The Nematode Caenorhabditis Elegans As a Model Of

THE NEMATODE CAENORHABDITIS ELEGANS AS A MODEL OF

ORGANOPHOSPHATE INDUCED MAMMALIAN NEUROTOXICITY

RUSSELL DAVID COLE

(Under the Direction of Phillip L. Williams)

ABSTRACT

Research has shown a number of basic similarities between the nervous systems

of the nematode Caenorhabditis elegans and higher animals including mammals. The purpose of this study was to investigate the toxicity of a chemical class known to be neurotoxic in C. elegans and compare this toxicity to that seen in mammals. The behavioral response of C. elegans to 15 organophosphate (OP) pesticides was characterized using computer automated tracking. Toxicity was ranked and compared to the ranked toxicity of the same compounds in rats and mice. Toxic mechanism was also examined through cholinesterase activity assays performed on worms exposed to 8 of the

15 OPs. A significant correlation was found between rank order of toxicity in C. elegans and rats and mice. Cholinesterase inhibition by some OPs was also confirmed in C. elegans. Specific comparisons and implications concerning C. elegans’ potential as a

mammalian neurotoxicity model are discussed.

INDEX WORDS: Caenorhabditis elegans, Organophosphate, Neurotoxicity, Rats, Mice, Behavior, Cholinesterase, Biological Model

THE NEMATODE CAENORHABDITIS ELEGANS AS A MODEL OF

ORGANOPHOSPHATE INDUCED MAMMALIAN NEUROTOXICITY

RUSSELL DAVID COLE

B.S., The University of Georgia, 1998

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2003

Russell David Cole

THE NEMATODE CAENORHABDITIS ELEGANS AS A MODEL OF

ORGANOPHOSPHATE INDUCED MAMMALIAN NEUROTOXICITY

RUSSELL DAVID COLE

Major Professor: Phillip L. Williams

Committee: Cham E. Dallas Jeffrey W. Fisher

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia August 2003

DEDICATION

I have heard that J.S. Bach dedicated every piece of music he wrote to the glory of

God. I do not wish to draw comparisons, but only to follow the wisdom of his actions. I dedicate this thesis to the glory of my God, Jesus Christ. Over the last two years, as in

the rest of my life, his love has been unwavering, his grace overwhelming, his mercy

sustaining, his enablement sufficient, and his provision complete. It is he who has

brought me this far, and it is he who will take me on. v

ACKNOWLEDGEMENTS

I would like to thank Dr. Phillip Williams for serving as my advisor for this thesis

work, and to Dr. Jeffery Fisher and Dr. Cham Dallas for serving on my thesis committee.

I would also like to thank Dr. Gary Anderson, Dr. Windy Boyd, and Meagan Kane for their help in the lab. Thanks is also due to Deanna Conners for her help with the cholinesterase assay protocol, and to Dr. Marsha Black for the use of her lab during this portion of my investigation. Thanks to Sandra McPeake, Ella Willingham, and Regina

Davis for all of their help in various administrative and accounting endeavors. I would also like to offer a general thanks to the faculty, staff, and students of the Environmental

Health Science Department for an enjoyable environment in which to work over the past two years.

I would like to thank my wife, Sandy, for her love, steady encouragement,

unwavering support, and patience in sharing me with nematodes while I have pursued

this degree. I thank my family for their support and willingness over the last two years to

frequently sacrifice my presence in deference to this work. I would also like to thank my

church family at the Athens Vineyard for their steadfast prayers and support. I owe a

particular debt to Dr. Steve Holloway for his listening ear, ready encouragement, sound

advice, and overall friendship over these last years.

Lastly, I would like to thank Dr. Michael Petelle who taught me 10th grade

Biology with such excellence and enthusiasm as to set the standard for my studies in the

biological sciences. vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... v

LIST OF TABLES...... viii

LIST OF FIGURES ...... ix

CHAPTER

1 INTRODUCTION ...... 1

References ...... 4

2 LITERATURE REVIEW...... 5

References ...... 28

3 THE NEMATODE CAENORHABDITIS ELEGANS AS A

MODEL OF ORGANOPHOSPHATE INDUCED MAMMALIAN

NEUROTOXICITY...... 49

Abstract ...... 50

Introduction ...... 51

Materials and Methods ...... 54

Results ...... 60

Discussion ...... 62

Acknowledgements ...... 72

References ...... 73

4 CONCLUSIONS...... 86 vii

LIST OF TABLES

Page

Table 2.1. Basic Description of Chemicals Tested...... 41

Table 3.1. Toxicity values and results for Spearman’s Rank Order Correlation comparison………………………..……………….79 viii

LIST OF FIGURES

Page

Figure 3.1. Comparison of toxicity values for C. elegans (behavioral EC50s) to rats and mice (LD50s) ...... 80

Figure 3.2. Graphical comparison of ChE activity levels in nmols activity/mg protein/minute...... 81

Figure 3.3. Response of movement behavior to decreasing exposure solution pH ...... 82

Figure 3.4. Graphical comparison of ChE activity levels expressed as % control activity………………………………………...... 83

Figure 3.5. Oxidative desulfuration of the parathion...... 84

Figure 3.6. Organophosphate induced change in movement versus exposure solution pH...... 85

CHAPTER 1

INTRODUCTION

In the decades since the so called “Green Revolution,” our ability to synthesize organic chemicals has grown at a rate beyond our ability to assess the benefits and dangers of newly discovered compounds. As our understanding of biology and chemistry has grown, we have also been faced with the need to return to previously studied chemicals to better understand their actions and fates in the body and the environment. In this setting there is an ever present need for testing the toxicity of chemicals to humans.

Because of the unethical nature of conducting most toxicity tests in humans, the majority of testing is carried out using animals, primarily mammals, or their tissues. This solution also creates problems. Animal testing is expensive, time consuming, and is in some cases deemed unacceptable in its toll on animal life. In an effort to limit the number of animal tests carried out, three main types of alternative testing are often undertaken as preliminary toxicity screening tools. Structure-activity relationships seek to predict toxicity based upon the toxicity of well characterized compounds with similar chemical structure. This does not directly require the use of animals, but can also be unreliable. In vitro testing provides for a more efficient use of animals and is selective for the particular tissues in question. In vitro tests, however, completely eliminate many pharmacokinetic factors important to toxicity such as absorption, distribution, and biotransformation.

Toxicity testing using alternative organisms includes aspects of pharmacokinetics and 2

pharmacodynamics into the toxicity model, but care must be taken to ensure that these

factors in the test organism accurately model the chemical behavior, target organ, and site

of action in humans.

This purpose of this thesis is to continue in the exploration of the nematode

Caenorhabditis elegans as an alternative testing organism for some forms of

neurotoxicity in mammals. This question is addressed through toxicity testing of the

known neurotoxic organophosphate pesticides in C. elegans, and comparison of the

results of this testing to published mammalian toxicity values. The second chapter of this

thesis is a review of the literature pertinent to this modeling question. It includes

discussions of C. elegans and its neurological similarities to mammals as well as a

discussion of organophosphate toxicity in mammals and C. elegans. Chapter 3 describes

a series of experiments undertaken to compare acute organophosphate behavioral toxicity

in C. elegans to lethality in mammals.

The hypothesis of this work was that sublethal behavioral toxicity in C. elegans

exposed to organophosphates should bear enough similarity to mammalian toxicity for C.

elegans to be useful as a model in OP toxicity in mammals. To address this question, 15

organophosphates of varying mammalian toxicity were tested for their affect on C.

elegans movement behavior following 4-hour aquatic exposures. Triplicate measures of movement by worms exposed to five OP concentrations and a blank control were used to characterize the concentration-response of C. elegans to each compound. Assessment of movement behavior was carried out with a computer automated tracking program interfaced with a video camera continuously collecting images of nematodes under dark field illumination. Concentration-response data were used to estimate the chemical 3

concentration effectively reducing C. elegans movement by 50% (EC50). These EC50 values were compared to rat and mouse LD50 values obtained from the RTECS database

using Spearman’s Rank Order Correlation Coefficient.

Organophosphate insecticides exert toxicity primarily by the inhibition of acetylcholinesterase (AChE) in the nervous system. Although the literature includes tests with AChE mutants which seem to indicate that AChE inhibition also occurs in C.

elegans, this experiment measured directly cholinesterase activity in worms. Worms

were exposed to eight of the fifteen OPs tested at concentrations approximating the EC50

and high concentration used in behavioral toxicity tests. Cholinesterase activity was

quantified in control and exposed worms following 4-hour exposures using a

modification of Ellman’s procedure (1961). Exposed groups were compared to controls

using analysis of variance. This chapter also addresses the affect of different

organophosphate compounds on exposure solution pH and the effect of pH on worm

movement. Similarities and differences in toxicity are discussed.

Chapter 4 summarizes the conclusions that can be drawn from the presented

study, and suggests possible ideas for future experiments. 4

Reference

Ellman, G.L., Courtney, K.D., Andres, Jr. V., Featherstone, R.M. 1961. A new and

rapid colorimetric determination of acetylcholinesterase activity. Biochem.

Pharmacol. 7, 88-95.

CHAPTER 2

LITERATURE REVIEW

Organophosphorus Esters

Organophosphorus esters (organophosphates or OPs) are a broad and important class of pesticidal chemicals which serve as the active ingredient in many insecticides, acaricides, nematocides, helminthicides, herbicides, and fungicides (Chambers, 1992).

Virtually unknown until the late 1930s, the German chemist Gerhard Schrader was the first to explore OP synthesis and utility. Schrader’s work lead to the discovery of a number of insecticides as well as two extremely toxic OPs (tabun and soman) that have since been employed as chemical warfare agents (Chambers, 1992). Primarily due to their high efficacy and low environmental persistence, organophosphates became the insecticide of choice in the 1970s (Pope, 1999). The utility and risk of OPs comes from their potential to poison the nervous system of invertebrate and vertebrate animals by inhibiting the essential enzymatic activity of acetylcholinesterase. This has resulted not only in the control of pestilent insect species, but also in the accidental poisoning of nontarget animal species, most notably humans. Movement in recent years to greater use of the more selective pyrethroid and carbamate insecticides has reduced the dependence on OP usage, but these chemicals still account for a large and important part of pest control efforts in the world today. 6

The term organophosphate is used to describe a group of chemicals in which there is a pentavalent phosphorus sharing a double bond with either oxygen or sulfur, and some combination of carbon containing substituents, traditionally denoted X, Y, and Z.

Though the X and Y substituents can display almost endless variety of alkyl or aryl identities, their attachment to the central phosphorus atom is typically through a direct bond with an oxygen, nitrogen or sulfur (Ecobichon, 1982). The Z designation is typically reserved for that substituent which is most easily replaced through chemical substitution. Enormous variation in the Z substituent is also possible, but in the case of organophosphate pesticides, this group is most commonly a halogen, aryl group, phosphate, cyanide, thiocyanate, carboxylate, phenoxy, or thiophenoxy group

(Ecobichon, 1982). The nomenclature associated with organophosphates (OPs) is complicated and historically inconsistent. An in-depth discussion of this nomenclature can be found in O’Brien (1960).

The physicochemical properties of a particular organophosphate ester are driven by the nature of its substituent groups. The vast array of possible substituent combinations creates an even greater variety of overall physicochemical characteristics.

The individual and combined qualities of these component groups are crucial in determining the rate of absorption, distribution, biotransformation and target site interactions which culminate in the potency and selectivity of each chemical (Ecobichon,

1982). Attempts have been made to predict how changing substituents and the resulting alterations in electronic, steric, and hydrophobic properties modify biological activity.

These efforts have met with some success, and are discussed by Hansch (1969).

Acute Toxicity

The acute toxicity of organophosphate compounds is classically attributed to

inhibition of the nervous system enzyme acetylcholinesterase (AChE). Under normal

circumstances, AChE serves to end the transmission of cholinergic nervous impulses by

degrading the neurotransmitter acetylcholine (ACh) at neural synapses and

neuromuscular junctions. The inactivation of ACh is accomplished via transesterification

whereby free choline is released, and the active serine of the enzyme is acetylated.

Subsequent hydrolysis rapidly restores the enzyme to the active state. In the presence of

an organophosphate, the active serine displaces the Z substituent and becomes

phosphorylated. Hydrolytic restoration of the phosphorylated enzyme to the active state

proceeds at a rates typically orders of magnitude slower than the acetylated case

effectively inactivating the enzyme. Instead of reactivation, the phosphorylated enzyme

can alternatively undergo a process termed aging. In aging, dealkylation occurs at the

phosphoester X or Y position stabilizing the phosphorylated enzyme to the point that

hydrolytic reactivation becomes virtually impossible (Ecobichon, 1982). Several factors

determine the degree of AChE recovery in the hours and days following inhibition.

Rapid and virtually complete recovery is characteristic with some exposures while

inhibition by other OPs results in the slow reestablishment of AChE activity through de

novo enzyme synthesis.

The degree to which AChE inhibition occurs in vivo depends upon factors

specific to the particular chemical and animal species in question. Variability in toxicity is frequently observed between taxonomic groups and among species within those groups

(Benke et al., 1974; Moss and Fahrney, 1978; Chambers and Carr, 1995). Hutson and 8

Millburn (1991) describe these differences as being rooted in the multifactorial nature of

OP absorption, bioavailability, metabolism and inhibitory potential. A number of

structural factors have been proposed by Wallace (1992) as modifiers of the pharmacodynamic efficiency of AChE inhibition. After comparing the hydrolytic rates

for several substrates by the AChE of rats, hens, and rainbow trout, Kemp and Wallace

(1990) concluded that steric occlusion and nucleophilic strength at the enzyme’s active

esteratic site play important roles in vulnerability to phosphorylation. Proximity of the

AChE active site to its choline-coordinating anionic region, and allosteric interaction

between the anionic and active sites may also be important to inhibitory dynamics within

and between species (Wallace, 1992).

Recent studies indicate that some OPs cause secondary effects which may modify the impact of AChE inhibition. Pope (1999) reviewed several studies reporting pre- and postsynaptic interactions of different OPs with both nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR, respectively). Kahn et al. (2000) found acute sarin exposures in rats not only caused increased binding of mAChRs and nAChRs, but also persistent stimulation of choline acetyltrasnferase, an enzyme involved in the biosynthesis of ACh. These effects combine into a complicated picture. The OP binding of postsynaptic nAChRs could enhance the effects of heightened ACh stimulation, but significant binding may only occur at OP concentrations well above what is necessary to cause AChE inhibition (Pope, 1999). Stimulation of choline acetyltransferase could increase ACh release, but presynaptic mAChRs regulate choline reabsorption which is the rate limiting step in ACh production. In contrast to these studies, Camara et al.

(1997) concluded that methamidophos exposure did not impact neurotransmitter release 9 or post-synaptic nAChRs. It seems, therefore, that the potential for OPs to affect ACh release and neurotransmitter receptors may be both chemical and concentration specific.

The biotransformation of organophosphate compounds is critical to their overall toxicity. There are numerous accounts of metabolism causing dramatic toxicity differences between chemicals and between species (Gaines, 1966; Hutson and Millburn,

1991; Chambers and Carr, 1995). In many cases, metabolism is required for OP bioactivation as well as detoxication. The most common groups requiring bioactivation are the phosphorothioates (e.g. parathion, Table 2.1) and phosphorodithioates (e.g. dimethoate, Table 2.1) which must undergo oxidative desulfuration to the corresponding oxon in order to exhibit any significant anticholinesterase behavior (Meyers and Mendel,

1952; Metcalf and March, 1953; Gaines et al., 1966). Activation is predominantly carried out in mammals by Cytrochrome P-450 isozymes in liver and other tissues

(Davison, 1955; Murphy and Dubois, 1957; Ecobichon, 2001). Chambers and Carr

(1995) found that induction of rat microsomes with phenobarbital increased the rate of chlorpyrifos desulfuration but decreased overall toxicity. Their conclusion was that microsomal induction shifted the activity ratio between two or more P450s competitively carrying out the desulfuration and dealkylation of chlorpyrifos. This case demonstrates the dynamic nature of the bioactivating and/or detoxifying pathways that work in series and in parallel to modify the concentration of different OP species in vivo (Kemp and

Wallace, 1990).

Organophosphate detoxication results from a large collection of enzymatic reactions. Phase I reactions include the activities of Cytochrome P450s, glutathione-S- transferases, and variety of phosphotases, esterases, and amidases (Dicowsky and 10

Morello, 1971; Ecobichon, 1982,). Degradation reactions can involve phosphoester bonds or side chain components (Matsumura, 1985). As with other xenobiotics, these reactions depend greatly upon the physicochemical nature of the substrate with some pathways displaying considerable specificity. Serine esterases and proteases other than

AChE are believed to play a protective role in low to moderate level OP exposures

(Richardson, 1995). As with AChE, phosphorylation of these enzymes is usually irreversible raising concerns over the potential effects of their inactivation. Phase II conjugation reactions have also been reported for OP compounds in the literature, but appear to be much less common than phase I reactions. Hutson et al. (1967) identified glucuronide and glycine conjugates in the breakdown of chlorfenvinphos in rats and dogs. Trichlorfon and some related compounds have also been reported to be susceptible to glucuronidation (Yang, 1976). While accounts can be found in the literature, they seem overshadowed by phase I reactions. A more comprehensive treatment of OP biotransformation is given by Yang (1976).

Clinical Manifestations and Treatments

The physiologic effects of acute OP intoxication are predominantly due to the

buildup of ACh in the central nervous system and at the nicotinic and muscarinic

synapses of the peripheral nervous system and neuromuscular junctions (Marrs, 1993).

The clinical symptoms are numerous and vary to some degree in occurrence and severity

between individuals and type of exposure. Classic symptoms of acute OP poisoning

include miosis, headache, confusion, tremors, convulsions, rhinorhea, lachrymation,

diarrhea, bradycardia, tachycardia, muscular weakness, and fasiculations (Marrs, 1993). 11

Death is usually the result of asphyxiation due to some combination of hypotension,

bronchoconstriction, diaphragmatic paralysis, and depression of the respiratory centers in

the brain (Ecobichon, 1982). Though they seem to bear little correlation to actual

toxicity, depression of erythrocyte AChE, plasma AChE, and butyrylcholinesterase

(BChE) can be measured as confirmation of exposure to an anticholinesterase agent

(Richardson, 1995). Treatment of acute poisoning can entail the treatment of symptoms

as well as attempts to reverse the inhibition of AChE. The muscarinic acetylcholine

receptor blocking agent atropine is often employed to mute the effects of ACh buildup,

and artificial respiration is employed when needed. Attempts can also be made to

chemically promote the dissociation of the phosphodiester-enzyme complex by

administration of oximes such as pralidoxime (Marrs, 1993).

Organophosphates are also reported to have acute effects not clearly linked to

AChE inhibition. Acute cognitive and sensory effects have been described, but it has been unclear whether these symptoms resulted from the direct action of the OPs, or secondarily from anoxia associated with intoxication (Marrs, 1993). Abdel-Rahman et al. (2002) report acute sarin exposures at the LD50 level in rats increased permeability of the blood-brain barrier and caused extensive damage to the cerebral cortex, hippocampus, and cerebellum in addition to inhibiting brain AChE and plasma BChE. Exposure at the

LD50 level also caused degeneration in the motor and sematosensory cortex, and doses down to 0.5 (LD50) caused death of cerebellar Purkinje fibers. With respect to non- nervous system organs, postmortem histological evidence has drawn possible links between acute OP intoxication and cardoiotoxicity (Marrs, 1993). There have also been anecdotal, and somewhat circumstantial, reports of OP disruption of the immune system 12

(Richardson, 1995). Recent experiments lend some mechanistic credibility to these

observations. Li et al. (2002) describe in vitro tests where dichlorvos exposure resulted

in the dose dependent inhibition of natural killer cells, cytotoxic T lymphocytes, and

lymphokine-activated killer cells. These cells function in directed cell death in part by

the release of cytolytic granzymes containing serine proteases. Tests by Li et al. (2002)

also showed the dose dependent inhibition of granzymes offering an explanation for

decreased immunocell function.

A so called intermediate syndrome has been described as resulting from some cases of acute OP intoxication. The common symptoms of this condition include the onset of proximal muscle weakness and paralysis 1-4 days following intoxication (Mars,

1993; Ray, 1998). The syndrome commonly culminates in acute respiratory failure, but is unresponsive to the usual treatments (atropine and oximes) of OP poisoning (Marrs,

1993).

Organophosphate Induced Delayed Neuropathy (OPIDN) is another syndrome

associated with acute poisoning by some OP compounds. OPIDN is characterized by

retrograde axonal degeneration of the long, large diameter nerve fibers approximately 10-

14 days following an episode of acute distress. Secondary to the axonal degeneration,

demyelination, Schwann cell proliferation, and macrophage accumulation usually occur

(Barrett et al., 1985). The result is a long term, usually permanent paralysis of the legs

and less commonly the arms (Richardson, 1995). The onset of OPIDN has been linked to

the phosphorylation and aging of NTE (neurotoxic esterase or neuropathy target

esterase), but this relationship is still poorly understood (Marrs, 1993). NTE is an

esterase protein primarily of neuronal origin with decreasing concentrations in the brain, 13 spinal cord, peripheral nerves, and some other tissues (Richardson, 1995). NTE is less vulnerable to phosphorylation than AChE explaining the development of neuropathy almost exclusively after exposures sufficient to produce acute intoxication (Marrs, 1993).

A thorough treatment of OPIDN and the compounds and processes associated with its onset is given by Barrett et al. (1985).

Chronic Effects of Organophosphate Chemicals

There is a general lack of clinical evidence indicating persistent effects from chronic or subchronic OP exposures. The absence of clear long term effects may in part be due to the difficulty of drawing conclusions from uncontrolled and poorly characterized exposures, but even long term clinical usage of select OPs has failed to raise significant cause for concern (Ray, 1998). A few studies, however, have shown persistent changes in brain electrical activity. Korsak and Sato (1977) performed EEGs and a battery of neuropsychological tests on a group of workers had received chronic and clinically nontoxic exposures to organophosphate compounds. Their findings indicated asymptomatic EEGs in the exposed group, distinguishing the left frontal lobe as the brain region most highly impacted. Duffy et al. (1979) also conducted EEGs on a group which had been occupationally exposed one or more times at least one year previous. Using computer analysis of the EEGs, they were able to distinguish increased beta-wave activity suggestive of a long term impact on brain ACh receptors. In both studies, it seems that any effects were too subtle to produce gross clinical signs. In a well defined subchronic study, Sheets et al. (1997) found no additional effects on brain ChE after 13 weeks of feeding exposures compared to levels after 1 and 2 years. No additional 14

behavioral effect was found after the first 4 weeks. This evidence supports the idea that

OPs have little long term effect.

Sublethal Organophosphate Exposure and Mammalian Behavior

Comparisons between studies characterizing the behavioral response of mammals

to sublethal OP exposures can be difficult not only because they differ in chemical and

animal species, but also because of a general lack of standardization in behaviors

measured, measurement unit, route of administration, and time course for observation.

Despite these differences, a picture of overall behavioral depression emerges from the

literature. One of the more consistent behavioral changes observed in response to acute

sublethal exposures is decreased locomotor activity (Lynch et al., 1986; Padilla et al.,

1992; Llorens et al., 1993; Dell‘Omo and Shore, 1996; Dell’Omo et al., 1997; Sheets et

al., 1997) . Padilla et al. (1992) saw decreased movement along with miosis, diarrhea,

and tremors in response to intraperitoneal (IP) injections of paraoxon. Two studies of

behavior in the field following a single dimethoate administration to wood mice

(Dell’Omo and Shore, 1996) and shrews (Dell’Omo et al., 1997) observed decreased

locomotor activity along with grooming, sniffing, and rearing behaviors for a six hour

period followed by a total behavioral recovery. In both cases there was a temporal

correlation between the behavioral changes and decreased ChE activity measurements

taken in the laboratory. They also found that behavior returned to normal six hours after

administration which is significantly faster than the recovery of AChE activity. Subacute

and subchronic studies have produced similar findings to acute experiments. Llorens et

al. (1993) observed the coincidence of classic clinical signs and decreased locomotion in 15

rats following repeated exposures to disulfoton. While the clinical signs quickly

developed a tolerance response, movement behavior remained depressed over the 30 day

exposure period. These results were in keeping with those of Padilla et al. (1992).

Sheets et al. (1997) performed a study where six OPs were incorporated into rat diets for

13 weeks. Increasing behavioral and enzymatic depression was observed for 4 weeks,

but no further cumulative response was detected.

Due to the high risk of subjectivity in measuring behavioral changes, several

researchers have used standardized observation procedures and automated measurement

systems in determining behavioral response. Several researchers have used the

Functional Observational Battery (FOB) of Moser (1988, 1989) to characterize certain

behaviors in different settings or following certain stimuli (Padilla et al., 1992; Sheets et

al., 1997). Sheets et al. (1997) found the combination of clinical observations with the

FOB to be effective at detecting affects on the central nervous system as well as in the

periphery. Rodent movement behavior has been effectively quantified using a figure- eight maze equipped with infrared motion sensors automated to keep track of beam interruptions (Padilla, 1992; Llorens et al., 1993; Sheets et al., 1997). The FOB and automated measurement were able to detect behavior changes correlating to brain ChE depression of approximately 20% leading to the judgment that these techniques are more

sensitive than simple observation (Padilla et al., 1992; Sheets et al., 1997). 16

Caenorhabditis elegans

The nematode Caenorhabditis elegans is a free living bactiverous soil nematode

of the order Rhabditida. In the mid to late 1960s, it was isolated and characterized by

Sidney Brenner for use in the laboratory research. Since that time, it has become one of

the most studied animal species in history being used as a biological model in genetics,

molecular biology, developmental biology, and neurobiology.

Caenorhabditis elegans has many traits that make it suitable for use as a model

organism. The adult is approximately 1mm in length allowing large numbers of

individuals to be kept in a limited space. C. elegans has a life span of approximately

three weeks and a life cycle from egg to reproductive adult of 3-4 days at 200C. It easily

lives in aqueous culture or on agar filled petri dishes with Escherichia coli as a food

source (Wood, 1988). The adult is typically a self fertilizing hermaphrodite with a

lifetime fecundity of approximately 300 eggs per individual. Males infrequently develop and can mate with hermaphrodites allowing for some gene flow within a population

(Wood, 1988). Age synchronous populations are easily obtained through the collection and culturing of eggs. C. elegans is highly tolerant to ranges of temperature (12-250C),

pH (3.2-11.8), salinity (up to 15.46 g/L NaCl), and water hardness (0.236-0.246 g/L

NaHCO3) (Kahanna et al, 1997). In the absence of food, C. elegans can exist for several

months as dauerlarvae, a facultative dormant life stage from which it can reemerge with

the restoration of sufficient resources.

Pertinent aspects of C. elegans anatomy, physiology, and behavior

The basic anatomy of C. elegans consists of two concentric tubes. The inner tube

is made up of the intestine and gonad while the outer tube includes the cuticle,

hypodermis, musculature, and nervous tissues (Wood, 1988). The two tubes are

separated by the potential space of the pseudocoelom. The adult hermaphrodite has 810

somatic cells. Each cell has been characterized from differentiation to death by Sulston et al. (1983) and found to show invariant patterns of cell development and migration

(Schnabel and Priess, 1997).

C. elegans is one of the simplest organisms with a centralized nervous system. As in higher organisms, the system’s basic functional unit is the neuron. The adult hermaphrodite has 302 neurons along with 56 glial and associated support cells (Chalfie and White, 1988). The entire system has been mapped by White et al. (1986) through the reconstruction of serial dissection electron micrographs. As a whole, the nervous system includes approximately 2000 neuromuscular junctions, 5000 interneural chemical synapses and 600 gap junctions (Thomas, 1994). Most of the neurons are bundled into secondary structural units. The most important of these are the nerve ring, and dorsal and ventral nerve cords (Rand and Nonet, 1997).

C. elegans neural morphology is quite simple. Neurons typically possess one or two unbranched processes that make connections en passant with little specialization of the process terminals (Thomas, 1994). Presynaptic terminals show little structural distinction, but characteristically display areas concentrated with neurotransmitter filled vesicles. Post synaptic terminals also show little structural specialization. The type of 18

structural complimentarity displayed by terminal invagination in mammalian neuromuscular junctions is completely absent in C. elegans (Rand and Nonet, 1997).

It has been shown that C. elegans neurons do not exhibit classical action

potentials, and do not possess the sodium channels or myelin sheaths needed for rapid

electrochemical conduction over long distances (Goodman et al., 1998). It has been

hypothesized that C. elegans lost its sodium channels over time because action potentials

were not required for the short distances of transmission (Davis and Stretton, 1989).

Instead, membrane currents are conducted using a wide variety of potassium and calcium

channels (Wei et al., 1996). These membrane currents mediate tonic neurotransmitter

release capable of graded increases or decreases in response to shifts in electric potential

(Davis and Stretton, 1989). This type of constant electrochemical signal modification

may support less drastic but more sensitive responses to sensory inputs.

C. elegans uses wide array of classical neurotransmitters to chemically span the

synaptic cleft. These neurotransmitters are produced and handled in a manner very

similar to vertebrates (Bargmann, 1998). Enzymatic biosynthesis of neurotransmitters

from common metabolites is followed by microtubule mediated vesicular transport to

neuronal terminals and calcium induced exocytosis (Rand and Nonet, 1997).

Neurotransmitters that have been described in C. elegans include acetylcholine (Lewis et

al., 1980; Hosono et al., 1987), GABA (McIntire, 1993), serotonin (Horvitz et al., 1982),

dopamine (Sulston et al., 1975), glutamate (Li et al., 1997), and several neurogenic

peptides (Rand and Nonet, 1997).

Acetylcholine (ACh) is the primary excitatory neurotransmitter utilized in C.

elegans motor function. The genes controlling its biosynthesis by choline 19

acetyltransferase (cha-1) and vesicular transport (unc-17), show significant similarities

between nematodes and higher vertebrates (Hosono et al., 1987; Erickson et al., 1994;

Bargmann, 1998). Proteins involved in ACh exocytosis including syntaxin, synaptobrevin, and synaptotagmin also show homologies to mammals (Bargmann, 1998).

The work of Lewis et al. (1980) demonstrated the presence of nicotinic acetylcholine

receptors (nAChRs) in C. elegans. Since that time genetic analysis has predicted the

presence of approximately 40 different subunits belonging to the nicotinic class

(Bargmann, 1998). Muscarinic ACh receptors (mAChRs) have also been identified in C.

elegans. These receptors are similar but not identical to vertebrate types in their

pharmacological characteristics (Culotti and Klein, 1983).

As in higher animals, ACh in C. elegans is degraded between neurons or in the

neuromuscular junction by acetylcholinesterase (AChE). Both C. elegans and vertebrates

have more than one form of AChE, but whereas vertebrates have one AChE gene whose

product undergoes alternative splicing posttranscriptionally, C. elegans possesses four

distinct AChE genes (Combes et al., 2001). The ace-1 and ace-2 genes encode the two

major forms of AChE in C. elegans which account for approximately 95% of the overall

AChE activity. The ace-1 gene has been traced to chromosome X (Arpagaus et al.,

1994). A study linking green fluorescent protein (GFP) production to the ace-1 promoter

localized its expression almost exclusively to the worm’s musculature (Combes et al.,

2001). The ace-2 gene is part of chromosome I, and is expressed in the sensory and

motor neurons of the ganglia and nerve cords (Combes et al., 2001). The ace-3 gene was

discovered by Johnson et al. (1988), and encodes a minor form of AChE. A fourth ace

gene was found by Grauso et al. (1998). Its presence has been confirmed through RT- 20

PCR analysis of transcription products, but it seems to be translated into little, if any, active product in vivo (Combes, 2001).

Studies using C. elegans strains exhibiting null mutations in the ace-1, ace-2, and ace-3 genes have revealed different aspects of their relationships to each other. A high degree of functional overlap has been described between ACE-1 and ACE-2 enzymes.

Inactivation of either gene alone results in very little phenotypic change while the ace-1, ace-2 double mutant displays highly impaired behavior (Culotti et al., 1981; Johnson et al., 1981; Johnson et al, 1988). This functional redundancy is interesting given the

differing nature and distribution of ACE-1 and ACE-2 enzymes. The level of

incoordination displayed by ace-1, ace-2 double mutants compared to either single

mutant reveals the subordinate role ACE-3 plays compared to the two major forms. The

ace-1, ace-2, ace-3 triple mutant is not viable (Rand and Nonet, 1997). This demonstrates that some AChE activity is essential for C. elegans survival, and that the activity of the ace-4 gene product is negligible.

The protein products of the different AChE genes have also been studied.

Johnson and Russell (1983) described five protein forms of various sizes which they

divided into two classes (traditionally denoted as A and B) according to physicochemical

characteristics. Further study has shown the "class A" proteins are components of the

ACE-1 form of AChE and products of the ace-1 gene. Likewise, the class B proteins

have been shown to be components of the ACE-2 enzyme encoded by the ace-2 gene

(Combes, 2001). Johnson and Russell (1983) found the ACh binding affinities for ACE-

1 and ACE-2 to be similar to vertebrate AChE, but both were less substrate specific.

They also demonstrated ACE-1 and ACE-2 to be vulnerable to a variety of cholinesterase 21

inhibitors including two organophosphate pesticides. ACE-3 accounts for approximately

5% of C. elegans’ AChE activity, and displays different pharmacological characteristics

than ACE-1 or ACE-2. Kolson and Russell (1985a, 1985b) found ACE-3 to have a much

greater affinity for ACh than the two major forms, and also to be considerably less vulnerable to inhibition by anticholinesterase agents. ACE-1, ACE-2, and ACE-3 account for virtually all AChE activity in C. elegans. Difficulties in isolating the ACE-4 protein have lead to the speculation that ace-4 is essentially a non-functional gene

(Combes et al., 2000; Combes et al., 2001).

Early investigations caused researchers to think there would be little commonality

between C. elegans and vertebrate AChE (Johnson and Russell, 1983). Since that time

an increasing number of similarities have emerged between the different forms. Using

genetic analysis, Arpagaus et al. (1994) discovered 42% identity between ACE-1 and

mammalian AChEs. The highest degree of homology is between ACE-1 and the C-

terminal region of vertebrate T-peptide. Sedimentation studies by Combes et al. (2001)

found that the major form of ACE-1 in vitro was a tetramer likely bearing some

association with a smaller monomer. This was consistent with previous investigations by

Arpagaus et al. (1992) which showed the product of ace-1 in the nematode Stinernema

carpocapsae (a rhabditidae related to C. elegans) was a catalytic subunit which

oligomerized into an amphiphilic tetramer. This structural arrangement bears

resemblance to the T-peptide G4 form of AChE found in the vertebrate brain (Combes et

al., 2001). Evidence from studies using ace-1 mutants indicate that the product of ace-2

is a hydrophilic monomer which can dimerize and associate with a glycolipid moiety

(Combes et al., 2001). This quaternary structure is similar to dimeric H-peptides of 22

vertebrates which are anchored to membrane surfaces by a glycolipid unit added to the

dimer posttranslationally (Combes et al., 2001). The differential expression of ace genes by cell type is also interestingly similar to the AChE arrangement in vertebrates.

Whether these and other similarities are due to homology or analogy remains to be determined.

Despite its simplicity, C. elegans’ neuromuscular system supports a variety of behaviors. More than 250 genes have been identified with some role in regulating behavior (Thomas, 1994). Muscular contractions resulting in sine wave type motion allow for forward and backward movement. The worms feed, defecate, lay eggs, and in the case of males exhibit a fairly complex mating behavior (Wood, 1988). C. elegans also displays complex chemosensory behavior exhibiting attraction and repulsion responses to a well over 100 chemical stimuli (Bargmann, 1993; Thomas, 1994).

Reviews of these behaviors and the research concerning them can be found in Riddle et al. (1997).

Metabolism

Most research on how C. elegans deals with xenobiotics has been devoted to the

detoxification of heavy metals. It is only in recent years that pathways for the

metabolism of organic contaminants have received attention. Unlike higher organisms,

C. elegans does not have an organ primarily devoted to the biotransformation of

xenobiotics. Much of this activity is concentrated in the intestine (McGhee, 1987;

Menzel et al., 2001). Only reports of phase I type biotransformations in C. elegans have

been published, but there has been at least one account of gene induction for phase II 23 enzymes like UDP-glucuronosyltransferases (Menzel et al., 2001). Approximately 80 cytochrome P450 genes have been identified in C. elegans (Nelson, 1999). Although little is known about C. elegans’ CYP450s, it is believed that many of them developed in order to cope with the variety of conditions nematodes face (Gotoh, 1998). A large portion of these enzymes belong to a pure group of nematode P450 isozymes designated as the C. elegans clan. This clan appears to share a common ancestor with the 2 clan of

P450s which include many mammalian enzymes known to be active in drug metabolism

(Nelson, 1999). Using RT-PCR to characterize RNA expression, Menzel et al. (2001) looked for evidence of P450 induction following exposures to known xenobiotic inducers. They found four subfamilies of C. elegans CYP450s to be particularly responsive to such compounds as beta-napthoflavone, phenobarbital, atrazine, clofibrate, lansoprazole, and fluoranthene. Other metabolic enzymes likely to be important to OP toxicity have also been identified. McGhee et al. (1987) characterized a nonspecific esterase from the gut of C. elegans that showed little affinity for ACh, but was stoichiometrically inhibited by several organophosphates. Several forms of glutathione-S- transferase have also been identified (van Rossum et al., 2001).

The use of C. elegans as a Model Organism

In thinking about the directions he should pursue in his research, Sydney Brenner in the early 1960s had the idea of using genetic techniques in a simple model organism to study development and the nervous system (Brenner, 1988). In his initial description of the genetics of C. elegans, Brenner (1974) cites the potential for determining the structure of its entire nervous system as a primary reason for choosing this nematode as his model 24 organism. Since that time, all cell lineages of C. elegans have been traced (Sulston et al.,

1983), its nervous system has been mapped (White et al., 1986), and its genome has been sequenced (C. elegans Sequencing Consortium, 1998). These important accomplishments have combined in a unique way to allow for Brenner’s initial ambitions to become a reality. In the study of developmental and neurological questions, these databases can be used in conjunction with mutant and transgenic strains of C. elegans to decipher what structures are affected and what processes are altered.

C. elegans has been used as a model for ageing for more than twenty years

(Zuckerman and Himmelhoch, 1980). The transgenic capabilities of C. elegans have also made it useful as a model for human degenerative neurological diseases. Invertebrate models allow researchers to use a variety of powerful genetic tools in the analysis of transgenic lines expressing disease associated proteins over a short lifespan (Link et al.,

2001). Human beta-amyloid peptide, a peptide associated with the onset of Alzheimer’s disease, has been expressed in C. elegans by Link et al. (2001). Worms expressing the protein displayed a progressive onset of paralysis. A gene encoding multiple polyglutamine repeats of the sort associated with Huntington’s disease have also been expressed in C. elegans sensory neurons (Faber et al., 1999). Cells expressing the longest of such repeats were found to exhibit an age dependent decrease in function. The deleterious impacts of human beta-amyloid peptide and polyglutamine repeats in nematode cells strongly imply that the pathology of these diseases lie to a great extent in the basic cellular processes conserved between vertebrates and invertebrates (Link,

2001). 25

C. elegans has also found use as a model in the biological response to anesthetic

agents. A number of researchers have used behavioral endpoints to characterize the

response of C. elegans to a host of volatile anesthetic agents (Morgan and Cascorbi,

1985; Crowder et al., 1996; Kayser et al., 1998). Work with anesthetics in higher animals

has long established a correlation between anesthetic potency and lipophilicity called the

Meyer-Overton correlation. Early work by Morgan and Cascorbi (1985), and Morgan et

al. (1988) confirmed that the wild type N2 strain of C. elegans adheres to the Meyer-

Overton relationship, but also described mutant strains deviating from this response.

Subsequent work with these mutant strains demonstrated that contrary to the traditional paradigm, the anesthetic response was controlled by several pathways at multiple sites of action (Morgan and Sedensky, 1994, 1995; Kayser et al., 1998). Working with a more common anesthetic, Anton et al. (1992) found ethanol to have a general effect on worm movement very similar to that observed in higher animals including mammals. Morgan and Sedensky (1995) were able to replicate these results. Critical evaluations of C. elegans by Anton et al., (1992) and Morgan and Sedensky (1995) lead to the conclusion that despite certain limitations, C. elegans’ strict adherence to important aspects of the basic anesthetic response makes it a useful as a mammalian model.

Efforts have also been made to evaluate C. elegans as a model for mammalian

xenobiotic toxicity. Williams and Dusenbery (1988) found that acute lethality values in

C. elegans exposed to eight heavy metal salts predicted overall mammalian acute

lethality as consistently as tests in rats and mice individually. Williams and Anderson

(2000) conducted blind tests on five gadolinium-based compounds used in magnetic

resonance imaging utilizing movement behavior as the measured endpoint. These tests 26

correctly ordered the toxicity for four out of the five compounds compared to median lethal doses for the same compounds in mice. Anderson et al., (submitted for publication) found five compounds across three chemical classes used in behavioral

toxicity tests to have an identical order of toxicity in C. elegans as in mammals.

Transgenic and wild type strains of C. elegans have been used in environmental

toxicity testing. Most common has been its use in assessing the toxicity of heavy metal

contamination in soil (Black and Williams, 2001; Boyd and Williams, 2003) and aquatic

environments (Hitchcock et al., 1997). Williams and Dusenbery (1990) found salts of

lead (Pb) and aluminum (Al) to display toxicity dynamics consistent with neurotoxicity.

Nematode behavior has been shown to be much more sensitive than lethality in the

characterization of toxins (Dhawan, et al., 2000; Anderson et al., 2001). The

development of computer automated tracking systems has made possible the accurate

characterization of nematode movement as a toxic endpoint (Dusenbery, 1996; Dhawan

et al., 1999; Boyd et al., 2000). With the idea that movement behavior may be

preferentially sensitive to neurotoxicants, several researchers have used locomotion in the

examination of chemicals known to be neurotoxic. Anderson et al. (2001) demonstrated

that movement in C. elegans was more sensitive to Pb than to Cu over a 4-hr exposure

period while this difference in sensitivity was masked at exposures lasting 24-hr. Recent

work by Anderson et al. (submitted) indicated that this preferential sensitivity may be

indicative of mechanistic differences between the known neurotoxin Pb and Cu which is

not believed to be neurotoxic. They also conclude that the combination of 4-hr exposures

with locomotor measurements is well suited to the detection of neurotoxicity. 27

The research reviewed here leaves outstanding the question of how similar the

dynamics of neurotoxicity are between C. elegans and mammals. Answering this question will require a more rigorous evaluation of C. elegans’ response to neurotoxic chemicals than has been undertaken to this point. The purpose of this thesis work was to begin to address this question of comparability by characterizing the toxic response of C.

elegans to a class of neurotoxins and making comparisons to the corresponding response

displayed by mammals. 28

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Table 2.1. Basic Description of Chemicals Tested.

Compound aUse/Accepted bRat LD50 aWater Solubility cStructure Mechanism (mg/kg) (mg/L)

Demeton-S-methylsulfone dInsecticide C6H15O5PS2 Metabolite 32.4 > 2.2x104 FW: 262.3 Cholinesterase Inhibitor CAS: 1740-19-6

Dichlorvos Insecticide and C4H7Cl2O4P Acaricide 17 1.8x104 FW: 221.0 Cholinesterase Inhibitor CAS: 62-73-7

a The Pesticide Manual, 12th ed. 2000. Tomlin, C.D (Ed.). 2000. . British Crop Protection Council, Farnham, UK. b RTECS Database, National Institute for Environmental Health. c Images obtained from Chemfinder.com (http://www.chemfinder.com). d International Program on Chemical Safety. Environmental Health Criteria 197 (http://www.inchem.org/documents/ehc/ehc/ehc197.htm) 42