Citation
The effects of permethrin and DDT on the neurite outgrowth of cultured neurons of lymnaea stagnalis and embryonic chickens

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Title:
The effects of permethrin and DDT on the neurite outgrowth of cultured neurons of lymnaea stagnalis and embryonic chickens an attempt to develop an in vitro model for testing toxicity
Creator:
Ferguson, Charles A
Publication Date:
Language:
English
Physical Description:
xvi, 200 leaves : illustrations ; 29 cm

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Subjects / Keywords:
Toxicity testing ( lcsh )
Neurons -- Cultures and culture media ( lcsh )
DDT (Insecticide) -- Toxicology ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 170-178).
General Note:
Submitted in partial fulfillment of the requirements for the degree of Master of Arts, Department of Integrative Biology.
Statement of Responsibility:
by Charles A. Ferguson.

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Source Institution:
|University of Colorado Denver
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Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
17882261 ( OCLC )
ocm17882261

Full Text
THE EFFECTS OF PERMETHRIN AND DDT ON THE
NEURITE OUTGROWTH OF CULTURED NEURONS
OF Lymnaea stagnalis AND EMBRYONIC CHICKENS:
AN ATTEMPT TO DEVELOP AN in vitro MODEL FOR
TESTING TOXICITY
by
Charles A. Ferguson
B.A., University of Colorado at Denver, 1985
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Arts
Department of Biology
1987


This thesis for the Master of Arts degree by
Charles A. Ferguson
has been approved for the
Department of Biology
by
Gerald Audesirk
Janis Driscoll
Date
/'/.To/>-7


Ill
Ferguson, Charles A. (M.A., Biology)
The Effects of Permethrin and DDT on the Neurite
Outgrowth of Cultured Neurons of Lymnaea
stagnalis and Embryonic Chickens: An Attempt to
Develop an in vitro Model for Testing Toxicity.
Thesis directed by Associate Professor Gerald
Audesirk.
Isolated neurons from the mollusc L.
stagnalis and embryonic chicks were cultured to
determine the effect on neurite outgrowth of two
insecticides, permethrin and DDT.
These were exposed to concentrations of
25uM to 500uM permethrin dissolved in alcohol and
DDT concentrations of 500nM to 25uM. The effect of
these concentrations of toxins on the ability of
the neuron to extend new neurites was studied.
The number of L. stagnalis cells with
neurites as compared to two controls decreased as
concentrations of permethrin increased from 25uM to
250uM. An LC,-q of 50uM for permethrin was found.
Cells exposed to levels of 500uM or greater did not
survive.


IV
L. stagnalis cells exposed to DDT showed a
decrease in neurite growth as concentrations
increased from 500nM to lOuM. An LC,-q of 750nM was
found. Cells did not survive concentrations
greater than lOuM.
Embryonic chick cells exposed to
permethrin showed a decreased growth rate as
concentration increased from 25uM to 250uM with
permethrin and lOuM to 500nM with DDT.
Effects of delayed permethrin exposure to
existing neurites of L, stagnalis was studied.
Regression of neurites occurred in a dose-dependent
and time-dependent manner, with total regression of
existing neurites occurring more rapidly as
concentration increased.
Electrical activity of neurons of L.
stagnalis was recorded after exposure to
permethrin. This exposure ranged from 20 minutes to
20 hours. It was found that as concentration
increased, a significantly shorter exposure time
was necessary to elicit repetitive spontaneous
action potentials with an eventual total
depolarization of the cell for several seconds
before returning to the original resting potential.
These studies indicate the potential


usefulness of these models as assays for the
determination of neurotoxicity of different
substances in the environment. Further, our data
suggest that in vitro models may be useful in
determining response between species to specific
environmental contaminants. Electrophysiological
data support previous work that Na+ channels are
being affected by this toxin.


VI
DEDICATION
To Bill Seiwald, my second father who died before
being able to see the result of his love,
compassion, and faith. I will be eternally
grateful.


Vll
ACKNOWLEDGMENTS
Robert Frost once said, "I am not a teacher,
but an awakener". I have had the sincere privilege of
working with a person who has been a friend, a teacher,
a mentor; a person who has helped "awaken" in me a sense
of self worth. To Dr. Gerald Audesirk my most sincere
thanks for your time, your help, your support, and your
confidence in me.
I would like to thank Dr. Alan Brockway and Dr.
Janis Driscoll for their help and constructive criticism
of this project from it's inception to it's completion.
To my dear wife, Lynn, a very deep thank you
for your sacrifices, help, love, and support. In spite
of how inadequate it may sound, I could not have
accomplished this without your understanding and love.
I also need to thank those in the lab who have
helped with various parts of this project including
Michelle Lomme, Dave Shugarts, and James Frank.
This work was supported by a grant awarded to
Dr. Gerald Audesirk from the Environmental Protection
Agency.
I have four things to learn in life:
To think clearly without hurry or confusion;
To love everybody sincerely;
To act in everything with the highest motives;
To trust in God unhesitatingly.
Helen Keller


CONTENTS
CHAPTER
I. INTRODUCTION................................ 1
General Overview/Purpose.................. 1
Preface to Animals........................ 4
Lymnaea stagnalis as a Test Animal. 4
Embryonic Chicks as Test Animals.......... 5
Advantages/Disadvantages of
Experimental Animal..................... 6
Permethrin versus DDT..................... 9
Permethrin as a Toxin.................... 10
DDT as a Toxin........................... 16
Statement of Hypotheses/Research
Questions.............................. 20
Limitations and Delimitations of the
Experimental Model..................... 28
II. MECHANISMS................................. 30
Morphology/Physiology of Nerve Fibers
and Growth Cones....................... 30
Role of Cyclic AMP and Calcium in
Neurite Growth......................... 32
Pyrethroid Mechanism of Action........... 37
Effects on Voltage-Dependent Sodium
Channels............................. 40
Effects of TTX on Neurite Outgrowth.. 46


ix
Effects of Commercial Pyrethroids
on Cell Function.................... 47
Differences in Effect of Similar
Pyrethroids.......................... 48
Effects on Ca^+-ATPase................. 49
Effects on Norepinephrine Release.... 50
DDT Mechanism of Action.................. 51
III. METHODS AND PROCEDURES..................... 53
Lymnaea stagnalis........................ 53
Preparation of Solutions and Culture
Media................................ 53
Conditioning of Media.................. 56
Culture Plate Preparation.............. 56
Dissection Procedure................... 57
Cell Dissociation...................... 58
Data Collection Methods/Criteria..... 60
Immediate Exposure................... 60
Gross Morphology..................... 61
Delayed Exposure..................... 62
Embryonic Chickens....................... 63
Preparation of Solutions/Culture
Media................................ 63
Culture Plate Preparation.............. 65
Dissection Procedure................... 65
Cell Count and Dilution.............. 66
Data Collection Methods/Criteria..... 67
Methods Used to Analyze Data
67


X
Electrophysiological Recordings.......... 68
Intracellular Recording of
Spontaneous Activity................. 68
IV. OBSERVATIONS AND RESULTS................... 71
Effects of Permethrin on Neurite Out-
growth of L. stagnalis Neurons....... 71
Effects of DDT on Neurite Outgrowth of
L. stagnalis Neurons.................. 82
Comparison of Effects of Permethrin
and DDT on Neurite Outgrowth of L.
stagnalis Neurons....................... 93
Effects of Delayed Exposure of
Permethrin on the Neurite Outgrowth
of L. stagnalis Neurons............... 96
Effects of Permethrin on Neurite
Outgrowth of Embryonic Chicken
Neurons................................. 99
Effects of DDT on the Neurite Out-
growth of Embryonic Chicken Neurons.. 114
Comparison of Effects of Permethrin
and DDT on the Neurite Outgrowth of
Embryonic Chicken Neurons............. 117
Comparison of Permethrin and DDT Effect
on Neurite Outgrowth of L, stagnalis
and Embryonic Chicken Neurons........... 123
Analysis of Variance.................... 131
Effects of Permethrin on the Electro-
physiological Characteristics of
Individual Neurons of L. stagnalis. . 133
V. DISCUSSION................................ 151
Final Conclusions....................... 168
BIBLIOGRAPHY..................................... 170
APPENDIX
A. Solution and Media Composition
179


xi
Lymnaea stagnalis....................... 179
Embryonic Chicks........................ 181
B. Dissection Procedure...................... 186
Lymnaea stagnalis....................... 186
Procedure for Conditioning Media
(LL-15)............................... 187
Embryonic Chicks........................ 188
C. Cell Dissociation Procedure............... 192
D. Environmental Toxicology Data............. 194
E. Accidental Poisioning/Antidote............ 195
F. Storage/Disposal Data..................... 196
G. Warning Statements........................ 197
H. Biological Activity....................... 198
I. Cell Types
200


TABLES
Table
1. Culture Conditions Permethrin in
Alcohol Lymnaea....................... 73
2. Total Growth Versus Concentration
Permethrin in Alcohol Lymnaea......... 74
3. Cell Type Versus Concentration Lymnaea
Permethrin in Alcohol................... 78
4. Average Number of Neurites Per Cell And
Number of Branches Per Neurite
Lymnaea Permethrin in Alcohol......... 81
5. T-Scores for Lymnaea Neurite Outgrowth -
Permethrin in Alcohol Toxins Versus
LL-15 Control and ETOH Control.......... 83
6. Culture Conditions DDT in Alcohol
Lymnaea................................. 84
7. Total Growth Versus Concentration -
Lymnaea DDT in Alcohol................ 86
8. Cell Type Versus Concentration Lymnaea
DDT in Alcohol............................ 89
9. T-Scores for Lymnaea Neurite Outgrowth
DDT in Alcohol Toxins versus LL-15
Control and ETOH Controls............... 92
10. Summary of Delay Data Lymnaea with
Permethrin................................ 97
11. Total Growth Versus Concentration -
Permethrin in DMSO Embryonic Chicks.. Ill
12. T-Scores for Chick Neurite Outgrowth
Permethrin in DMSO Toxins Versus
LL-15 Control and DMSO Control
116


Xlll
13. Total Growth Versus Concentration -
DDT in DMSO Embryonic Chicks.......... 118
14. T-Scores for Chick Neurite Outgrowth -
DDT in DMSO Toxins versus LL-15
Control and DMSO Control................ 122
15. Total Growth versus Concentration -
Chicks Exposed to Permethrin and DDT... 126
16. 2x2x5 Analysis of Variance-L. stagnalis
and Embryonic Chicks x Permethrin and
DDT x 5 Concentrations of Toxin......... 132
17. Average Spikes per Minute Permethrin in
Alcohol RPD-1......................... 134


FIGURES
Figure
1. Diagram of Structure of Type I and
Type II Pyrethroids....................... 12
2. Structure of Permethrin..................... 13
3. Structure of DDT............................ 17
4. The Second Messenger Concept................ 33
5. Cyclic AMP Pathway.......................... 35
6. Mechanism of Action Potential Generation. 38
7. Repetitive Discharges Induced by a
Single Stimulus........................... 41
8. Repetitive Discharges Induced by a
Single Stimulus........................... 41
9. Effects of luM (+)-trans Allethrin on
Sodium Current............................ 43
10. Hypothetical Model for Interaction of
Tetramethrin with Sodium Channels......... 45
11. Lymnaea Neurite Outgrowth Permethrin
vs Controls.............................. 75
12. Lymnaea Neurite Outgrowth Permethrin
vs Solvent Controls...................... 76
13. Cell Types Lymnaea Permethrin in
Alcohol................................... 79
14. Cell Types Lymnaea Permethrin in
Alcohol................................... 80
15. Lymnaea Neurite Outgrowth DDT vs
Controls................................. 87
16. Lymnaea Neurite Outgrowth DDT vs
Solvent Controls......................... 88


XV
17. Cell Types Lymnaea DDT in Alcohol....... 90
18. Cell Types Lymnaea DDT in Alcohol....... 91
19. Lymnaea Neurite Outgrowth Permethrin
and DDT vs Controls.................... 94
20. Lymnaea Neurite Outgrowth Permethrin
and DDT vs ETOH Controls............... 95
21. Lymnaea Delayed Exposure to
Permethrin Sum of Neurite Length Over
Time.................................... 98
22-26. Pictures of Delayed Permethrin Exposure
to Lymnaea Neurites...................... 100
27. Embryonic Chick Neurite Outgrowth -
Permethrin vs Controls.................. 112
28. Embryonic Chick Neurite Outgrowth -
Permethrin vs DMSO Controls............. 113
29. Other growth parameters Embryonic
chicks Permethrin in DMSO............. 115
30. Embryonic Chick Neurite Outgrowth DDT
vs Controls............................. 119
31. Embryonic Chick Neurite outgrowth DDT
vs DMSO Controls........................ 120
32. Other growth parameters Embryonic
chicks DDT in DMSO.................... 121
33. Embryonic Chick Neurite Outgrowth -
Permethrin and DDT vs Controls.......... 124
34. Embryonic Chick Neurite Outgrowth -
Permethrin and DDT vs Solvent Controls. 125
35. Comparison of Neurite Outgrowth L.
stagnalis vs Embryonic Chicks,
Permethrin vs Controls and Solvent
Controls................................ 127
36. Comparison of Neurite Outgrowth L.
stagnalis vs Embryonic Chicks,
DDT vs Controls and Solvent Controls... 129


XVI
37. Number of action potential after
exposure to permethrin.................. 135
38-43. Electrophysiological Records of Lymnaea
Neurons Exposed to Permethrin.......... 136


CHAPTER ONE
INTRODUCTION
General Overview/Purpose
Many government agencies are charged with
the responsibility to test man-made substances and
determine their toxicity. Unfortunately, these
agencies are rarely able to do any significant
testing for chronic effects of many compounds
because of the large number of them and the expense
of long-term in vivo testing procedures necessary
for the evaluation of chronic effects for most
chemicals. Therefore, although a great deal is known
about the acute effects of many toxic compounds, not
much is known about their chronic effects.
Current testing methods and procedures for
chronic exposure suffer from two major drawbacks.
First, the majority of testing uses live animals
that must be sacrificed to obtain the desired
information. Such in vivo testing is expensive,
because of the cost of maintenance and feeding.
This limits any testing for long-term, low-dose
effects to small inexpensive experimental animals.


2
Secondly, since the animals are usually sacrificed
so that tissues can be analyzed for toxic effects,
you can't get data on the progression of toxicity
with time of exposure. This makes it very difficult
to determine cellular mechanisms.
In an attempt to improve on in vivo
studies, a number of researchers have used nerve
tissue preparations for the study of different
neurotoxins. These include nerve-muscle preparation
of the clawed frog, Xenopus laevis (Ruigt, et
al.,1986), isolated crayfish nerve cords, (Narahashi,
1985), ganglia from Lymnaea stagnalis (Audesirk,
1985), the cereal sensory nerve of the adult male
cockroach, Periplaneta americana (Gammon, et al.,
1983), and the giant squid axon (Lund, 1982).
These studies were short-term exposure studies of
seconds to hours exposure only. For this reason,
they may not be good models for long-term exposure
studies.
With the advent of reliable techniques,
several investigators have turned to neuronal cell
cultures in the search for more appropriate,
accurate, and faster techniques for cellular studies
(Wong, et al., 1984; Kostenko, et al., 1983; Wong,
et al., 1983; Kostenko, et al., 1982; Dagan, et al.,


3
1981; Wong, et al., 1981; Kostenko, et al., 1974).
The use of cell culture models decreases the number
of animals sacrificed to do research, has the
potential for allowing more direct observation and
elicitation of data, as well as limiting the effects
of extraneous factors such as extracellular
metabolites, genetic predisposition to growth and
metabolism, and environmental factors. The ability
to control these factors could lead to the use of
cell cultures as screening tests for the toxicity of
new substances. This would most likely take a
considerable amount of time, as acceptance of new
concepts is occasionally met with a large degree of
reluctance until such time as the new method has
proven itself to be as adequate as the prevailing
"gold" standard. For cultures to be readily accepted
several criteria should probably be met. They should
give results that agree with current toxicology data
and that agree with in vivo results. They should
help work out some of the mechanisms of action, and
they should be an accurate predictor of toxicity.
I will begin by introducing the test
animals and toxins used in this project, the
advantages and disadvantages of the experimental
model, the specific hypotheses being considered, and


4
any limiting factors that had to be considered.
Since not all readers of this thesis may be familiar
with cell culture techniques and toxicology, terms
will be defined as they are encountered as well as
abbreviations.
Preface to Animals
Two specific animals were used; Lymnaea
stagnalis. and embryonic chicks. For any toxicity
model to be appropriate for wide use, it should be
applicable to determination of human toxicity. For
this reason, embryonic chicks were used. However,
many toxins also affect animals in natural
ecosystems, and therefore a variety of species may
be best to use to determine the extent of toxicity
that may be encountered.
Lymnaea stagnalis As A Test Animal
Lymnaea stagnalis is a fresh water pond
snail that has a circumboreal distribution. This
particular animal was used for this project for
several reasons. The neurons of Lymnaea are very
large, upwards of 110 micrometers in diameter and
thus are very visible under minimum magnification.
This size facilitates intracellular recordings, and


5
the observation of growth in culture. It also helps
in the cell isolation since the cells can be
visualized as they are dissociated. Secondly, these
animals reproduce relatively rapidly in optimum
conditions with an animal reaching an average of one
gram in weight in approximately ninety days. They
also have a very high fecundity. They can be
manipulated in the lab to produce isogenetic
strains, thus reducing potential variability caused
by using cells from genetically different animals.
Kostenko et al. (1974, 1982, 1983) have
worked on culture media composition, cell isolation
techniques, and plating techniques for culture of
Lymnaea. Similar techniques have been developed and
used with two other molluscan species, Helisoma and
Aplysia (Wong, et al., 1983; Wong, et al., 1981;
Dagan, et al., 1981). In this project, several new
methods were developed. These are outlined in
Chapter Three.
Embryonic Chicks as Test Animals
The use of invertebrates as a test animal
indeed has its advantages. However, to be able to


6
appropriately apply any data obtained, it is
necessary to be able to show that these same data
can be obtained from other species more related to
humans. Ultimately it would be useful to be able to
perform these same types of experiments on primates
or other mammals. In lieu of this, we did do some
experiments on embryonic chickens to determine if any
significant species differences existed with respect
to these toxins. Embryonic chicks were used for
several reasons. First, they are easy to obtain and
maintain. Second, the procedure for culturing chick
neurons has been well worked out by previous
researchers (Freshney, 1983), and only had to be
adapted to our experimental design. Last, it is much
easier to get these cells to grow in culture than
invertebrate cells. Due to the ease of the isolation
procedure and the vast number of cells one obtains,
it is possible to do several experiments with the
cells obtained from one animal, thus dramatically
reducing the number of animals that are sacrificed.
Advantages/Disadvantages of Experimental Model
There are numerous advantages to doing this
type of testing using this model. Various authors


7
have stated the positive aspects of using
neuroculture models for the investigation of various
neurotoxic agents as well as the possibility that
these types of models could very well replace the
current testing procedures such as the Draize Ocular
Allergy Test (Borenfreund, et al., 1984; Yonezawa,
et al., 1983; Damstra, et al., 1983; Hooisma, 1982).
There are several advantages to doing this
study with growing neurites. Because of the ease in
observation, it was felt that this would be a more
efficient method to attempt to determine a toxicity
versus neurite outgrowth curve. Embryonic animals
are very capable of extending new neurites in an in
vitro system. Using this model allows a study of the
influences of toxins on neurite outgrowth, which is
in contrast to an in vivo model where influences on
growth are not easily studied. It is much easier to
quantitate growth and to define the causes of toxins
directly in an in vitro system. Finally, there are
many toxins which exert an effect in developing
embryos. More specifically, it was felt that
measuring neurite outgrowth and observing some
aspects of neurite gross morphology in a culture
system would be much easier than making these
observations from whole animals. It would also


8
enable me to do sequential temporal observations of
growth. Having a "naked" cell might allow the more
direct examination of channel electrophysiology
through intracellular recording and correlation to
morphologic data and toxin/environmental factors.
Last, this model using vertebrate species reduces
the number of animals that are sacrificed for each
experiment in that one animal is able to provide a
large enough quantity of cells to do several
experiments.
There are a number of disadvantages to this
model as well, and must be taken into consideration.
Since many if not all of the toxic substances we
encounter are taken into the body through oral
ingestion, inhalation, or dermal absorption, it is
possible that some type of metabolic interaction may
take place between the toxic substance and other
molecules within the body. It could be that this
interaction synthesizes some intermediate toxin
that is the actual causative agent in altered growth.
There is no known way at present to test this
possibility in an invertebrate cell culture system.
Second, this model did not allow for the
determination of what effect or relationship the
blood-brain barrier may have had with these toxic


9
substances. It is a well documented fact that due to
the tight junctions between the pia mater and
capillary bed of the choroid plexus, between the
capillary endothelial cells and as a result of
reduced endothelial pinocytosis, a great number
of substances are selectively screened out and never
enter the brain cavity, except in embryonic and
diseased brains, which have a leaky blood brain
barrier. Third, with the neurons being dissociated
from the brain, it was not possible at this time to
study the effect or action any of the neuro-support
cells such as oligodendrocytes, astrocytes, or
microglia may have on neurons exposed to these
toxins. Last, there are numerous technical problems
associated with cell isolation, particularly the
Lymnaea neurons. One of major importance is the need
for a protein currently called conditioning factor
that must be present for neurite outgrowth to occur.
This factor's potential effect on the toxin-cell
interaction is unknown at present.
Permethrin versus DDT
These two toxins were used in this project
due to the differences one observes with respect to
their toxicity. Permethrin is considered


10
to be a relatively "safe" insecticide with a very
low mammalian toxicity level. DDT on the otherhand,
is well known for its extreme toxicity, particularly
within many avian and fish ecosystems. Since one of
the objectives of this project is to develop a
toxicity model for these types of toxins, the use of
a low versus high toxic compound will provide data
as to whether or not the necessary sensitivity will
be present.
Permethrin As A Toxin
Synthetic pyrethroids were first employed in
agriculture in the mid-ninteen seventies. They are
an analog of the natural pyrethrums, an extract of
chrysanthemums. They have rapidly become one of the
most commonly used types of insecticides in today's
agricultural market. Although previous toxicology
studies seem to indicate that there is a rather
large saftey margin associated with pyrethroid use,
neurotoxic symptoms have been observed in several
experimental animals (Penick Corp.).
Pyrethroids are divided into two categories,
based on biochemical structure and symptomology
observed in experimental animals (Narahashi,
1985; Narahashi, 1982a; Narahashi, 1982b;


11
Wouters, et al., 1978; Verschoyle et al., 1982).
Verschoyle and Aldridge originally created
two categories of pyrethroids based on the clinical
symptoms exhibited by affected animals: tremor (T)
which is a condition of very fine, rapid muscle
contractions, and choreoathetosis with salivation
(CS) which is a condition of slow, wide, extremity
movement and weakness with an associated excessive
salvation. Lawerence and Casido (1982) independently
classified pyrethroids based on structural
differences of the primary structure. Type II has
a cyano- group located at the alpha carbon of the
pyrethroid molecule and Type I does not (Fig. 1).
Lawerence and Casido (1982) found that the
Type I pyrethroids corresponded symptomatically with
the T pyrethroids of Verschoyle and Aldridge, while
Type II corresponded with CS pyrethroids. The Type
I-II terminology of pyrethroid classification is the
most commonly accepted form used today.
In rats, Type I pyrethrin toxicity causes
such physical symptoms as sparring, aggression, and
a gradual tremor leading to full extension of the
body. Death is usually associated with clonic
seizures (Verschoyle and Aldridge, 1980; Verschoyle
and Barnes, 1972).


12
PYRETHROIDS
TYPE I
TYPE II
Fenvolerote
0
Fig. 1 Structures of Type I and Type II pyrethroids.
The major structural difference between these two
classes of pyrethroids is the presence of a cyano-
group in the Type II pyrethroids.


13
Type II toxicity causes distinctly different
symptoms including excessive salivation, whole body
tremor, splayed gait of the hind legs, and
choreoathetosis (Gray, 1985). For a more detailed
explanation of the proposed mechanisms of action and
molecular events associated with pyrethroids, please
see Chapter Two, Mechanisms.
The specific pyrethroid employed in this
project is called permethrin. Formally, permethrin
is 3-(phenooxyphenyl)methyl(+)-cis,trans-3-(2,2-
dichloroethenyl)-2,2-dimethylcyclopropane-
carboxylate. It's chemical structure is (Fig. 2);
Permethrin
It is a Type I pyrethroid discovered by Michael
Elliott and associates at Rothamsted Experimental
Station in England during their work on synthetic
analogs of natural plant products. Permethrin has an
extremely high level of activity against a broad
spectrum of insect pests while having a low level of


14
acute mammalian toxicity (Penick Corp). Due to its
photostability it is much more persistent on inert
and plant surfaces than the natural form, pyrethrum,
or several of the other synthetic forms such as
resmethrin, allethrin, or tetramethrin. Its
empirical formula is ^21'^20^'*'2^*3 ^ ^as a mo-*-ecu-*-ar
weight of 391.3. It is water white to pale yellow in
color and occurs as a colorless crystal to a viscous
fluid. It has a viscosity of 340 at 25 C (in CPS),
O
and boils at 220 C at 0.05mmHg. It has a vapor
O
pressure of less than 10 Torr at 50 C and a specific
gravity of 1.190-1.272 at 20 C. It is soluble or
miscible in most organic solvents except ethylene
glycol and is only soluble less than lppm in water.
O
It has a flash point of >102 C, a viscosity of 340
at 25C (in CPS) and is moderately explosive when
exposed to heat or flame and reacts with oxidizing
material. It has a cis/trans ratio of approximately
65/35, with a half-life in soil of 10-25 days at
O
25 C depending on soil type (Penick Corp.).
Soil stability studies show permethrin is
hydrolyzed in soil by present micro-organisms, while
soil mobility studies indicate it is immobile in a
wide range of soil types (Penick Corp.).
When administered orally to rats, permethrin


15
is rapidly metabolized and almost completely
eliminated from the body in a few days. Only three to
six percent of administered doses were excreted
unmodified in the feces of the animals.
Animal toxicology studies have yielded the
following data. Acute oral toxicity of technical
material resulted in a LD^-q of >4000 mg/kg for each
of the following animals; rats, mice, guinea pigs,
and rabbits. Acute dermal toxicity resulted in a
LD^q >4000 mg/kg in rats and >2000 mg/kg in rabbits.
Acute inhalation resulted in a of 23.4mg/L
(four hour exposure) in rats. Sub-acute toxicity
testing of ten daily doses of lOOOmg permethrin per
kg/day to rats caused no toxic signs, abnormalities,
or deaths. A 90-day no effect level of lOOmg/kg/day
equivalent to 4000ppm in the diet has been
established in dogs. Permethrin is not known to be
teratogenic or mutagenic (Penick Corp.).
Environmental toxicology data, accidental
poisioning/antidote, storage/disposal data, and
warning statements as well as biological activity and
manufacturers suggested treatment application are
included in appendices D through H.


16
DDT as a Toxin
DDT, dichlorodiphenyltrichloroethane (Fig.
3), has a long and quite interesting history. It was
first synthesized in 1874 by Othmar Ziedler who was
working on substitution products of aromatic
hydrocarbon compounds. In 1930, on a hunch, Paul
Muller of Geigy used it against a variety of insect
pests including potato beetles and clothes moths and
found it worked very well in controlling these
pests. As World War II broke out, there became an
obvious need for something that would combat insect
borne diseases such as malaria, and typhus.
DDT was the chemical of choice.
In 1942 the firm of J.R. Geigy supplied the
Department of Agriculture with a product trade-named
Gesarol. It turned out on analysis to be DDT. Further
testing at that time revealed that it worked very well
against a broad spectrum of insect pests as well
as appearing not to have the harmful side effects of
many of the arsenic based insecticides being used
commercially at the time.
DDT became the overall agricultural chemical
of choice after showing its effectivness in
controlling the Naples Typhus epidemic of 1943. DDT
started being used more and more commercially as well


Fig. 3 Structure of DDT


18
as within the military. It was only after being used
on a broad level that the true hazards started to
become obvious. Application of DDT to forests and
fields resulted in significant bird and fish
death as well as a dramatic reduction in the breeding
populations of these animals.
It was shortly after this that the potential
danger to humans also started to become evident. It
was found that DDT was very readily and easily
absorbed into the body fat of humans, and that
lactating mothers passed significant levels of this
compound on to their children during breast feeding.
DDT exerts its effects neurologically
by slowing the closure of the voltage dependent
sodium channels once they have been opened by an
action potential, resulting in an increased
hyperexcitable state.
The symptoms of DDT toxicity include
hyperexcitability in vertebrates as well as
invertebrates, increased sensitivity to external
stimuli, convulsions, tremors, and ataxia. There are
repeated discharges on a cellular level in both
motor and sensory nerve fibers. Skeletal muscles
exhibit tetanic contractions (Narahashi, 1984).


19
There are several forms of DDT available
with varying degrees of toxicity. The particular
form that I used is the p,p'- dichloro-
diphenyltrichloroethane form which is the
most toxic form of this compound based on EPA
studies. DDT has a molecular weight of 354.50, with
an empirical formula of C^HgCl^. It has a melting
point of 108.5-109C. It is practically
insoluble in water, dilute acids, and dilute bases.
It's solubilities (g/lOOml) are: acetone 58, benzene
78, carbon tetrachloride 45, chlorobenzene 74,
cyclohexanone 116, and 95% alcohol 2. It is freely
soluble in pyridine and dioxane. Solubility in
organic solvents increases dramatically with an
increase in temperature. It is resistant to
destruction by light and oxidation and this has
resulted in difficulties in residue removal from
water, soil, and foodstuffs.
Human toxicity: Poisioning may occur by
ingestion or by absorption through the skin or
respiratory tract. Acute poisioning symptoms include
tremors of head and neck muscles, tonic and clonic
convulsions, cardiac or respiratory failure and
death. The estimated oral fatal dose is 500mg/kg body
weight of the solid material. Solvents such as
kerosene increase toxicity. Death occurs in two to


20
twenty-four hours. Chronic poisioning symptoms
include hepatic damage, CNS degeneration, agranulo-
cytosis (an increase in granular leukocytes),
dermatitis, weakness, convulsions, coma, and death.
Statement of Hypothesis/Research Questions
Do pyrethrins and/or DDT have any direct effects
on the growth of neurites in a cell culture system?
A great deal of work has been done on the
effects of permethrin and DDT on axonal growth in
different experimental animals, both during
development and after delayed exposures. It would
appear that one of the primary mechanisms of action
of these toxins is to cause severe distal axonal
degeneration. Herbert E. Lowndes and Thomas Baker
define distal axonopathy as follows:
Distal axonopathies are, by definition,
degenerative events in axons (sensory or
motor) that may appear as primary
manifestations of neurotoxicity and are
not secondary to demyelination, compromise
of Schwann/glial cell function or impairment
of vascular integrity; pathological changes
predominantly in the distal portions of the
axon, with little or no corresponding
alterations in the cell body. It is not
necessary, however, to consider pathological
changes as the sole criteria of the onset
of distal axonopathy, because neural
dysfunction often precedes the apperance
of morphological alterations (Chapter 13,
page 193).
There are three theories that have been proposed as


21
to the mechanism underlying distal axonopathies. They
are (i) neuron somal compromise, (ii) abnormal
anterograde axonal transport, and (iii) direct axonal
damage. Neuron somal compromise is the currently
favored hypothesis. It is felt that the axons suffer
from a lack of the necessary essential materials
needed for survival that are transported from the
neuron soma down to the most distal portions. This
would in turn lead one to think that these toxins
are in some way exerting their effect
intracellularly by altering some essential
metabolic biochemical pathway directly or through a
second messenger system such as the calcium-
calmodulin or cyclic AMP pathways.
The abnormal anterograde axonal transport
theory states that the "neurotoxin interferes either
with the mechanism by which materials are exported
from the neuron soma, or with the axonal transport
system by which materials are conveyed along the axon
to distal sites of utilization" (Sabri, et al.,
1982; Chapter 14, pp206-207). Again, when you
realize the importance of specific ions such as
calcium to the synthesis of microtubules and other
cytoskeletal associated structures, it could be
entertained that these toxins are affecting ionic


22
balances between intracellular and extracellular
compartments either directly or indirectly, and thus
altering the cell's capability of transporting the
necessary molecules down the axon. The direct
axonal damage theory states that toxins exert a
direct local toxic effect along the entire axon.
This is supported in part by work done by Spencer
and Schaumburg (1974) where they feel that many of
the distal axonopathies that are seen cannot be
fully explained by either one of the other two
theories. This is also based on the assumption that
vulnerabilites of the soma are not the same as those
experienced by the axons of the soma (Sabri, M.I.,
et al., 1982). This could be of some significance
particularly when one considers the difference found
with respect to ion channel populations between
neuronal somas and axons. This is especially
important with respect to calcium channels, which
are found in high concentration on cell bodies and
are virtually absent on axons.
Morphologic indications of toxicity include
symmetrical distal axonal degeneration occurring
concurrently in peripheral and select tracts of the
central nervous system. It is thought that changes
are caused by a degeneration of long, large diameter


23
axons in the peripheral nervous system and spinal
cord (Davis, and Richardson, 1979).
The spinal cord tracts, dorsal columns of the
fasiculis gracilis, corticospinal pathways, and
spinocerebellar tracts show distal axonal
degeneration in response to acute toxicity.
Degeneration has also been found in the white matter
of the cerebellar vermis of cats. It appears that
damage increases from proximal to distal with
respect to the cell body of the neuron. With this in
mind, one would suspect that evidence of toxicity
should be observed in vitro when neurons are exposed
to various concentrations of toxin. This would most
likely be seen with either numbers of neurites
and/or length of neurites seen in vitro. One would
also have to suspect the cell survival in general
would be affected if doses of toxin reach high
enough levels.
Are there solvent/pesticide interactions that
might cause altered growth patterns
in neurites?
Research has shown that there is a definite
synergistic/antagonistic relationship between
pesticides and the solvents used to dissolve them.
This work was done using the fungus Pythium ultimum
and several solvents including acetone, ethanol,


24
methanol, hexane, dimethyl sulfoxide, and N,N-
dimethylformamide. The fungicide captan was mixed with
these solvents and the fungus was then exposed to this
mixture. This mixture was considered toxic when growth
of the fungus was reduced by fifty percent or more. In
summary, it was shown that ethanol, methanol, and N,N-
dimethyIformamide were the most toxic with EC,-q values
of 0.51 to 2.29% (v/v), acetone and dimethyl sulfoxide
were moderately toxic with EC,_q values of 1.99 to
12.07% (v/v) and the least toxic solvent was hexane
with an EC^q value of 36.60% (v/v). There were also
some synergistic as well as antagonistic interactions
noted dependent on both the amount of the fungicide
and the amount of the solvent used (Stratton,
1985). Therefore, with this in mind, I felt it was
necessary to be sure that there were not some type of
interactions occuring as noted above with
permethrin or DDT and the two solvents used, ethanol
and DMSO respectively.
Alcohol has been shown to be toxic to
neurite growth. Exposure of rat myocardial cells to
600, 800, and 1000mg% ethyl alcohol resulted in
several findings. Toxicity was evaluated by
measuring the amount of lactate dehydrogenase and
succinate dehydrogenase leakage after exposure to


25
ethyl alcohol. Exposure of one hour, four hours, and
twenty-four hours all showed increases in LDH and
SDH leakage as well as a decrease in the beating
rates (Butler et al., 1985). Dow et al. (1985) found
that ethyl alcohol inhibits process formation by
chick embryo sensory and spinal cord neurons. This
was dose-dependent. Neurotrophic factor production
was also decreased. Adhesion, survival and receptor
interaction of sensory neurons with nerve growth
factor were not affected (Dow, 1985).
Do permethrin and/or DDT have any delayed effect on
existing neurites?
It will be important to determine
whether or not permethrin and DDT have any
effect on neurites that already exist. If isolated
cells were to be grown in a culture system and allowed
to gain significant neurite growth, would exposure to
permethrin and DDT significantly alter these neurites?
One would suspect that if permethrin and/or DDT are
altering the cytoskeletal components in such as way as
to decrease growth, that if neurites are already in
existenance then perhaps these microtubule structures
would not be affected by delayed exposure. On the
other hand, it could be that permethrin and DDT would
alter the cells ability to synthesize necessarily


26
essential macromolecules or alter the transport system
of the axon, thus causing retraction and eventual
death of the neurite and/or cell.
Is there any significant difference between the
effects of permethrin and/or DDT on different
species ?
It would be conceivable that these two toxins
would have different levels of toxicity with respect
to different species tested. This would be important
to determine if this model is to be used for any type
of toxicity testing. It is also conceivable that
different species may have differing physiological
patterns that would render one or the other more or
less susceptible to these toxins.
Do these toxins alter neuronal physiology?
Is there any correlation between concentrations
causing decreased growth in a cell culture model
and concentrations causing abnormal
electrophysiological patterns in exposed neurons?
Numerous researchers have found through
techniques such as single electrode intracellular
recording, voltage clamping, and patch clamping that
DDT and Permethrin do affect the voltage dependent
sodium channels in neuronal membranes. The bulk of
this work has been done using isolated axonal
preparations from squid, crayfish, and cockroach


27
(Narahashi, 1980; Vijverberg, et al., 1985; Gammon,
et al., 1985). The exact mechanism of these toxins'
action on the appropriate channels is discussed in
detail in the next Chapter. This relates to the
question of whether or not these channels are
necessary for the extension of new neurites in a
cell culture model. If sodium channels are being
altered, is this in turn causing other physiological
changes resulting in a faulty mechanism for
cytoskeletal formation and decreasing in neurite
outgrowth?
Does the in vitro neurotoxicity found in cell
culture correlate with in vivo toxicity?
In order for this model to be considered an
adequate assay for neurotoxicity, it must correlate
with known data with respect to in vivo toxicity. If
the data that is obtained from an in vitro assay
varies significantly from data obtained from an in
vivo model, or if there are significant differences
between species, then this model would not be
acceptable. It must be determined that this
correlation exists.


28
Limitations and Delimitations of the Experimental
Model
There are several factors that could not be
controlled by the experimentor that had to be taken
into consideration when developing this project.
First, there may be some type of predisposition
determined genetically with respect to neurite
outgrowth that would be difficult to determine or
control. It is for this reason that having an animal
such as Lymnaea which is capable of being genetically
controlled to produce an isogenetic strain would be
helpful. Secondly, it has been determined that the
location of neurite outgrowth from the cell membrane
is an intrinsic property of the cell and as of now is
not understood well enough to begin to control. If a
cell were to adhere to the substrate in such a way as
to block this exit site, neurite outgrowth
theoretically would not occur. Last, the substrate
used for cell adhesion is critical as research has
shown that this is necessary for cytoskeleton-related
structures to extend and facilitate axonal outgrowth
(Hammarback, et al., 1986; Bunge, 1986; Edelman,
1983; Johnston, et al., 1980).
There were several delimiting factors, or
factors under the control of the experimentor that
need to be considered as well. First, due to the


29
nature of the difficulty of cell isolation of Lymnaea
there were steps in this procedure that could
ultimately affect how well isolated cells grow.
This would be independent of the effects of any
substances in the culture media, or associated with
the test animals. These problems could arise during
dissection, during removal of the desired tissue from
the organism being used, or as a result of pipetting
of the ganglia for dissociation. Second, there was the
potential for outside contamination of the media thus
resulting in contaminated surrounding for the growing
neurites. The metabolic products of these unwanted
organisms would have a dramatic effect on the pH of
the media and could alter the growth of neurites, as
well as affect neuron survival. Third, with respect to
the electo-physiologic recording, identification
and/or successful penetration of the exact same cell
in every brain was quite difficult. Although attempts
were made to use the same cell in every recording,
this was not always possible due to congenital
variations of the animal, excess connective tissue
which did allow successful penetration of the cell, or
experimentor error. When this occurred, other cells
were used to obtain some data rather than discard the
animal or tissue without any data being gathered.


CHAPTER TWO
MECHANISMS
Morphology/Physiology of Nerve Fibers and
Growth Cones
Neurites grow as a result of the extension of
a morphologic structure called a growth cone. This is
a fan-shaped structure found at the end of an
extending neurite. Growth cones are composed of several
structures. These include microtubules, which are 24-
28nm in diameter, and neurofilaments, which are 9-
llnm in diameter. Both microtubules and
neurofilaments lie in a longitudinal manner in the
cell and are associated with mictrotubule associated
proteins (MAP's). MAP's may promote the lateral
interaction of microtubules and organelles of the
neurite as well as permit transport of vesicles and
mitochondria down the neurite (Johnston, et al.,
1980). This is critical in that axonal processes of
neurons lack synthetic capabilities and are dependent
on the transport of necessary molecules and nutrients
down the axon to the area of need (Bunge, et al.,
1986) .


31
Growth cones also contain microfilaments,
which are 4-6nm in diameter and form a mesh just below
the plasma membrane. These structures may play a
critical role in the attachment mechanism.
There are five groups of axonal components
that move at maximum time average velocities of l-4mm
day ^ and move in an anterograde fashion. These
include the slowest, neurofilaments and microtubules,
and the next slowest, actin. The fast moving
components move 100-400mm day ^ and move either
anterograde or retrograde. The anterograde moving
components include most of the vesicular structures
whereas most of the retrograde components are larger
in size, 0.1-0.5um in diameter, and are mostly
lysosme structures (Bunge, et al., 1986).
Neurite outgrowth rates vary considerably,
varying from 50um per hour to lOOum per hour. They
are capable of stopping and starting again without
any apparent external signal.
Neurite outgrowth is also dramatically
affected by the substrate. Several researchers using
in vitro culture systems with molluscs have employed
several different substrates for neural attachment.
These include using chick plasma on glass coverslips
(Dagan, 1981), polylysine on cover slips (Wong, et


32
al 1984), collagen or polylysine (Wong, et al.,
1981) and primaria dishes (Audesirk, 1985).
Although the exact mechanism of neurite
outgrowth is not worked out as yet, a great deal is
known. Neurites begin their growth when the growth cone
"feels around" for an appropriate substrate. Once this
is found, the growth cone attaches, (a function of the
microfilaments), and extending microtubules then
stabilize the neurite. The growth cone then continues as
before, feeling around for an appropriate substrate and
the cycle repeats.
Role of Cyclic AMP and Calcium in Neurite Growth
Cyclic AMP is thought to act as a second
messenger in neurons. Fig. 4 summarizes this second
messenger concept at synapses. A neurotransmitter (T)
interacts with its appropriate receptor in the plasma
membrane (A). This triggers cAMP production through
adenylate cyclase (B). Cyclic AMP then phosphorylates
a membrane protein via a protein kinase. This
protein, being linked to an ion channel, triggers a
synaptic potential (Bradford, 1986).
This entire chain of events is reversed via
phosphoprotein phosphatase and phosphodiesterase.


33
Presynaptic nerve terminal
Neurotransmittcr
(T)
Postsynaptic neuron
ATP
Adenyl cyclase
r,
Cyclic AMP
(2d messenger)
Protein kinase
A. Transmitter receptor
B. Closely associated
adenyl cyclase
C. Ion conductance
channel in membrane
Phosphoprotein
phosphatase
Fig. A The second messenger concept. Neurotransmitter
receptor (A) interacts with neurotransmitter (T) and
initiates cyclic nucleotide formation via a cyclase
enzyme (B). The cyclic nucleotide is the second
messenger. It phosphorylates a membrane protein via a
protein kinase. This protein is closely linked to an ion
conductance channel (C) leading to a membrane potential
change (synaptic potential). (Bradford, 1985; page 168)


34
Phosphoprotein phosphatase dephosphorylates the
protein kinase and substrate protein and
phosphodiesterase inactivates cAMP (Bradford,
1986)(Fig. 5).
Cyclic AMP may act as a second messenger in
most other cells as well. This second messenger has
several manifestations in cells including protein
phosphorylation, changing of membrane permiability to
specific ions, and control of intracellular levels of
calcium. It can also influence rates of
neurotransmitter synthesis, regulate microtubule
function, and accelerate carbohydrate and lipid
metabolism that supplies the necessary cellular
energy for physiologic responses (Bradford, 1986).
Cyclic AMP is also important in gene activation.
The polymerization of microtubules and
assembly of the cytoskeleton are influenced by cAMP
(Browne, et al., 1982; Nigg et al., 1985). A great
number of interactions between intracellular messengers
and microtubules are mediated by cAMP-dependent
protein kinase and calcium-calmodulin-dependent
protein kinase, which phosphorylates both tubulin
(Burke and DeLorenzo, 1981; Goldenring, et al., 1983)
and microtubule-associated protein (Tsuyama, et al ,
1986; Yamamoto, et al.,
1983; Yamauchi and Fujisawa,


35
Fig. 5 Pathways of information flow for the
mobilization of glycogen by skeletal muscle. Binding of
epinephrine to its receptor specifically stimulates cAMP
synthesis, which activates cAMP-dependent protein
kinase. Phosphorylation activates phosphorylase kinase
but inactivates o^her enzymes of glycogen metabolism.
The effects of Ca ions are synergistic with those of
cAMP. Solid arrows, stimulatory signals; open arrows,
inhibitory signals. (Harold, 1987; page 493)


36
1983). This would lead one to predict that increases
in cAMP or calcium would activate cellular kinases
inhibiting microtubule polymerization. Decreases in
cAMP or calcium would have the opposite effect, and
promote polymerization of microtubules.
It has been found that the addition of
millimolar amounts of cAMP analogs, such as dibuturyl
cAMP, to primary neurons causes increased morphologic
differentiation (Schubert, et al., 1978). It has also
been found that cAMP restores the ability to
polymerize microtubules in certain microtubule
assembly-deficient cells (Means et al., 1982B).
Calcium and calmodulin interact with cAMP
also. Calcium currents have been stimulated in
several cell types where cAMP has been used (e.g.,
cardiac cells: Reuter, 1983; snail neurons: Green and
Gilette, 1984; Hockberger and Connor, 1984). Calcium
currents stimulated by cAMP should also create an
activation of calmodulin. On the other hand,
calmodulin may alter cAMP metabolism through
stimulation of adenylate cyclase and/or
phosphodiesterase (Chao, et al., 1984).
Anglister, et al. (1982), using mouse N1E-115
neuroblastoma cells have shown that there are a
large number of calcium channels located on the


37
growth cones of cells. They have found further that
growth cone expansion and elongation are promoted by
the injection of intracellular current. Application
of cobalt and/or cadmium to block calcium channels
also stops growth of neurites. Contradictory
information from Cohan and Kater (1986) would
indicate that action potentials inhibit neurite
growth in neurons cultured from the pond snail
Helisoma trivolvis. They have found that large
transient increases in calcium in the growth cone
occur along with action potentials, which would
suggest that calcium fluxes may inhibit neurite
outgrowth. One must take into consideration that the
rate of generated action potentials was quite
different between these two studies.
Pyrethroid Mechanism of Action
It would be helpful at this point to review
action potential generation so that further
discussion will make more sense. Fig. 6 summarizes
this phenomenon graphically, where E, is the
1C
equilibrium potential for potassium, E^ is the
equilibrium potential for sodium, gNa is the membrane
sodium conductance, gK is the membrane potassium
conductance and AP is the generated action potential.


38
Passive Fluxes Na-K Pump
Fig. 6 Mechanism of action potential generation. Upper
half illustrates changes in membrane sodium conductance
(gNa) and potassium conductance (gK) during an action
potential (AP). Resting potential (RP) is close to the
potassium equilibrium potential (E^), and the peak of
the action potential approaches the sodium equilibrium
potential (E^ ). Lower half illustrates ionic fluxes
during the action potential and recovery. (Narahashi,
1985; page 5)


39
In summary, it is an increase in gNa+ or opening of
the voltage dependent Na+ channels that accounts for
the rising phase of the action potential and closing
of or decrease in gNa+ along with an opening of the
potassium channels or an increase in gK+ that causes
the descending portion of the action potential.
Therefore, an increase in the depolarization after-
potential with pyrethroids could be due in part to
inhibition of gNa+ decrease, and/or a decrease in
gK+. It is important to note that all pesticide work
has been done on isolated axons which contain few
calcium channels.
Classical pyrethroids are esters of
cyclopropane carboxylic acids with alkenylmethyl
cyclopentenolone alcohols. Their activity depends on
the intact ester, and derivations of either the
alcoholic or acidic components are inactive. Activity
is influenced by the absolute configuration of the
asymmetrical carbon atom at C-l of the
cyclopropane ring and at C-4 of the cyclopentenolone
ring. All very active pyrethroids have an (R)-
configuration at C-l and an (S)-configuration at the
C-4 location (Wouter, et al., 1978).


40
Effects on Voltage-Dependent Sodium Channels
Pyrethroids have been shown to elicite
repetitive firing in muscle end plates (Fig.7).
Studies have shown that this is not as a result of
responses evoked in end-plate membranes, but are a
result of presynaptic nerve fiber firing as shown by
extracellular recording of end-plates (Narahashi,
1985) .
Tissues from experimental animals have been
exposed to various pyrethroids and examined
electrophysiologically. It has been shown that a
single stimulus delivered to many of these
preparations elicits repetitive firing in the
stimulated axon. Further work has shown that this is
due in part to an elevated after-potential and
eventual depolarization of the resting potential and
a new action potential (Fig. 8).
If one were to look at the anatomy of an
action potential in one of these test animals, it
would be nearly impossible to say
what the causative agent of the delayed
after-potential may be. The use of voltage clamping


5ms
20mV (_
2ms
Fig. 7 & 8 Repetitive discharges induced by a single
stimulus in a crayfish giant axon exposed to lOuM (+)-
trans tetramethrin. Intracellular recording. A, control;
B, 5 min after application of tetramethrin; C and D, 10
min. (Narahashi, 1985; page 4)


42
has allowed the investigation of the channels in the
plasma membrane and researchers have determined that
the most likely candidate for this action is the
voltage dependent sodium channels.
Fig. 9 shows the membrane currents associated
with step depolarizations with voltage clamping,
where I is the capacitative current, I is the total
membrane ionic current, I is the sodium current, I
Na K
is the potassium current, and I the tail current.
Here, the ionic current flows in an inward direction
and is transient in nature. This transient current is
carried by the sodium ions and the steady-state
current is carried by the potassium ions.
Fig. 9 is an example of the effects of a
specific pyrethroid, allethrin, on the sodium
currents of a squid axon. It is easy to see that
after application of allethrin, the tail current,
which is the resultant resting potential after the
step-depolarization associated with the closing of
the sodium channel at the repolarized potential level
is increased and prolonged in its decay time course.
All of these phenomena have been observed in
various species such as lobster, squid, and giant
cockroach axons as well as N1E-115 neuroblastoma
cells (Lund, et al., 1982; Gammon, 1983; Yamamoto,


43
Fig. 9A Membrane currents associated with a step
depolarization of the membrane under voltage clamp
conditions. I capacitative current; I total membrane
ionic current; I sodium current; I,rapotassium
current; tail current (Narahasni, 1985; page 7)
sodium current of a squid giant axon. A, a transient
inward sodium current is followed by a steady-state
outward potassium current before application of
allethrin. B, the potassium current was blocked by
replacing internal potassium with cesium. C, sodium
currents as in B, but before and after application of
allethrin. Note that the inward steady-state sodium
current during a step depolarization are both increased
markedly by allethrin. (Narahashi, 1985; page 8)


44
1983; Nishimura, 1985; Vijverberg, et a1., 1985;
Yamamoto, et al., 1986).
A mechanism has been proposed for the
interaction of pyrethroids with the sodium channels.
Most pyrethroids exist as two optical isomers and two
geometrical isomers. The (-)-trans and (-)-cis
isomers have little or no effects. However, the (+)-
trans and (+)-cis are effective in a dose-dependent
manner .
Based on these observations, a scheme has
been developed for the site of binding of
various isomers in the sodium channel (See
Figure 10). There are trans and cis agonistic
sites to which (+)-trans and (+)-cis
tetramethrin bind with a high affinity. In
addition, there is a negative allosteric
site to which (-)-trans and (-)-cis
tetramethrin bind causing inhibition of the
action of active (+) isomers. TTX binds
to a site seperate from any of the above.
(Narahashi, 1982)
Based on the work of Anglister et al. (1982)
one might expect the effects of pyrethroids on
neurite outgrowth to be both quantitatively and
qualitatively related to the pesticide and its
concentration. Neurotransmitter release is direcly
related to and dependent on calcium. An influx of
calcium occurs when action potentials occur and the
cell is depolarized. The greater the number of action
potentials, or the greater the magnitude of action


45
(+)-cis (-).cis
(high affinity) (high affinity)
Fig. 10 Hypothetical model for the interaction of
tetramethrin with the sodium channel. (Narahashi, 1985;
page 19)


46
potentials, the greater the influx of calcium. Lower
concentrations or weakly active pesticides that
induce only moderately increased firing should
stimulate neurite growth through small increases in
calcium influx through calcium channels; high
concentrations or very active pesticides should
inhibit growth by causing massive calcium influx
during high-frequency firing.
Effects of TTX on Neurite Outgrowth
Van Huizen and Romijn (1987) have shown that
tetrodotoxin (TTX), a known blocker specific for
sodium channels can initially alter several
parameters of neurite outgrowth in vitro. The
addition of 10 TTX to culture of isolated fetal
rat cerebral cortex cells resulted in (1) an
increased percent of cells and cell aggregrates with
neurites as compared to controls, (2) an increase in
the number of neurites per cell as compared to
controls, and (3) an increase in the length and
branching of neurites as compared to controls. These
effects were seen during the first forty-eight hours
of exposure. It is important to note that beyond this
time, growth parameters showed a steady decrease with


47
time .
It is also interesting to note that a TTX
concentration of 10 TTX had similar effects as 10
^ TTX, only not as pronounced which would tend to
make one believe this effect may be dose or
concentration related.
In any case, this creates several questions
with respect to the role of sodium channels and the
initial outgrowth of neurites in vitro.
Effects of Commercial Pyrethroids on Cell Function
It has been shown that different commercial
derivatives and mixtures of Deltamethrin, another
pyrethroid, have considerably different effects when
applied to cells in vitro. Baeza-Squiban et al.
(1987) tested the effect of technical deltamethrin
and a commercial form of the pyrethroid, DECIS on
growth parameters of both plant and animal cells.
Using the marine phytoplankton Dunaliella biocylata,
a fresh water algae Chlamydomonas reinhardii, and a
mouse fibroblast cell line, NCTC clone L929, these
investigators looked at several factors. These
included effects of pyrethroids on cell
proliferation, timing of the cell cycle, cellular


48
uptake of the toxin, and it's location within the
cell on uptake. Concentrations ranging between 5 x
-5 -4
10 M to 5 x 10 M had no effect on either plant
species' growth rate or morphology. Fibroblast
proliferation was decreased. The addition of DECIS
markedly decreased plant growth as well as fibroblast
proliferation. It appeared the cell cycle was
undisturbed. It was found that the matrix of the
DECIS greatly enhanced the transport of the toxin
across the membrane of the cell and thus increased
toxicity. This creates many questions with respect to
what or how these toxins are being transported or
facilitated across the cell membrane.
Differences in Effect of Similar Pyrethroids
As more work has been done on pyrethroids
and their mode of action, it has become increasingly
obvious that individual pyrethroids exert very
different effects on exposed tissues with respect to
biochemical and well as electrophysiological
parameters. There may be more going on than simply an
alteration of the sodium channels of these cells.
Work done recently by Tippe (1987) shows
that there are differences between the effects of


49
three pyrethroids, deltamethrin, cypermethrin, and
fenvalerate, on the excitability thresholds in
isolated axons. IOOuM deltamethrin is capable of
changing the membrane potential to +30mV within ten
minutes. Voltage is shifted toward the more negative
or hyperpolarized direction. This would in effect
decrease the cell's ability to generate an action
potential. 500uM fenvalerate shifts the membrane
potential (V ) in a depolarizing direction. 200uM
cypermethrin had no effect on V There is no
evidence as yet to suggest why these three similar
toxins appear to exert such dramatically different
effects.
Effects on Ca^+-ATPase
Many pesticides inhibit Ca^+-ATPase (Miller
et al., 1976; Yamaguchi, et al., 1980; Huddart et
al., 1974). It has been shown that both types of
pyrethroids, Type I and Type II, have very distinctly
different effects on the Na-Ca and Ca+Mg ATPase
mechanisms in cockroaches. This research has shown
several things; (1) These Ca^+ dependent ATPase pumps
in brain were very sensitive to pyrethroid
insecticides in vitro. (2) The Type I (non-cyano)


50
pyrethroids had a much greater effect on Na-Ca
ATPase than on Ca+Mg ATPase, whereas the Type II had
a reverse effect with respect to these two pumps.
Permethrin in particular had a very large effect on
all Ca^+-stimulated ATPase pumps in vivo and the
concentration needed to cause this effect (10 ^M) was
similar to the concentration needed to produce this
effect in vitro The in vivo inhibition of these
pumps increased as the calcium concentration
decreased.
Inhibition of Ca^+-ATPase should increase
intracellular [Ca^+]. This in turn could lead to
calmodulin activation and inhibition of microtubule
polymerization and neurite growth inhibition.
Effects on Norepinephrine Release
Studies of norepinephrine (NE) release from
rat brain synaptosomes has again raised the question
of the significance of calcium to the mechanism of
these toxins(Brooks et al., 1987). The cyano-
pyrethroids greatly enhanced the calcium-dependent,
potassium stimulated release of NE. The non-cyano
pyrethroids had little or no effect on this release.
Concentrations less than 10 had no effect at all.


51
Calcium uptake was directly proportional to the toxin
stimulated release of NE. It is interesting to note
that Parathion, DDT and DDE, an analog of DDT had no
appreciable effect on NE uptake or release (Brooks et
al., 1987).
DDT Mechanism of Action
DDT acts in a very similar manner as the
pyrethroids. It increases and prolongs the
depolarizing after-potential and as a result creates
multiple action potentials in nerve fibers
(Narahashi, T. 1982). Further work done with
voltage clamp techniques again showed that the effect
was primarily due to a partial inhibition of sodium
inactivation mechanisms.
It has been further demonstrated that DDT
inhibits calmodulin action. This was shown
using bovine heart phosphodiesterase-calmodulin
systems. It was found that at concentrations less
than 10 the inhibitory action of DDT was on the
calmodulin portion of the pathway alone. This was in
direct contrast to permethrin in this same model.
Permethrin was capable of affecting not only
calmodulin but also phosphodiesterase. This results


52
in an inhibitory action on a number of systems
including cyclic nucleotide phosphodiesterase, brain
adenylate cyclase, brain membrane kinase, and Ca^+-
Mg^+-ATPase (Rashatwar et al., 1985).
In summary, it would be unwise to assume that
only one mechanism could be responsible for the
observed morphologic and electrophysiologic effects
seen with these toxins and their interaction with
neurons both in vivo and in vitro. A great deal of
work remains to be done to ascertain all of the
interactions that may occur with respect to these
toxins and neurons.


CHAPTER THREE
CULTURE METHODS AND PROCEDURES
Lymnaea stagnalis
Preparation of Solutions and Culture Media
All solutions used for this project were
prepared in the lab with the exception of the
starting culture media. These solutions were prepared
and placed into sterile storage containers and kept
cold for extended use. When necessary, as with the
culture media, and all solutions placed on the tissue
being cultured, sterilization filtering was done a
0.2um syringe filter. All sterilization was done in a
laminar flow hood after 10/86. Up to that date,
these procedures were carried out in the lab without
the aid of this sterile atmosphere.
Solutions used for culturing of Lymnaea cells
include Leibowitz-15 media (L15), Lymnea Leibowitz-
15 culture media (LL-15), stock solutions of NaCl,
KC1, MgCl2*6H20, CaCl2*6H20, TRIS, HEPES, BaCl2> and
K-acetate, 4X Lymnaea ringers solution Lymnea ringers
solution, antibiotic saline (ABS), a concentrated
solution of gentamycin, amphoteracin, and


carbenicillin (GAC), poly-lysine solution,
permethrin with alcohol (PE), and DDT with alcohol
(DDTE).
54
Leibowitz-15 medium. Culture media were based
on a salt-free Leibowitz L-15 medium (Gibco special
order). It was stored in sterile one liter bottles
and refrigerated after being made. Medium was not used
if greater than two weeks old.
LL-15 culture medium. LL-15 culture medium
consisted of 50% L-15 with Lymnaea salts and GAC (See
appendix A). After preparation, the LL-15 was
sterilized through a 0.2um syringe filter. Medium was
stored in sterile 20mL serum bottles at 4 C. Medium
was discarded after two weeks.
Antibiotic saline. This was used for
dissection and preparation of most of the enzymes
used during cell dissociation. It was prepared from
Lymnaea ringers solution with the addition of lmL
antibiotic/antimycotic (Sigma) per lOOmL of saline.
This was also filtered into sterile bottles for
storage and further use.


55
GAC. This is a 100X concentration of
Gentamycin (50mg/ml), amphotercin (2mg/ml), and
carbenicillin (50mg/ml). This was prepared by mixing
5mL of gentamycin, 0.2mL (200uL) of amphotercin, and
0.05g of carbenicillin. This was then diluted to lOmL
with deionized water for a total volume of lOmL, and
kept frozen until used. The exact final
concentrations of each antibiotic are located in
appendix A.
Poly-lysine. Polylysine was used to coat
the culture dishes used for Lymnaea cells. It is a
highly charged molecule that created a coating for
the adherence of neurons and potential new neurites.
The exact procedure for the preparation of this
solution is located in appendix B.
Permethrin and DDT test solutions. Those used
in the culture dishes were prepared using absolute
ethyl alcohol and preparing 1 molar stock solutions
from which further dilutions were made. These were
stored in a separate area to avoid the possibility of
contamination or poisoning of other solutions and to
minimize personnel contact within the lab. Attempts


56
to syringe sterilize these solutions resulted in a
large majority of the toxin being removed from the
solvent. Therefore, no sterilization was done on
these solutions.
Conditioning of Medium
As was previously discussed, Lymnea neurons
do not extend neurites well unless they are cultured
in medium that has been previously conditioned.
Three brains per 2ml provides optimum amounts of
conditioning factor (Wong, R.G. et al., 1984). To
condition medium, the appropriate number of brains
were carefully dissected out and placed into a
sterile petri dish containing ABS. After these brains
had soaked in this ABS for five minutes, they were
then placed into two succeeding ABS rinses of five
minutes each before being placed into the culture
medium. Brains were transferred with flame sterilized
forceps. The dish containing the brains and culture
medium was then allowed to incubate at 20 C for
seventy-two hours before being used. (Please see
specific procedure in Appendix B).
Culture Plate Preparation
Because neurites and neurons in general need


57
an adhesive surface for growth, it was necessary to
coat culture plates so that they had the necessary
surface for adhesion. 25mg of polylysine was mixed
with 3.75mL of 1M TRIS and enough deionized water to
bring the final volume to 25mL. This was adjusted to
a pH of 8.4. This solution was then applied to the
culture dishes using a syringe and filtering the
solution through a 0.2um filter. It was allowed to
remain on the dishes for 60 minutes at which time it
was aspirated off, and replaced with 2mL of sterile
deionized water. The water was left for 15 minutes,
aspirated, and replaced again with new sterile
deionized water. This was left in place for an
additional 15 minutes, after which it was aspirated.
A marked grid was placed on the bottom of the culture
dishes for cell location and then they were placed
into an air tight container containing a desssicant.
These dishes were used within two weeks. If
contamination was evident on the plates, they were
discarded.
Dissection Procedure
A detailed description of brain dissections
from L. stagnalis is presented in Appendix B.
Only a brief summary is presented here. I used


58
mature snails that weighed no less than 0.5g, removed
the shell, and cut the exposed portion of the snail
off into a dissection dish filled with ABS. Pins were
placed through the anterior and posterior portions of
the snail to secure it to the dissection dish. After
visualization of the brain had been accomplished, it
was removed using microscissors and placed into a
smaller dissection dish where it was fully pinned out
for further work. An example of this is located in
Appendix B.
Cell Dissociation
Methods for cell dissociation are modified
from those of Kostenko et al., (1974), Dagan et al.,
(1981), and Wong et al., (1981). A specific detailed
description of this procedure is located in Appendix
C.
I dissected out two brains for every culture
dish I wished to seed using the dissection procedure
described earlier. The brains were pinned out and
incubated in a one percent solution of protease
(Dispase, Boehringer Mainheim) for thirty minutes.
This was then washed off and the loose connective
tissue around the ganglia removed. A 0.1% solution of
trypsin was then placed on the brains for sixty


59
minutes, followed by a 0.1% solution of trypsin
inhibitor for thirty minutes. During the time the
trypsin was on the brains, the sheaths surrounding
the ganglia had been mechanically removed using two
pair of microforceps.
At the end of the ninety minutes, the ganglia
to be used were cut from the rest of the brain and
aspirated into a micropipette and placed into the
culture dishes. The ganglia were gently aspirated up
and down several times through the micro-pipette to
separate the cells from the ganglia. After a majority
of the cells were separated, the culture dishes were
O
placed into the incubator at 20 C and allowed to grow
for seventy-two hours.
Other researchers have used fire-polished
glass pipettes to dissociate the cells, gently
teasing them apart from the ganglia. This method was
attempted, but I felt it was damaging too many of the
cells and that other techniques should be attempted.
The use of the aspirator worked much better,
providing that an appropriate diameter pipette could
be made and polished enough to avoid the adhesion of
the remaining connective tissue or ganglia to the
pipette .
Kostenko et al , (1974) tested various


60
isolation media in which they varied the concentrations
of calcium and/or magnesium, isolated the cells into
the dish, and then replaced this medium with their
final culture medium. This was also tried, but again I
felt that I was loosing a large number of cells that
had not had time to adhere to the substrate, and the
time necessary to allow them to adhere was damaging
them enough that growth rates were far below optimum
as compared to cells isolated into standard media and
left alone without changing the media.
Various concentrations of protease and
trypsin were used, as well as varying times of
exposure. Qualitatively it was felt that the method
used above provided both optimum cell seperation and
cell growth. When stronger solutions or longer times
were used, it appeared that cell survival and/or
adherence, and/or neurite outgrowth were dramatically
reduced .
Data Collection Methods/Criteria
Immediate Exposure
The following data were collected during the
immediate exposure portion of this project. When
possible this was done with a single blind technique,
where another investigator labeled pre-prepared tubes


61
with letters for which a key was made and kept until
after data collection had been completed. Only then
were the concentrations known to me. In addition, to
be certain no bias occurred with respect to the grids
counted, a series of pregenerated random numbers were
used to determine which grid to count for which
experiment. A minimum of 100 viable cells were
counted in each dish. Criteria used or modified for
determination of viability were developed by Wong et
al . (1981). A living neuron is round and phase bright
and adhering to the substrate (Wong, R.G. et al.,
1981). Cells were exposed from the onset of the
experiment to either L-15 media (control), L-15 media
with solvent (solvent control), or L-15 media with
solvent and toxin (experimentals).
Gross Morphology
The following data were collected after the
cells had been growing for seventy-two hours. Some of
these data were collected using photographs taken of
these cells.
1) Number of neurites growing per cell body.
2) Average number of branches growing per
neurite.


62
3) Living neurons were classified into four
categories:
i) Type one cells showed no growth.
ii) Type two cells had one neurite with
no branching of the neurite.
iii) Type three cells had multiple neurites
with branching.
iv) Type four cells had no appreciable
neurites growing but exhibited a
large growth cone structure that
circumscribed the soma (haloing).
Delayed Exposure
The following procedure for data collection
was followed during the delayed exposure portion of
this project. Cells were plated following standard
procedure as outlined previously. They were allowed
to grow in standard medium without toxins for a
minimum of three days. After three days,
measurements and photographs of all neurites of a
chosen cell were taken. Cells were chosen by a quasi-
random procedure. The grids to look at were chosen
again by random number. However, after a cell was
located within this grid, I chose to use it only if
it had a minimum of two neurites growing both of which


63
were at least twice the diameter of the cell
body in length. The position of this cell in the grid
was recorded so that it could be found again on
subsequent observations.
The culture media was then removed from the
plates, and the desired toxic media placed on the
plate. Measurements of neurite length and
photographs were taken at 24,48, and 72 hours after
exposure to the toxic media.
Culture Methods and Procedures
Embryonic Chickens
Preparation of Solutions/Culture Media
All solutions were prepared commercially and
mixed in the lab under sterile conditions in a
laminar flow hood. After preparation, these solutions
were kept refrigerated. None were used after they
were two weeks old. (See Appendix B for components).
Hanks Basic Salt Solution. This was supplied
by Sigma Co. in powder form and was prepared as
needed. We prepared one liter at a time, buffering
for pH, and sterilized it using positive pressure
filtration. It was placed into sterile containers and
refrigerated.


64
Ca-Mg free HBSS. Trypsin for cell
dissociaton was dissolved in HBSS. A 1% trypsin
solution was prepared by mixing the appropriate
quantity of trypsin in lOmL of Ca-Mg free HBSS and
then kept frozen until use.
MEM Culture Media. The actual culture media
was prepared using MEM, FCS, and GLU. A lOOmL
alloquot of MEM was mixed with lOmL of FCS, lmL of
GLU, and lmL of antibiotic/antimycotic. This solution
was then refrigerated for further use.
Vitrogen Preparation. This is by far the most
difficult and critical portion of solution
preparation. Vitrogen (Collegen Corp.) is pure
collogen and must be kept cold to remain dissolved in
liquid. Once it reaches room temperature or above, it
rapidly becomes quite solid. This preparation was
used to embed the cells in for growth. This was done
to control cell migration, and helped to keep the
cells single for neurite measurement. It was prepared
by mixing 1600uL of Vitrogen with 200uL of 10X MEM
which contained phenol red for pH indication, and
200uL of 0.125M HEPES. This was kept in an ice bath
to keep it liquid. Sterile NaOH or HC1 were used to
adjust pH if necessary. (Please see Appendix C).


65
Permethrin and DDT solutions. One molar stock
solutions dissolved in DMSO were prepared from which
further dilutions were made as necessary. DMSO was
used in this instance because it was found that
concentrations of alcohol necessary for dissolving
the DDT were toxic to embryonic chick cells. These
were kept far from other solutions to avoid the
possibility of comtamination.
Culture Plate Preparation
In order for the vitrogen solution to
properly flow on the culture plates, it is necessary
to pre-wet the plates with plain MEM (no FCS or GLU)
and allow them to set for several minutes. This
solution is then aspirated off immediately prior to
application of the vitrogen-cell suspension. It is
critical that all the MEM be removed from around the
edges of the culture dish, or else the vitrogen may
not adhere to the culture dish.
Dissection Procedure
A detailed description of this procedure is
located in Appendix B. A brief summary is presented
here. A 7-10 day old fertilized egg was used. The
shell was wiped off with alcohol, and a small opening


66
made in the top of the egg. The embryo was then
removed and placed into a sterilized dissection dish
containing HBSS, and the brain tissue removed. This
was then placed into a sterile tube containing HBSS
and 1% trypsin to digest the connective tissue around
the cells. The brain tissue was left in this solution
for 20 minutes. The HBSS was then removed and
replaced with 2-3 milliliters of clean culture media,
where upon the FCS will inactivate the trypsin. Using a
pasteur pipette with a narrowed orifice and that had
been fire-polished, the brain tissue was aspirated
several times until the cells were totally
dissociated from one another.
Cell Count and Dilution
3
The number of cells per m was determined in
order to place the appropriate number of cells per
dish. A drop of the cell suspension was placed on a
hemocytometer. Cells were counted in the appropriate
grids and total number per milliliter calculated. A
more detailed account of this procedure is located in
appendix B.


67
Data Collection Methods/Criteria
The following data were collected. When
possible this was done with a single blind technique
to decrease the potential for bias. In addition, as
with the Lymnaea culture, random numbers were used to
determine which grids to count when counting cells.
Three fields were counted in each dish and the
following data obtained; (1) Number of cells alive
per field, (2) Number of cells growing per field, (3)
Total neurite length per field, (A) total number of
neurites per field, (5) average length of neurites
per growing cell, and (6) average number of neurites
per growing cell. All of these data were obtained
using a Jandel Scientific digitizing tablet.
Methods Used to Analyze Data
The appropriate factorial analysis of
variance was employed to determine significance with
these data. All of these analysis' were done with the
assistance of pre-prepared computer enhanced
statistical programs. Error bars on graphs will
represent standard deviations. One-way error bars
will be employed to avoid overlap.


68
Electrophysiological Recordings
For electrophysiologic recording, brains were
dissected out using standard procedures and were
placed in culture media with the appropriate toxin
for the necessary time before recording. No other
enzymatic digestion was used to loosen or remove any
remaining connective tissue. Solutions containing
toxin were prepared immediately prior to dissection
and placed into air tight sterile tubes.
Intracellular Recording of Spontaneous Activity
All recordings of intracellular activity were
done using glass electrodes, with an average
resistance of 60 MegOhms. These were filled with 2M
potassium acetate. Recordings were done using a Dagan
8800 amplifier/stimulator, a Grass stimulator, and a
Gould polygraph. The recording chamber was placed
within an electrically neutral field to eliminate
aberent electrical interference. The desired
media-toxin solution was perfused over the brain
while recordings were done.
After the brain was pinned out, the
connective tissue located around the cell being
recorded from was very carefully removed. The


69
electrode was then placed into the cell and the
recording done. Certain criteria had to be met to
consider a cell acceptable for recording. These were
(i) The initial magnitude of any spontaneous action
potential had to be no less than -60mV. (ii) The
resting potential of the cell was no less than -50mV.
(iii) Stimulation of the cell with a (+/-) 4nA
current resulted in no repetative firing in control
cells.
Recordings were made continuously for 2
minute intervals every 4-5 minutes as long as the
cell remained viable. Data collected include
number of action potentials per minute, magnitude,
and duration of the action potential and recovery
time of the resting potential.
After each recording, the chamber was rinsed
for several minutes with absolute ethanol to remove
any residual toxin and then rinsed with sterile LL-15
media to remove the ethanol.
Delayed exposure data were obtained in much
the same manner. Brains were dissected out and placed
in sterile vials containing LL-15 media with the
appropriate toxin added. These brains were allowed to
incubate in this media for the desired length of time
before recording. During the recording session, the


70
same concentration of LL-15 with toxin was perfused
over the brain during recording.
When possible, the cell labeled RPD-1 which
is located in the right parietal ganglion was used
for recording (Benjamin, P.R. el al., 1978). When
this cell was not available for recording, I
attempted the following cells in the order presented
until such time as an acceptable recording was made;
RPD-2, VD-1, and the B cells located in the right
parietal ganglion. If no successful recordings were
obtained after attempting these cells, then the brain
was discarded.


CHAPTER FOUR
OBSERVATIONS AND RESULTS
Effects of Permethrin on neurite ougrowth of L.
stagnalis neurons
Neurite growth dose-response curves were
generated after exposing isolated neurons of L.
stagnalis to concentrations of permethrin ranging
from 25uM to 250uM. These curves were generated
against a defined medium control and a solvent
control. For this phase of the experiment, growth
was defined as any neurite extension from a phase
bright, round, attached neuron that exceeded the
diameter of the neuron cell body in length. 100
cells were counted. Counting of cells was done by
finding a quadrant on the bottom of the culture
dish. To decrease bias, a random number generator
was used to determine which of a possible twenty
quadrants was to be counted. Living cells were
counted and categorized as to the type of neurite
growth that was apparent (Appendix I). Type I cells
were alive but had no apparent signs of new neurite


72
outgrowth. Type II cells had a single neurite, Type
III had multiple neurites and type IV had veiling,
but no new neurites. A sample was considered
complete once one hundred separate live cells had
been counted. If multiple grids were necessary to
find one hundred cells that met minimum cirteria,
random numbers were again used. No cell was counted
more than once.
Conditioning times of the medium, the number
of hours cells were allowed to grow, the age of the
medium, and pH of the medium were kept as constant
as possible. Table One is a summary of these culture
conditions. The minimum vs maximum conditioning time
varied by only 1.39 hours. The medium age varied by
less than one day while pH varied 0.01 units. Hours
grown varied by twelve hours at its extreme points.
Table Two summarizes the percent growth of
neurites versus both defined medium (LL-15) and
solvent plus medium (LL-15 + ETOH). Percent growth
was determined by adding the total of type II-IV
cells and dividing that by the total number of cells
counted. This is summarized graphically in Figs. 11
and 12. As concentration increases, from 25uM
permethrin in ETOH to 250uM permethrin in ETOH,
growth decreases both in defined medium as well as


73
TABLE ONE
CULTURE Lymnaea s CONDITIONS - tagnalis PERMETHRIN IN ALCOHOL
TOXIN HRS COND HRS GROWN MEDIA pH OF
AGE MEDIA
CONTROLS MEAN 70.14 75.36 9.00 7.57
STD DEV 6.16 17.93 2.48 0.01
N 14 14 14 14
ETOH CONT MEAN 70.14 75.36 9.00 7.57
STD DEV 6.16 17.93 2.48 0.01
N 14 14 14 14
25uM PE MEAN 68.75 72.13 8.38 7.57
STD DEV 7.87 2.37 3.00 0.01
N 8 8 8 8
50uM PE MEAN 68.75 69.13 8.13 7.56
STD DEV 7.87 14.61 2.98 0.01
N 8 8 8 8
lOOuM PE MEAN 68.75 72.13 8.25 7.57
STD DEV 7.87 12.23 2.95 0.01
N 8 8 8 8
200uM PE MEAN 68.75 81.13 8.38 7.56
STD DEV 7.87 16.8 3.00 0.01
N 8 8 8 8
250uM PE MEAN 68.75 78.13 8.50 7.57
STD DEV 7.87 10.59 3.08 0.01
N 8 8 8 8


74
TABLE TWO
TOTAL GROWTH VERSUS CONCENTRATION
PERMETHRIN IN ALCOHOL LYMNAEA
TOXIN TOTAL GROWTH % OF CONTROL % OF ETOH
CONTROL MEAN 30.07 100.00 94.17
STD DEV 3.62
N 7.00
ETOH CONT MEAN 31.93 106.15 100.00
STD DEV 5.91
N 7.00
25uM PERM MEAN 24.75 80.20 77.51
STD DEV 3.15 10.48 9.62
N 7.00
50uM PERM MEAN 14.12 46.80 44.22
STD DEV 4.37 14.50 13.69
N 7.00
lOOuM PERM MEAN 8.37 27.75 26.21
STD DEV 3.72 12.54 11.81
N 7.00
200uM PERM MEAN 5.75 19.00 18.00
STD DEV 2.82 9.36 8.82
N 7.00
250uM PERM MEAN 4.62 15.34 14.46
STD DEV 2.18 7.23 6.81
N 7.00


Percent Growth
75
Lymnaea Neurite Outgrowth
Permethrin vs Controls
Fig. 11 Isolated neurons of L. stagnalis were
exposed to concentrations of permethrin ranging
from 25uM to 250uM, and compared to defined LL-15
media controls. Growth of new neurites is
altered in a dose-dependent manner. Cells did not
survive in concentrations of permethrin above
250uM.


76
Lymnaea Neurite Outgrowth
Permethrin vs Solvent Controls
Concentration(uM)
Fig. 12 Isolated neurons of L. stagnalis were
exposed to concentrations of permethrin ranging
from 25uM to 250uM, and compared to solvent
controls containing 1% ETOH. Growth of new neurites
is altered in a dose-dependent manner. Cells did
not survive in concentrations of permethrin above
250uM.


77
the defined medium plus solvent control with average
growth decreasing from 80% with 25uM to less than
15% with 250uM. Concentrations greater than 250uM
permethrin resulted in non-viable conditions for
cell survival, and no neurite outgrowth was
observed .
Table three as well as fig. 13 and 14
summarize a further break down of growing cells into
the type previously described. As concentration
increases from 25uM to 250uM permethrin, there is a
proportional decrease in each of the growing cell
types (II-IV) as well as a corresponding increase in
alive but non-growing cells (I). Of the cells
growing in the controls, the majority (53.24%) were
type II while type III composed 35.41% and type IV
8.78%. If one looks at the percent growth of each
cell type against its corresponding controls, one
can see the percent growth follows a similar,
predictable curve as seen with the total growth
curve and absolute number of cells for each type.
Table four summarizes data gathered with
respect to the number of neurites per cell and the
number of branches per neurite found. There is no
definite correlation between controls and any of the
toxin levels with respect to neurite number or


78
TABLE THREE
CELL TYPE VERSUS CONCENTRATION
LYMNAEA PERMETHRIN IN ALCOHOL
TOXIN TYPE I TYPE II TYPE III TYPE IV
CONTROLS MEAN 69.14 16.43 10.93 2.71
STD DEV 3.62 3.31 5.04 2.05
N 14 14 14 14
ETOH CONT MEAN 68.07 19.14 10.00 2.79
STD DEV 5.91 4.94 4.61 2.57
N 14 14 14 14
250uM PE MEAN 95.38 2.13 2.50 0.00
STD DEV 2.18 0.78 1.73 0.00
N 8 8 8 8
% OF CONT 137.9 12.9 22.8 0
% OF ETOH 140.1 11.1 25.0 0
200uM PE MEAN 94.28 4.13 1.38 0.25
STD DEV 2.82 2.03 1.49 0.43
N 8 8 8 8
% OF CONT 136.3 25.1 12.6 9.2
% OF ETOH 138.5 21.5 13.8 15.4
lOOuM PE MEAN 91.63 4.00 3.38 1.00
STD DEV 3.77 1.66 1.93 1.00
N 8 8 8 8
% OF CONT 132.5 24.3 30.9 36.9
% OF ETOH 134.6 20.8 33.8 35.8
50uM PE MEAN 85.88 7.50 5.25 1.25
STD DEV 4.37 4.18 2.28 1.48
N 8 8 8 8
% OF CONT 124.2 45.6 48.0 46.1
% OF ETOH 126.1 39.1 52.5 44.8
25uM PE MEAN 75.25 12.55 8.25 4.00
STD DEV 3.15 3.64 1.85 2.83
N 8 8 8 8
% OF CONT 108.8 76.3 75.4 147.6
% OF ETOH 110.5 65.5 82.5 143.3


Cell Types Lymnaea
Permethrin in Alcohol
79
Fig. 13 Growing neurons were further broken down
into categories based on the type of neurite
growing (see text). Type I neurons, alive but with
no new neurites, increased in number as
concentrations of permethrin increased from 25uM to
250uM. These are compared to control values.


80
Cell Types Lymnaea
Permethrin in Alcohol
Fig. 14 Growing neurons were further broken down
into categories based on the type of neurite
growing (see text). Type II neurons, neurons with
one neurite, type III neurons, neurons with
multiple neurites, and type IV neurons, neurons
with no neurites but with haloing, all decreased as
the concentration of permethrin increased from 25uM
to 250uM. This decrease in the number of type II-IV
cells is similar to the overall growth curve
observed with permethrin against both defined media
control and solvent controls. These are compared to
control values.


TABLE FOUR
81
AVERAGE NUMBER OF NEURITES PER CELL
AND NUMBER OF BRANCHES PER NEURITE
LYMNAEA - - PERMETHRIN IN , ALCOHOL
TOXIN NUMBER OF NEURITES NUMBER OF BRANCHES
CONTROLS MEAN 1.52 1.50
STD DEV .19 .83
N 10 10
ETOH CONT MEAN 1.59 1.32
STD DEV .29 .77
N 10 10
500uM PE MEAN .89 .00
STD DEV .34 .00
N 4 4
% OF CONT 58.50 0.00
% OF ETOH 55.90 0.00
250uM PE MEAN 2.10 2.27
STD DEV .40 1.36
N 4 4
% OF CONT 138.10 151.30
% OF ETOH 132.00 171.90
200uM PE MEAN 1.47 1.03
STD DEV .34 .21
N 4 4
% OF CONT 96.70 68.60
% OF ETOH 92.40 78.00
lOOuM PE MEAN 1.83 1.98
STD DEV .44 1.57
N 4 4
% OF CONT 120.30 132.00
% OF ETOH 115.00 150.00
50 uM PE MEAN 1.53 .65
STD DEV .27 .28
N 4 4
% OF CONT 100.60 43.30
% OF ETOH 96.20 49.20
25 uM PE MEAN 1.62 1.72
STD DEV .69 .79
N 4 4
% OF CONT 106.50 114.60
% OF ETOH 101.80 130.30


82
branching per cell. In fact the highest toxin level,
250uM shows the largest mean. However, very few
cells grew in this concentration, so the sample size
is small .
Table five summarizes the probability
scores for all culture conditions. The largest p
value found was 3.76 x 10 This was found using
the Students T-Test and a one-way ANOVA.
Effects of DDT on the Neurite Outgrowth of L.
stagnalis neurons
Neurite outgrowth dose-response curves were
generated after exposing isolated neurons of L.
stagnalis to concentrations of DDT in ETOH ranging
from 500nM to 25uM. These curves were generated
against a defined medium control and a solvent
control. Growth was defined as previously stated for
permethrin as were the counting procedures.
Conditioning time of medium, the number of
hours the cells were allowed to grow, medium age, and
medium pH were kept as constant as possible. Table
Six is a summary of these culture conditions. The
variance in conditioning time was 2.5 hours.
Hours grown ranged from 73.67 to 76.25 hours with a
variance of 3.19 hours. Medium age differed by


TABLE FIVE
83
T-SCORES FOR LYMNAEA NEURITE OUTGROWTH PERMETHRIN IN ALCOHOL
TOXINS VERSUS LL-15 CONTROL AND ETOH CONTROL
TOXIN AVG GROWTH STD DEV VARIANCE
CONTROL 30.86 3.62 13.10
ETOH CONT 31.93 5.91 34.93
25uM PE 24.75 3.15 9.92
50uM PE 14.12 4.37 19.10
lOOuM PE 8.375 3.72 14.21
200uM PE 5.75 2.82 7.95
250uM PE 4.62 2.18 4.75
CONDITION T-SCORE p-SCORE
25uM PE VS CONTROL 12.98 1.680E-11
50uM PE VS CONTROL 29.96 <1.00E-14
lOOuM PE VS CONTROL 43.96 Cl.OOE-14
200uM PE VS CONTROL 128.31 Cl.OOE-14
250uM PE VS CONTROL 66.26 Cl.OOE-14
25uM PE VS ETOH 10.88 3.761E-10
50uM PE VS ETOH 24.68 Cl.OOE-14
lOOuM PE VS ETOH 34.11 Cl.OOE-14
200uM PE VS ETOH 40.75 Cl.OOE-14
250uM PE VS ETOH 44.02 Cl.OOE-14
ANALYSIS OF VARIANCE - PERMETHRIN IN ALCOHOL
Number of Cases = 7 Number of Variables = 7
GROUP MEAN N GROUP MEAN N
1 32.250 7 5 8.375 7
2 32.000 7 6 14.125 7
3 4.625 7 7 24.125 7
4 5.750 7
GRAND MEAN 17.321 56
SOURCE SUM SQRS df MEAN SQRS F RATIO
Between 6959.714 6 1159.952 95.285
Within 596.500 49 12.173
Total 7556.214 55
PROBABILITY = Cl.OOE-14


84
TABLE SIX
Lymnaea stagnalis
CULTURE CONDITIONS DDT IN ALCOHOL
TOXIN HRS COND HRS GROWN MEDIA pH OF
AGE MEDIA
CONTROLS MEAN
STD DEV
N
ETOH CONT MEAN
STD DEV
N
500nM DDT MEAN
STD DEV
N
750nM DDT MEAN
STD DEV
N
lOOOnM DDT MEAN
STD DEV
N
lOuM DDT MEAN
STD DEV
N
MEAN
STD DEV
N
66.25 76.25
7.77 12.02
8 8
66.25 76.25
7.77 12.02
8 8
67.50 74.50
4.56 13.22
4 4
65.00 73.67
8.62 10.86
6 6
65.00 73.67
8.62 10.86
6 6
65.00 73.67
8.62 10.86
6 6
67.14 76.86
7.92 12.73
7 7
3.88 7.56
1.83 0.01
8 8
4.00 7.56
1.80 0.01
8 8
5.25 7.56
1.48 0.01
4 4
4.33 7.56
1.97 0.01
6 6
4.33 7.56
1.97 0.01
6 6
4.33 7.56
1.97 0.01
6 6
3.57 7.56
1.50 0.01
7 7
25uM DDT