphenylhydantoin in vitro did not induce
conformational changes as measured by ANS and intrinsic fluorescence.
iv
-------
CONTENTS
Disclaimer ii
Foreword iii
Abstract iv
I ' ';
1. INTRODUCTION . . . 1
General 1
Objectives 2
2. CONCLUSIONS 3
3. MATERIALS AND METHODS A
4. EXPERIMENTAL PROCEDURES 7
5. RESULTS 9
References 16
-------
SECTION 1
INTRODUCTION
GENERAL
A large number of industrial and agricultural chemicals including
pesticides, detergents, solvents, plastics, chemical Intermediates, and
plasticizers may be introduced into the environment as effluents from
manufacturing plants, or during their use and disposal. Among the major
concerns associated with the presence of these chemicals in the environment
is their potential toxicity to man. From a toxicological standpoint, the
nervous system is one of the more sensitive sites of action of various
environmental contaminants.
At present, a toxicological evaluation of neurotoxic chemicals usually
consists of measuring a given organism's behavioral response, neurophysio-
logical activity and examining pathological changes in the nervous system.
These methods are suitable for detecting neurotoxic disorders resulting
from acute or chronic toxicity, but may not be able to detect subtle effects
or a minimal degree of damage resulting from low-level exposure to these
chemicals. Evidence has been presented which shows that subclinical nerve
damage was detected in workers exposed to lead who showed no clinical
neurological symptoms. » Therefore, reliable, sensitive techniques are
needed to detect early damage resulting from a variety of neurotoxic
substances.
-------
Although various neurotoxic substances like DDT, nicotine, pyrethroids,
tetrodotoxin and methylmercury act via different mechanisms, an effect upon
the nerve membrane as a whole is implicated with all of them.3'4 It
is generally believed that these chemicals interact specifically or non-
specifically with components of the nerve membrane, thereby leading to
possible changes in membrane function. Such changes may account for the
neurophysiological effects of these chemicals. It is conceivable that such
alterations may be preceded by changes in the conformation or structural
organization of the nerve membrane. The conformational changes could there-
fore be used as an indicator of neurotoxicity.
In recent years, fluorescent probes have been employed for detecting
structural or conformational changes in membranes and other biological
structures.8'9'10'11'12'13 The high sensitivity of fluorescent probes to their
microenvironment permits one to detect relatively low levels of alteration
which cannot be discerned by biochemical or physiopathological methods.
The objective of this investigation was to explore the feasibility of using
fluorescent probes as a sensitive technique for detecting effects produced
by the action of neurotoxic chemicals.
OBJECTIVES
1. To examine if selected neurotoxic chemicals induce conformational
changes in nerve cells or their components in vivo, using fluorescent
probes.
2. To establish the usefulness of fluorescent probes as a tool for
predicting neurotoxicological hazards.
-------
SECTION 2
CONCLUSIONS
The present investigation was undertaken to determine whether DDT,
a neurotoxic pesticide, produces conformational changes in nerve membranes
and if these changes can be detected by fluorescent probes. It has been
shown that DDT alters ion permeability in nerve membranes.5 Hilton et al.3
and Hilton and O'Brien14 have postulated that the mechanism by which DDT
alters nerve membrane permeability involves modification of the membrane
structure. It may be postulated that DDT, on account of its high lipid
solubility, may expand cellular membranes leading to an alteration in their
structure as has been reported for certain nonspecific lipid-soluble drugs.6
In the present study, using brain synaptosomes as a model system, we
have demonstrated that any structural or conformational changes produced
by DDT can not be detected by fluorescent probe technique suggesting that
the technique is not a sensitive indicator of DDT-induced toxicity. This
evaluation of the fluorescent probes is based on the assumption that the
pesticide at the concentrations tested did induce changes in the structure
of the synaptosomes.
-------
SECTION 3
MATERIALS AND METHODS
Preparation of Brain Subcellular Fractions
Brain synaptosomes were prepared from male Sprague-Dawley rats fed
ad_ libitum and weighing 200-250g. The animals were sacrificed by decapita-
tion, the brain was excised and placed in cold (4°C) homogenizing medium.
' -
Rat brain synaptosomes were prepared according to the method of Gray and
Whittaker. ** The brain tissue was disrupted by homogenization in 0.32 M
sucrose. The crude mitochondrial fraction was separated from the cell
debris, red blood cells, nuclei and soluble fraction by means of differ-
ential centrifugation. Synaptosomes, free mitochondria, and myelin were
then separated by means of density-gradient centrifugation. The scheme
for the isolation of various subcellular fractions is outlined in
Figures 1 and 2.
The protein content of synaptosomes was determined according to the
method of Lowry et^ al.
Fluorescence .Measurements
Fluorescence measurements were made with an Aminco-Keirs spectro-
photofluorometer (American Instruments Company) using an X-Y recorder. The
light source was a Xenon lamp. Measurements were made at 25°C in a temper-
ature-controlled cell holder connected to a circulating water bath.
Aliquots of synaptosomal preparations were resuspended in 0.32 M sucrose
-------
in 15 mM Trls-HCl buffer (pH 7.4) before making fluorescence measurements.
The final synaptosome concentration was approximately 75 wg/ml unless
indicated otherwise. An aliquot of ANS (8-anilino-l-naphthalene sulfonic
acid) stock solution was then added to give a final probe concentration of
3.2 X 10 M, unless indicated otherwise. The fluorescence measurements
were carried out at wavelengths corresponding to excitation and emission
maxima determined by analysis of the spectrum of ANS bound to the
synaptosomes.
ANS was obtained from Eastman Kodak and was recrystallized twice from
boiling water before use.
Brain homogenate
-Pi (nuclei and cell debris)
-P2
~A (ravelin fragments)
-B (nerve-ending particles)
_C (mitochondria)
—P3 (microsomes)
—Pi+ (ribosomes: post-microsomal fraction)
Figure 1. Nomenclature used in describing subcellular fractions.
-------
Homogenate of brain tissue 10% (w/v) in 0.32 M sucrose
centrifuged at lOOOg for 10 minutes
I
Precipitate
V
Washed twice by resuspension
in 0.32 M sucrose and
re-centrifuging as above
f
Separated in a density
gradient to give
three fractions
Layer between
0.32 and 0.8 M
sucrose
centrifuged
at 105g for
60 min.
Layer between
0.8 and 1.2 M
sucrose
diluted with
equal vols.
of water,
centrifuged
at 105g for
60 min.
Washings added
to supernatant
->
Supernatant centrifuged
at 17000g for 55 min.
\/
Supernatant centrifuged
at 105g for 60 min.
Pellet
below
1.2 M
sucrose
Supernatant
centrifuged at 10bg
for 120 min.
1
*
Final supernatant
(discarded)
B
Figure 2. Scheme summarizing preparation of subcellular fractions.
6
-------
SECTION 4
EXPERIMENTAL PROCEDURES
Method of Tissue or Cell Treatment
Since absorption, distribution, excretion, and metabolism of a chemical
determine its toxicity to the animal, it is desirable to expose the animal
to the test chemical in vivo followed by in vitro testing to determine if
the chemical produces any effect. The rats were exposed to the toxic
substance, following which suitable tissues were removed and examined for
possible conformational changes.
Selection of Cell and/or Cell Components Examined for Possible Conformational
Changes
The techniques for measuring conformational changes using fluorescent
probes, reproducibly and without complicated instrumentation requires that
the biological material be in suspension. This precludes the use of brain
slices or axons. In the present studies, which were designed to examine
an overall effect of a given chemical on cell structure, it was desirable
to use an isolated intact cell system in order to detect the possible
conformational changes occurring in the whole nerve cell as well as its
components. However, since techniques for the preparation of intact
nerve cells are not currently available, we used nerve cell synaptosomes
(presynaptic nerve-ending), as the test system because this fraction
represents a well-characterized and functionally important part of the
-------
neuron. There Is evidence to show that synaptosomes retain most of the
metabolic and membrane properties of the nerve ending in situ. As Whittaker
has pointed out, synaptosomes can be regarded as miniature, non-nucleated
neurons, or even as model cells.
Type of Fluorescent Probe Used
Both intrinsic and extrinsic fluorescent probes were used in these
studies to detect any possible conformational changes. In these studies, a
fluorochrome, 8-anilino-l-naphthalene sulfonic acid (ANS) was used as an
extrinsic probe since it has been most commonly used in detecting conforma-
tional changes in membranes. Anesthetics and certain drugs which are known
to act on the cell membrane have been found to cause a change in the ANS
fluorescence in the presence of microsomal, erythrocyte and mitochondrial
membranes.18'19'20'21'22 Spero et al.7reported that tetrodotoxin, a
neurotoxin, reduced the ANS fluorescence increase in guinea pig brain
synaptosomes.
Fluorescence parameters including (i) fluorescence intensity, (ii)
fluorescence spectra, and (iii) energy transfer from intrinsic membrane
chromophores to ANS were measured to determine if any conformational
changes in the test system had occurred.
-------
SECTION 5
RESULTS
Interaction of ANS with Rat Brain Synaptosomes
Addition of synaptosomes to a dilute solution of ANS resulted in a
marked enhancement of the fluorescence measured at 490 nm, using light of
390 nm wavelength for excitation. The enhancement of ANS fluorescence due
to synaptosomes was accompanied by a shift of the emission maximum from
520 to 490 nm wavelength. Since ANS does not fluoresce in aqueous solution,
the appearance of fluorescence on addition of synaptosomes indicates an
interaction of the dye molecules with synaptosomes. An enhancement of ANS
fluorescence and a shift of the maximum emission to shorter wavelengths
is characteristic of a change in ANS environment from one of high to low
dielectric constant. On the basis of the above observations, it is reason-
able to assume that ANS is bound to hydrophobic phase when this compound
interacts with synaptosomes.
Figure 3 indicates a titration of 6 x 10 M ANS with brain synapto-
somes (390 nm excitation, 490 nm measurement). Figure 4 illustrates the
titration of 75 yg/ml of synaptosomal protein/ml with ANS.
Effect of an Acute Dose of DDT on Synaptosome Fluorescence
In these experiments the effect of DDT was studied following adminis-
tration of a single dose which produced overt neurological symptoms in the
animals. Sprague-Dawley rats (250-300g) were fasted overnight before being
-------
c
8
I
2
o
3
75 100 125
Protein (/jg/ml)
175
200
Figure 3. Tltration of ANS (6 x 10~ M) with
brain synaptosomes
10
-------
c
5
§
50
75 100
ANS(/LiM)
125
150
175
Figure A. Titration of brain synaptosomes
(75 yg protein/ml) with ANS
11
-------
given high purity UDT in corn oil by oral intubation at a rate of 150 mg/kg
body weight. Control animals were given corn oil. The treated animals
showed tremors within 4 hours. They were then sacrificed by decapitation,
the brain was excised and the synaptosomes were prepared as described
earlier.
Aliquots of synaptosomal preparation were resuspended in 0.32 M sucrose
in 15 mM Tris-HCl buffer (pH 7.4). The final membrane concentration was
approximately 75 yg of protein/ml. An aliquot of ANS stock solution was
_c
then added to give a final probe concentration of 3.2 x 10 M and fluor-
escence measurements were performed in an Aminco-Keirs spectrofluorometer.
The excitation and emission wavelengths were 390 and 490 nm, respectively.
The emission spectrum of membrane-bound ANS was also measured.
Fluorescence Intensity and Spectra
The results showed that the fluorescence intensity and spectra of the
ANS bound to synaptosomes from control and DDT-treated rats did not differ.
DDT treatment also caused no change in the fluorescence intensity of ANS
bound to brain mitochondria. These findings represent the results of three
separate experiments.
Effect of Energy Transfer from Intrinsic Membrane Chromophores to ANS
The excitation spectrum of ANS overlaps the emission spectrum of
tryptophan and tyroslne. One might expect a measurable transfer of excita-
tion energy from membrane tryptophane and tyrosine to ANS molecules inserted
in close proximity to these amino acids. Therefore, in the presence of ANS,
excitation of protein fluorescence by 280 nm light produces energy transfer
from tryptophan emission to bound ANS resulting in ANS fluorescence with an
12
-------
emission peak at about 490 nm. It has been shown that energy transfer is
sensitive to even a small perturbation in membrane conformation.
To determine if DDT treatment had an effect on energy transfer, DDT-
exposed or control membrane suspensions (protein content 75 pg/ml) were
placed in cuvettes and the intrinsic protein fluorescence peak (340 nm) was
read following excitation with 280 nm light. ANS was then added to the
cuvette and the emission was recorded following excitation at 280 nm. Treat-
ment with DDT did not cause a change in ANS emission resulting from excita-
tion at 280 nm, suggesting that the pesticide does not affect the transfer
of tryptophan emission energy to ANS.
Effect of DDT on Calcium-dependent Enhancement in Fluorescence
The action of DDT on synaptosome-bound ANS fluorescence was further
evaluated by examining the effect of the pesticide on a calcium-dependent
2+
increase in ANS fluorescence. Divalent cations including Ca cause an
increase in the red cell membrane-bound ANS.20'23 It is likely that if
DDT induces any conformational changes in synaptosomes, such changes may
2+ 2+
modify the ANS-fluorescence in response to Ca . We observed that Ca
enhanced the fluorescence intensity of synaptosome-bound ANS by about 60%.
2+
When synaptosomes from DDT-treated rats were incubated with Ca , no change
in fluorescence intensity or quantum yield was observed as compared to
2+
untreated synaptosomes also incubated with Ca
Effect of DDT on Synaptosomal Protein (Intrinsic)Fluorescence
The intrinsic chromophores of the proteins in the membrane originate
e\ 1,
from the amino acids, tyrosine and tryptophan. A change in fluorescence
intensity or a shift in the position of the peak of intrinsic fluorescence
13
-------
can be used as a measure of conformational changes resulting from treatment
with membrane perturbants. The intrinsic fluorescence technique has been
used to study the interaction of anesthetics and hormones with erythrocyte
n c Of
membranes and proteins.^ '
For measurement of intrinsic protein fluorescence, the fluorescence
emission was scanned from 300-400 nm following excitation with 280 nm light.
The peak height, which was usually at 340 nm in control synaptosomes was
recorded. Our results showed that treatment with DDT did not alter the
intensity or emission spectrum of the native protein fluorescence.
Effect of Repeated Doses of DDT on Synaptosomes Fluorescence
The above studies showed that a single dose of DDT which produced overt
neurological symptoms in rats, did not induce conformational changes in
brain synaptosomes. Additional studies were undertaken to examine whether
or not administration of repeated sublethal doses of the pesticide to rats
over an extended period induced conformational changes in brain synaptosomes.
In these studies, two groups of rats (200-250 gm) were given DDT at
two different dosage levels daily for eight consecutive days. One group
of animals was administered the pesticide daily in corn oil by oral intuba-
tion at a rate of 25 mg/kg body weight. These experiments were repeated
with the second group using a dose of 50 mg/kg. Control animals were given
corn oil. None of the animals in either of the treated groups showed
obvious symptoms of toxicity at the end of the treatment period. They were
then sacrificed by decapitation, the brain was excised and the synaptosomes
were prepared according to the method of Gray and Whittaker. The effect
of DDT on synaptosomes as measured by extrinsic and intrinsic fluorescence
was determined as described in single-dose studies.
14
-------
The results showed that like the single dose of DDT, administration of
repeated doses of the pesticide at the. levels used in the study did not
induce any conformational changes in brain synaptosomes as measured by ANS
fluorescence (intensity, emission spectrum and energy transfer). DDT
treatment also did not alter the intensity or emission spectrum of intrinsic
fluorescence.
Effect of Diphenylhydantoic on Synaptosomal Membranes Exposed to the Drug
in vitro
Diphenylhydantoic (dilantiir^v is an Important drug used to protect
SO
epileptic patients against seizure. It has been suggested that Dilantin^
interacts with plasma membranes and exerts a stabilizing effect on all
excitable cell membranes.27*28 It was therefore of interest to determine
if the drug induces conformational changes in synaptosomal membranes as
measured by intrinsic and ANS fluorescence.
fa
In these studies, the in vitro effect of Dilantiir^ on the fluorescence
of ANS bound to synaptosomes isolated from untreated rats was examined.
The synaptosomes were incubated with the sodium salt of diphenylhydantoin
(10~ to 10" M) for fifteen minutes. An aliquot of ANS solution was then
_c
added to give a final probe concentration of 5 x 10 M and fluorescence
(S)
measurements were made as described above. We observed that the Dilantin^
-4
up to a concentration of 10 M did not cause any change in the fluorescence
•*
intensity or spectrum of ANS. The chemicals also showed no effect on
intrinsic fluorescence.
15
-------
REFERENCES
1. Catton MJ, Harrison MJG, Fullerton PM, et al: Br Med J 2:80, 1970
2. Seppalainen AM, Hernberg S: Br J Ind Med 29:243, 1972
3. Hilton BD, Bratkowski TA, Yaraada M, et al: Pestic Biochem Physiol
3:14, 1973
4. Hilton BD, O'Brien RD: Pestic Biochem Physiol 3:206, 1973
5. Narahashi T: Bull WHO 44:337, 1971
6. Segall HJ, Wood JM: Nature 248:456, 1972
7. Spero, L, Siemens AJ, Toulis, GA: Mol Pharmacol 9:330, 1973
8. Azzi A: Methods Enzymol 32:234, 1974
9. Brand L, Gohlke JR: Anna Rev Biochem 41:843, 1972
10. Chignell CF: Methods in Pharmacology Edited by CF Chignell. 1972,
2: pp 33
11. Radda GK: Current Topics in Bioenergetics. Edited by DR Sanadi.
1971, 4pp81
12. Radda GK, Vanderkooi J: Biochim Biophys Acta 265:509, 1972
13. Seeman P: Pharmacol Rev 24:583, 1972
14. Gray EG, Whittaker VP: J Anat 96:79, 1962
15. Lowry OH, Rosebrough NJ, Farr AL, et al: J Biol Chem 193:265, 1951
16. Whittaker VP, Barker LA: Methods in Neurochemistry. Edited by
R Fried. 1972, 2ppl
17. Narahashi T, Hess HG: J Gen Physiol 51:177, 1968
18. Azzi A, Chance B, Radda GK, et al: Proc Nat Acad Sci 62:612, 1969
19. Chance B, Azzi A, Mela L, et al: FEES Lett 3:10, 1969
20. Feinstein MB, Spero L, Felsenfield: FEBS Lett 6:245, 1971
16
-------
21. Freedmuu KB, Kadda CK: FEBS Lett 5:150, 1969
22. Vanderkooi J, Martonosi A: Arch Bloc hem Biophys 133:153, 1969
23. Rubaclava B, Munoz 1), Gltler C: Biochemistry 8:2742, 1969
24. Teale FWJ, Weber G: Biochm J 65:476, 1956
25. Sonnenberg M: Proc Natl Acad Sci USA 68:1051, 1971
26. Spero L: Proc. Canad Fed Biol Sco 14:32, 1971
27. O'Donnell JM, Kovacs T, Szabo B: Pflugers Arch 358:275, 1975
28. Wilensky AJ, Lowden JA: Can J Physiol Pharmacol 50:346, 1972
17
-------
TECHNICAL REPORT DATA
(Please read Instructions on the rcrcrse before c
1. REPORT NO.
EPA-6QO/1-78-QU
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
SCREENING METHODS FOR TOXIC SUBSTANCES
5. REPORT DATE
February 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Harish C. Sikka
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Syracuse Research Corporation
Syracuse, New York 13210
10. PROGRAM ELEMENT NO.
1LA629
11. CONTRACT/GRANT NO.
68-02-1786
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP.NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The present report deals with the application of fluorescent probe technique
to the detection of conformational changes in rat brain synaptosomes under the
influence of certain neurotoxic chemicals, chlorophenothane (DDT) and
diphenylhydantoin, which are expected to produce functional changes in nerve
membranes. Pretreatment of the rats with (i) a single dose of DDT capable of
producing neurological symptoms and (ii) repeated sub-lethal doses of DDT did
not alter the fluorescence characteristics of the hydrophobic probe 8-anilino-l-
naphthalene sulfonic acid (ANS) bound to the synaptosomes. DDT treatment also showed
no effect on native protein fluorescence of synaptosomes.
Exposure of synaptosomes to diphenylhydantoint in vitro did not induce
conformational changes as measured by ANS and intrinsic fluorescence.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
toxicity
fluorescent penetration tests
sieve analysis
neurochemistry
brain
laboratory animals
toxic substances
screening tests
06 A, T
14C
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport}
UNCLASSIFIED
21. NO. OF PAGES
23
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
18
------- |