Molecular Mechanisms of Action of Nickel in the Nematode

MOLECULAR MECHANISMS OF ACTION OF NICKEL IN THE NEMATODE

CAENORHABDITIS ELEGANS

DEAN VALLEN MEYER

(Under the Direction of Phillip L. Williams)

ABSTRACT

Caenorhabditis elegans is a bacterivorous nematode used as a model organism in biosciences research. Its characteristics make it suitable for toxicological investigations of environmental exposures. It has been used extensively to assess the effects of exposure to metals. Nickel is a ubiquitous metal present in the soil, air, and water, to which all living organisms are exposed daily. In this dissertation, the effects of nickel on C. elegans are examined, with particular emphasis on characterizing cellular detoxification pathways involved in counteracting the effects of high concentrations of nickel. Pathways studied included metallothioneins, divalent metal transporters, a heat shock protein, an ABC transporter, phytochelatin, and coelomocytes. In addition, this dissertation evaluated the differential toxicity of seven soluble nickel salts, and investigated whether nickel caused degeneration of cholinergic and dopaminergic pathways in C. elegans.

INDEX WORDS: Nickel, Caenorhabditis elegans, detoxification

MOLECULAR MECHANISMS OF ACTION OF NICKEL IN THE NEMATODE

CAENORHABDITIS ELEGANS

DEAN VALLEN MEYER

BA, Emory University, 1999

MPH, University of South Florida, 2009

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

Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2015

Dean Vallen Meyer

MOLECULAR MECHANISMS OF ACTION OF NICKEL IN THE NEMATODE

CAENORHABDITIS ELEGANS

DEAN VALLEN MEYER

Major Professor: Phillip L. Williams

Committee: Kevin K. Dobbin Travis C. Glenn Robert M. Gogal, Jr. Jia-Sheng Wang

Electronic Version Approved:

Suzanne E. Barbour Dean of the Graduate School The University of Georgia August 2015

ACKNOWLEDGEMENTS

I would like to extend my gratitude and appreciation to my husband, Karl L.

Schaller, and to my mentor, John F. Risher, for their support and encouragement.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER

1 INTRODUCTION ...... 1

References ...... 8

2 LITERATURE REVIEW ...... 11

References ...... 24

3 TOXICITY TESTING OF NEUROTOXIC PESTICIDES IN

CAENORHABDITIS ELEGANS ...... 31

Abstract ...... 32

Introduction ...... 32

Endpoints Assessed ...... 35

Exposure ...... 37

Insecticides ...... 40

Fungicides ...... 71

Herbicides ...... 72

Discussion ...... 81

Acknowledgments...... 88

References ...... 89

4 DIFFERENTIAL TOXICITIES OF NICKEL SALTS TO THE NEMATODE

CAENORHABDITIS ELEGANS ...... 98

Abstract ...... 99

Introduction ...... 99

Materials and Methods ...... 100

Results ...... 104

Discussion ...... 108

References ...... 110

5 DETOXIFICATION MECHANISMS IN CAENORHABDITIS ELEGANS

EXPOSED TO NICKEL...... 112

Abstract ...... 113

Introduction ...... 113

Materials and Methods ...... 119

Results ...... 121

Discussion ...... 133

Acknowledgments...... 136

References ...... 136

6 ASSESSING DIRECT DOPAMINERGIC AND CHOLINERGIC NEURON

DAMAGE POTENTIAL IN CAENORHABDITIS ELEGANS FOLLOWING

ACUTE AND SUBCHRONIC NICKEL EXPOSURE ...... 140

Abstract ...... 141

Introduction ...... 141

vii

Materials and Methods ...... 144

Results ...... 145

Discussion ...... 157

Acknowledgements ...... 160

References ...... 160

7 CONCLUSIONS...... 163

viii

LIST OF TABLES

Page

Table 3.1: Toxicity studies of organophosphates using a C. elegans animal model ...... 42

Table 3.2: Toxicity studies of carbamates using a C. elegans animal model ...... 59

Table 3.3: Toxicity studies of pyrethroids using a C. elegans animal model ...... 66

Table 3.4: Toxicity studies of other pesticides using a C. elegans animal model ...... 73

Table 3.5: Advantages and disadvantages of the three standardized testing media for C.

elegans toxicity testing ...... 83

Table 4.1: Nickel compounds used in this study ...... 103

Table 4.2: Median lethal concentration values for seven nickel compounds ...... 106

Table 4.3: Nickel salt pairs significantly different from each other ...... 107

Table 5.1: Lethal concentrations for strains lacking metallothionein genes ...... 123

Table 5.2: Lethal concentrations for strains lacking smf genes ...... 126

Table 5.3: Lethal concentrations for Hsp90 mutant strain, daf-21 knockdown, compared

to wild type ...... 129

Table 5.4: Lethal concentrations for strains lacking one or more of the following:

Coelomocytes, hmt-1, pcs-1 ...... 132

LIST OF FIGURES

Page

Figure 3.1: Proposed toxicity testing protocol ...... 85

Figure 4.1: Lethality curves for seven nickel compounds ...... 105

Figure 5.1: Model of expected lethality curves ...... 118

Figure 5.2: Lethality curves for strains lacking metallothionein genes ...... 122

Figure 5.3: Lethality curves for strains lacking smf genes...... 125

Figure 5.4: Lethality curve for Hsp90 mutant strain, daf-21 knockdown, compared to

wild type...... 128

Figure 5.5: Lethality curves for strains lacking one or more of the following:

Coelomocytes, hmt-1, pcs-1 ...... 131

Figure 6.1: Live C. elegans nematode mounted on an agarose pad after 8 hours of

immersion in K-medium (control) ...... 146

Figure 6.2: Live C. elegans nematode mounted on an agarose pad after 8 hours of

immersion in 4.44 mM nickel ...... 147

Figure 6.3: Live C. elegans nematode mounted on an agarose pad after 8 hours of

immersion in 9.62 mM nickel ...... 148

Figure 6.4: Live C. elegans nematode mounted on an agarose pad after 24 hours of

immersion in K-medium (control) ...... 149

Figure 6.5: Live C. elegans nematode mounted on an agarose pad after 24 hours of

immersion in 4.44 mM nickel ...... 150

Figure 6.6: Live C. elegans nematode mounted on an agarose pad after 24 hours of

immersion in 9.62 mM nickel ...... 151

Figure 6.7: Live C. elegans nematode mounted on an agarose pad after 8 hours of

immersion in K-medium (control) ...... 152

Figure 6.8: Live C. elegans nematode mounted on an agarose pad after 8 hours of

immersion in 4.44 mM nickel ...... 153

Figure 6.9: Live C. elegans nematode mounted on an agarose pad after 8 hours of

immersion in 9.62 mM nickel ...... 154

Figure 6.10: Live C. elegans nematode mounted on an agarose pad after 24 hours of

immersion in K-medium (control) ...... 155

Figure 6.11: Live C. elegans nematode mounted on an agarose pad after 24 hours of

immersion in 4.44 mM nickel ...... 156

Figure 6.12: Live C. elegans nematode mounted on an agarose pad after 24 hours of

immersion in 9.62 mM nickel ...... 157

CHAPTER 1

INTRODUCTION

Metals are a subset of the chemical elements which make up our world. Their

physical and chemical properties make them highly useful in the manufacture of products

in multiple industries – construction, appliances, information technology,

communications, transportation, utilities, power generation, medicine, and decorative

accessories.

Nickel (Ni) is a transition metal, number 28 on the periodic table, with an atomic

mass of 58.69. It is an essential trace element for plants and some vertebrates, though its

physiological role in humans has not been elucidated, and it is not yet known whether it

is essential in humans (Sunderman 1977; Maroney and Ciurli, 2014). Nickel is widely

used worldwide in steel alloys, batteries, computer components, paint, ceramics, jewelry,

coins, and as a coating over other metals. Nickel is attractive as an industrial component

because of its durability, resistance to corrosion, and ready interaction with other metals

(Kornik and Zug, 2008). As such, occupational exposures to nickel ore and dusts are

common. One common effect is skin irritation and sensitization known as contact

dermatitis, a cell-mediated hypersensitivity involving macrophages and lymphocytes

(Silvennoinen-Kassinen 1980; Nordling 1985). Sensitization may also cause asthma and conjunctivitis (Bencko 1983).

Nickel compounds have been classified as Class 1 carcinogens by the

International Agency for Research on Cancer (IARC), indicating that they have been 2 found to be carcinogenic to humans (IARC 2012). Carcinogenicity results generally from nickel compounds which are water-insoluble, while water-soluble nickel compounds are not believed to be carcinogenic in experimental animals (Kasprzak et al., 2003). The carcinogenicity of water-insoluble compounds in rodents seems to be related to long-term sequestration of nickel particulates in the intracellular matrix, building up to a concentration of ionic nickel that can produce chromosomal aberrations, sister chromatid exchanges, and DNA-protein crosslinks (Costa and Heck, 1984; Sunderman 1984). Thus, carcinogenic activity of nickel particulates is proportional to their cellular sequestration, whether through phagocytosis or dissolution of particulates in physiological fluids

(Hansen and Stern, 1983). This sequestration appears to be related to the molecular structure or the surface characteristics of the nickel compound in question (Costa and

Mollenhauer, 1980). In addition, it has been suggested that epigenetic mechanisms of mutagenicity, such as DNA hypermethylation, histone hypoacetylation, or silencing of transcription factors, may also be involved (Kasprzak et al., 2003; Maroney and Ciurli,

2014).

In humans, cancers of the lungs, the nasal sinuses, or the larynx are the only neoplasia associated with long-term occupational exposure to nickel, through inhalation of fumes and dusts containing nickel (Bencko 1983). The main route of non-occupational exposure is ingestion, as nickel leaches into food from stainless steel cooking pots and cutlery (Kuligowski and Halperin, 1992). It is also present in some foods, such as soybeans and whole grains; the average dietary intake of nickel is estimated at 100-600

µg/day (Schroeder et al., 1962). Internal levels of nickel are measurable in body fluids, especially blood and urine, and may reach 1.5 µg/L in blood and 6.5 µg/L in urine in 3

healthy adults (Angerer and Lehnert, 1990). However, most ingested nickel is readily

excreted, and only a small proportion is absorbed (Sunderman 1977). It is believed that

the human gastrointestinal tract absorbs only an estimated 5% of ingested nickel

compounds (Léonard et al., 1981).

Nickel is naturally present in soil and in sea water; combustion of fossil fuels

contributes to its presence in the air, particularly in urban areas (Bencko 1983). Industrial

and household pollution can affect all living species, their habitats and ecologies. To

regulate intracellular levels of metals, organisms evolved multiple mechanisms, such as

glutathione and its derivatives, metallothioneins, heat shock proteins, and metal

transporters. It is not yet known, however, whether knockdown or saturation of one

mechanism leads to a compensatory reaction from other detoxification pathways

(Martinez-Finley and Aschner, 2011).

The effects of nickel have been studied in multiple species, and those results are summarized in chapter 2. Mammalian laboratory organisms such as rodents have been the preferred species for study, because they are closer to humans than invertebrates are.

Testing assays for mammals, however, can take several years and incur large expenses.

Accordingly, protocols which leverage alternative animal models have emerged in recent years, alongside the more traditional mammalian models. Although invertebrates do not appear at first sight to offer valuable contributions of data relevant to our understanding of human disease, genetic and biochemical similarities indicate otherwise (Ballatori and

Villalobos, 2002).

Caenorhabditis elegans is a bacterivorous nematode (family: Rhabditidae) which has been used as a laboratory organism for over four decades. Its short lifespan (2-3 4 weeks), ease of culturing, and fully sequenced genome have made it an attractive, useful model organism in multiple fields, such as toxicology, genetics, developmental and reproductive biology, cellular biology, and pharmacology (Leung et al., 2008). Research studies utilizing C. elegans include investigations into areas such as organ development, neurodegenerative disorders, programmed cell death, lifespan and the aging process,

RNA interference, DNA damage, and oxidative stress.

In toxicology, C. elegans is utilized both in ecotoxicological studies, intended to evaluate real-world exposures and their effects, and in mechanistic studies aimed at predicting mammalian toxicity. Endpoints tested include lethality, growth, reproduction, lifespan, behavior, disruption of cellular processes, biochemical and molecular changes, gene expression, and DNA mutation (Leung et al., 2008).

C. elegans is utilized as a model organism to research a wide range of health effects of relevance. Molecular mechanisms of human disease may be understood and elucidated even in the absence of clinical presentation in the laboratory model. Research involving invertebrate models may also illuminate possibilities for novel drug targets (Sin et al., 2014). Currently, researchers utilize C. elegans to investigate human health conditions which include neurodegenerative diseases, diabetes, some aspects of cancer, and obesity. Data generated from invertebrate models can contribute insights to mechanisms of toxicity in mammalian systems, including the cellular causes of disease.

These findings can serve as the first step in such investigations, before engaging in experiments which involve mammalian laboratory models or human cell lines. The relevance of the invertebrate model is strengthened when findings point to a similar cellular pathology or toxicity as that observed in the corresponding human disease 5

(Teschendorf and Link, 2009). Aquatic and soil toxicity tests utilizing C. elegans have

been shown to have a high degree of reproducibility (Höss et al., 2012).

The ability to conduct forward and reverse genetic screens in C. elegans adds to its value as a model organism for mechanistic research into molecular pathways underlying toxication and disease progression (Silverman et al., 2009). Many C. elegans

genes have recognized counterparts in vertebrates, adding to the nematode’s value in

ecotoxicological research, in monitoring the production of specific gene products and

activation of molecular pathways in response to an environmental exposure, as well as for

predicting threats to human health (Lagido 2009).

Moreover, engineering a particular mutation in C. elegans, or generating a

transgenic line which expresses a fluorescent reporter in a gene of interest, is a fast and

inexpensive undertaking, allowing for the generation of results which may not be readily

attainable in a comparable rodent assay, due to cost and time constraints (Teschendorf

and Link, 2009). The use of transgenic fusion constructs, such as luciferase, β-

galactosidase, or green fluorescent protein (GFP), is an effective method for investigating

expression patterns in C. elegans (McKay et al., 2003). It permits rapid, reliable measurement of sublethal toxicological endpoints, furthering our understanding of the cellular mechanisms involved (Candido and Jones, 1996; Lagido et al., 2001; Lagido et al., 2009). We used transgenic strains containing expressing GFP for the study described

in chapter 6.

The advantages of using C. elegans in toxicological research include:

- The sharing of basic biological functions with higher organisms (such as DNA

replication and repair, as well as cellular processes – respiration, autophagy,

endocytosis, etc.)

- The structure and functionality of the nematode’s nervous system are a simplified

version of mammalian nervous systems. All of C. elegans’ neurotransmitters have

human homologues (including dopamine, serotonin, GABA, acetylcholine, and

glutamate.)

- Testing assays are rapid and low-cost, and permit testing large numbers of

nematodes.

C. elegans can survive a pH range of 3.1 – 11.9, making it more suitable to aquatic tests than some other organisms, such as daphnids (Khanna et al., 1997).

Limitations of utilizing C. elegans in toxicity testing include:

- The absence of many specific organs central to toxicology studies in mammals –

liver, kidneys, lungs, skin, and a circulatory system.

- Routes of exposure and detoxification may vary from mammalian ones. Systemic

exposure is usually limited to passive diffusion.

- The nematode may not be a suitable model for testing the effects of volatile

organic compounds, viruses, and some mycotoxins.

- Toxicity testing may be contingent of the solubility of the chemical tested – in

some cases, the addition of a vehicle such as acetone or ethanol may be required.

- Some conclusions may not be simple enough to extrapolate to higher animals. 7

This dissertation set out to characterize nickel toxicity to C. elegans, and examine

the routes of nickel’s mechanism of action in the nematode. Nickel was selected because

it is a ubiquitous, environmentally-relevant metal, and has not been the sole focus of prior work in C. elegans. This dissertation sought to investigate the nematode’s suitability as a model organism in toxicological testing involving nickel.

Chapter 2 is a high-level survey of the scientific literature pertaining to the known

physiological and cellular effects of nickel in living systems. In addition, it surveys

published studies of nickel exposure in C. elegans.

Because some metals are known to cause neurotoxicity, a thorough survey was

performed of the literature on C. elegans as a model organism for testing chemicals

which exert neurotoxic action. Much of the published literature centers on pesticides. The

survey therefore focused on studies of pesticides with direct neurotoxic action in C.

elegans, to summarize and present the available information. That literature review,

which was published in the Journal of Toxicology and Environmental Health, Part B, is

included here in its entirety as chapter 3.

To assess whether the toxicity of nickel compounds to C. elegans is influenced by

the structure of the molecule, and to select a nickel compound for further testing, lethality

curves for seven nickel salts were generated in chapter 4. The results were compared with

published data from other organisms.

Chapter 5 examines the role of multiple metalloreactive mechanisms in protecting

C. elegans from the adverse effects of nickel. Knockout (null) strains of the nematode

were used. A broad spectrum of metal-regulating and -detoxifying factors were 8

examined, which are highly conserved across the phylogenetic tree of life –

metallothioneins, phytochelatins, metal transporters, and heat shock proteins.

In chapter 6, the neurotoxicity of nickel was investigated, by testing its ability to

cause direct neuronal degeneration in C. elegans. The objective of the study was to determine whether exposure to nickel caused direct structural alterations to dopaminergic

and cholinergic neurons.

Chapter 7 summarizes the major findings and conclusions from the three studies,

and offers suggestions for future research.

References

Angerer J, Lehnert G. 1990. Occupational chronic exposure to metals. Nickel exposure of stainless steel welders--biological monitoring. International Archives of Occupational and Environmental Health 62:7-10.

Ballatori N, Villalobos AR. 2002. Defining the molecular and cellular basis of toxicity using comparative models. Toxicology and Applied Pharmacology 183:207-220.

Bencko V. 1983. Nickel: A review of its occupational and environmental toxicology. Journal of Hygiene, Epidemiology, Microbiology, And Immunology 27:237-247.

Candido EP, Jones D. 1996. Transgenic Caenorhabditis elegans strains as biosensors. Trends in Biotechnology 14:125-129.

Costa M, Mollenhauer HH. 1980. Phagocytosis of nickel subsulfide particles during the early stages of neoplastic transformation in tissue culture. Cancer Research 40:2688- 2694.

Costa M, Heck JD. 1984. Perspectives on the mechanism of nickel carcinogenesis. Advances in Inorganic Biochemistry 6:285-309.

Hansen K, Stern RM. 1983. In vitro toxicity and transformation potency of nickel compounds. Environmental Health Perspectives 51:223-226.

Höss S, Ahlf W, Bergtold M, Bluebaum-Gronau E, Brinke M, Donnevert G, et al. 2012. Interlaboratory comparison of a standardized toxicity test using the nematode Caenorhabditis elegans (iso 10872). Environmental Toxicology and Chemistry / SETAC 31:1525-1535.

International Agency for Research on Cancer (IARC). 2012. A review of human carcinogens. C: Metals, arsenic, fibres and dusts. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 100(C):169-218.

Kasprzak KS, Sunderman FW, Jr., Salnikow K. 2003. Nickel carcinogenesis. Mutation Research 533:67-97.

Khanna N, Cressman CP, Tatara CP, Williams PL. 1997. Tolerance of the nematode Caenorhabditis elegans to pH, salinity, and hardness in aquatic media. Archives of Environmental Contamination and Toxicology 32:110-114.

Kornik R, Zug KA. 2008. Nickel. Dermatitis: Contact, Atopic, Occupational, Drug 19:3- 8.

Kuligowski J, Halperin KM. 1992. Stainless steel cookware as a significant source of nickel, chromium, and iron. Archives of Environmental Contamination and Toxicology 23:211-215.

Lagido C, Pettitt J, Porter AJ, Paton GI, Glover LA. 2001. Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors. FEBS Letters 493:36-39.

Lagido C. 2009. Transgenic Caenorhabditis elegans as biosensors. in the book "Nematodes as Environmental Indicators":225-252.

Lagido C, McLaggan D, Flett A, Pettitt J, Glover LA. 2009. Rapid sublethal toxicity assessment using bioluminescent Caenorhabditis elegans, a novel whole-animal metabolic biosensor. Toxicological Sciences: An Official Journal of The Society of Toxicology 109:88-95.

Léonard A, Gerber GB, Jacquet P. 1981. Carcinogenicity, mutagenicity and teratogenicity of nickel. Mutation Research 87:1-15.

Leung MCK, Williams PL, Benedetto A, Au C, Helmcke KJ, Aschner M, et al. 2008. Caenorhabditis elegans: An emerging model in biomedical and environmental toxicology. Toxicological Sciences: An Official Journal of The Society of Toxicology 106:5-28.

Maroney MJ, Ciurli S. 2014. Nonredox nickel enzymes. Chemical Reviews 114:4206- 4228.

Martinez-Finley EJ, Aschner M. 2011. Revelations from the nematode Caenorhabditis elegans on the complex interplay of metal toxicological mechanisms. Journal of Toxicology 2011:895236-895236.

McKay SJ, Johnsen R, Khattra J, Asano J, Baillie DL, Chan S, et al. 2003. Gene expression profiling of cells, tissues, and developmental stages of the nematode C. elegans. Cold Spring Harbor Symposia on Quantitative Biology 68:159-169.

Nordlind K. 1985. Nickel binding and uptake in thymocytes and peripheral blood lymphocytes of nickel-allergic and control subjects. International Archives of Allergy and Applied Immunology 78:364-367.

Schroeder HA, Balassa JJ, Tipton IH. 1962. Abnormal trace metals in man--nickel. Journal of Chronic Diseases 15:51-65.

Silvennoinen-Kassinen S. 1980. Lymphocyte transformation in nickel allergy: Amplification of t-lymphocyte responses to nickel sulphate by macrophages in vitro. Scandinavian Journal of Immunology 12:61-65.

Silverman GA, Luke CJ, Bhatia SR, Long OS, Vetica AC, Perlmutter DH, et al. 2009. Modeling molecular and cellular aspects of human disease using the nematode Caenorhabditis elegans. Pediatric Research 65:10-18.

Sin O, Michels H, Nollen EAA. 2014. Genetic screens in Caenorhabditis elegans models for neurodegenerative diseases. Biochimica Et Biophysica Acta.

Sunderman FW, Jr. 1977. A review of the metabolism and toxicology of nickel. Annals of Clinical and Laboratory Science 7:377-398.

Sunderman FW, Jr. 1984. Recent advances in metal carcinogenesis. Annals of Clinical and Laboratory Science 14:93-122.

Teschendorf D, Link CD. 2009. What have worm models told us about the mechanisms of neuronal dysfunction in human neurodegenerative diseases? Molecular Neurodegeneration 4:38-38.

CHAPTER 2

LITERATURE REVIEW

Nickel is a ubiquitous transition element, found in soil, air, and water, and widely used in multiple industries. The following pages are intended to give a review of nickel’s kinetics and dynamics in living systems, as well as its toxicological effects and its contribution to various disease states. This overview is succeeded by a brief survey of published literature pertaining to nickel’s effects in Caenorhabditis elegans, then by a summary of the salient points.

A. Overview nickel and its effects on living systems

Essentiality of nickel. In bacteria and plants, nickel is an essential constituent of several enzymes (Joho et al., 1995). In higher animals, it is considered an essential element for chickens, rats, goats, sheep, and swine, although its specific physiological function is unknown (Spears 1984; Coogan et al., 1989). Bone strength, reproduction, energy metabolism, and sensory function have been shown to be influenced favorably by dietary nickel in some species (Maroney and Ciurli, 2014). Supplementing the diet of ruminants with nickel has been shown to have beneficial effects. Four functions for nickel have been proposed: (a) metabolism of some proteins, (b) hormonal regulation,

(c) influence of enzyme activity, and (d) influence of cell membrane structure and function. To date, however, no mammalian enzyme has been identified as requiring nickel or as being activated specifically by nickel (Spears 1984). 12

Kinetics in unicellular organisms. At least 9 microbial enzymes that require nickel as a co-factor have been identified, though its import and homeostasis must be actively controlled to avoid toxicity. In bacteria, nickel import occurs through one of two mechanisms: ABC-type transporters (such as in Escherichia coli), or HoxN-type permeases (such as in Helicobacter pylori). Once inside the cell, nickel is stored by the histidine-rich HPN protein, which is similar to metallothionein (Dosanjh and

Michel, 2006).

The transport of nickel, in its divalent cation form (Ni2+), into cells occurs against

concentration gradients. An economical solution for most bacterial cells is the uptake

through high-capacity, low-specificity uptake systems, such as the magnesium (Mg2+)

uptake system. However, these systems are expressed constitutively, and cannot be

downregulated when metal accumulation inside the cell exceeds desirable levels. At

high levels, even essential metals can disrupt membrane and enzymatic activity and

damage DNA (Bruins et al., 2000). To avoid interference with cellular functions by

excess metal cations, microbes have had to develop metal regulation factors. A

reduction to the metallic form of nickel would require too much energy, while

keeping the reduced metal inside the cell, where it could later reoxidize. Methylation

or other covalent modification of the divalent metal cations would not be favored

either, as the resulting compounds would be unstable and mutagenic. Therefore

bacteria rely on active efflux systems which are plasmid-encoded to expel excess

metal cations (Nies 1992). In yeasts and other fungi, metals are usually sequestered into histidine-rich vacuoles (Joho et al., 1995). Microorganisms can also resist metals by exclusion barriers, intracellular sequestration by protein binding, extracellular 13

sequestration by glutathione, or biotransformation to a less toxic form, energy

permitting (Bruins et al., 2000).

Several mechanisms of nickel toxicity in bacteria have been proposed. These

include (a) replacement of essential metals in metalloproteins – such as iron, zinc, and

magnesium; (b) protein binding to inhibit enzymatic activity, and (c) causing

oxidative stress by inducing the generation of reactive oxygen species (Macomber

and Hausinger, 2011).

Effects in plants. Plants synthesize metallothioneins and phytochelatins to bind

to metals and prevent cellular damage (Tito et al., 2011). Hörger et al. (2013)

proposed that metal-accumulating plants, in which uptake and storage of metals is

unusually high, developed this trait as a defense against herbivores and pathogenic

microorganisms.

While nickel is an essential micronutrient in some plants, required for activation

of the enzyme urease, which catalyzes the hydrolysis of urea into ammonia and

carbon dioxide, nickel can affect the activity of plant enzymes such as amylase,

protease, and ribonuclease, resulting in retarded seed germination and growth of some

crops (Schaumlöffel 2012; Sethy and Ghosh, 2013).

Effects in animals. Nickel toxicity is modulated by multiple variables, including

animal species, chemical species, chemical concentration, route of exposure, and

solubility. Distributed by the blood, ingested water-soluble nickel compounds are largely eliminated in the feces. Inhaled soluble compounds are dissolved by mucus and removed via ciliary transport. In contrast, non-water-soluble nickel particles can be phagocytosed into the epithelial cells of the lung, where they may remain at high 14

levels for long periods (Cameron et al., 2011; Schaumlöffel 2012; Das et al., 2008;

Coogan et al., 1989).

In multiple mammalian and avian species, exposure to nickel has been shown to produce the following effects: (a) nephrotoxicity; (b) embryotoxicity; (c) teratogenicity; (d) hematotoxicity; (e) neurotoxicity; (f) hepatotoxicity; (g) cardiovascular toxicity, and (h) pulmonary toxicity (Coogan et al., 1989; Das et al.,

2008).

In addition, nickel is largely associated with immunotoxicity. It suppresses many

immune functions, such as interferon production, natural killer (NK) cell activity,

phagocytosis by macrophages, antibody production, and T-lymphocyte-mediated reactions, by altering the activity of these specific cell types. It can also elicit an immune response which leads to asthma or to contact dermatitis, by forming allergens. Nickel is considered the most common dermal contact sensitizer in industrialized nations (Coogan et al., 1989; Thierse et al., 2005; Cameron et al.,

2011).

Cellular processes. Much of the cellular damage caused by nickel can be traced not to a direct toxic insult, but rather to nickel’s influence over factors which regulate cellular processes. This results in inappropriate activation of some signaling pathways or in the inhibition of others. Nickel crosses cell membranes by active transport through calcium channels and magnesium channels (Forgacs et al., 2012; Eitinger and

Mandrand-Berthelot, 2000; Coogan et al., 1989), and can then compete with calcium

and magnesium for specific receptors, leading to decreased intracellular levels of

these essential elements (Das et al., 2008). 15

Nickel has been shown to affect other cellular processes by causing the following effects: (a) impairment of cellular heme-dependent metabolism, both by hampering heme synthesis and by inducing its degradation (Maines and Kappas, 1977); (b) substituting for zinc, thus impairing the activity of some zinc finger proteins, a class of DNA-binding proteins acting as transcription factors to regulate the fidelity of transcription (Sarkar 1995); (c) generating free radicals to cause DNA damage

(Forgacs et al. 2012; Sarkar 1995); (d) mimicking hypoxia by depleting intracellular ascorbate and preventing the HIF-1 α protein from degradation by the ubiquitin- proteasome pathway (Forgacs et al. 2012; Barchowsky and O’Hara, 2003; Salnikow and Kasprzak, 2005); (e) depletion of glutathione (Das et al., 2008); (f) inhibition of thrombospondin, which is a regulator of angiogenesis (Zhang et al., 2009), and (g) eliciting inflammatory processes (Barchowsky and O’Hara, 2003).

Nickel has also been shown to induce gap junction closure in cultured cells, inhibiting gap junction intercellular communication (Vinken et al., 2010). Moreover, it may interfere with the metabolism of essential metals, such as iron, manganese, calcium, zinc, and magnesium (Lu et al., 2005).

Oxidative damage. Oxidative stress, the imbalance between generation of highly-reactive oxygen species and regulation of their levels and products, can exert toxic effects and interfere with normal cell signaling pathways. Metals may mediate pathogenic effects such as lipid peroxidation, inflammation, and inhibition of DNA repair. Nickel promotes oxidative damage by inducing oxygen species that can target

DNA. It is known to deplete glutathione and protein-bound sulfhydryl groups, leading to the production of the reactive oxygen spcies (ROS) superoxide ion, hydrogen 16

peroxide (H2O2), and the hydroxyl radical (˙HO) (Stohs and Bagchi, 1995). ROS can

modify DNA bases, produce DNA-protein cross-links, and cause depurination and strand breaks (Kasprzak 1995).

ROS may serve as physiological signal transduction messengers. Nickel is known

- to induce ROS such as H2O2, ˙HO, superoxide anion radicals (O2 ), and 8-

hydroxydeoxyguanosine (8-oxo-dG) (Kasprzak 1995; Beyersmann and Hartwig,

2008; Cameron et al., 2011). Nitric oxide (NO) is a radical and a signaling molecule,

regulating the expression of diverse genes and the function of their products. NO may

react with metals, enabling them to modify its activities.

NF-kappa β is a transcription factor which regulates the expression of genes

mediating the early response to inflammation and apoptotic reactions. Oxidative

stress potentiates or amplifies the activation of NF-kappa β (Chen et al., 2001). Nickel

activates the NF-kappa β transcription complex. The activated NF-Kβ complex activates a number of genes, including the iNOS gene (inducible nitric oxide synthase). The potential to mediate oxidative damage may lead to both genotoxic and epigenetic consequences, resulting in the advancement of carcinogenesis (Buzard and

Kasprzak, 2000).

DNA damage. In cultured mammalian cells, DNA has been shown to be one of the main targets of nickel, which can induce strand breaks, form DNA-protein crosslinks, inhibit DNA polymerase, and precipitate sister-chromatid exchanges. The parts of the cell cycle most sensitive to nickel’s effects appear to be the late G1 phase and

early S-phase (Fischer and Škreb, 2001). 17

Nickel is known to interfere with various DNA repair pathways (Beyersmann and

Hartwig, 2008). Though there is no single mechanism accounting for metal-induced

inhibition of DNA repair systems, potential targets identified include some zinc-

finger proteins, such as the mammalian XPA protein, which are essential for damage

recognition during nucleotide excision repair (Hartwig and Schwerdtle, 2002).

Another gene essential to DNA repair, O6-methylguanine DNA methyltransferase

(MGMT), has been shown to be silenced in vitro in nickel-treated cells (Cheng et al.,

2012), and in a dose-dependent manner (Iwitzki et al., 1998).

Besides causing DNA damage directly, in the presence of hydrogen peroxide, nickel can damage DNA indirectly through inflammation reactions, paving the way for chemical carcinogenesis (Kawanishi et al., 2002).

Epigenetics. The carcinogenic potential of nickel appears to result from both genotoxic and epigenetic mechanisms, though our understanding of the process is incomplete (Arita et al., 2012). Epigenetics is the study of heritable changes in gene expression which are not caused by changes in the DNA sequence. Nickel has been shown to affect cells and promote cell transformation by the following epigenetic mechanisms: (a) causing increased methylation of DNA, particularly cytosine bases, leading to transcriptional repression of genes (Beyersmann and Hartwig, 2008; Arita and Costa, 2009); (b) binding to histones in the cell nucleus and inhibiting acetylation, thus blocking some transcription factors and resulting in gene silencing

(Beyersmann and Hartwig, 2008; Costa 2002; Arita and Costa, 2009; Bal et al.,

2000); (c) increasing chromatin condensation, thereby pulling actively-expressed genes from euchromatin into heterochromatin and silencing their genetic activity, 18

adversely affecting the cell if these genes include regulators of cell senescence or

tumor suppressor genes (Costa 1995; Beyersmann and Hartwig, 2008; Cangul et al.,

2002; Rojas et al., 1999), and (d) inducing structural changes in chromatin which may

cause the expression of previously-inactive oncogenes (Costa 1989).

Nickel activates hypoxia signaling pathways by blocking iron uptake. Hypoxic

responses decrease histone acetylation, leading to gene silencing. Hypoxia results in

low levels of acetyl CoA, a substrate for acetylation of histones and other proteins

(Costa et al., 2005).

The DNA repair gene O6-methylguanine DNA methyltransferase (MGMT), for example, is known to be silenced both at the mRNA and the protein level in nickel- treated cell cultures (Cheng et al., 2012). In addition, nickel’s ability to mimic a hypoxic state in the cell, as mentioned above, may lead to metabolic conditions that select for transformed cells which are resistant to apoptosis (Beyersmann and

Hartwig, 2008).

Carcinogenesis. Nickel, although a known carcinogen, is non-mutagenic or only

weakly mutagenic in classical in vivo laboratory assays (Cheng et al., 2012). Direct

genotoxic effects (through binding to DNA) appear limited to exposures to

comparatively high concentrations (Hartwig 1998). Metal genotoxicity is generally

caused by indirect mechanisms, such as interactions with proteins and formation of

complexes that perturb cellular homeostasis and promote conditions for the

proliferation of an altered phenotype (Beyersmann and Hartwig, 2008; Salnikow and

Kasprzak, 2005). The three predominant mechanisms of nickel carcinogenesis appear

to be: (a) deregulation of normal cell processes and induction of oxidative stress, 19

leading to stimulation of cell growth, (b) perturbation of DNA repair systems, leading to mutation accumulation, and (c) promotion of cell proliferation by inactivation of apoptosis and tumor suppressor genes (Beyersmann and Hartwig, 2008).

Nickel’s carcinogenic potential depends on the metal’s increased intracellular

levels (Cameron et al., 2011). Nickel’s bioavailability and reactivity with cellular

targets is modulated by its speciation (Beyersmann 1994). Water-soluble nickel salts

are usually excreted rapidly, while non-water-soluble, particulate nickel compounds,

such as nickel sulfide (Ni3S2) or nickel oxide (NiO), are only gradually dissolved in

the lysosomes, and their intracellular concentrations remain high, resulting in

oxidative stress (Hartwig and Schwerdtle, 2002; Costa et al., 2002). Soluble nickel

could act as a tumor promoter, or not contribute to carcinogenicity at all. Generally,

soluble nickel salts cannot deliver enough nickel ions to the nuclei of target cells

(Goodman et al., 2009).

DNA repair systems in particular have been shown to be sensitive to nickel in its

divalent cation state (Ni2+), leading to the accumulation of unremoved DNA lesions

and increasing the risk of tumor formation. Nickel is known to inhibit the DNA

damage recognition step in the nucleotide excision repair system through its

displacement of divalent magnesium, and the excision of damaged bases during base

excision repair processes (Hartwig 1998). Costa et al. (2001) concluded that nickel-

induced carcinogenesis is greatly influenced by gene silencing, in particular tumor suppressor genes and cell senescence genes.

Other effects. Recent studies have suggested novel roles for nickel in advancing specific disease states. Chen et al. (2009) postulated that nickel could be involved in 20

the development of non-familial diabetes; they propose that since ROS induced by

nickel could lead to lipid peroxidation and the impairment of DNA repair enzymes,

downstream effects could lead to irreversible glucose deregulation.

It has been suggested that nickel could act as a metalloestrogen – a metal that binds to estrogen receptors and mimics estrogen’s actions. Byrne et al. (2013) found that nickel can activate the estrogen receptor in the absence of estradiol, inducing a proliferation of estrogen-dependent breast cancer cells. Aquino et al. (2012), examining breast cancer patients, found higher levels of nickel in their tissues than are found in the general population.

B. Effects of nickel on Caenorhabditis elegans

The effects of nickel on C. elegans have been investigated to some extent, usually as part of a screening involving multiple metals. Harrington et al. (2012) tested the utility of various chelating agents in ameliorating metal-induced toxicity in C. elegans, and found that cyclam, EDTA, and histidine were effective in binding nickel when present in the liquid test medium alongside the metal salt.

For sub-lethal effects, Cai et al. (2010) observed germline cell apoptosis in young adult C. elegans exposed to 0.01 mM nickel for 12 hours. Using transgenic nematodes carrying β-galactosidase (lacZ) reporters, Cioci et al. (2000) exposed C. elegans to nickel sulfate hexahydrate, and found expression of mtl-2, but not hsp-16 or hsp70, which did respond to cadmium and mercury. Anbalagan et al. (2012) found some expression of both mtl-1 and mtl-2 and of heat shock protein genes, including hsp-16, in C. elegans GFP transgenic strains exposed to nickel chloride. It is possible 21

that the different responses by heat shock proteins were modulated by the salt form

tested. This dissertation found that nickel chloride was more toxic to C. elegans than

nickel sulfate (see chapter 4); it is possible that the heat shock proteins induced by

Anbalagan et al. (2012) responded more to the chloride anion than to the nickel

cation.

Rudel et al. (2013) tested the effects of nickel on C. elegans growth, development,

lifespan, and reproduction, and found a decrease in reproduction. Wang and Wang

(2008) found that sublethal toxicity of nickel in C. elegans could also be transferred

to progeny, affecting lifespan, development, and locomotory behavior.

Testing for lethality, Tatara et al. found a nickel LC50 value of 68.8±1.2 mM for

the nitrate salt (1998), and 63.4±8.96 mM for the chloride salt (1997). Williams and

Dusenbery (1990), using nickel chloride in K-medium, found an LC50 of 2,916 mg/L

(95% CI: 1,661-5,118). Peredney and Williams (2000) reported an LC50 of 2,490 mg/L for a 24 hour exposure to the nitrate salt. Since they did not report the formulation of the salt, the unit of milligrams per liter (mg/L) cannot be converted to millimolars. Chu and Chow (2002), using nickel sulfate hexahydrate, found an LC50

of 436.02 μM (95% CI: 418.78-453.71).

C. elegans’ utility in toxicity testing is contingent on the degree to which its toxic responses are predictive of those in higher organisms. Hunt et al. (2012) exposed C. elegans to the chloride salts of cadmium, mercury, copper, and potassium, and found correlations to toxicity rankings in rats. Williams and Dusenbery (1998), exposing C. elegans to various metals, compared the median lethality values to published LD50

values in rats and mice, and found that the nematodes generated lethality values 22

parallel to the rodent acute values. When comparing the effects of carbamate

insecticides on locomotion in C. elegans, Melstrom and Williams (2007) also found

significant correlation to data in rat and mouse models. This provides additional

support to the validity of carrying out testing in C. elegans as a tool in toxicology.

C. Summary

The metal’s most prevalent effect, nickel sensitivity, may be treated in several

ways – ingestion avoidance, chelation therapy, and immune modulation (Veien et al.,

2011). Sodium diethyldithiocarbamate (DDTC) and disulfiram have been shown to be

the most effective chelators against an acute exposure to nickel (Blanuša et al., 2005).

Multiple studies have demonstrated that nickel can induce apoptosis in cultured

cells. Nickel depletes glutathione, one of the scavengers of hydroxyl radicals, leading

to an increase in ROS, which can result in the activation of NF- kappa β, a transcription factor with apoptotic properties (Pulido and Parrish, 2003). Cells can sense reduced oxygenation conditions (hypoxia) and respond by activating the hypoxia-inducible factor-1 (HIF-1). Exposure to nickel has been shown to produce hypoxia-like reactions. It is hypothesized that the metal blocks gene-regulation reactions that are oxygen-dependent. Nickel probably achieves this effect by substituting for divalent iron (Fe2+) in regulatory actions by dioxygenases, deactivating the enzymes. Arresting cell metabolism to a state mimicking hypoxia triggers cellular responses which may lead to a malignant cell phenotype which is resistant to apoptosis. HIF-1 activation after nickel exposure may therefore play an important role in the neoplastic process (Denkhaus and Salnikow, 2002; Maxwell and 23

Salnikow, 2004). Nickel may also inhibit the expression of Bcl-2, a regulator of apoptosis (Rana 2008).

The solubility of the nickel compound directly influences its deposition. Costa et al. (2005) showed that in human lung cells exposed to insoluble nickel particles, the nickel was contained in the nucleus; whereas in cells exposed to soluble nickel chloride, the nickel ions localized in the cytoplasm.

Though nickel is a weak mutagen, it enhances the cytotoxicity and genotoxicity of multiple agents, usually through the inhibition of DNA repair. This inhibition has been attributed both to structural changes in DNA molecules and to interaction with repair enzymes, perhaps by displacement of essential metal ions (Hartwig et al.,

1994). In cultured mammalian cells, nickel was shown to interfere with the incision step in nucleotide excision repair; the addition of magnesium partially reversed the inhibition of repair. This indicates that competition between divalent cations of nickel and magnesium may explain some of nickel’s effects. The repair inhibition was observed at concentrations below cytotoxic levels of nickel (Hartwig et al., 1994).

Predominant mechanisms of metal carcinogenicity appear to be:

1. Induction of oxidative stress, leading to stimulation of cell growth and DNA

damage.

2. Inhibition of DNA repair, leading to accumulation of mutations.

3. Inactivation of growth controls and a mimicking of a hypoxic state, which may

provide metabolic conditions favorable for the selection of altered cells resistant

to apoptosis (Beyersmann and Hartwig, 2008). 24

In vitro studies have shown that nickel nanoparticles may cause cell apoptosis, leading to mutagenesis. Another indirect path to carcinogenesis has been demonstrated by the inflammation reactions to nickel-based nanoparticles in

mammalian studies, including human subjects (Magaye and Zhao, 2012). Nickel may

enhance tumor progression by inhibiting the activity of NK cells (Sunderman 1989).

Some other factors should be considered when evaluating conditions of nickel

exposure. Attig et al. (2014), working with the mussel Mytilus galloprovincialis,

postulated that temperature may play a modulating role in invertebrates’ ability to

detoxify nickel and counter its oxidative effects. In addition, species differences have

been observed in cell mutation responses to nickel (Oller et al., 1997).

In conclusion, nickel exerts adverse effects on multiple organ systems and

multiple cellular processes. Its toxicity derives mostly from the indirect effects of

exposure, and both genotoxic and epigenetic pathways are involved in its promotion

of carcinogenicity. Since nickel cannot be eliminated from our environment,

understanding its physiological effects and our cellular defense systems is a matter of

ongoing relevance and importance.

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CHAPTER 3

TOXICITY TESTING OF NEUROTOXIC PESTICIDES IN CAENORHABDITIS

ELEGANS1

______

1 Meyer, D., and Williams, P. L. 2014. Journal of Toxicology and Environmental Health,

Part B, 17(5):284-306. Reprinted here with permission of the publisher. 32

Abstract

The use of pesticides is ubiquitous worldwide, and these chemicals exert toxic effects on both target and non-target species. Understanding the modes of action of pesticides, as well as quantifying the exposure concentration and duration, is an important goal of clinicians and environmental health scientists. Some chemical exposures result in adverse effects on the nervous system. The nematode Caenorhabditis elegans (C. elegans) is a model laboratory organism well established for studying neurotoxicity, since the components of its nervous system are mapped and known, and most of its neurotransmitters correspond to human homologs. This review covers published studies in which C. elegans nematodes were exposed to pesticides with known neurotoxic actions. Endpoints measured include changes in locomotion, feeding behavior, brood size, growth, lifespan, and cell death. From data presented, evidence indicates that C. elegans can serve a role in assessing the effects of neurotoxic pesticides at the sublethal cellular level, thereby advancing our understanding of the mechanisms underlying toxication induced by these chemicals. A proposed toxicity testing scheme for water- soluble chemicals is also included.

Introduction

Numerous pesticides are in common use for agricultural, veterinary, household, and landscaping applications. These pesticides, which include insecticides, rodenticides, fungicides and herbicides, exert their toxic effects on both target and non-target species.

Understanding their modes of action (MOA), as well as quantifying exposure concentration and duration, is an important goal of clinicians and environmental health 33

scientists. Many pesticides function by affecting the nervous system. At sufficiently high

concentrations, they produce paralysis and subsequent necrosis and death. While the

neurotoxic effects of acute poisonings of pesticides in humans are well-documented, data are lacking on the effects of low-level, long-term exposures (Ross et al., 2013).

The use of mammalian models to study neurotoxicity is well established. The time and expense involved, however, have recently led to the adoption of non- mammalian models, along with the ongoing development of toxicity testing assays which are rapid and affordable (Peterson et al., 2008). The biologist Sydney Brenner (1974) first brought scientists’ attention to the utility of the nematode Caenorhabditis elegans as a test organism. C. elegans was the first multicellular organism to have its entire genome sequenced. Its genes and metabolic pathways are highly conserved with those of mammals (Levin et al., 2009; Blaxter, 2011). Its small size (approx. 1 mm), ease of culturing and maintenance, short lifespan and large brood size make it a viable animal model for lab studies. In addition, as a relatively simple animal with a limited repertoire of behavior, work with C. elegans may offer fewer confounding factors that work with higher animals (Williams and Dusenbery, 1990a). Since neurotransmitters are well- conserved from nematodes to vertebrates (Villatte et al., 1998), and C. elegans possesses only 302 neurons, most of which have known human homologs, it is positioned as an appropriate and convenient testing organism for neurological research. For information regarding the relationship between particular neurotransmitters and essential functions in

C. elegans, please refer to McVey et al. (2012). Studies of neurodegenerative diseases use C. elegans to shed light on the causes and progression of such disorders as 34

Alzheimer’s disease and Parkinson’s disease, as well as for testing therapeutic drugs

(Aschner et al., 2010; Ruan et al., 2010).

Because the nervous system of C. elegans is a well-established tool for studying

neurological effects which are applicable to mammals, this review focused on the use of

C. elegans in studies of pesticides which exert neurotoxic action. For reviews of the general use of C. elegans as a tool in neurotoxicological research, please refer to Leung et al. (2008), Helmcke et al. (2010), and Avila et al. (2012). The manner in which toxicity

testing is conducted in C. elegans, which endpoints are commonly measured, and which types of effects are usually seen is presented here. A list of studies, which have been published using C. elegans to evaluate the effects of neurotoxic insecticides, is provided.

Sections on fungicides and herbicides are also included. Though fungi and plants do not possess nervous systems, some of the chemicals used to control their growth exert neurotoxic effects on organisms which come into contact with them, from soil nematodes to the agricultural workers applying the chemicals. A section on rodenticides was not included because published reports of C. elegans work with rodenticides which produced specifically neurotoxic effects were not found. In addition, the pesticide rotenone was excluded because it does not affect the nervous system directly. Rotenone acts by disrupting the electron transport chain in mitochondria, leading to oxidative stress.

Though the cascading effects of oxidative stress may eventually damage dopaminergic neurons, and some studies linked rotenone to the development of parkinsonism (Jadiya and Nazir [2012]), this study focused only on those pesticides with a direct mechanistic effect on the nervous system. This is also the reason that insecticidal crystal proteins have not been included, since they act by damaging the gut, and do not exert neurotoxic action. 35

The studies referenced below underscore the utility and relevance of data gathered from

C. elegans research for the study of neurotoxicity, therefore we have also included a proposed toxicity testing protocol for water-soluble chemicals.

Endpoints Assessed

Toxicologists study multiple endpoints when characterizing the effects of

chemicals on living systems. These endpoints range from those observed at the whole-

organism level (such as lethality, lifespan, behavior, and fecundity), to endpoints at the

tissue level (tumor formation, morphological abnormalities), or at the cellular and

subcellular levels (apoptosis, gene expression, formation of DNA adducts). Researchers

working with C. elegans have established protocols for studying these endpoints in the nematode.

Lethality is a common endpoint frequently assessed in pesticide studies.

Nematodes are exposed to known concentrations of the chemical for a given number of hours, and the live ones are then counted under a microscope. This allows for the establishment of a concentration-response curve. Variables in this method include the medium of exposure, the length of exposure, the age of the nematodes, and whether a food source is provided during the exposure.

Many chemicals, however, including pesticides, produce sublethal changes at low concentrations (Williams and Dusenbery, 1990a). These changes, such as modifications in behavior, growth, reproduction, and phenotype, as well as the development of disease, are of great interest to scientists in suggesting molecular pathways and gene functions.

Some common endpoints frequently assessed in C. elegans after chemical exposures 36

include brood size, time to propagation, body length, lifespan, generation of reactive

oxygen species, reduction in body lipid content, or induction of cellular apoptosis.

Although these endpoints are not specifically neurotoxic, they may be explained by

indirect effects on neuronal cells. A decrease in feeding, for instance, may be attributed to

slower rates of pharyngeal pumping; a decrease in brood size may be the result of

paralysis of the vulval muscles. Studying alterations in these endpoints may point to the

pathways involved in such cascade effects.

In particular, the assessment of an organism’s behavior following a chemical

exposure may serve as a biosensor of adverse effects. In an invertebrate like C. elegans,

change in movement is a neurological effect (whether direct or indirect), and one that is easily observed (Anderson et al., 2004). Related endpoints include a decrease in number of body bends, reversals, or head thrashing, a decrease in feeding, inhibition of the activity of neurotransmitters (cholinergic, dopaminergic, or GABAergic), or degeneration of neurons.

“Behavior” encompasses 4 primary endpoints in C. elegans:

1. Movement

2. Attractive-repellent type behavior

3. Feeding

4. Thrashing

Locomotory endpoints are measured by either manual observation or automated

(computer) observation. Feeding is measured by capturing the optical density of food

source bacteria in the test medium over time. These endpoints are influenced by the

combination of the nematode’s environment, the properties of its nervous system, and its 37

experience (Bargmann, 1993). Genetic mutants of C. elegans enable researchers to

identify genes which affect its movements, leading to a greater understanding of these

mechanisms and pathways in humans (Cronin et al., 2005).

Exposure

The main exposure route in C. elegans is ingestion through pharyngeal pumping.

The nematode possesses no respiratory system, so inhalation is not an exposure route in

C. elegans. As for exposures correlating with surface contact by the chemical, the question of whether the collagenous cuticle of the nematode is permeable to pesticides is an open one. Only a few, limited studies are available. Jackson et al. (2005) found ingestion solely through the gut.

There is currently no method for determining the internal amount of a chemical in

the nematode, which is why the term “dose” does not apply. What is measured is the

concentration to which the nematode is exposed, but the actual internal concentration is

unknown.

Many factors modulate a chemical exposure in C. elegans, including the age of

the nematode, duration of the exposure, concentration of the chemical, water-solubility

and bioavailability of the chemical, presence or absence of food, and nature of the test

medium. Almost all C. elegans testing is conducted in one of three media:

1. Agar: C. elegans is usually cultured and maintained on nematode growth medium

(NGM) agar (Brenner, 1974). The chemical of interest may be added to the liquid

agar prior to pouring it into Petri dishes, or smeared on the agar along with the

bacterial preparation used as food. The main disadvantage of this approach lies in 38

the inability to control the amount of chemical to which the nematode are

exposed, as its bioavailability in the agar is difficult to quantify, and as the

nematodes are likely to remain in the film of condensation on top of the agar. N2,

the strain selected for designation as the wild-type, is typically non-burrowing

under normal maintenance conditions, and these nematodes remain on the surface

of the agar. One cannot quantify the exposure concentration, only the chemical

that was added to the medium. And the more complex the medium, the more

likely that the chemical would bind to one or more of the constituents.

2. Liquid medium: A liquid medium allows for precise quantification of the

chemical tested, though a hydrophobic chemical may first require dissolution in a

vehicle such as ethanol, acetone, or dimethyl sulfoxide (DMSO). The possible

effect of the vehicle itself on the nematodes introduces another layer of

complexity to the study. M9 buffer is sometimes used as a testing medium for C.

elegans, however the more common approach is to use K-medium, a solution of

0.032M potassium chloride and 0.051M sodium chloride (Williams and

Dusenbery, 1990b). When testing metallic compounds, the phosphate in M9 may

chelate some of the metals out of the solution, lowering their bioavailability to the

nematodes; K-medium was developed specifically to avoid such binding, though

Ma et al. (2009) found that K-medium may affect the toxicity of nanoparticles to

C. elegans. Since real-world exposure of C. elegans to pesticides occurs in soil,

testing these chemicals in a liquid medium may not take into account possible soil

sorption of chemicals, and thus may overestimate the bioavailability of chemicals

to the nematodes (Sese et al., 2009). 39

3. Soil or aquatic sediment: This is a lesser-used medium for testing chemical

exposures, and is usually leveraged to model exposures in agricultural settings,

where biodegradation or soil sorption of the chemical is expected to influence its

bioavailability to a significant extent. Sediments in bodies of water absorb

pesticides through runoff from nearby fields. Testing in soil is relevant for

assessing the toxicity of pesticides, many of which are applied to the soil or

sprayed on plants, ending up in the soil. Variables include the organic content of

the soil being tested, the chemicals used to recover the nematodes, and the

presence of indigenous nematode populations. For protocols on the use of soil for

testing chemical exposures in C. elegans, please see Freeman et al. (1999),

Peredney and Williams (2000), and Boyd et al. (2001). Though soil is the most

environmentally-relevant of the three testing media, it is also the most labor-

intensive to perform, and introduces the greatest opportunity for the chemical to

interact with the medium.

Brinke et al. (2011) described a semi-fluid gellan gum medium for testing sublethal endpoints in nematodes, to support three-dimensional distribution of the nematodes and food bacteria. This medium is presented as a solution to the problem of dissolving hydrophobic chemicals in exposure studies. To date, it has not been used in other published C. elegans studies. It still involves the use of some vehicle to aid in dissolving the chemical, and leaves open the question of the concentration actually absorbed by the nematodes.

Insecticides

Insecticides are generally divided into groups of chemicals with related structures

and MOA. Even though some of these chemicals have been banned in some countries

and are no longer applied to crops there, studies of these chemicals may still be of

toxicological relevance if long-term persistence in soils or sediments is a possibility, such

as with DDT or lindane (gamma-HCH) (Shegunova et al., 2007). The chemicals listed

below produce neurotoxic activity, and were evaluated in lab settings in relation to their

effects on the non-target organism C. elegans.

Organophosphates

In use since the 1940s (Casida and Durkin, 2013), organophosphate (OP) pesticides exert their effects by inhibiting acetylcholinesterase (AChE) activity in the nervous systems of insects. The inhibition of AChE leads to a buildup of acetylcholine

(Ach) and a subsequent failure of transmission (Combes et al., 2001). OPs have also been shown to inhibit AChE in higher eukaryotes, including humans (Casida and Durkin,

2013). The binding process by which OPs inhibit cholinesterase is irreversible (Lee et al.,

2011). As a group, OPs are structurally-related compounds, but they can elicit a variety

of neurotoxic effects which may not be identical for all compounds (Mileson et al.,

1998). This variation in response may be due to additional targets for some OP

chemicals, which might modify the adverse response and affect other biological systems

(Pope 1999). Factors such as the route of entry and the duration of exposure may also

play a part in the variability of toxic responses (Kwong, 2002).

Among vertebrates, only one gene is known to encode AChE, but C. elegans possesses at least four (Combes et al., 2001). The enzymes encoded by one of the C. elegans AChE genes may be more or less sensitive to a given insecticide that those encoded by another of the AChE genes (Melstrom and Williams, 2007b). Abou-Donia

(1993) postulated that some OPs produce a delayed neurotoxicity, by acting on the cytoskeleton and leading to a degeneration of axons and myelin. Some OP insecticides may also produce impairment of dopaminergic neurons (Ali and Rajini, 2012). Viñuela et al. (2010) found transcriptional responses related to stress, innate immunity, and metabolism following C. elegans exposure to chlorpyrifos and diazinon. Table 3.1 outlines studies of the neurotoxic effects of OP compounds on C. elegans. 42

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Malathion

LC50 = 195 mg/L; dichlorvos

Williams LC50 = 0.51 and Malathion, Specific mg/L; Dusenbery, vapona concs. not 24 hr for Lethality, locomotion 1990a (dichlorvos) N2 Agar reported lethality 3 day Yes locomotion was reduced Normal behavior and AChE N2, activity Opperman PR946, Recovery were and Chang, GG202, M9 Not from restored 1991 Fenamiphos GG201 buffer 1 x 10-4 M 24 hr reported Yes exposure within 24 h 43

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Specific Movement Anderson Chlorpyri- K- concs. not was et al., 2004 fos N2 medium reported 4 hr 3 day No Movement decreased AChE Dichlorvos, activity was parathion, Specific reduced for Cole et al., and 11 K- concs. not AChE 5 of the 2004 others N2 medium reported 4 hr 3 day No activity chemicals Parathion Saffih- (and its Mobility Hdadi et metabolite Not Multiple 3 hr and 6 All life Not was al., 2005 paraoxon) reported Soil concs. hr stages reported Mobility inhibited 44

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Feeding was reduced by N2 and 50% at Boyd et Chlorpyri- 18 other K- concs. of al., 2007 fos strains medium Up to 8 µM 24 hr 3 day Yes Feeding 1.0-2.2 µM Movement and AChE activity were reduced after exposure to each Lethality, chemical; Specific movement, dichlorvos Rajini et Dichlorvos K- concs. not AChE was most al., 2008 and 9 others N2 medium reported 4 hr 3 day No activity toxic 45

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results

LC50 was calculated as 0.966 mg/L; AChE was inhibited; no significant Lethality, effect growth, observed on fecundity, body length Roh and Chlorpyri- K- All life Not AChE or number Choi, 2008 fos N2 medium Four concs. 24 hr stages reported inhibition of eggs 46

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Locomo- tion, All Yes (for propaga- endpoints 72 h test); tion, decreased Ruan et al., Chlorpyri- K- 0.003-3 24 hr and no (for 24 develop- after 2009 fos N2 medium mg/L 72 hr L4 h test) ment exposure Feeding Both Jadhav and behavior, endpoints Rajini, K- AChE were 2009 Dichlorvos PC72 medium 5-80 µM 4 hr L4 No activity inhibited 47

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results

EC50 was 0.141 µM for feeding, 0.09 µM for reproduction, 22.9 L1 – µM for 4 – 48 hr, adults, Feeding, growth, and depen- depen- reproduc- 1.1 microns Multiple ding on ding on tion, per second Boyd et Chlorpyri- N2 and K- sublethal endpoint endpoint growth, for al., 2010 fos CB5584 medium concs. measured measured Yes locomotion locomotion 48

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Mobility, growth, Mobility, fecundity growth, were all Specific fecundity, reduced; Roh and Fenitro- N2, K- concs. not Not AChE AChE was Choi, 2011 thion GK317 medium reported 24 hr 3 day reported inhibition inhibited 49

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Brood sizes were only Brood size; decreased in multiple the higher endpoints concs., and N2, related to after 48 h of MD701, spermatids, exposure or him-5 oocytes, longer; mutant, and gameto- Ruan et al., Chlorpyri- fog-2 0.003-3 24 hr, 48 apoptotic genesis was 2012 fos mutant Agar mg/L hr, 72 hr L4 Yes cells inhibited Reduction Larval of 16-42% Jones et Chlorpyri- 0.385-0.643 stage to in brood al., 2012 fos N2 Agar µM adulthood L1 Yes Brood size size 50

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Locomotion and AChE Locomo- activity, tion, AChE dopamine activity, content, and dopamine lifespan content, were all lifespan, reduced; degenera- some tion of neuronal Ali and dopami- degenera- Rajini, Monocroto- N2, K- nergic tion was 2012 phos BZ555 medium 50-200 µM 48 hr L1-L2 No neurons observed 52

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Brood size was decreased; AChE Brood size, activity was AChE decreased, Leelaja activity, levels of and Rajini, Monocroto- K- 0.85-3.4 levels of ROS were 2013 phos N2 medium mM 4 hr L4 No ROS increased Feeding was inhibited by 48.75% for 400 mg/L dichlorvos dichlorvos; and by Anbalagan Dichlorvos, K- 300 mg/L Mixed Feeding 19.67% for et al., 2013 chlorpyrifos N2 medium chlorpyrifos 28 hr stages Yes inhibition chlorpyrifos

Table 3.1. Toxicity studies of organophosphates using a C. elegans animal model. hr – hours. The units of measurement are provided here as they were reported in the original studies. Due to differences in chemical formulations, the units cannot be converted to one standardized format. 54

Measurement of acetylcholinesterase (AChE) activity in C. elegans following

exposure to OPs confirmed that these compounds affected the nematodes similarly to

their effects on target species. A reduction in AChE activity was observed following

exposure to monocrotophos (Cole et al., 2004; Rajini et al., 2008; Ali and Rajini, 2012;

Leelaja and Rajini, 2013), fenitrothion (Roh and Choi, 2011), dichlorvos (Cole et al.,

2004; Rajini et al., 2008; Jadhav and Rajini, 2009), chlorpyrifos (Roh and Choi, 2008),

and parathion (Cole et al., 2004).

Overall, lethality is not used as much as the more sensitive sublethal endpoints in

studying the effects of OP insecticides on C. elegans. Williams and Dusenbery (1990a)

found a median lethal concentration (LC50) value of 195 mg/L for malathion and 0.51

mg/L for vapona (dichlorvos) after 24 hr of exposure on agar, while Roh and Choi (2008)

found an LC50 of 0.966 mg/L for chlorpyrifos after 24 hr of exposure in an aquatic

medium. Testing the lethality of 10 different OP insecticides after a 4 hr exposure in K-

medium, Rajini et al. (2008) found LC50 values ranging from 0.039 mM (for dichlorvos) to 472.83 mM (for methamidophos).

Changes in locomotion represent a measurable effect of a chemical on the nervous system of C. elegans. These changes may be captured and analyzed visually or with a

computerized tracking system, which records the frequency and speed of body bends,

head-thrashing, and movement across agar or a microscope slide. Williams and

Dusenbery (1990a) were the first to track and analyze locomotive changes in nematodes

exposed to pesticides, and observed a reduction in movement in nematodes exposed to

malathion and dichlorvos. A decrease in rate and speed of movement due to the inhibition

of AChE activity following exposure to OP insecticides is to be expected, and has since 55 been observed in C. elegans exposed to chlorpyrifos (Anderson et al., 2004; Ruan et al.,

2009; Boyd et al., 2010), parathion (Saffih-Hdadi et al., 2005), dichlorvos (Rajini et al.,

2008), fenitrothion (Roh and Choi, 2011), and monocrotophos (Ali and Rajini, 2012).

Feeding behavior, like locomotion, represents a quantifiable effect of OP exposure on the neuromuscular system of C. elegans. It is usually measured by exposing the nematodes to the pesticide in a liquid medium in the presence of OP50, a uracil- deficient strain of Escherichia coli used as a food source (Brenner, 1974). Periodically, the bacterial density of the solution is measured and plotted on a graph. Jadhav and Rajini

(2009) observed an inhibition in feeding by L4 nematodes exposed to dichlorvos for 4 hr.

Working with a longer exposure of 28 hr and with nematodes of various life stages,

Anbalagan et al. (2013) found that feeding was inhibited by 48.75% for dichlorvos and by 19.67% for chlorpyrifos. With an 24 hr exposure, Boyd et al. (2007) found that feeding was reduced by 50% at concentrations of 1.0-2.2 µM of chlorpyrifos.

Body length can be used to measure the effect of chemicals on the growth of C. elegans. Roh and Choi found a reduction in body length of nematodes exposed to fenitrothion (2011), but not to chlorpyrifos (2008), possibly due to the low concentrations used. Ruan et al. (2009) and Boyd et al. (2010), however, did find a reduction in body length among nematodes exposed to chlorpyrifos, though only at the higher concentrations (22.9 µM in the case of Boyd et al.).

Brood size, also referred to as fecundity or propagation, has been a popular endpoint of investigation in studies of OPs and C. elegans. It is a relatively simple matter to expose young adults (usually the fourth larval stage) to the chemical of interest, then place one nematode on each agar plate, and count the progeny at specified time frames. 56

Ruan et al. (2009) found a decrease in brood size after exposing young (L4) wild-type

nematodes to chlorpyrifos in K-medium; subsequently, Ruan et al. (2012) narrowed the decrease in brood size to exposure to higher concentrations of chlorpyrifos and a longer exposure period (48 hr), although Boyd et al. (2010) found that only 0.09 µM of chlorpyrifos was needed to elicit a 50% reduction in brood size for the same length of time. Jones et al. (2012) found a reduction in brood size of 16-42% after exposing L1 larvae to chlorpyrifos on agar until they reached adulthood.

Roh and Choi (2008), working with chlorpyrifos and K-medium, did not observe a significant effect on the number of eggs. These results might be due to using older, fully-developed nematodes besides the L4 life stage. However, a decrease in brood size was noted following exposure of adult (three day old) nematodes to fenitrothion. Data suggest that some OP insecticides exert compound-specific effects. Leelaja and Rajini

(2013) observed a decrease in brood size after exposing L4 nematodes to monocrotophos for only 4 hr.

Induction of reactive oxygen species (ROS) following an exposure, or measurement of ROS levels in the exposed group versus the controls, represents an endpoint that has only recently attracted the attention of scientists studying the effects of pesticides in C. elegans. Jadiya and Nazir (2012) found that chlorpyrifos induced ROS, while Leelaja and Rajini (2013) showed that monocrotophos increased ROS levels.

Other endpoints have also been explored in the study of exposure to OPs.

Opperman and Chang (1991) measured the recovery of C. elegans in a clean environment after a sublethal exposure to fenamiphos, finding that normal behavior and AChE activity resumed with 24 hr. Ruan et al. (2012) exposed nematodes to chlorpyrifos, and noted 57

reproductive defects such as abnormal characteristics in spermatids and oocytes, as well

as an increase in cellular apoptosis. Jadiya and Nazir (2012) observed that exposure to

chlorpyrifos induced aggregation of alpha synuclein proteins, and a reduction in the

overall lipid content of exposed nematodes.

Following recent interest in using C. elegans to model neurodegenerative disorders such as Parkinson’s disease (PD), Ali and Rajini (2012) evaluated the potential of the OP monocrotophos to elicit changes in dopaminergic neurons and dopamine content in C. elegans. The authors concluded that dopaminergic features of PD may be elicited by exposure to monocrotophos.

Studies not represented in Table 1 have involved exposing C. elegans to OP insecticides for different objectives. Schouest et al. (2009), while investigating oxygen respiration rates in C. elegans, noted a 24-hr LC50 value for paraoxon (a metabolite of the

chemical parathion) greater than 10 µM. Boyd et al. (2009) described the effects of

chlorpyrifos on C. elegans growth using a mathematical model. For the effect of

chlorpyrifos on egg-laying, Martin et al. (2009) found a median effective concentration

(EC50) value of 3.5 mg/L. Saffih-Hdadi et al. (2005) estimated the EC50 of paraoxon to be 1.1 µg/ml in C. elegans. In addition, different forms of AChE are known to be encoded by multiple genes in nematodes (Selkirk et al., 2005; Melstrom and Williams,

2007b). Lewis et al. (2009) investigated alterations in global gene and protein expression following exposures to dichlorvos and fenamiphos, to identify the genes that respond specifically to OP treatment; they observed upregulation in the expression of detoxification genes of neuronal and muscle tissues. Moreover, the two chemicals, which are structurally dissimilar, produced two distinguishable expression profiles, so while the 58

results were consistent with known effects of OPs, they highlighted variations in the

mechanisms of toxicity which were likely due to the differing structures of the two

chemicals.

Overall, C. elegans demonstrated marked effects on multiple endpoints as the

result of exposure to OP insecticides, although in some cases, significant effects were

only observed at the higher concentration levels.

Carbamates

Carbamates (also known as methylcarbamates) were introduced in the 1960s to

control crop pests (Casida and Durkin, 2013). Like OPs, carbamate pesticides target

AChE, the enzyme responsible for termination of cholinergic nerve transmission. Unlike

OPs, the chemical bond which inhibits cholinesterase is reversible in carbamate

compounds (Leibson and Lifshitz, 2008). Mahoney et al. (2006) found that genetic

mutants with decreased ACh release take a longer time to paralysis than wild-type, while mutants with increased ACh release take less time, supporting our understanding of the mode of action of carbamates. Melstrom and Williams (2007b) used the carbamate insecticide propoxur to measure differences in the inhibition of the two main classes of

AChE. Table 3.2 summarizes studies of the neurotoxic effects of carbamate pesticides on

C. elegans. 59

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Normal behavior and Opperman N2 and AChE and Aldicarb, 3 Recovery activity were Chang, carbofuran, mutant M9 Not from restored 1991 oxamyl strains buffer 1 x 10-4 M 24 hr reported Yes exposure within 24 h N2 and Mahoney 8 Wild-type et al., mutant NGM Young became 2006 Aldicarb strains agar 0.25 - 2 mM 6 hr adults Yes Paralysis paralyzed 60

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Toxicity ranges and changes in movement were Melstrom comparable and Aldicarb K- Specific with Williams, and 10 medi- concs. not Mortality, mammalian 2007a others N2 um reported 4 hr 3 day No locomotion data Movement is influenced more by Melstrom N2 and Class B and 2 K- Specific AChE than Williams, mutant medi- concs. not by Class A 2007b Propoxur strains um reported 4 hr 3 day No Movement AChE 61

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Effects noted in both media; nematode Semi- growth was fluid affected gellan 0.2 - 96 more in the gum; mg/L for Growth, gellan gum Brinke et M9 both types reproduc- medium than al., 2011 Aldicarb N2 buffer of media 4 d Juveniles Yes tion in M9 62

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Reduced movement at doses 4 mg/L and above; time to first egg extended; brood size Lifespan, reduced; time to body size reproduc- reduced at all Hatching tion, brood concs.; to size, lifespan Wren et NGM Hatching to reproduc- growth, variable by al., 2011 Aldicarb N2 agar 1 - 16 mg/L L4 tion Yes movement concentration 63

Chemicals Strains Concentra- Exposure Age Nema- Endpoints Reference tested tested Media tion range duration tested todes fed? assessed Results Alpha synuclein Alpha protein synuclein aggregation protein and aggregation, induction induction of of reactive reactive oxygen oxygen species; species, and Jadiya and reduction a reduction Nazir, N2, NGM Not in lipid in lipid 2012 Aldicarb NL5901 agar 10 µM 48 hr reported Yes content content Table 3.2. Toxicity studies of carbamates using a C. elegans animal model. hr – hours. The units of measurement are provided here as they were reported in the original studies. Due to differences in chemical formulations, the units cannot be converted to one standardized format. 64

The majority of studies involved aldicarb, which has been shown to produce

paralysis in mammals such as humans and rats. The paralysis may be reversible once the

exposed organisms are removed from the source of the exposure, with no side effects or

carcinogenic or teratogenic sequelae. Although acutely toxic, aldicarb is generally

metabolized and excreted rapidly (Risher et al., 1987; Mahoney et al., 2006).

In the studies we reviewed, based on the concentrations to which the nematodes

are exposed, as well as on the duration of exposure, normal behavior and AChE activity

may be resumed when the nematodes are transferred to a clean environment (Opperman

and Chang, 1991). Other endpoints observed in C. elegans following carbamate exposure

included changes in movement (Melstrom and Williams, 2007a; Wren et al., 2011);

alterations in lifespan, time to reproduction, brood size, and growth (Wren et al., 2011),

and induction of reactive oxygen species (ROS), reduction in body lipid content, and

aggregation of alpha synuclein protein (Jadiya and Nazir, 2012).

In addition to standard media preparations, Brinke et al. (2011) tested C. elegans

sublethal exposure to aldicarb in a semi-fluid nematode growth gellan gum. This medium

was used in order to enable the nematodes to move in three dimensions, as well as to

support the testing of hydrophobic chemicals which may not dissolve well in aqueous

media. For the chemical’s effect on the nematodes’ reproduction, data showed an EC50

value of 1.3 mg/L in gellan gum, compared with 1.8 mg/L in M9 buffer. For effects on

nematode growth, an EC50 value of 5.4 mg/L in gellan gum, compared with 12.5 mg/L in

M9 buffer was noted. The authors of the study attributed the higher toxicity values in the

M9 medium to a wider range of variation between replicates.

Pyrethroids

Pyrethroids induce paralysis by keeping voltage-gated channels open in neuronal membranes (Soderlund et al., 2002). Table 3.3 summarizes the studies of neurotoxic pyrethroids which utilized the C. elegans animal model. There are relatively few such studies, mostly involving the pyrethroid cypermethrin. Ruan et al. (2009) noted a reduction in locomotion, brood size, and development following exposure to cyhalothrin.

Both Shashikumar and Rajini (2010) and Anbalagan et al. (2013) found some feeding inhibition following exposure to pyrethroids; in the former case, however, the cause was uncertain, and in the latter case, the inhibition seemed small (2.34% inhibition for cypermethrin, and 6.39% inhibition for deltamethrin). 66

Chemicals Strains Concentration Exposure Age Nema- Endpoint Reference tested tested Media range duration tested todes fed? s assessed Results Locomo- tion, propaga- All Yes (72 h tion, endpoints Ruan et K- 24 hr and test); no develop- were al., 2009. Cyhalothrin N2 medium 0.002-2 mg/L 72 hr L4 (24 h test) ment decreased Shashiku- Feeding mar and inhibited, Rajini, K- Feeding cause 2010 Cypermethrin PC72 medium 5-15 mM 4 hr 3 day Yes behavior uncertain Shashiku- Both mar and Brood endpoints Rajini, K- size, were 2011 Cypermethrin N2 medium 15 mM 4 hr L4 No lifespan decreased 67

Chemicals Strains Concentration Exposure Age Nema- Endpoint Reference tested tested Media range duration tested todes fed? s assessed Results Alpha synuclein protein aggregation, induction of reactive oxygen species, Reduc- Jadiya and N2, Not reduction tion in Nazir, NL590 repor- in lipid lipid 2012 Cypermethrin 1 Agar 5 mg/L 48 hr ted Yes content content 68

Chemicals Strains Concentration Exposure Age Nema- Endpoint Reference tested tested Media range duration tested todes fed? s assessed Results Feeding was inhibited by 2.34% for N2 and cyperme- 24 thrin and Anbalagan Cypermethrin transge- 6.39% et al., and nic K- Mixed Feeding for delta- 2013 deltamethrin strains medium 100 mg/L 28-40 hr stages Yes inhibition methrin Table 3.3. Toxicity studies of pyrethroids using a C. elegans animal model. hr – hours. The units of measurement are provided here as they were reported in the original studies. Due to differences in chemical formulations, the units cannot be converted to one standardized format. 69

Jadiya and Nazir (2012) exposed C. elegans to cypermethrin and found a reduction in the nematodes’ lipid content. It is possible, however, that this reduction could be explained by an inhibition of feeding (which they did not test), as their exposure window was long (48 hr), and they had blended the cypermethrin into the agar. It is not out of the question that the nematodes stopped feeding at some point, or that the OP50 bacteria seeded on the agar had interacted in some adverse way with the cypermethrin.

Taken together, we are inclined to think that feeding inhibition, as an endpoint in toxicity testing of pyrethroid chemicals, is less sensitive than changes in locomotion or propagation.

Organochlorines

Organochlorine (OC) insecticides act by interfering with cation exchange across the nerve cell membranes. Introduced in the 1940s (Casida and Durkin, 2013), but now mostly banned in developed countries, their persistence in the environment keeps their effects relevant nonetheless. Bezchlebová et al. (2007) exposed C. elegans to the OC pesticide toxaphene in natural soil, and reported LC50 values of 466 mg/kg for a 24-hr exposure (95% confidence interval: 326-607) and 261 mg/kg for a 48-hr exposure (95%

CI: 173-349). Sochová et al. (2007), also working with toxaphene but in a different natural soil, found 1,285 mg/kg (90% CI: 587-1983) and 376 mg/kg (90% CI: 247-504), respectively. Both studies used 3-4 days old wild-type (N2) nematodes. Two factors may account for the variations between their findings. First, Sochová et al. provided the nematodes with a bacterial food source during the exposure, while Bezchlebová et al. did not. Second, the soils used in the exposures differed in their organic and clay contents. It 70 can be expected that higher organic content coated on a higher proportion of fine particles will lead to more effective soil sorption of the toxaphene, lowering its bioavailability to the nematodes.

Sochova et al (2007), who compared the results of the exposure with published data on other invertebrate species as well, concluded that C. elegans was less sensitive to toxaphene in soil than were the soil model organisms Folsomia candida (springtail) and

Enchytraeus crypticus. Although the group also tested C. elegans exposure to toxaphene on agar and in K-medium, it was concluded that the results were not statistically significant, and confidence intervals could not be determined in those tests.

Anbalagan et al. (2013) conducted a feeding assay on C. elegans exposed to 200 mg/L of endosulfan, a GABA-gated chloride channel antagonist. After 28 hr of exposure, nematodes showed a negligible inhibition of feeding behavior (1.09±0.12%).

Table 3.4 summarizes the findings of these three studies. Overall, it appears that

C. elegans are less sensitive to organochlorine insecticides than other model organisms.

Neonicotinoids

Neonicotinoids act as agonists of insect nicotinic ACh receptors, the ligand-gated ion channels that mediate synaptic transmission (Millar and Denholm, 2007; Jones and

Sattelle, 2010). One would therefore expect locomotion to be an effective endpoint in measuring toxicity of these chemicals. Ruan et al. (2009) exposed C. elegans to imidacloprid for 24 hr, and noted a significant, concentration-dependent reduction in locomotion and head-thrashing behavior, as well as an increase in propagation time and decrease in body length (precise numbers were not reported). Gomez-Eyles et al. (2009) 71

exposed wild-type C. elegans young adults to two widely-used neonicotinoid insecticides, imidacloprid and thiacloprid. The nematodes were exposed on treated agar for 48 hr and scored for effects on reproduction. The study found an EC50 of 519 mg/L

for imidacloprid and 289 mg/L for thiacloprid. Table 3.4 summarizes these studies.

Fungicides

Most fungicides exert their effects in a manner which is not neurotoxic to non-

target organisms. The exception, however, is a class of fungicides known as

thiocarbamates and dithiocarbamates. These are organosulfur compounds which induce

distal peripheral neuropathy. Table 3.4 summarizes related studies in C. elegans.

Negga et al. (2012) suggested that the dithiocarbamate pesticide mancozeb

promoted neuronal degeneration in both GABAergic and dopaminergic neurons in C.

elegans. Brody et al. (2013) concurred that mancozeb impaired the function of

dopaminergic neurons, and demonstrated inhibition of serotonin-mediated behaviors, still

within LC50 concentrations. Mancozeb contains manganese, which has been linked to the onset of non-familial Parkinson’s disease (Benedetto et al., 2010). Previously, Negga et al. (2011) exposed wild-type C. elegans young adults to mancozeb, and found an acute

(30 min) LC50 value of 0.22% of the active ingredient, and a chronic (24 hr) LC50 value

of 0.5% of active ingredient, still within the recommended concentrations for agricultural

application.

Guven et al. (1999) assessed the effects of mancozeb and the related compound

maneb on the stress response in C. elegans, finding strong responses at a concentration of

500 µg/ml. Easton et al. (2001) assessed the growth of transgenic C. elegans exposed to 72

these compounds, and observed that nematode growth was reduced at a concentration of

100 ppm (1 µg/ml).

Caito et al. (2013) examined whether thiocarbamate compounds and

dithiocarbamate metabolites could cause neuronal degeneration in C. elegans. The study results demonstrated a selective neurotoxicity – the compounds induced dopaminergic neurodegeneration and a decrease in dopamine content, while not affecting glutamatergic, cholinergic, or GABAergic neurons.

Herbicides

Herbicides of various formulations are widely used around the world. The herbicide glyphosate was shown to reduce the activity of AChE in fish, as well as the activity of catalase and superoxide dismutase (Modesto and Martinez, 2010). Table 3.4 summarizes related studies in C. elegans. 73

Chemical(s) Exposure Endpoints Reference Pesticide class tested details assessed Results

LC50 was 466 mg/kg for 24 24 hr and hr and 261 Bezchlebová 48 hr in mg/kg for 48 et al., 2007 Organochlorines Toxaphene soil Lethality hr

LC50 was 1,285 mg/kg 24 hr and for 24 hr and Sochová et 48 hr in 376 mg/kg for al., 2007 Organochlorines Toxaphene soil Lethality 48 hr Feeding inhibition was Anbalagan negligible at et al., 2013 Organochlorines Endosulfan 28 hr Feeding 200 mg/L 74

Chemical(s) Exposure Endpoints Reference Pesticide class tested details assessed Results Reduction in locomotion and head thrashing; increase in Locomotion, time to time to propagation; Ruan et al., propagation, decrease in 2009 Neonicotinoids Imidacloprid 24 hr growth body length

EC50 was 519 mg/L for Gomez- Imidacloprid imidacloprid Eyles et al, and 48 hr on Reproductio and 289 mg/L 2009 Neonicotinoids thiacloprid agar n for thiacloprid Strong Guven et al., Mancozeb Stress response at 1999 Dithiocarbamates and maneb responses 500 µg/ml 75

Chemical(s) Exposure Endpoints Reference Pesticide class tested details assessed Results Growth was reduced at a Easton et al., concentration 2001 Dithiocarbamates Mancozeb Growth of 1 µg/ml

LC50 was 0.22% of the active ingredient for 30 min and 0.5% of the active Negga et al., ingredient for 2011 Dithiocarbamates Mancozeb Lethality 24 hr 76

Chemical(s) Exposure Endpoints Reference Pesticide class tested details assessed Results Degeneration observed in GABAergic and Negga et al., Neuronal dopaminergic 2012 Dithiocaebamates Mancozeb impairment neurons Impaired function of dopaminergic neurons; inhibition of serotonin- Brody et al., Neuronal mediated 2013 Dithiocarbamates Mancozeb impairment behaviors 77

Chemical(s) Exposure Endpoints Reference Pesticide class tested details assessed Results EPTC, Degeneration molinate, observed in Caito et al., and Neuronal dopaminergic 2013 Dithiocarbamates MeDETC impairment neurons Reported an

Menzel et Reproductio EC50 of 86.64 al., 2005 Herbicides Atrazine n mg/L 78

Chemical(s) Exposure Endpoints Reference Pesticide class tested details assessed Results A reduction in head- thrashing was observed at a concentration of 7 mg/L; length of time to propagation Movement was increased and length after an Ruan et al., of time to exposure of 2009 Herbicides Glyphosate propagation 72 hr 79

Chemical(s) Exposure Endpoints Reference Pesticide class tested details assessed Results Neurodegener ation observed in GABAergic and Negga et al., Neuronal dopaminergic 2012 Herbicides Glyphosate 24 hr impairment neurons Feeding was impaired by 24.6% at a Anbalagan concentration et al, 2013 Herbicides Glyphosate 28 hr Feeding of 104 mg/L Table 3.4. Toxicity studies of other pesticides using a C. elegans animal model. hr – hours. The units of measurement are provided here as they were reported in the original studies. Due to differences in chemical formulations, the units cannot be converted to one standardized format. 80

Ruan et al. (2009) found a reduction in head-thrashing movement of C. elegans

after exposure to a high concentration of glyphosate (7 mg/L); in addition, length of time

to reproduce was increased after an exposure of 72 hr. Negga et al. (2012) noted

neurodegeneration in both GABAergic and dopaminergic neurons in C. elegans exposed

to sublethal concentrations of a glyphosate-containing herbicide for 24 hr. Anbalagan et

al. (2013) conducted a feeding test on C. elegans, and observed a 24.6% inhibition in

feeding after 28 hr of exposure to a 104 mg/L solution of glyphosate.

Though now banned in the European Union, atrazine is still the most commonly

applied herbicide in the world. The chemical may produce dopaminergic neurotoxicity

and oxidative damage in mammals (Zhang et al., 2011). Menzel et al. (2005) found that

atrazine inhibited reproduction in C. elegans in a concentration-dependent manner, with

an EC50 value of 86.64 mg/L.

The herbicide paraquat (methyl viologen) is not directly neurotoxic. Paraquat acts

by inhibiting the mitochondrial electron transport chain (Saha et al., 2009) and producing

superoxide, which induces oxidative damage (Cochemé and Murphy, 2008). In C.

elegans studies, paraquat is often used as a tool to induce oxidative stress and generate

ROS. The objects of these studies are wide-ranging, from examinations of the causes of specific diseases, such as familial Parkinson’s Disease (Saha et al., 2009), amyotrophic lateral sclerosis (Oeda et al., 2001), or dominant optic atrophy (Kanazawa et al., 2008), to investigations into drug resistance (Zubovych et al., 2010), DNA repair (Skjeldam et al.,

2010), cancer (Koon and Kubiseski, 2010), germ cell apoptosis (Salinas et al., 2006), patterns of stress responses (Anbalagan et al., 2013), longevity (Fujii et al., 2005; Vertino et al., 2011), and genetics of aging (Hartman et al., 1995). 81

Discussion

We have evaluated the efficacy of C. elegans as a model organism for testing pesticides which exert neurotoxic action on target or non-target species. Among the different classes of pesticides reviewed, carbamates are considered far less toxic than organophosphates to organisms of multiple phyla (Lee et al., 2011). With the limited data available for C. elegans, similar findings have been reported. In general, of the OPs and carbamates tested, OPs show effective concentration (for sublethal endpoints) and lethal concentration ranges lower than those in carbamates, denoting a higher toxicity. The ranges generally follow those in mammals. For both classes, the mechanism of action has been identified as the same as in higher organisms. We believe that C. elegans is a good model for both OP and carbamate toxicity, and can serve as a predictor of relative toxicity in these classes of chemicals.

Organochlorines may be more toxic than is evident in C. elegans studies, while pyrethroids and neonicotinoids have not been studied extensively in nematodes.

Exposures to fungicides and herbicides have yielded information on neuron degeneration and generation of reactive oxygen species, and these chemicals will no doubt continue to be studied in C. elegans for the physiological and molecular changes they elicit.

From the data discussed above, it appears that locomotion and reproduction are the most sensitive endpoints for testing the toxicity of pesticides in C. elegans, followed by feeding inhibition and growth. Lifespan and lethality are less sensitive, and while oxidative stress and gene expression can be sensitive measures of the exposure, their testing protocols are more involved and incur greater expense. We therefore recommend 82 locomotion and reproduction as the endpoints most likely to offer data relevant for comparisons with mammals.

The three exposure media allow for various methods of toxicity testing with C. elegans. The soil testing matrix is the most environmentally-relevant, but testing in liquid is the best controlled, reproducible protocol, offering the fewest inaccuracies. One cannot compare the results of a soil exposure with those of a liquid exposure experiment directly, and recovering 90% or more of the test nematodes from soil can be challenging when learning the method. We believe that the aquatic medium is the best for making extrapolations to higher eukaryotes, however, the ability to test in soil is one reason why

C. elegans is a good model for evaluating pesticide toxicity in an environmentally- relevant setting. We do not recommend conducting toxicity tests on agar plates, since there is no way to quantify the chemical concentration absorbed by the nematodes. Table

3.5 summarizes the advantages and disadvantages of the three testing media. 83

Medium Advantages Disadvantages

Soil Environmentally-relevant; Labor-intensive method; test standardized protocols available chemical may sorb to the soil, decreasing its availability; nematode recovery may be difficult; presence of indigenous nematode populations may skew test data Agar Easy and inexpensive; Cannot quantify the exposure standardized protocols available concentration; the test chemical may bind to the constituents of the agar; hydrophobic test chemicals may require dissolution in a vehicle such as acetone Aquatic Greatest bioavailability of the Hydrophobic test chemicals may test chemical – offers the best require dissolution in a vehicle estimation of exposure such as acetone concentration; represents the nematodes’ micro-environment; easy and inexpensive; offers control and reproducibility; standardized protocols available Table 3.5. Advantages and disadvantages of the three standardized testing media for

C. elegans toxicity testing.

The presence of a bacterial food source adds a complication of possible interaction between the bacteria and the chemical, but feeding the nematodes is necessary during exposures of 24 hr or longer. If a short exposure (4 hr) in an aquatic medium, without food, is possible, we believe that it is the best way to conduct toxicity testing in

C. elegans.

We propose a toxicity testing protocol in C. elegans for water-soluble chemicals, recommending the following steps (Figure 3.1): 84

(a) Aqueous exposure for 4 hr without a food source, to determine a range of lethal

and sublethal concentrations.

(b) Aqueous exposure for 4 hr without a food source, using a sublethal exposure,

followed by removal to a clean environment to observe behavioral changes by

tracking movement. A similar protocol is employed to observe reproductive

effects, by counting progeny after removing exposed nematodes to clean Petri

dishes and observing for up to 72 hr.

endpoints – lethality, locomotion, reproduction.

(d) Longer-term soil testing, if the chemical is believed to be an environmental

contaminant in the soil.

(e) Comparison of results for this chemical, or chemicals of similar structure, with

test data from mammals.

(f) Mechanistic studies of specific molecular targets and cellular processes related to

the exposure.

Figure 3.1. Proposed toxicity testing protocol. To investigate the effects of a chemical, we suggest beginning with a 4 hr aqueous exposure without food to evaluate lethality; behavior and reproductive effects may be observed by tracking the exposed nematodes after removal to a clean environment. The next step is a longer aqueous exposure with food, followed by a soil exposure if the chemical is believed to be an environmental contaminant. Finally, design mechanistic studies to probe for the molecular and cellular targets and processes involved, including those of the nervous system. These studies should be informed by mammalian data, if available.

Using C. elegans in toxicological research presents numerous advantages. The nematode shares basic biological functions, such as cellular and DNA processes, with higher organisms. The structure and functionality of its nervous system are a simplified human version, and most of the nematode’s neurotransmitters correspond to human homologs. Its embryogenesis and organogenesis pathways are well-defined and invariant.

It possesses microsomal enzymes which carry out biotransformation reactions. C. elegans

lab assays are rapid, low-cost, and amenable to high-throughput analysis of large

numbers of nematodes.

C. elegans does possess several limitations as a model organism. It lacks many

specific organs, such as kidneys, liver, lungs, skin, and a circulatory system. Ingestion is

possibly the only route of exposure, and systemic cellular exposure is usually limited to

passive diffusion. Moreover, toxicity testing is contingent on the solubility of the

chemical tested. In addition, it may not be as sensitive to some classes of chemicals, such

as polycyclic aromatic hydrocarbons (PAHs), as other invertebrates (Sese et al., 2009).

The redundancy of having four genes encoding AChE, rather than the single gene in

mammals, may make C. elegans less sensitive to pesticides that disrupt AChE activity.

But results in C. elegans have been promising with OPs and carbamates in terms of

predicting effects.

Direct comparisons between classes of insecticides, and even within one class,

yield a wide range of toxicity levels, making prediction difficult even within one species,

let alone across species (Chambers and Carr, 1995). Though the lethality of pesticides to

non-target organisms is simple to measure, it is the sublethal endpoints which are more

sensitive, more useful to researchers, and probably more likely to correlate with 87 mammalian data. Endpoints such as changes in movement, a reduction in AChE activity or dopamine, effects on body length or brood size, induction or elevation of ROS, a shortened lifespan, or developmental defects, contribute to our knowledge of the effects of chemicals at the cellular level, before they are detectable at the whole-organism level, pointing the way to investigations into likely causes and to molecular pathways underlying them.

Much of the data reviewed in this paper originated from the testing of the older, established classes of pesticides – the AChE-inhibiting OPs and carbamates. However, the ongoing trend of phasing them out in favor of newer classes, such as pyrethroids, may yield new results across multiple endpoints in C. elegans. As new lab technologies are developed, concurrently with research advances at the proteome and metabolome levels, one may also see new testing assays. Besides the sensitivity of the assay, one important consideration is the ability to automate the sorting and evaluation of multiple nematodes.

One instrument which has received attention recently for its contribution to high- throughput screening is the COPAS BIOSORT (Union BioMetrica, Holliston, MA), a large-particle flow cytometer which can sort and analyze thousands of nematodes rapidly

(Boyd et al., 2010).

One topic which has received growing attention in recent years is the toxicity of mixtures, since most chemical agents are not found singly in the environment, but in combinations. Svendsen et al. (2010) exposed C. elegans to mixtures of pesticides from different classes, and measured the effects on reproduction against those of individual pesticides. They observed synergism when neonicotinoids (imidacloprid or thiacloprid) were mixed with OPs (diazinon or chlorpyrifos), some antagonism when a pyrethroid 88

(permethrin) was mixed with OPs (diazinon or chlorpyrifos), but no interaction when

permethrin was mixed with the neonicotinoids. Mixture toxicity will no doubt continue to

be a focus of investigation due to its environmental relevance.

The data are presented as they were reported in the studies. Because of different

formulations of chemicals by separate manufacturers, and different units of measurement

reported in the studies, as well as varying levels of detail and a lack of clarity as to

whether the reported concentrations represented formula weight values or chemical-

specific values, it was not possible to present the findings in one unified format. Other

difficulties in making comparisons include the variations in the ages of the nematodes

tested, the different lengths of exposures, and the dilution factor of the active ingredient

in multiple preparations of a commercially-available pesticide.

The end goals of using C. elegans to study pesticide neurotoxicity are the elucidation of the mechanisms of toxication, and applicability of results to understanding specific human health conditions. C. elegans offers the convenience of a rapid, low-cost, whole-animal approach in lab settings, and researchers continue to rely on it in mechanistic studies of neurotoxicity. No doubt it will continue to play a role in the examination of scientific problems of relevance, such as acquired resistance to chemicals, or effects of chronic, low-level exposures on non-target species.

Acknowledgements

Dean Meyer (graduate student) is supported by The Georgia Power Company and

Foundation.

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CHAPTER 4

DIFFERENTIAL TOXICITIES OF NICKEL SALTS TO THE NEMATODE

CAENORHABDITIS ELEGANS1

______

1 Meyer, D., Birdsey, J.M., Wendolowski, M.A., Dobbin, K.K., and Williams, P.L. To be submitted to the journal Environmental Toxicology and Chemistry.

Abstract

This study focused on assessing whether nickel toxicity to the nematode

Caenorhabditis elegans was affected by the molecular structure of the nickel salt used.

Nematodes were exposed to seven nickel salts [nickel sulfate hexahydrate

(NiSO4∙6H2O), nickel chloride hexahydrate (NiCl2∙6H2O), nickel acetate tetrahydrate

(Ni(OCOCH3)2∙4H2O), nickel nitrate hexahydrate (N2NiO6∙6H2O), anhydrous nickel

iodide (NiI2), nickel sulfamate hydrate (Ni(SO3NH2)2∙H2O), and nickel fluoride tetrahydrate (NiF2∙4H2O)] in an aquatic medium for 24 hours, and lethality curves were

generated and analyzed. Nickel fluoride, nickel iodide, and nickel chloride were most

toxic to C. elegans, followed by nickel nitrate, nickel sulfamate, nickel acetate, and nickel

sulfate. The LC50 value of the least toxic salt, nickel sulfate, was statistically different

from the corresponding values in the halogen-containing compounds. In order to

eliminate the contribution to toxicity of the non-nickel component of the molecule, we

recommend that the sulfate salt be used in future nickel testing involving aquatic

invertebrates.

Introduction

Nickel is a ubiquitous metal, present in the soil, air, and water. It is widely used in

multiple industries. High intracellular concentrations can lead to multiple deleterious

health effects. The divalent cation (Ni2+) readily complexes with other elements to form molecules with differing chemical and physical properties. The toxicity of nickel molecules depends on their route of absorption and their solubility. Even within one route 100

of exposure, it is conceivable that these disparate nickel complexes could cause differing

toxicities for living organisms.

C. elegans is a non-parasitic, bacterivorous nematode used as a model organism in life sciences research. It is suitable for such studies because of its ease of culturing and genetic tractability. In toxicology, it is often used to evaluate toxicological responses to chemicals of environmental relevance and concern. It has been shown to be well-suited for characterizing cellular and molecular disruptions by environmental toxicants (Leung et al., 2008). Few studies to date, however, have focused on nickel’s effects on C. elegans.

The objective of this study was twofold. First, to determine whether different nickel salts resulted in differing toxicities to C. elegans. It was important to carry out

such a comparison in order to assess whether the non-metallic components of the nickel

salts played a part in the toxicity, skewing our understanding of nickel’s effects. Second,

to provide guidance on selecting a nickel compound for use in subsequent C. elegans

toxicological work, one whose toxicity could reasonably be attributed to the nickel

cation.

Materials and Methods

Nematode culturing and maintenance. Strain N2 (wild-type) was obtained from

the Caenorhabditis Genetics Center (Minneapolis, MN), which is funded by the NIH

Office of Research Infrastructure Programs (P40 OD010440). Nematodes were

maintained at 20°C on K-agar plates (Williams and Dusenbery, 1988) seeded with a lawn

of Escherichia coli strain OP50 (Brenner 1974). Nematodes were age-synchronized as 101 described by Emmons et al. (1979). Four day old nematodes were washed off the plates with K-medium (0.051 M NaCl and 0.032 M KCl [Williams and Dusenbery, 1990]) and allowed to pellet in centrifuge tubes before being added to the assay well plates.

Chemicals. Stock solutions of the following reagent-grade compounds were obtained from Sigma-Aldrich (St. Louis, MO) and prepared in K-medium: Nickel sulfate hexahydrate (NiSO4∙6H2O), nickel chloride hexahydrate (NiCl2∙6H2O), nickel acetate tetrahydrate (Ni(OCOCH3)2∙4H2O), nickel nitrate hexahydrate (N2NiO6∙6H2O), anhydrous nickel iodide (NiI2), nickel sulfamate hydrate (Ni(SO3NH2)2∙H2O), and nickel fluoride tetrahydrate (NiF2∙4H2O). These seven nickel salts were selected because they were water-soluble – while mammals can be dosed by oral gavage, aquatic toxicity testing of invertebrates is limited by the solubility of the compounds of interest. All solutions were maintained at 20°C away from light. Table 1 presents a summary of the nickel compounds used in this study.

Lethality assay. Immediately prior to exposure, OP50 bacteria were pelleted by centrifugation at 3,000g for 7 minutes at 20°C, and suspended in the test concentrations at a 1:1 ratio (20 mL test concentration for each pellet from a 20 mL solution containing

OP50). This was done to ensure that the nematodes had a food source during the exposure, as tests without food can yield control mortality above the standard acceptable levels of 10% (Cressman and Williams 1997). The test solutions were vortexed to disperse the bacteria evenly. 1 mL aliquots of the test concentrations with OP50 were added to 12-well untreated polystyrene culture plates (BD Biosciences, San Jose, CA), with three wells for each concentration. K-medium served as the control. 10±1 nematodes were then added to each well by pipette. The culture plates were incubated at 20°C for 102

24±1 hours. The nematodes were counted and assessed for survival by gently prodding

with a metal wire to elicit independent movement. The test was replicated 4-6 times and

the results aggregated. A lethality curve was then charted from the results.

Data analysis. Median lethal concentrations (LC50 values) were calculated by

probit analysis using SAS 9.3 (SAS Institute, Cary, NC). An unpaired t-test and the

Wilcoxon signed-rank test were used to compare LC50 values of the three compounds containing halogen anions with the other four. Tukey’s honest significant difference

(HSD) test was used to compare all seven salts in pairs. p ≤ 0.05 values were considered statistically significant. 103

Percent of molecular mass comprised by Compound Symbol CAS Atomic weight nickel

Nickel sulfate NiSO4∙6H2O 10101-97-0 262.85 22.33% hexahydrate Nickel chloride NiCl2∙6H2O 7791-20-0 237.71 24.69% hexahydrate Nickel acetate Ni(OCOCH3)2∙4H2O 6018-89-9 248.78 23.59% tetrahydrate Nickel nitrate N2NiO6∙6H2O 13478-00-7 290.80 20.18% hexahydrate Nickel iodide NiI2 13462-90-3 312.51 18.78% (anhydrous) Nickel sulfamate Ni(SO3NH2)2∙H2O 13770-89-3 268.96 21.82% hydrate Nickel fluoride NiF2∙4H2O 13940-83-5 168.77 34.78% tetrahydrate

Table 4.1. Nickel compounds used in this study. 104

Results

Figure 4.1 presents the lethality curve for each compound. Table 4.2 gives the median lethal concentration (LC50) values of each compound. Two LC50 values are given for each chemical – one representing the whole molecule, and one representing only the nickel fraction of the molecule.

Tukey’s HSD test, used to compare differences between pairs of nickel salts, found significant differences between five pairs (Table 4.3). It is noteworthy that nickel sulfate, the least toxic salt of the seven tested, was significantly different from all four halogen-containing salts (nickel iodide, nickel fluoride, and nickel chloride).

Of the seven salts tested, the three compounds containing halogen anions displayed higher toxicity than the other four. A t-test comparing the LC50 values of the three halogen-containing salts against those of the four others yielded a two-tailed p- value of 0.001, signifying that the two groups were statistically different from each other.

The 95% confidence interval for the difference between the two groups did not contain zero. Wilcoxon’s signed-rank test concurred that the two groups were significantly dissimilar.

105

Figure 4.1. Lethality curves for seven nickel compounds.

106

Compound LC50 of whole molecule (with 95% confidence intervals) LC50 of nickel only (with 95% confidence intervals) Ni fluoride 10.37 mM (7.89 – 13.06) 3.61 mM (2.74 – 4.54) Ni iodide 14.77 mM (10.82 – 22.00) 2.77 mM (2.03 – 4.13) Ni chloride 16.52 mM (11.39 – 21.26) 4.08 mM (2.81 – 5.25) Ni nitrate 26.88 mM (22.45 – 31.53) 5.42 mM (4.53 – 6.36) Ni sulfamate 42.16 mM (30.79 – 73.23) 9.20 mM (6.72 – 15.98) Ni acetate 49.14 mM (37.43 – 58.60) 11.59 mM (8.83 – 13.82) Ni sulfate 51.77 mM (39.13 – 77.56) 11.56 mM (8.75 – 17.32)

Table 4.2. Median lethal concentration values for seven nickel compounds. 107

Pair Tukey’s HSD p-value nickel iodide – nickel sulfate 0.017 nickel fluoride – nickel sulfamate 0.024 nickel fluoride – nickel sulfate 0.006 nickel fluoride – nickel acetate 0.026 nickel chloride – nickel sulfate 0.018

Table 4.3. Nickel salt pairs significantly different from each other. 108

Discussion

This study found that the three halogen-containing compounds (nickel fluoride,

nickel iodide, and nickel chloride) were the most toxic to adult C. elegans nematodes,

while nickel sulfate and nickel acetate were the least toxic. The LC50 values of the sulfate

and acetate forms exceeded those of the halogens by a factor of three.

Comparative studies of sulfate and chloride salts in other species, though not

involving nickel, concur that chloride ions are far more toxic to mammals (Stokinger et

al., 1953) and to wild rice (Fort et al., 2014) than sulfate salts are. Henderson et al. (2012)

ranked the lethality of multiple nickel compounds to female rats, to evaluate the

differences in toxicity based on the element’s speciation and molecular form. They found

a wide range of responses between compounds, with nickel fluoride ranking as the most

toxic compound, as it did in this study. Of the seven compounds used in this study,

Henderson et al. tested five. Their toxicity ranking for these compounds, from most to

least toxic, was nickel fluoride > nickel sulfate > nickel chloride > nickel acetate > nickel

sulfamate. A comparison between the studies using Spearman’s rank correlation

coefficient yielded a value of 0.3, indicating no linear correlation. Possible reasons for the differences with this study may be attributable to the differing methodologies employed.

Henderson et al. exposed rats to a single oral dose, followed by 14 days of observation, or until death occurred, and calculated the half-maximal lethal dose (LD50) from an acute exposure. In this study, adult C. elegans nematodes were immersed in the test medium for a period of 24 hours, and then scored for life signs without undergoing a follow-up period in a clean environment. In addition, while Henderson et al. used about 10 rats for each test concentration of each nickel compound, this study utilized between 90 and 180 109 nematodes per test solution per compound. Moreover, Henderson et al. were not able to establish a confidence interval for their LD50 value for the sulfate salt, indicating insufficient numbers of test organisms.

Our study results concurred with those of Tatara et al. (1997; 1998), who found no statistically significant difference between the lethality to C. elegans of nickel chloride and nickel nitrate. Of all possible pairings, we found significant differences between five pairs only.

The second objective of this study was to select a nickel salt for use in subsequent studies. Previous nickel testing in C. elegans had mainly utilized nickel chloride or nickel sulfate (Anbalagan et al., 2012; Ma et al., 2009). The concern in the present study was to select a compound where there was reasonable certainty that toxicity could be attributed to the nickel fraction of the molecule, rather than to the non-metallic elements. As the three compounds containing halogen anions displayed differentially higher toxicity than the other four compounds, they were ruled out, as presumably the higher toxicity resulted from the halogen anions of the molecules. Nickel sulfate’s last-place ranking of LC50 values in the present study, along with its high water solubility (650 g/L), made it the nickel compound of choice for subsequent studies.

This study demonstrated that the structure and non-metallic components of a metal-containing molecule can affect its toxicity. These physicochemical differences can result in increasing or decreasing the ion’s bioavailability to target organs (Henderson et al., 2012), as well as in altering the toxicokinetics of the absorbed dose. In assessing nickel toxicity, it is important to ensure that the observed toxicity is derived from nickel, rather than from the non-metallic elements in the molecule. For this reason, we 110

recommend using nickel sulfate in future tests involving aquatic exposure to nickel. As

the most common contact dermatitis-causing allergen in humans (Zug et al., 2009), and

owing to its omnipresence in the environment, nickel research will remain relevant for a

long time to come.

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Emmons SW, Klass MR, Hirsh D. 1979. Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America 76:1333-1337.

Fort DJ, Mathis MB, Walker R, Tuominen LK, Hansel M, Hall S, et al. 2014. Toxicity of sulfate and chloride to early life stages of wild rice (Zizania palustris). Environmental Toxicology and Chemistry / SETAC 33:2802-2809.

Henderson RG, Durando J, Oller AR, Merkel DJ, Marone PA, Bates HK. 2012. Acute oral toxicity of nickel compounds. Regulatory Toxicology and Pharmacology: RTP 62:425-432.

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Ma H, Glenn TC, Jagoe CH, Jones KL, Williams PL. 2009. A transgenic strain of the nematode Caenorhabditis elegans as a biomonitor for heavy metal contamination. Environmental Toxicology and Chemistry / SETAC 28:1311-1318.

Stokinger HE, Spiegl CJ, Root RE, Hall RH, Steadman LT, Stroud CA, et al. 1953. Acute inhalation toxicity of beryllium. IV. Beryllium fluoride at exposure concentrations of one and ten milligrams per cubic meter. AMA Archives of Industrial Hygiene and Occupational Medicine 8:493-506.

Tatara CP, Newman MC, McCloskey JT, Williams PL. 1997. Predicting relative metal toxicity with ion characteristics: Caenorhabditis elegans LC50. Aquatic Toxicology 39:279-290.

Tatara CP, Newman MC, McCloskey JT, Williams PL. 1998. Use of ion characteristics to predict relative toxicity of mono-, di- and trivalent metal ions: Caenorhabditis elegans LC50. Aquatic Toxicology 42:255-269.

Williams PL, Dusenbery DB. 1988. Using the nematode Caenorhabditis elegans to predict mammalian acute lethality to metallic salts. Toxicology and Industrial Health 4:469-478.

Williams PL, Dusenbery DB. 1990. Aquatic toxicity testing using the nematode Caenorhabditis elegans. Environmental Toxicology and Chemistry / SETAC 9:1285- 1290.

Zug KA, Warshaw EM, Fowler JF, Jr., Maibach HI, Belsito DL, Pratt MD, et al. 2009. Patch-test results of the North American Contact Dermatitis Group 2005-2006. Dermatitis: Contact, Atopic, Occupational, Drug 20:149-160.

112

CHAPTER 5

DETOXIFICATION MECHANISMS IN CAENORHABDITIS ELEGANS EXPOSED TO

NICKEL1

______

1 Meyer, D., Samuel, R.S., Asef, C.K., Morris, G.H., Dobbin, K.K., Glenn, T.C., and

Williams, P.L. To be submitted to the Journal of Applied Toxicology.

113

Abstract

We characterize the detoxification pathways of nickel in the nematode

Caenorhabditis elegans by systematically examining multiple known metalloprotective

pathways, including metallothioneins CeMT-1 and CeMT-2, the divalent metal transporters SMF-1 and SMF-3, the heat shock protein Hsp90, phytochelatin synthase

(PCS-1), the ABC transporter hmt-1, and the nematode’s coelomocytes. The divalent metal transporters SMF-1 and SMF-3, and the heat shock protein Hsp90, appear not to be involved in nickel detoxification in C. elegans. An examination of metallothionein yielded a suggestion of potentiation of toxicity, rather than detoxification, as a double knockout strain was far less sensitive to nickel than the wild-type. The absence of phytochelatin synthase was sufficient to affect toxicity significantly. Similar toxicity was observed when both coelomocytes and hmt-1 were deleted. We conclude that nickel is likely detoxified in C. elegans by phytochelatin synthase, which is derived from glutathione, and by an interaction of hmt-1 with the nematode’s coelomocytes, and that these two detoxification pathways act independently of each other.

Introduction

Nickel is a transition metal present in soil, water, and air. It is widely used in industrial settings. Nickel can exert multiple adverse health effects on biological systems, and is known to be carcinogenic (Das et al., 2008). It is also the most common contact allergen in the world (Zug et al., 2009). Nickel is considered an essential element in some mammals and birds, although its specific physiological function is unknown (Spears 114

1984). For these reasons, the mechanisms of nickel detoxification are important to

understand.

Caenorhabditis elegans is a bacterivorous nematode that has been used

extensively as a model organism for examining the toxicological effects of metals on

cellular mechanisms (Leung et al., 2008; Martinez-Finley and Aschner 2011). C. elegans testing has been leveraged to investigate the mechanisms underlying the toxicity of multiple metals. To date, however, these studies have not focused extensively on the molecular mechanisms of nickel toxicity in the nematode.

Organisms have evolved varied metal-responsive pathways, resulting in sequestration, biotransformation, or interruption in the trafficking of metal ions across cell membranes (Cui et al., 2007). Cellular detoxification systems in bacteria, plants, and animals include heat shock proteins, metallothioneins, glutathione, and various transporters (Martinez-Finley and Aschner 2011). While organisms possess multiple metalloprotective systems, the question of whether saturating or knocking out one system leads to compensatory action by other systems remains open.

Metal-responsive genes and proteins in C. elegans

Metallothioneins are cysteine-rich molecules involved in binding metal ions, and can be used as biomarkers of exposure to some heavy metals (Ma et al., 2009). C. elegans possesses two metallothionein isoforms, CeMT-1 and CeMT-2, which may act independently of each other (Swain et al., 2004). Transcription of metallothionein is induced within 30 minutes of exposing C. elegans to high concentrations of metal, although mtl-2 transcription is limited to intestinal cells (Freedman et al., 1993). A 115

possible limitation of metallothionein binding as a detoxifying element may be its

transience, as metallothioneins can release metal ions shortly after binding them (Vallee

1995). In addition, C. elegans metallothioneins are more sensitive to some metals, such

as cadmium, than to others – such as copper or zinc, indicating that other mechanisms for

detoxifying metals are likely present in the nematode (Swain et al., 2004).

The divalent metal transporter (DMT-1) is a protein, highly conserved from

bacteria to mammals, which mediates metal ion transport (Settivari et al., 2009). Soluble

nickel ions can enter the cell via the divalent metal transporter (Lu et al., 2005). The C.

elegans orthologs of DMT-1 are termed SMF-1, SMF-2, and SMF-3. Deletion of one of the smf genes may lead to increased sensitivity to a given metal, or, conversely, to increased tolerance (Au et al., 2009). As with C. elegans metallothioneins, the three divalent metal transporters are not interchangeable in their functionality, and vary in their localization as well. While SMF-1 and SMF-3 are found mainly on the apical membrane of intestinal cells, SMF-2 is mainly localized in the cytoplasm of the pharyngeal epithelium (Anderson and Leibold 2014).

Heat shock proteins (Hsp) are molecular chaperones that facilitate protein synthesis when activated by perturbations in cellular conditions (Martinez-Finley and

Aschner 2011). While some of C. elegans’ 60 heat shock proteins are upregulated following exposure to some metals, the overriding limitation of using heat shock proteins as biomarkers of exposure is their non-selectivity for metals (Cioci et al., 2000). We used a nematode strain containing a point mutation in the gene which transcribes Hsp90.

Nematodes in which the gene transcribing Hsp90 (daf-21) has been knocked out do not survive long, indicating the importance of daf-21 in the animal, but the mutation greatly 116

decreases the synthesis of the protein (Birnby et al., 2000). Wang et al. (2014) found that

these mutants were more sensitive to lead exposure than wild-type.

Phytochelatins are a class of glutathione-derived peptides that play a role in metal

homeostasis by binding metal ions (Vatamaniuk et al., 2001). They are synthesized by the

enzyme phytochelatin synthase (PCS-1) in response to toxic metal exposures (Ha et al.,

1999; Clemens et al., 1999; Clemens et al., 2001). In C. elegans, phytochelatins are

known to confer tolerance to cadmium, mercury, and arsenic (Vatamaniuk et al., 2002).

An increase in cytosolic concentrations of some metal ions has been shown to be

sufficient for the induction of phytochelatin synthase (Bundy and Kille 2014).

ATP-binding cassette (ABC) transporters are involved in many physiological

processes (Hanikenne et al., 2005). The ABC transporter CeHMT-1 has been shown to

mediate the detoxification of cadmium, arsenic, and copper, without depending

exclusively on the synthesis of phytochelatins (Vatamaniuk et al., 2005; Schwartz et al.,

2010).

Although pcs-1 and hmt-1 are expressed in different cell types in C. elegans, they

are co-expressed in the nematode’s coelomocytes. These are six cells, arranged in pairs

along the body cavity, whose function is as yet unknown. The coelomocytes endocytose

fluid from the nematode’s body cavity continuously. While the endocytosis action itself

has been shown to be unnecessary for the nematode’s growth or fertility under normal

conditions, researchers have speculated that the coelomocytes have a similar function to a

liver in higher animals, to detoxify the fluid they internalize (Fares and Greenwald 2001;

Schwartz et al., 2010).

117

Objective of the study

This study comprises a systematic examination of multiple routes of heavy metal detoxification as they pertain to C. elegans’ response to a nickel exposure. The objective of this study was to investigate the extent to which each of the metal-detoxifying mechanisms above played a role in nickel tolerance in C. elegans. The study compared the effects of nickel exposure in wild-type nematodes with those in null (knockout) and almost-null (knockdown) mutants, to clarify the mode of action of nickel in C. elegans.

Figure 5.1 presents a model of the expected lethality curves, where concentration of the exposure chemical is plotted against mortality levels in the test organism. The wild-type strain serves as the control. Strains containing mutations that reduce toxicity, by providing some detoxifying benefits, are expected to yield a lethality curve to the right of the wild-type curve; the detoxifying mechanism confers protection on the test organism, thereby increasing the concentration required to cause death. Conversely, strains that contain mutations that potentiate toxicity, by removing detoxification mechanisms, are expected to yield a lethality curve to the left of the wild-type curve – absent the protective factor, the concentration required to cause death is much lower. We tested a variety of C. elegans strains with known mutations to specific metal-responsive genes to determine the response and infer whether the response was consistent with toxicity-potentiation, toxicity-reduction, or not significantly different from wild-type. 118

Figure 5.1. Model of expected lethality curves. 119

Materials and Methods

Nematode strains and maintenance. Strains N2 (wild-type), JT6130 (point

mutation in daf-21, the gene transcribing Hsp90), VF2 (pcs-1 null), VF3 (hmt-1 null),

VF14 (coelomocyte and hmt-1 double knockout), VF9 (coelomocyte, hmt-1, and pcs-1 triple knockout), and VC128 (mtl-2 null) were obtained from the Caenorhabditis

Genetics Center (Minneapolis, MN), which is funded by the NIH Office of Research

Infrastructure Programs (P40 OD010440). Strains MAB23 (smf-1 null) and MAB37 (smf-

3 null) were supplied by Michael Aschner (Albert Einstein College of Medicine). Strains

JF97 (mtl-1 null) and JF81 (mtl-1 and mtl-2 double knockout) were supplied by Jonathan

H. Freedman (NIEHS). Strain NP717 (no coelomocytes) was supplied by Hana Fares

(The University of Arizona). Nematodes were maintained at 20°C on agar plates seeded with a lawn of Escherichia coli strain OP50 as a food source (Brenner 1974). Age- synchronized adult nematodes were obtained by rinsing a pellet of mixed-stage nematodes with a hypochlorite solution which killed all but the eggs, as described by

Cressman and Williams (1997). These harvested eggs were transferred to clean agar plates and allowed to reach maturity. Four-day-old adult nematodes were used in all tests.

Chemicals. Stock solutions of nickel sulfate hexahydrate (NiSO4∙6H2O) (Sigma-

Aldrich, St. Louis, MO) were prepared with K-medium (0.032 M potassium chloride and

0.051 M sodium chloride) as the diluting agent (Williams and Dusenbery 1990). The

sulfate salt was used because, unlike nickel chloride or nickel fluoride, nickel sulfate’s

toxicity is probably derived from the nickel cation, rather than from the non-metallic

elements in the molecule (Meyer et al., in submission). Solution concentrations were

verified by inductively coupled plasma optical emission spectrometry (ICP-OES). Test 120

concentrations were then diluted from the stock solutions. Solutions were maintained at

20°C until needed. Immediately prior to exposure, OP50 bacteria were pelleted by

centrifugation and suspended in the test concentrations at a 1:1 ratio (20 mL test

concentration for each pellet from a 20 mL solution containing OP50). This was done to

ensure that the nematodes had a food source during the exposure, as tests without food

can yield control mortality above the accepted standard level of 10% (Cressman and

Williams 1997). The test solutions were vortexed to disperse the bacteria evenly.

Lethality assay. One mL aliquots of the test concentrations with OP50 were added to 12-well untreated polystyrene culture plates (BD Biosciences, San Jose, CA), with

three wells for each concentration. K-medium served as the control. 10±1 nematodes

were then added to each well by pipette. The culture plates were incubated at 20°C for

24±1 hours. The nematodes were counted and assessed for survival by gently prodding

with a metal wire to elicit independent movement. The test was replicated 4-6 times and

the results aggregated. A lethality curve was then charted from the results, plotting nickel

concentrations against percent mortality. Central estimates of lethality were calculated

from the data.

Data analysis. Lethal concentrations (LC10, LC25, and LC50 values) were

calculated by probit analysis using SAS 9.3 (SAS Institute, Cary, NC). Comparisons of

lethality values between wild-type and other strains were performed by t-test, one-way analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) test. p≤0.05 values were considered statistically significant.

121

Results

Metallothioneins. A lethality curve was generated for each of the null strains

(Figure 5.2). The differences between the strains are greatest at the lowest concentration

of nickel. Lethal concentrations for varying proportions of C. elegans populations were

then determined (Table 5.1). At the LC50 level, there were no significant differences

between the knockout strains and the wild-type; however, some differences were found

between the strains at lower lethality levels. At the LC10 level, each single knockout was

statistically different from the double knockout (p<0.01), and the double knockout was

different from wild-type (p<0.01). At the LC25 level, each single knockout was

statistically different from the double knockout (p<0.05 and p<0.01), and the double knockout was different from wild-type (p<0.01). At the lower concentrations, the double knockout was less susceptible to the effects of nickel than either of the single knockouts.

These differences became insignificant at the LC50 level. 122

Figure 5.2. Lethality curves for strains lacking metallothionein genes.

123

Strain LC10 (with 95% CI) LC25 (with 95% CI) LC50 (with 95% CI) mtl-1 knockout 5.68 mM (3.83 – 7.38) 10.78 mM (8.48 – 13.28) 21.99 mM (17.63 – 29.62) mtl-2 knockout 4.07 mM (0.98 – 7.53) 8.53 mM (3.52 – 13.87) 19.40 mM (11.63 – 34.26)

Double knockout 15.75 mM (12.12 – 18.66) 20.91 mM (17.46 – 24.17) 28.63 mM (24.78 – 34.04)

Wild-type 2.65 mM (1.62 – 3.62) 5.33 mM (3.96 – 6.92) 11.56 mM (8.75 – 17.32)

Table 5.1. Lethal concentrations for strains lacking metallothionein genes. 124

Divalent metal transporters. Only two of the three null strains (Au et al., 2009)

were available for testing. The lethality curves for both null strains, smf-1 and smf-2 knockouts, lie to the left of the wild-type (Figure 5.3). Again, the difference between the knockouts and the wild-type is greater at low concentrations of nickel. There were, however, no statistical differences between either of these null strains and the wild-type at the LC10, LC25, or LC50 levels (Table 5.2). 125

Figure 5.3. Lethality curves for strains lacking smf genes.

126

Strain LC10 (with 95% CI) LC25 (with 95% CI) LC50 (with 95% CI) smf-1 knockout 0.76 mM (0.15 – 1.51) 1.93 mM (0.76 – 3.10) 5.37 mM (3.37 – 9.36) smf-3 knockout 0.66 mM (0.02 – 1.62) 1.57 mM (0.19 – 3.04) 4.08 mM (1.71 – 8.45) Wild-type 2.65 mM (1.62 – 3.62) 5.33 mM (3.96 – 6.92) 11.56 mM (8.75 – 17.32)

Table 5.2. Lethal concentrations for strains lacking smf genes.

127

Heat shock protein (Hsp90). A lethality curve was generated for the daf-21 mutant in which Hsp90 is barely expressed, and compared with wild-type (Figure 5.4).

Although the curve for the daf-21 mutant is slightly to the right of the wild-type, there

were no statistical differences between the mutant strain and the wild-type at the LC10,

LC25, or LC50 levels (Table 5.3). 128

Figure 5.4. Lethality curve for Hsp90 mutant strain, daf-21 knockdown, compared to wild-type.

129

Strain LC10 (with 95% CI) LC25 (with 95% CI) LC50 (with 95% CI)

Hsp90 point 5.77 mM (2.32 – 8.50) 9.84 mM (5.80 – 12.98) 17.78 mM (13.55 – 24.59) mutation

Wild-type 2.65 mM (1.62 – 3.62) 5.33 mM (3.96 – 6.92) 11.56 mM (8.75 – 17.32)

Table 5.3. Lethal concentrations for Hsp90 mutant strain, daf-21 knockdown, compared to wild-type. 130

Phytochelatin synthase, hmt-1, and coelomocytes. A lethality curve was generated

for each of the strains tested (Figure 5.5) and lethal concentrations for all strains were summarized (Table 5.4). Again, differences were most pronounced at the lowest concentrations. At the LC50 level, none of the mutant strains were statistically different

from the wild-type. At the LC10 and LC25 levels, however, three strains were

significantly different from the wild-type: VF9 (no coelomocytes, pcs-1, or hmt-1), VF2

(no pcs-1), and VF14 (no coelomocytes or hmt-1). As VF2, which lacks pcs-1, did not differ from the triple deletion mutant, one can infer that the absence of pcs-1 is sufficient to affect the toxicity of nickel to C. elegans significantly. VF14, which lacks both coelomocytes and hmt-1, did not differ from the triple deletion mutant. 131

Figure 5.5. Lethality curves for strains lacking one or more of the following: Coelomocytes, hmt-1, pcs-1.

132

Strain LC10 (with 95% CI) LC25 (with 95% CI) LC50 (with 95% CI)

VF14 (no coelomocytes 0.23 mM (0.04 – 0.59) 1.04 mM (0.34 – 2.07) 5.66 mM (2.98 – 11.16) or hmt-1) VF3 (no hmt-1) 2.50 mM (1.05 – 4.13) 5.72 mM (3.30 – 8.63) 14.37 mM (9.54 – 24.06) VF2 (no pcs-1) 0.09 mM (0.04 – 0.17) 0.49 mM (0.30 – 0.70) 3.09 mM (2.31 – 4.41) NP717 (no 1.41 mM (0.44 – 2.31) 3.63 mM (2.15 – 5.18) 10.38 mM (7.01 – 23.04) coelomocytes) VF9 (triple deletion 0.09 mM (0.03 – 0.16) 0.36 mM (0.22 – 0.52) 1.78 mM (1.36 – 2.40) mutant) Wild-type 2.65 mM (1.62 – 3.62) 5.33 mM (3.96 – 6.92) 11.56 mM (8.75 – 17.32)

Table 5.4. Lethal concentrations for strains lacking one or more of the following: Coelomocytes, hmt-1, pcs-1. 133

Discussion

Overall, some knockouts resulted in toxicity reduction, while others appeared to

be toxicity-potentiating. In all cases, the differences were most clear at the lowest

concentrations tested.

At lower concentrations, the metallothionein knockout strains were all statistically

different from the wild-type, and, unexpectedly, much less susceptible to nickel.

Although the central estimates of lethality respond in parallel for wild-type, the mtl-1 knockout, and the mtl-2 knockout, the differences are not statistically significant at the

LC50 level. Because the central estimates of lethality respond similarly, we suggest that

future studies increase sample size to ensure sufficient power to demonstrate statistical

significance. In contrast, the double knockout of mtl-1 and mtl-2 has a far steeper slope

than the other three. It is possible that in C. elegans, mtl-1 and mtl-2 potentiate or

increase sensitivity to nickel; removing one gene sequence results in higher tolerance to

nickel, and removing both yields an even larger difference. It is also possible that

uncharacterized additional mutations in these lines affected the nematode’s sensitivity. It

is unknown why this study encountered an apparent potentiation of toxicity by

metallothionein, when most metal studies in C. elegans find metallothionein to be a

detoxifier (Swain et al., 2004; Ma et al., 2009; Jiang et al., 2009; Zeitoun-Ghandour et al.,

2010; Anbalagan et al., 2012).

It is also possible that neither metallothionein gene is induced by nickel, and that

in creating the double knockout strain used in this study, an unrelated sequence was

inadvertently removed, resulting in a mutant more hardy to some exposures. 134

The knockout strains lacking the divalent metal transporter genes smf-1 and smf-3

were not statistically different from the wild-type at the three lethal concentration values

tested, indicating that these two molecules are not involved in the detoxification of nickel

in C. elegans. A smf-2 knockout was not available for this study. Response was in the

expected direction, and the curves were similar in shape, with greater differences at lower

concentrations. Again, future studies will want to increase sample sizes to ensure

sufficient power for detecting differences if they occur.

The mutant strain JT6130, which contains a point mutation in daf-21 and consequently translates only minimal levels of the heat shock protein Hsp90, was not statistically different from the wild-type.

The true function of the nematode’s coelomocytes is so far unknown (Schwartz et al., 2010). Although it has been speculated that they are metalloprotective by continuously endocytosing the fluid in the body cavity (Fares and Greenwald, 2001), this study did not find a conclusive role for coelomocytes in detoxifying nickel. A mutant strain lacking coelomocytes was not statistically different from the wild-type in sensitivity to nickel. Interestingly, though, a strain lacking both coelomocytes and hmt-1 was more sensitive to nickel than the wild-type, while a strain lacking only hmt-1 was not. This implies that in the presence of nickel, the C. elegans coelomocytes are capable of maintaining homeostasis without hmt-1, but that the reverse is not true.

A strain lacking pcs-1 was more sensitive to nickel, as was a mutant lacking all three – coelomocytes, hmt-1, and pcs-1. It can be concluded that pcs-1 plays an important role in nickel detoxification in C. elegans, independently of hmt-1 or of coelomocytes.

Knocking out pcs-1 appears to be sufficient to affect the toxicity of nickel to C. elegans 135

significantly. In addition, because VF14, which lacks both coelomocytes and hmt-1, did not differ from the triple deletion mutant, one could also argue that the double absence of coelomocytes and hmt-1 is sufficient to affect the toxicity of nickel, independent of pcs-1.

This indicates two parallel detoxification pathways for nickel, acting independently of

each other. Schwartz et al. (2010), exposing C. elegans to cadmium, arsenic, and copper,

also concluded that detoxification occurred by these two pathways.

All differences disappeared at the LC50 level. We speculate that this concentration

and exposure priod (24 h) are consistent with the point when cellular levels of reactive

oxygen species are expected to have depleted glutathione production and overwhelm

repair mechanisms.

It is clear that C. elegans possesses multiple routes of sequestering and binding metal ions, and that these routes depend on the exposure concentrations, the metal itself, and the characteristics of the molecular compound ingested. As these molecular pathways are highly conserved between phyla, continued investigations using the nematode are of value and relevance to research in mammalian biology and health.

The effects of disabling a given detoxification pathway may be masked by compensatory action through another pathway. The result may be increased tolerance or increased sensitivity. It is also possible that nickel induces the expression of genes that are not actually involved in its detoxification.

The findings from this study suggest that the main route of nickel detoxification in the nematode C. elegans is through the oligomers of glutathione produced by the enzyme phytochelatin synthase in response to oxidative stress, which act as chelators.

This concurs with the conclusion of Schwartz et al. (2010) that pcs-1 acts independently 136

of hmt-1 and the nematode’s coelomocytes, and that both pathways play a role in metal

detoxification.

To our knowledge, this study is the first to attempt to characterize mechanisms of

action of a metal in C. elegans by systematically examining multiple molecular pathways that may be involved. The vast library of C. elegans mutants available to researchers, comprising knockout, knockdown, and transgenic strains, along with the simplicity of creating new mutants to investigate the function of one cell, gene, or protein of interest, enable such systematic evaluations in a rapid, inexpensive, and easily-replicated manner.

The nematode readily lends itself to mechanistic work on metals of public health and toxicological interest, and will no doubt continue to function as a valuable tool in such research endeavors.

Acknowledgments

The authors thank the researchers and institutions listed in the Materials and

Methods section for sharing the nematode strains used. We are also grateful for the assistance of Rebecca Auxier of the Center for Applied Isotope Studies at the University of Georgia.

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CHAPTER 6

ASSESSING DIRECT DOPAMINERGIC AND CHOLINERGIC NEURON DAMAGE

POTENTIAL IN CAENORHABDITIS ELEGANS FOLLOWING ACUTE AND

SUBCHRONIC NICKEL EXPOSURE1

______

1 Meyer, D., Birdsey, J.M., and Williams, P.L. To be submitted to the journal Cellular and Molecular Neurobiology.

141

Abstract

Multiple heavy metals can cause neurodegenerative disorders in both vertebrates and invertebrates. C. elegans is a nematode which has been utilized extensively in neurobiological research and as a model for toxicological effects. We investigated whether exposure to nickel for 8 hours and 24 hours, followed by a two day incubation period in a clean environment, resulted in degeneration of dopaminergic and cholinergic neurons in C. elegans. Results suggest that nickel is not directly neurotoxic in C. elegans for these two neurotransmitters.

Introduction

Multiple heavy metals have been linked to the development of neurodegenerative disorders. Examples include lead, manganese, aluminum, zinc, and iron (Martinez-Finley et al., 2011). Nickel is a ubiquitous metal present in the soil, air, and water, to which all organisms are naturally exposed. In mammals, nickel has been linked to the formation of some neurofibrosarcomas (IARC 2012), as well as to the loss of cell viability through increased production of reactive oxygen species (Xu et al., 2010).

Nematodes possess many of the same neurotransmitters as mammals, including dopamine (DA), acetylcholine (ACh), glutamate, and γ–amino butyric acid (GABA)

(McDonald et al., 2006). Caenorhabditis elegans, a nematode of the family Rhabditidae, has been used as a scientific tool of biological and genetics investigations since the

1970s, and is well-suited for studying metal-induced neurodegeneration due to its ease of culturing, limited set of behaviors, and a completely mapped nervous system, comprised of only 302 neurons in the adult hermaphrodite (Leung et al., 2008). Much work has been 142

done in the field of C. elegans neurotoxicity, particularly with pesticides (for a review,

see Meyer and Williams 2014).

Dopamine (DA) is a neurotransmitter involved in cell-cell signaling. The

dopamine transporter (DAT) pumps dopamine from synapses into the cytosol,

terminating its signal; DAT dysfunctions have been linked to Parkinson’s disease and

schizophrenia. Four of the five mammalian G-protein coupled receptors on which dopamine acts have been identified in C. elegans, and more dopamine receptors have been hypothesized but not identified to date. The C. elegans hermaphrodite possesses

eight dopaminergic neurons, arranged in pairs along its body. They are known to regulate

locomotion (Chase and Koelle 2007).

The role of dopamine in C. elegans has been investigated by analyzing both loss-

of-function assays and gain-of-function assays. Dopamine has been found to play a part

in several C. elegans behaviors, including locomotion, chemosensation, and egg-laying

(McDonald et al., 2006; Vidal-Gadea et al., 2011). Similarities have been found between

dopaminergic neurodegeneration in C. elegans and mammalian models (Nass and

Blakely 2003). Dopaminergic neuron degeneration assays have become standard among

C. elegans researchers who study susceptibility factors contributing to the development

of Parkinson’s disease (PD); for examples of these assays, please see Berkowitz et al.

(2008), and Tucci et al. (2011). Several published studies report metal-induced

degeneration of dopaminergic neurons in C. elegans. Benedetto et al. (2010) observed

degeneration after a 24 h exposure to manganese, while VanDuyn et al. (2010) found

similar damage caused by methylmercury after 48 h of exposure. 143

Acetylcholine (ACh) is an excitatory neurotransmitter acting at neuromuscular junctions of both vertebrates and invertebrates (Duerr et al., 2008). Acetylcholine is synthesized from choline and acetyl-CoA, and its action is terminated by the enzyme acetylcholinesterase (Mullen et al., 2007). More than a third of C. elegans nerve cells are cholinergic, and acetylcholine plays a role in the nematode’s pumping, locomotion, and egg-laying activities (Richmond and Jorgensen 1999). Further, cholinergic signaling in C. elegans has been shown to be affected by exposure to the metalloid arsenic (Liao et al.,

2010), and neurodegeneration was observed after exposure to lead and mercury (Xing et al., 2009). Both dopamine and acetylcholine play important roles in the nervous system, and have been investigated extensively in C. elegans.

Researchers use genetic mutants of C. elegans to study neurodegeneration following environmental exposures (Aschner et al., 2010). The practice of tagging specific neurons with GFP has been utilized extensively to observe dysfunction or loss of neuronal cells over time due to protein aggregation or environmental exposures

(Teschendorf and Link, 2009). GFP is easily observed through the transparent cuticle of the nematode (Graves et al., 2005), allowing for in vivo examination of the nerve cells of interest (Dexter et al., 2012). Degenerative damage to the GFP-tagged cells in these strains result in the complete loss of GFP fluorescence. Using C. elegans’ neuronal map, where the location of each nerve cell is known and invariant, tagging or ablation of a given cell can yield information about its function (Bargmann and Avery 1995).

This study utilized transgenic strains of C. elegans to assess whether exposure to nickel resulted in degeneration of dopaminergic or cholinergic neurons in C. elegans.

144

Materials and Methods

Nematode culturing and maintenance. Strains BZ555 (dat-1p::GFP, expresses

fluorescence in all dopaminergic neurons) and LX929 (unc-17::GFP, expresses

fluorescence in all cholinergic neurons) were obtained from the Caenorhabditis Genetics

Center (Minneapolis, MN), which is funded by the NIH Office of Research Infrastructure

Programs (P40 OD010440). Strains were maintained at 20°C on K-agar plates (Williams and Dusenbery, 1988) seeded with a lawn of Escherichia coli strain OP50 (Brenner

1974). Nematodes were age-synchronized as described by Emmons et al. (1979). Three

day old (young adult) nematodes were washed off the plates with K-medium (0.051 M

NaCl and 0.032 M KCl [Williams and Dusenbery, 1990]) and allowed to pellet via

gravity in centrifuge tubes before being added to the assay well plates.

Neuronal degeneration assay. For each strain, nematodes were divided into three

groups: (a) K-medium controls; (b) exposure to 9.62 mM nickel, and (c) exposure to 4.44

mM nickel. The two exposure concentrations were selected as approximately

corresponding to the LC50 and LC25 values for the nickel fraction from the sulfate salt

solution, as determined previously in our lab. Solution concentrations were verified by

inductively coupled plasma optical emission spectrometry (ICP-OES). Each of the three

groups was subdivided into two exposure durations: 8 h and 24 h, representing an acute

and a subchronic exposure, respectively. Each exposure group numbered 30-40

nematodes, which were added in groups of 10 to well plates, each containing 1 mL of the test solution. Nematodes in the 24 h group were fed as follows: OP50 bacterial preparations were centrifuged at 5,000g for 7 minutes to yield bacterial pellets. The pellets were resuspended in the test solutions at a ratio of 1:1 (10 mL of test solution for 145

every pellet from 10 mL of OP50). The test solutions were vortexed to disperse the

bacteria evenly, and 1 mL aliquots were deposited into untreated 12-well plates.

Following the exposure, the nematodes were rinsed and removed to a clean environment

(K-agar plates) for a 48 h incubation period. After 48 h, the nematodes were fixed on 5% agarose pads treated with 10 mM sodium azide (NaN3) as an anesthesizing agent. The

pads were then imaged with a Zeiss LSM 710 confocal microscope (Carl Zeiss AG,

Germany) at varying magnification settings and the images examined visually for

presence of GFP fluorescence. All images were acquired with an argon laser excitation

set at 488 nm (green) and a 525 nm emission filter. The assay was repeated for a total of

three replicates.

Results

Status of dopaminergic neurons after an 8 h exposure and 48 h incubation period.

Figures 6.1, 6.2, and 6.3 show examples of live nematodes mounted on agarose pads after

immersion in control medium (6.1), 4.44 mM nickel (6.2), and 9.62 mM nickel (6.3). No

change in the fluorescent markers was observed in any of the three groups, indicating no

difference between exposed and control groups.

146

Figure 6.1. Live C. elegans nematode mounted on an agarose pad after 8 h of immersion in K-medium (control) and 48 h of incubation in a clean environment.

The pairs of dopaminergic nerve cells are well delineated by GFP markers, indicating viable cells. 147

Figure 6.2. Live C. elegans nematode mounted on an agarose pad after 8 h of immersion in 4.44 mM nickel and 48 h of incubation in a clean environment. The dopaminergic nerve cells with GFP markers are visible in the adult hermaphrodite, as well as in the embryos it contains.

148

Figure 6.3. Live C. elegans nematode mounted on an agarose pad after 8 h of immersion in 9.62 mM nickel and 48 h of incubation in a clean environment. The pairs of dopaminergic nerve cells are well delineated by GFP markers, indicating viable cells.

Status of dopaminergic neurons after a 24 h exposure and 48 h incubation period.

Figures 6.4, 6.5, and 6.6 show examples of live nematodes mounted on agarose pads after immersion in control medium (6.4), 4.44 mM nickel (6.5), and 9.62 mM nickel (6.6). No 149 change in the fluorescent markers was observed in any of the three groups, indicating no difference between exposed and control groups.

Figure 6.4. Live C. elegans nematode mounted on an agarose pad after 24 h of immersion in K-medium (control) and 48 h of incubation in a clean environment.

The GFP tags are visible in the dopaminergic neurons, indicating viable cells.

150

Figure 6.5. Live C. elegans nematodes mounted on an agarose pad after 24 h of immersion in 4.44 mM nickel and 48 h of incubation in a clean environment. The dopaminergic nerve cells with GFP markers are visible in the adult hermaphrodites, as well as in the embryos they contain.

151

Figure 6.6. Live C. elegans nematode mounted on an agarose pad after 24 h of immersion in 9.62 mM nickel and 48 h of incubation in a clean environment. The dopaminergic nerve cells with GFP markers are visible in the adult hermaphrodite, as well as in the embryos it contains.

Status of cholinergic neurons after an 8 h exposure and 48 h incubation period.

Figures 6.7, 6.8, and 6.9 show examples of live nematodes mounted on agarose pads after

immersion in control medium (6.7), 4.44 mM nickel (6.8), and 9.62 mM nickel (6.9). No 152 change in the fluorescent markers was observed in any of the three groups, indicating no difference between exposed and control groups.

Figure 6.7. Live C. elegans nematode mounted on an agarose pad after 8 h of immersion in K-medium (control) and 48 h of incubation in a clean environment.

The cholinergic nerve cells, tagged with GFP, are visible throughout the pharynx and along the body wall.

153

Figure 6.8. Live C. elegans nematode mounted on an agarose pad after 8 h of immersion in 4.44 mM nickel and 48 h of incubation in a clean environment. The cholinergic nerve cells, tagged with GFP, are visible throughout the pharynx and along the body wall.

154

Figure 6.9. Live C. elegans nematode mounted on an agarose pad after 8 h of immersion in 9.62 mM nickel and 48 h of incubation in a clean environment. The cholinergic nerve cells, tagged with GFP, are visible throughout the pharynx and along the body wall, as well as within the embryos the nematode carries.

Status of cholinergic neurons after a 24 h exposure and 48 h incubation period.

Figures 6.10, 6.11, and 6.12 show examples of live nematodes mounted on agarose pads after immersion in control medium (6.10), 4.44 mM nickel (6.11), and 9.62 mM nickel 155

(6.12). No change in the fluorescent markers was observed in any of the three groups,

indicating no difference between exposed and control groups.

Figure 6.10. Live C. elegans nematode mounted on an agarose pad after 24 h of immersion in K-medium (control) and 48 h of incubation in a clean environment.

The cholinergic nerve cells, tagged with GFP, are visible throughout the pharynx and along the body wall. 156

Figure 6.11. Live C. elegans nematode mounted on an agarose pad after 24 h of immersion in 4.44 mM nickel and 48 h of incubation in a clean environment. The cholinergic nerve cells, tagged with GFP, are visible throughout the pharynx and along the body wall.

157

Figure 6.12. Live C. elegans nematode mounted on an agarose pad after 24 h of immersion in 9.62 mM nickel and 48 h of incubation in a clean environment. The cholinergic nerve cells, tagged with GFP, are visible throughout the pharynx and along the body wall. The younger nematode is dead and already decomposing.

Discussion

This study, to the authors’ knowledge, is the first effort to evaluate direct morphological effects of nickel on dopaminergic and cholinergic neurons in C. elegans.

In this case, the sets of exposed transgenic strains did not differ from the corresponding 158

sets of controls. GFP fluorescence was present, indicating that the tagged dopaminergic

and cholinergic cells did not suffer degenerative damage, although we did not assess

differences in fluorescence intensity.

This study used the median lethal concentration (LC50) of nickel sulfate as the

highest exposure concentration; using an even higher concentration would require

doubling or tripling the number of nematodes exposed, to account for high mortality rates

while still maintaining sufficient numbers of survivors to reach the post-incubation

imaging stage. It may also be possible that an incubation period of 48 hours is not

sufficient for neuronal degeneration to develop. Caito et al. (2013), exposing C. elegans

to non-metal-containing pesticides, found some dopaminergic damage after an incubation

period of 5-6 days. To build a longer incubation period in a clean environment post-

exposure, however, would require lowering the exposure concentration in order to ensure

survival; since this study found no neuronal degeneration at the LC50 exposure

concentration, it is reasonable to suppose that a lower concentration would not yield

alterations in neuronal cell morphology, regardless of the length of the post-exposure incubation period. The sulfate salt was used because, unlike nickel chloride or nickel fluoride, nickel sulfate’s toxicity is probably derived from the nickel cation, rather than from the non-metallic elements in the molecule (Meyer et al., submitted for publication).

The nematode’s cell complement is one-third neurons, and a large body of scientific literature attests to its suitability as a model organism for studies in neurobiology and neurotoxicology (for reviews, see Leung et al., 2008; Meyer and

Williams 2014). If C. elegans dopaminergic and cholinergic neurons are less sensitive to 159

nickel than mammalian cells, it could prove a useful model for exploring the mechanisms

which confer neuronal resistance to nickel.

Behavioral effects after a 24 h exposure – changes in the nematode’s locomotion, feeding, and egg-laying – are a representation of general toxicity, and not specifically neurotoxicity. Short (4 h) exposures to heavy metals can result in observable neurotoxic effects in C. elegans, but 24 h exposures display effects of overall toxicity (Anderson et al., 2004). Metal-induced neurotoxicity, in general, is associated with perturbation of genes involved in regulation of the cell cycle (Yu et al., 2010). In mammals, some neurological impairment has been reported in humans following accidental exposure to nickel in drinking water, as well as in rat studies investigating the effects of chronic nickel exposure. These effects, however, were attributed to overall cellular toxicity (Das et al., 2008).

The immense library of C. elegans transgenic and knockout strains facilitates mechanistic studies into almost every component of the nematode’s anatomy and physiology. This study did not investigate the effects of nickel on other neurotransmitters

– such as GABA, serotonin, and glutamate. Future studies could undertake such an examination.

Altogether, this study adds to the C. elegans toxicological knowledge base by suggesting that nickel is unlikely to cause structural damage to the nematode’s dopaminergic and cholinergic neurons.

160

Acknowledgments

The authors are grateful for the assistance of M.K. Kandasamy of the Biomedical

Microscopy Core, and Rebecca Auxier of the Center for Applied Isotope Studies, at the

University of Georgia.

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CHAPTER 7

CONCLUSIONS

This dissertation characterized nickel toxicity in C. elegans, and investigated molecular pathways known to be involved in detoxifying other metals. First, it investigated whether different nickel salts caused varying levels of toxicity in C. elegans.

Next, it assessed the involvement of multiple mechanisms known to be involved in detoxifying metals in the nematode. The third study examined nickel’s potential to cause dopaminergic and cholinergic neuron degeneration in C. elegans.

The following bullets summarize the major findings and conclusions.

1. Nickel compounds containing halogen anions display differentially higher

toxicities than other nickel compounds. Accordingly, nickel sulfate was selected

as the recommended nickel salt for subsequent studies, because of its

comparatively low toxicity to C. elegans.

2. C. elegans harnesses multiple detoxification mechanisms to maintain metal

homeostasis. These can act in tandem or in compensatory ways, which makes

elucidation of a definitive path difficult. It appears that the main detoxifiers of

nickel in the nematode are phytochelatin, which is produced by the enzyme

phytochelatin synthase as a derivative of glutathione, and the ABC transporter

HMT-1, functioning in tandem with the nematode’s coelomocytes. These two

detoxification pathways act independently of each other. 164

3. Nickel concentrations as high as the LC50 value failed to elicit structural damage

to dopaminergic and cholinergic neurons in nematodes after 24 hours of exposure

and a 48 hour incubation period. It is therefore unlikely – but not impossible –

that nickel can cause direct degeneration of these nerve cells in C. elegans.

In view of these findings, the following avenues of investigation are suggested for

future research:

1. Develop null strains which lack two or more metalloprotective genes – such as

mutants which lack both metallothioneins and phytochelatin synthase, or mutants

which lack all three orthologs of the divalent metal transporter. The intent here

would be to remove the masking of toxic effects by reducing the nematode’s

ability to employ compensatory detoxification pathways, enabling researchers to

learn more about the function of specific pathways in chelating a given metal.

2. Investigate whether neuronal damage is contingent on the developmental stage of

the nematode by comparing the results in young adults with those in larvae. In

addition, experiment with longer periods of incubation post-exposure.

3. Expand nickel testing to other invertebrates, and in particular to other nematodes,

such as Pristionchus pacificus, which is currently experiencing resurgence in

popularity as a model organism. Comparisons with other organisms may yield

insights into molecular pathways in vertebrates, especially mammals.

As researchers attempt to move to some extent away from toxicity testing in mammalian species, the input from research involving invertebrates grows in its significance. C. elegans has become an accepted model organism for investigating the toxicity and adverse health effects of chronic exposures to metals, particularly in the field 165

of neurodegenerative disorders. This dissertation focused on characterizing to toxicity

and detoxification pathways of nickel in the nematode. More work on nickel, which is

certainly needed, will surely follow. Because of its extensive library of knockout and

transgenic mutants, along with its ease of culturing and the ability to collect data rapidly

from large numbers of nematodes, C. elegans is a suitable model organism for studies which survey molecular and cellular mechanisms in a systematic way, to assess adverse effects of environmental toxicants such as metals, pesticides, and the emerging field of metal nanoparticles.