Caenorhabditis elegans: An Emerging Model in Biomedical and Environmental Toxicology

Abstract

The nematode Caenorhabditis elegans has emerged as an important animal model in various fields including neurobiology, developmental biology, and genetics. Characteristics of this animal model that have contributed to its success include its genetic manipulability, invariant and fully described developmental program, well-characterized genome, ease of maintenance, short and prolific life cycle, and small body size. These same features have led to an increasing use of C. elegans in toxicology, both for mechanistic studies and high-throughput screening approaches. We describe some of the research that has been carried out in the areas of neurotoxicology, genetic toxicology, and environmental toxicology, as well as high-throughput experiments with C. elegans including genome-wide screening for molecular targets of toxicity and rapid toxicity assessment for new chemicals. We argue for an increased role for C. elegans in complementing other model systems in toxicological research.

Full text

TOXICOLOGICAL SCIENCES 106(1), 5–28 (2008)
doi:10.1093/toxsci/kfn121
Advance Access publication June 19, 2008
REVIEW
Caenorhabditis elegans: An Emerging Model in Biomedical and
Environmental Toxicology
Maxwell C. K. Leung,* Phillip L. Williams,† Alexandre Benedetto,‡ Catherine Au,‡ Kirsten J. Helmcke,‡ Michael Aschner,‡
and Joel N. Meyer*
,1
*Nicholas School of the Environment, Duke University, Durham, North Carolina 27750; †Department of Environmental Health Science, College of Public
University of Georgia, Athens, Georgia 30602; and ‡Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37240
Received April 30, 2008; accepted June 10, 2008
The nematode Caenorhabditis elegans has emerged as an
important animal model in various fields including neurobiology,
developmental biology, and genetics. Characteristics of this
animal model that have contributed to its success include its
genetic manipulability, invariant and fully described developmen-
tal program, well-characterized genome, ease of maintenance,
short and prolific life cycle, and small body size. These same
features have led to an increasing use of C. elegans in toxicology,
both for mechanistic studies and high-throughput screening
approaches. We describe some of the research that has been
carried out in the areas of neurotoxicology, genetic toxicology, and
environmental toxicology, as well as high-throughput experiments
with C. elegans including genome-wide screening for molecular
targets of toxicity and rapid toxicity assessment for new
chemicals. We argue for an increased role for C. elegans in
complementing other model systems in toxicological research.
Key Words: Caenorhabditis elegans; neurotoxicity; genotoxicity;
environmental toxicology; high-throughput methods.
Caenorhabditis elegans is a saprophytic nematode species
that has often been described as inhabiting soil and leaf-litter
environments in many parts of the world (Hope, 1999); recent
reports indicate that it is often carried by terrestrial gastropods
and other small organisms in the soil habitat (Caswell-Chen
et al., 2005; Kiontke and Sudhaus, 2006). Although scientific
reports on the species have appeared in the literature for more
than 100 years (e.g., Maupus, 1900), the publication of
Brenner’s seminal genetics paper (Brenner, 1974) signaled its
emergence as an important experimental model. Work with
C. elegans has since led in a short time span to seminal
discoveries in neuroscience, development, signal transduction,
cell death, aging, and RNA interference (Antoshechkin and
Sternberg, 2007). The success of C. elegans as a model has
attracted increased attention as well in the fields of in
biomedical and environmental toxicology.
Clearly, C. elegans will be a valuable toxicity model only if
its results were predictive of outcomes in higher eukaryotes.
There is increasing evidence that this is the case both at the
level of genetic and physiological similarity and at the level of
actual toxicity data. Many of the basic physiological processes
and stress responses that are observed in higher organisms
(e.g., humans) are conserved in C. elegans. Depending on the
bioinformatics approach used, C. elegans homologues have
been identified for 60–80% of human genes (Kaletta and
Hengartner, 2006), and 12 out of 17 known signal transduction
pathways are conserved in C. elegans and human (NRC, 2000;
Table 1). We discuss specific examples in the areas of
neurotoxicology and genetic toxicology in this review.
Caenorhabditis elegans has a number of features that make it
not just relevant but quite powerful as a model for biological
research. First of all, C. elegans is easy and inexpensive to
maintain in laboratory conditions with a diet of Escherichia coli.
The short, hermaphroditic life cycle (~3 days) and large number
(300þ) of offspring of C. elegans allows large-scale production
of animals within a short period of time (Hope, 1999). Since
C. elegans has a small body size, in vivo assays can be conducted
in a 96-well microplate. The transparent body also allows clear
observation of all cells in mature and developing animals.
Furthermore, the intensively studied genome, complete cell
lineage map, knockout (KO) mutant libraries, and established
genetic methodologies including mutagenesis, transgenesis, and
RNA interference (RNAi) provide a variety of options to
manipulate and study C. elegans at the molecular level (Tables 2
and 3; for a more detailed presentation of genetic and genomic
resources, see Antoshechkin and Sternberg, 2007). We address
the particular power of these genetic and molecular tools in
C. elegans at more length below.
Since reverse genetic and transgenic experiments are much
easier and less expensive to conduct in C. elegans as compared
1
To whom correspondence should be addressed at Nicholas School of the
Environment, Box 90328, Duke University, Durham, NC 27708-0328.
Fax: (919) 668-1799. E-mail: joel.meyer@duke.edu.
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to many other model systems, it is a useful model for molecular
analyses of the response of conserved pathways to in vivo
chemical exposure. As an in vivo model, C. elegans provides
several characteristics that complement in vitro or cellular
models. The use of whole-organism assays, first of all, allows
the study of a functional multicellular unit, such as a seroto-
nergic synapse, instead of a single cell (Kaletta and Hengartner,
2006). Caenorhabditis elegans also enables the detection of
organism-level end points (e.g., feeding, reproduction, life
span, and locomotion) and the interaction of a chemical with
multiple targets in an organism. Thus, C. elegans complements
both in vitro and in vivo mammalian models in toxicology.
Of note, these characteristics facilitate high-throughput experi-
ments that can examine both fundamental toxicity, which are
critical since so many chemicals have yet to be thoroughly tested,
and the gene-gene and gene-environment interactions whose
importance is just beginning to be appreciated in toxicology.
Here we review three major applications of C. elegans in
biomedical and environmental toxicology: (1) mechanistic
toxicology, with a focus on neurotoxicity and genotoxicity; (2)
high-throughput screening capabilities; and (3) environmental
toxicology and environmental assessment. We emphasize
studies of neurotoxicity because they are the area of toxicology
in which C. elegans has been most exploited to date. We
discuss research methods, recent advances, and important
considerations including limitations of the C. elegans model.
Caenorhabditis elegans AND NEUROTOXICITY
Caenorhabditis elegans Is Well Suited for
Neurophysiology Analysis of Neurotoxicity
With 302 neurons representing 118 characterized neuronal
subtypes (Hobert, 2005), C. elegans provides an in vivo model
TABLE 1
Signal Transduction Pathways Conserved in Nematodes and
Vertebrates
a,b
Pathways involved in early development
Wnt pathway via b-catenin
Receptor serine/threonine kinase (tumor growth factor-b receptor) pathway
Receptor tyrosine kinase pathway (small G-protein [Ras] linked)
Notch-delta pathway
Receptor-linked cytoplasmic tyrosine kinase (cytokine) pathway
Pathways involved in later development (e.g., organogenesis and tissue
renewal)
Apoptosis pathway (cell death pathway)
Receptor protein tyrosine phosphatase pathway
Pathways involved in the physiological function of differentiated cells of the
fetus, juvenile, and adult
G-protein–coupled receptor (large G-protein) pathway
Integrin pathway
Cadherin pathway
Gap junction pathway
Ligand-gated cation channel pathway
a
Adapted from NRC (2000).
b
Signal transduction pathways that are not conserved in nematodes and
vertebrates include the Wnt pathway via c-Jun N-terminal kinase, the
Hedgehog pathway (patched receptor protein), the nuclear factor kappa-B
pathway, the nuclear hormone receptor pathway, the receptor guanylate cyclase
pathway, and the nitric oxide receptor pathway.
TABLE 2
Examples of Mutational Analysis of Caenorhabditis elegans in Toxicology Research
Approach/toxin investigated Mutants used Major findings References
A. KO mutant analysis
Black widow spider
venom
lat-1: KO of latrophilin Latrophilin is the receptor responsible for the
toxicity of venom
Mee et al. (2004)
As asna-1: KO of ArsA ATPase ArsA ATPase is important in Ar resistance in both
bacteria and animals
Tseng et al. (2007)
Cd pgp-5: KO of a ABC transporter ABC transporter is required for resistance to
Cd toxicity
Kurz et al. (2007)
PCB52 cyp-35A1 to cyp-35A5: KOs of
cytochrome P450 35A subfamily
CYP35A is required for fat storage
and resistance to PCB52 toxicity
Menzel et al. (2007)
B. Forward genetics screen
BPA bis-1: mutant created from EMS mutagenesis Collagen mutants are hypersensitive to BPA Watanabe et al. (2005)
Phosphine pre-1, pre-7, pre-33: mutants created from
EMS mutagenesis
Uptake and oxidization of phosphone are directly
associated with oxidative stress in cells
Cheng et al. (2003)
Bt toxins bre-1 to bre-5: mutants created from EMS
mutagenesis
Five new genes involved in Bt toxicity are identified Marroquin et al. (2000)
bre-5:KOofb-1,3-galactosyltransferase Carbohydrate modification is involved in Bt toxicity Griffitts et al. (2001)
bre-2 to bre-5: KOs of glycolipid
carbohydrate metabolism
Glycolipid receptors are targets of Bt toxins Griffitts et al. (2005)
bre-1: KO of GDP-mannose 4,6-dehydratase The monosaccharide biosynthetic pathway is involved
in Bt toxicity
Barrows et al. (2007b)
Note. ABC, ATP-binding cassette; PCB52, polychlorinated biphenyl 52; EMS, ethane methyl sulfonate.
6 LEUNG ET AL.

for studying mechanisms of neuronal injury with resolution of
single neurons. It intially underwent extensive development as
a model organism in order to study the nervous system
(Brenner, 1974), and its neuronal lineage and the complete
wiring diagram of its nervous system are stereotyped and fully
described (Sulston, 1983; Sulston et al., 1983; White et al.,
1986). Each neuron has been assigned a code name
corresponding to its location. For example, ADEL describes
the dopaminergic (DAergic) head neuron ‘‘anterior deirid left.’’
This relatively ‘‘simple’’ nervous system is comprised of 6393
chemical synapses, 890 electrical junctions, and 1410 neuro-
muscular junctions (Chen et al., 2006). Additionally, the main
neurotransmitter systems (cholinergic, c-aminobutyric acid
(GABA)ergic, glutamatergic, DAergic, and serotoninergic)
and their genetic networks (from neurotransmitter metabolism
to vesicle cycling and synaptic transmission) are phylogenet-
ically conserved from nematodes to vertebrates, which allows
for findings from C. elegans to be extrapolated and further
confirmed in vertebrate systems.
Several genes involved in neurotransmission were originally
identified in C. elegans. This is exemplified by the GABA
vesicular transporter unc-47 and the regulatory transcription
factor unc-30 (for review on the GABAergic system [Jorgensen,
2005]), the vesicular acetylcholine (ACh) transporter unc-17
(for review on the cholinergic system [Rand, 2007]), the
glutamate-gated chloride channel subunits a1 and b (glc-1 and
glc-2, respectively, for review on the glutamatergic system
[Brockie and Maricq, 2006]), and the synaptic proteins unc-18,
unc-13, unc-26 (for review on synaptic function [Richmond,
2005]). Experiments challenging the C. elegans nervous
system by laser ablation of individual neurons/axons, exposure
to drugs, and other external stimuli have facilitated the design
of robust behavioral tests to assess the function of defined
neuronal populations (Avery and Horvitz, 1990; Bargmann,
2006; Barr and Garcia, 2006; Brockie and Maricq, 2006; Chase
and Koelle, 2007; Goodman, 2006; Morgan et al., 2007; Rand,
2007). For example, inhibitory GABAergic and excitatory
cholinergic motor functions are assessed by quantifying the
sinusoidal movement (amplitude and frequency of body bends)
and foraging behavior of the worm. Motor and mechanosen-
sory functions of glutamatergic neurons are evaluated by
measuring the pharyngeal pumping rate and the response to
touch. Mechanosensory functions of DAergic and serotoniner-
gic neurons are appraised by observing the ability of worms to
slow down when they encounter food. Furthermore, the
creation of transgenic strains expressing fluorescent proteins
in defined neurons allows in vivo imaging of any desired
neuron. While experimentally challenging in the cells of
microscopic animals, electrophysiology studies can be con-
ducted with relative ease and success in live worms and
cultured C. elegans neurons, establishing that they are
electrophysiologically comparable to vertebrate neurons in
their response to various drugs (Bianchi and Driscoll, 2006;
Brockie and Maricq, 2006; Cook et al., 2006; Schafer, 2006).
Given the relative ease with which gene KO and transgenic
animals can be generated, the ability to culture embryonic or
primary C. elegans cells offers unique perspectives for
neurotoxicology applications and study designs.
Caenorhabditis elegans Is a Potent Model to
Decipher Genetic Aspects of Neurotoxicity
The conservation of neurophysiologic components from
nematodes to humans largely relies on shared genetic networks
and developmental programs. Hence, the availability of
mutants for many of the C. elegans genes facilitated significant
progress in unraveling of evolutionarily conserved cellular and
genetic pathways responsible for neuron fate specificity
TABLE 3
Examples of Transgenic Caenorhabditis elegans Used in Toxicology Research
Field/target tagged Reporter used Applications References
A. Mechanistic studies
DAergic neurons GFP Detect neurodegradation caused by chemicals Jiang et al. (2007)
CYP14A3 and 35A3 GFP Detect intestinal CYP overexpression in response to
PCB52 as well as other xenobiotic CYP inducers
Menzel et al. (2007)
GST GFP Measure GST induction in response to acrylamide as
well as other inducers of oxidative stress
Hasegawa and van der Bliek (in press)
B. Environmental biomonitoring
Heat shock proteins GFP; b-galactosidase Widely used for measuring stress response associated
to toxicity of heavy metals, fungicides,
pharmaceuticals, as well as field samples
Dengg and van Meel (2004); Easton et al. (2001);
Mutwakil et al. (1997); Roh et al. (2006)
Metallothionein b-galactosidase Specifically used for monitoring the bioavailability
of heavy metals
Cioci et al. (2000)
ATP level Firefly luciferase Measure the reduction of metabolic
activity in response to environmental stressor
Lagido et al. 2001
Note. CYP, cytochrome P450; GST, glutathione S-transferase.
CAENORHABDITIS ELEGANS IN TOXICOLOGY RESEARCH
7

(Hobert, 2005), differentiation (Chisholm and Jin, 2005),
migration (Silhankova and Korswagen, 2007), axon guidance
(Quinn and Wadsworth, 2006; Wadsworth, 2002), and
synaptogenesis (Jin, 2002, 2005). Recently, laser axotomy in
C. elegans has been successfully applied to identify axon
regeneration mechanisms (Gabel et al., 2008; Wu et al., 2007),
which are of utmost importance in developing treatments to
reverse neurodegenerative processes and spinal cord injuries.
Essential cell functions relevant to neurotoxicity studies are
also conserved. This is best exemplified by the mechanistic
elucidation of the apoptotic pathway in C. elegans, for which
the 2002 Nobel Prize in Physiology or Medicine was awarded
(Hengartner and Horvitz, 1994; Horvitz, 2003; Sulston, 2003).
The pathway is of direct interest to neurotoxicologists since
apoptosis is implicated in many neurodegenerative diseases
and toxicant-induced cell demise (Bharathi et al., 2006; Hirata,
2002; Koh, 2001; Mattson, 2000; Ong and Farooqui, 2005;
Savory et al., 2003). Pathways relevant to oxidative stress–
related neuronal injuries, such as the p38 mitogen-activated
protein kinase and AKT signaling cascades, the ubiquitin-
proteasome pathway, and the oxidative stress response are also
conserved in the worm (Ayyadevara et al., 2005, 2008;
Daitoku and Fukamizu, 2007; Gami et al., 2006; Grad and
Lemire, 2004; Inoue et al., 2005; Kipreos, 2005; Leiers et al.,
2003; Tullet et al., 2008; Wang et al., 2007a).
The nematode model is also amenable to interesting genetic
alterations. Hence, it is very easy to generate transgenic worms
expressing any kind of mutant recombinant protein, providing
means for the study of neurodegenerative diseases (see
additional discussion below). Gene KO and altered function
mutations are in many cases available from the Gene Knockout
Consortium or the National BioResource Project of Japan
(currently ~1/3 of the ~20,000 total genes in C. elegans;
Antoshechkin and Sternberg, 2007) or alternatively are
conveniently generated using chemicals, radiations, or trans-
posons (discussed below under Caenorhabditis elegans and
Genotoxicity). Hence, classical approaches to elucidate in-
tracellular pathways in C. elegans include forward and modifier
screens following random mutagenesis (Inoue and Thomas,
2000; Malone and Thomas, 1994; Morck et al., 2003; Nass
et al., 2005; O’Connell et al., 1998). Finally C. elegans is
amenable to gender manipulation (possible generation of
males, feminized males, masculinized hermaphrodites, or
feminized hermaphrodites) permitting studies on sex specificity
mechanisms of neurotoxicants or disorders and ‘‘rejuvenation’’
by forcing development through the quiescent dauer larval
stage (Houthoofd et al., 2002).
Neurotoxicological Studies in C. elegans
Years before the latest technologic developments (RNAi and
high-throughput techniques), C. elegans was used to study
toxicity mechanisms of environmental factors affecting the
nervous system. The following section provides a synopsis of
the available literature on neurotoxicity-related issues
addressed in C. elegans. It is not meant to be exhaustive but
rather to illustrate typical studies that are amenable in the
C. elegans platform. We highlight studies with exposure
outcomes to various metals and pesticides, as well as general
considerations on studies of neurodegenerative diseases. We
emphasize the utility of C. elegans in addressing hypothesis-
driven mechanisms of neurotoxicity and extrapolations to
vertebrate systems.
Toxicity Mechanisms of Neurotoxic Metals in C. elegans
Caenorhabditis elegans has been used as a model system to
elucidate the toxicity and toxicological mechanisms of various
heavy metals, such as Aluminum (Al), Arsenic (As), Barium
(Ba), Cadmium (Cd), Copper (Cu), Lead (Pb), Mercury (Hg),
Uranium (U), and Zinc (Zn). In general, these studies focused
on various toxic end points, such as lethality, reproduction, life
span, and protein expression. Some focus has also been
directed to the effects of these metals on the nervous system by
assessing behavior, reporter expression and neuronal morphol-
ogy. We provide here a few examples of these approaches.
Investigators have performed numerous studies to assess
behavior-induced alterations following exposure of the worm
to heavy metals. Depending on the end point assessed,
neurotoxic effects on specific neuronal circuitries can be
inferred.
For instance, a defect in locomotion reflects an impairment
of the neuronal network formed by the interneurons AVA,
AVB, AVD, and PVC providing input to the A- and B-type
motor neurons (responsible for forward and backward
movement) and the inhibitory D-type motor neurons involved
in the coordination of movement (Riddle et al., 1997). By
recording short videos and subsequently analyzing them using
computer tracking software, it has been possible to quantify the
overall movement of C. elegans (distance traveled, directional
change, etc.), body bends and head thrashes, upon metal
treatments, allowing to further correlate the data with damages
to neuron circuitries. These computer tracking studies showed
that worms displayed a dose-dependent decrease in locomotory
movement upon exposure to Pb (Anderson et al., 2001, 2004;
Johnson and Nelson, 1991) and Al (Anderson et al., 2004),
while an increase in locomotion was observed upon exposure
to low concentrations of Hg as compared with Cu (Williams
and Dusenbery, 1988). Another study showed that exposure
to Ba impaired both body bend and head thrashing rates in
a dose-dependent manner (Wang et al., 2008), corroborating
mammalian data on the effect of Ba on the nervous system
attributed to its ability to block potassium channels (Johnson
and Nelson, 1991).
Feeding behavior has also been shown to be affected upon
heavy metal exposure. Feeding requires a different neuronal
circuitry including M3 (involved in pharyngeal relaxation), MC
8
LEUNG ET AL.

(control of pumping rate), M4 (control of isthmus peristalsis),
NSM (stimulate feeding), RIP, and I neurons (Riddle et al., 1997).
A decrease in feeding was observed when worms were exposed to
Cd or Hg (Boyd et al., 2003; Jones and Candido, 1999).
Behavioral research studying the effect of heavy metals on
C. elegans has also taken the route of assessing the ability of
the worm to sense the toxin and alter its behavior accordingly,
involving other neural circuitry, such as the amphid and
phasmid neurons responsible for chemosensation (Riddle et al.,
1997). By generating concentration gradient–containing plates,
Sambongi et al. (1999) discovered that C. elegans was able to
avoid Cd and Cu but not Ni and that the amphid ADL, ASE,
and ASH neurons were responsible for this avoidance as their
combined ablation eliminated the avoidance phenotype.
Furthering the investigation into the role of ASH neurons,
researchers found that a calcium (Ca
2þ
) influx could be elicited
upon exposing the C. elegans to Cu, which may provide insight
into the mechanism of the ability of the worm to display
avoidance behaviors (Hilliard et al., 2005).
Caenorhabditis elegans exhibits both short-term and long-
term learning-related behaviors in response to specific sensory
inputs (Rankin et al., 1990), which involve defined neuronal
networks. As an example, thermosensation-associated learning
and memory rely on the AFD sensory neuron sending inputs to
the AIY and AIZ interneurons, whose signals are integrated by
the RIA and RIB interneurons to command the RIM motor
neuron (Mori et al., 2007). When assessing the function of this
circuitry, worms grown and fed at a definite temperature are
moved to a food-deprived test plate exposed to a temperature
gradient. The ability of the worms to find and remain in the
area of the test plate corresponding to the feeding temperature
reflects the functioning of the thermosensation learning and
memory network aforementioned (Mori et al., 2007). In-
terestingly, worms exposed to Al and Pb exhibit poor scores at
this test, indicative of a significant reduction of the worms’
learning ability (Ye et al., in press). This recapitulates the
learning deficits observed in young patients overexposed to the
same metals (Garza et al., 2006; Goncalves and Silva, 2007).
While behavioral testing was informative of the neuronal
circuitries affected by heavy metals, additional experiments
uncovered the molecular mechanisms of their neurotoxic
effects. For example, in the previously described study, after
determining that Al and Pb induced memory deficits, the
investigators showed that the antioxidant vitamin E effectively
reversed these deficits, indicating a role of oxidative stress in
Al and Pb neurotoxicity (Ye et al., in press). The involvement
of oxidative stress in metal-induced toxicity was further
confirmed when worms mutated in glutamylcysteine synthetase
(gcs-1), the rate-limiting enzyme in glutathione synthesis
exhibited hypersensitivity to As exposure when compared to
wild-type animals (Liao and Yu, 2005).
Studies conducted in mammalian models found that Hg is
able to block Ca
2þ
channels. In neurons, this blockage can
induce spontaneous release of neurotransmitters (Atchison,
2003). In C. elegans, the Ca
2þ
channel blocker verapamil was
found to protect against Hg exposure, suggesting that Ca
2þ
signaling plays a role in the toxicity of Hg in this model
organism as in mammals (Koselke et al., 2007).
Observation of neuron morphology following heavy metal
exposure was also performed using C. elegans strains
expressing the green fluorescent protein (GFP) in discrete
neuronal populations. Tests using depleted U evoked no
alterations in the DAergic nervous system of C. elegans,an
observation corroborated with data from mammalian primary
neuronal cultures (Jiang et al., 2007). Meanwhile, kel-8 and
numr-1, which are involved in resistance to Cd toxicity, were
upregulated upon Cd exposure. In particular, GFP levels of
KEL-8::GFP and NUMR-1::GFP were increased in the
pharynx and the intestine in addition to the constitutive
expression observed in AWA neurons (Cui et al., 2007a;
Freedman et al., 2006; Jackson et al., 2006; Tvermoes and
Freedman, 2008). Furthermore, numr-1 was shown to be
induced in response to heavy metals, such as Cd, Cu, Cobalt
(Co), Chromium (Cr), Ni, As, Zn, and Hg. NUMR-1::GFP was
localized to nuclei within the intestine and the pharynx and
colocalized with the stress-responsive heat-shock transcription
factor HSF-1::mCherry (Tvermoes and Freedman, 2008). This
indicates that these particular genes were altered in response to
heavy metals and this may aid in the understanding of the
toxicity of or the protection against these agents.
Toxicity Mechanisms of Neurotoxic Pesticides in C. elegans
Currently, there are over a hundred types of pesticides
available and substantial efforts have been put forth to examine
the neurotoxicity of these agents. Similarity in neural circuitry
and the conservation in genetic makeup between C. elegans
and humans have led to a number of recent studies on pesticide
neurotoxicity in this species (summarized in Table 4). In this
section, we discuss the effects of three groups of pesticides on
neurological pathways in C. elegans and their relevance to
understanding mechanisms of human neurotoxicity.
Paraquat, also known as methyl viologen (mev), is mainly
used as an herbicide. Increased concerns for the potential
human risks associated with paraquat exposure stems from
studies indicating that subjects experiencing exposure to this
and other herbicides/insecticides have a higher prevalence of
Parkinson disease (PD) (Liou et al., 1997; Semchuk et al.,
1992) (Gorell et al., 1998) and increased mortality from PD
(Ritz and Yu, 2000). The use of C. elegans to study the
etiology of PD will be discussed in the later section. This is due
to the specificity with which these pesticides target the
nigrostriatal DAergic system via an elevation of dopamine
and amine turnover (Thiruchelvam et al., 2000a, 2000b). All
forms of paraquat are easily reduced to a radical ion, which
generates superoxide radical that reacts with unsaturated
membrane lipids (Uversky, 2004), a likely mechanism of
CAENORHABDITIS ELEGANS IN TOXICOLOGY RESEARCH 9

TABLE 4
Pesticides that Have Been Tested Using Caenorhabditis elegans as a Model Organism
Compound Strains investigated Observations References
Paraquat mev-1(kn1)
a
, mev-2(kn2)
a
Hypersensitive to oxygen and paraquat, decreased SOD activity
b
Ishii et al. (1990)
rad-8(mn162) Hypersensitive to oxygen and paraquat, reduced fecundity,
decreased life span
Ishii et al. (1993)
age-1(hx542), age-1(hx546) Increased catalase and Cu/Zn SOD activity, increased life span Vanfleteren (1993)
Vitamin E (antioxidant) inhibits oxidative damage from paraquat
b
Goldstein and Modric (1994)
mev-1(kn1), rad-8(mn162) Paraquat and high oxygen content inhibit development,
inversely proportional to life span
Hartman et al. (1995)
age-1(hx546), daf-16(m26),
mev-1(kn1)
a
Increased resistance to paraquat and heat, extended life span,
increased SOD, and catalase mRNA level only in age-1
mutant, but not daf-16 or mev-1
Yanase et al. (2002)
mev-5(qa5005)
a
, mev-6
(qa5006)
a
, mev-7(qa5007)
a
Longevity and sensitivity to paraquat, UV or heat do
not correlate
Fujii et al. (2005)
mev-1(kn1), gas-1(fc21) Overproduction of superoxide anion in submitochondrial
particles upon paraquat exposure
Kondo et al. (2005)
skn-1(zu67) Activation of SKN-1 transcription factor, localizes to the
nucleus following paraquat exposure
Kell et al. (2007)
daf-2(e1370) Extended animal life span and increased resistance to ROS
produced by paraquat
Kim and Sun (2007);
Yang et al. (2007)
Overexpression of GSTO,
gsto-1 RNAi
Increased resistance to paraquat-induced oxidative stress Burmeister et al. (2008)
Rotenone gas-1(fc21) Increased sensitivity to rotenone under hyperoxia Ishiguro et al. (2001)
pdr-1, djr-1.1 RNAi Increased vulnerability to rotenone Ved et al. (2005)
Overexpression of LRRK2,
lrk-1 RNAi
Overexpression of wild-type LRRK2 strongly protects
against rotenone toxicity
Wolozin et al. (2008)
Ops N2 Computer tracking system is a promising tool for assessing
neurobehavioral changes associated with OP toxicity
Williams and
Dusenbery 1990
Cholinesterase inhibition associated with high behavioral toxicity Cole et al. (2004)
Absorption effects are more prominent than biodegradation
in soil toxicity tests
Saffih-Hdadi et al. (2005)
Carbamates N2 Rank order of toxicity of carbamate pesticides in C. elegans
correlates well with values for rats and mice, and degree
of behavioral alteration correlates with AChE inhibition
Melstrom and
Williams (2007)
Bt toxin bre-1(ye4), bre-2(ye31), bre-3
(ye28), bre-4(ye13), bre-5(ye17)
Extensive damage to gut, decreased fertility, and death Marroquin et al. (2000)
bre-5(ye17) Increased resistance to Bt toxin Griffitts et al. (2001)
bre-2(ye31), bre-2(ye71),
bre-3(ye28), bre-4(ye13)
Bt toxin resistance involves the loss of glycosyltransferase
in the intestine
Griffitts et al. (2003)
glp-4(bn2), kgb-1(um3),
jnk-1(gk7), sek-1(km4)
Bt toxin reduces brood size and causes damage to the intestine Wei et al. (2003)
A p38 MAPK and a c-Jun N-terminal-like MAPK are both
transcriptionally upregulated by Bt toxin
Huffman et al.
(2004a, 2004b)
Survival rate, infection level, and behavior differred in
C. elegans isolated from geographically distinct strains
Schulenburg and
Muller (2004)
bre-2(ye31), bre-3(ye28),
bre-4(ye13), bre-5(ye17)
Bt toxin resistance entails loss of glycolipid carbohydrates and
the toxin directly and specifically binds to Glycolipids
Griffitts et al. (2005)
bre-3(ye28) Resistance to Bt toxin develops as a result of loss of glycolipid
receptors for the toxin
Barrows et al. (2006)
bre-1(ye4), bre-2(ye31) Resistance to toxin is achieved by mutations in
gylcosyltransferase genes that glycosylate glycolipid or with
a loss of the monosaccharide biosynthetic pathway
Barrows et al.
(2007a, 2007b)
daf-2(e1370), daf-2(e1368), age-1
(hx546), daf-16(mgDf50), daf-2(0(m26)
Mutations in the insulin-like receptor pathway lead to
distinct behavioral responses, including the evasion
of pathogens and reduced ingestion
Hasshoff et al. (2007)
Reproduction and growth significantly reduced by Bt toxin Hoss et al. (2008)
Captan hsp-16.48;hsp-16.1::lacZ Stress induction localized to muscle cells of the pharynx Jones et al. (1996)
Inhibits feeding, cessation of muscular contraction
Dithiocarbamate fungicides hsp-16.48;hsp-16.1::lacZ Induction of stress response Guven et al. (1999)
Organochlorinated pesticides N2 Decreased sensitivity to organochlorinated pesticide in C. elegans
than other soil invertebrates. Compared to other organic
pollutants tested, organochlorinated pesticides are the most
toxic substances in soil or aquatic medium
Bezchlebova et al. (2007);
Sochova et al. (2007)
Note. MAPK, mitogen-activated protein kinase; ROS, reactive oxygen species.
a
These mutants showed defective dye filling, indicative of chemosensory neuron damage.
b
SOD, superoxide dismutase.
10 LEUNG ET AL.

neurotoxicity. Caenorhabditis elegans, has a well-defined, yet
simple DAergic network, consisting of eight neurons in the
hermaphrodite and an additional six neurons located in the tail
of the male (Chase and Koelle, 2007) and four DA receptors.
Dopamine is known to be required in the modulation of
locomotion and in learning in C. elegans (Hills et al., 2004;
Sanyal et al., 2004; Sawin et al., 2000). To date, several
paraquat/mev–altered strains have been generated to study
potential pathways in which paraquat exerts its toxic effects.
mev-1 (mutated for the succinate dehydrogenase) (Hartman
et al., 1995; Ishii et al., 1990; Kondo et al., 2005) and mev-3
(Yamamoto et al., 1996) were generated first, and both strains
displayed increased sensitivity to paraquat- and oxygen-
mediated injury as a result of increased production of
superoxide radicals (Guo and Lemire, 2003; Ishii et al.,
1990) and hypersensitivity to oxidative stress. mev-4 (Fujii
et al., 2004), mev-5, mev-6, and mev-7 (Fujii et al., 2005)
displayed resistance to paraquat. However, since the proteins
that are encoded by these genes are currently unknown, future
mapping of these genes will likely reveal pathways involved in
paraquat toxicity.
Paraquat exerts oxidative damage in vertebrates, which has
also been corroborated in C. elegans. Mutants that lack
antioxidant enzymes such as cytosolic or mitochondrial
superoxide dismutases (sod-1 and sod-2) show increased
sensitivity to paraquat (Yang et al., 2007), whereas mutants
with increased superoxide dismutase levels, such as age-1
(encoding the catalytic subunit of phosphoinositide 3-kinase)
(Vanfleteren, 1993; Yanase et al., 2002) and worms over-
expressing the omega-class glutathione transferase gsto-1
(Burmeister et al., 2008) display increased resistance to paraquat
toxicity. Moreover, C. elegans mutants hypersensitive to
oxygen toxicity, such as rad-8 (Honda et al., 1993; Ishii et al.,
1990) or those with a prolonged life span, such as daf-2
(encoding insulin/insulin growth factor receptor) (Bardin et al.,
1994; Kim and Sun, 2007) show increased tolerance to paraquat.
Taken together, these results provide novel information on
mechanisms by which paraquat mediates its toxicity (by
enhancing sensitivity to oxygen toxicity with an elevation in
production of reactive oxygen species and shortening life span)
and provide directions for future investigations on mechanisms
that lead to DAergic neurodegeneration.
A second ubiquitous pesticide is rotenone; it is a naturally
occurring and biodegradable pesticide effective in killing pests
and fish (Uversky, 2004). Researchers first reported in 2000
that Iv exposure to rotenone may lead in humans to the
development of PD-like symptoms accompanied by the
selective destruction of nigral DAergic neurons (Betarbet
et al., 2000). Since rotenone acts by inhibiting mitochondrial
NADH dehydrogenase within complex I (Gao et al., 2003), the
development of a mutant C. elegans strain that exhibits
mitochondrial inhibition provided an experimental platform
where the role of this enzyme could be directly evaluated.
A mutation in a 49-kDa subunit of mitochondrial complex I in
C. elegans mutant gas-1 displays hypersensitivity to rotenone
and oxygen (Ishiguro et al., 2001), highlighting the importance
of a functional complex I in rotenone resistance. Moreover,
C. elegans with alterations in PD causative genes are highly
sensitive to rotenone toxicity, suggesting the ability of these
proteins to protect against rotenone-induced oxidative damage
in DAergic neurons (Ved et al., 2005; Wolozin et al., 2008)
(see neurodegenerative disease section below).
The organophosphates (OPs) are a group of insecticides that
target the cholinergic system. ACh is the primary neurotrans-
mitter involved in motor function in most organisms, including
the nematode (Rand and Nonet, 1997). Due to the involvement
of the neuromuscular system, a computer tracking system was
used to study the neurobehavioral changes in C. elegans
associated with two OP pesticides (malathion and vapona).
Caenorhabditis elegans showed a remarkable decline in
locomotion at a concentration below survival reduction
(Williams and Dusenbery, 1990b). Comparison studies using
similar behavioral analyses were later developed to assess
movement alteration as an indicator of the neurotoxity of 15
OP pesticides (Cole et al., 2004) and carbamate pesticides,
which unlike OP pesticides are reversible AChE inhibitors
(Melstrom and Williams, 2007). The LD
50
values in C. elegans
closely correlated with LD
50
in both rats and mice. Pesticides
(vapon, parathion, methyl parathion, methidathion, and fun-
sulfothion) that showed cholinesterase inhibition were associ-
ated with pronounced behavioral toxicity (i.e., decrease in
movement). A recent study has compared end points using OPs
and found AChE inhibition to be the most sensitive indicator of
toxicity but also the most difficult to measure (Rajini et al.,in
press). Reduction in movement for 10 OPs was found to
correlate to rat and mouse acute lethality data. Finally,
simulation studies examining the rate of absorption and
biodegradation of OP (parathion) also (Saffih-Hdadi et al.,
2005) establish the relevance and reliability of C. elegans as an
experimental model and predictor for soil toxicity.
Caenorhabditis elegans in the Study of Neurodegeneration
As previously stated, the C. elegans nervous system
functionally recapitulates many of the characteristics of the
vertebrate brain. In particular, it can undergo degeneration
through conserved mechanisms and is thus a powerful model
for uncovering the genetic basis of neurodegenerative dis-
orders. In this section, we will focus on PD, Alzheimer disease
(AD), Huntington disease (HD), and Duchenne muscular
dystrophy (DMD).
PD is a progressive, neurodegenerative disorder afflicting
~2% of the U.S. population (Bushnell and Martin, 1999).
Characteristic features include a gradual loss of motor function
due to the degeneration of DAergic neurons within the
substantia nigra pars compacta and loss of DAergic terminals
in the striatum (Wilson et al., 1996). At the cellular level,
CAENORHABDITIS ELEGANS IN TOXICOLOGY RESEARCH 11

deposition of cytoplasmic Lewy bodies composed of aggre-
gated protein, such as a-synuclein, is observed. PD cases are
referred as familial (FPD) or idiopathic (IPD) depending on
whether the disease is hereditary (FPD) or from unknown
origin, possibly due to environmental exposure to neuro-
toxicants (IPD) (Dauer and Przedborski, 2003; Samii et al.,
2004). Among 11 genomic regions (PARK1 to 11) associated
with FPD, 7 were narrowed down to single genes: PARK1
(a-SYNUCLEIN), PARK2 (PARKIN), PARK4 (a-SYNU-
CLEIN), PARK5 (UCHL1), PARK6 (PINK1), PARK7 (DJ1),
PARK8 (DARDARIN/LRRK2), and PARK9 (ATP13A2) (Wood-
Kaczmar et al., 2006). All but a-SYNUCLEIN are strictly
conserved in the nematode with most residue positions mutated
in PD patients encoding identical amino acids in C. elegans
orthologues (Benedetto et al., 2008). Worms overexpressing
wild type, mutant A30P, or A53T human a-SYNUCLEIN in
DAergic neurons show differential levels of injury, including
reduced DA content, DAergic neuron degeneration, motor
deficits reversible by DA administration, intracellular
a-SYNUCLEIN aggregates similar to Lewy bodies, and
increased vulnerability to mitochondrial complex-I inhibitors,
which is reversed by treatment with antioxidants (Kuwahara
et al., 2006; Lakso et al., 2003; Ved et al., 2005). Furthermore,
deletion (Springer et al., 2005) and knockdown of the
C. elegans PARKIN and DJ1 genes produce similar patterns
of pharmacological vulnerability as those described above for
a-SYNUCLEIN overexpression (Ved et al., 2005). Other PD
genes in C. elegans have been investigated. For example, ubh-1
and ubh-3 (Chiaki Fujitake et al., 2004) share similar functions
with the human PARK5/UCHL1 orthologue. Studies on other
genes have been instrumental in unraveling previously unknown
functions. For example, examination of the PARK8/DARDARIN
orthologue lrk-1 showed that the protein allows the proper
targeting of synaptic vesicle proteins to the axon (Sakaguchi-
Nakashima et al., 2007) and protects against rotenone-induced
mitochondrial injury (Wolozin et al., 2008). Recently, RNAi,
genomic, and proteomic approaches using human
a-SYNUCLEIN transgenic worms identified genetic networks
linking PD to G-protein signaling, endomembrane trafficking,
actin cytoskeleton, and oxidative stress (Cooper et al., 2006;
Gitler et al., 2008; Hamamichi et al., 2008; Ichibangase et al.,
2008; van Ham et al., 2008; Vartiainen et al., 2006), illustrating
the power of this transgenic model for PD study.
Nonhereditary PD cases have also been associated with
exposure to 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine,
a designer drug that is converted intracerebrally (by astrocytes)
to 1-methyl-4-phenylpyridinium (MPPþ) by the monoamine
oxygenase B. MPPþ damages the DAergic nervous system,
leading to a typical Parkinsonian syndrome (Kopin and
Markey, 1988; Langston et al., 1984). Similarly, MPPþ-
exposed C. elegans show specific degeneration of DAergic
neurons and associated behavioral defects (Braungart et al.,
2004), which is due to ATP depletion (Wang et al., 2007b).
Exposures to rotenone (see above) or 6-hydroxydopamine also
lead to PD syndromes that share similar features both in
humans and worms (Cao et al., 2005; Ishiguro et al., 2001;
Marvanova and Nichols, 2007; Nass et al., 2002, 2005; Ved
et al., 2005). Though the nematode does not truly exhibit PD-like
symptoms, results with transgenic and drug-exposed worms
emphasize the relevance of C. elegans as a model organism that
(1) permits rapid insights in the genetic pathways involved in PD
and (2) enables high-throughput screening methods for the
development of new anti-PD drugs (Schmidt et al., 2007).
Tauopathies and polyglutamine extension disorders have
also been investigated in the worm using mutants and
transgenic strains (Brandt et al., 2007; Dickey et al., 2006,
Link, 2001; Kraemer et al., 2003, 2006, and Kraemer and
Schellenberg, 2007). The first AD-associated proteins identi-
fied were the beta-amyloid peptide precursor (betaAPP) and the
presenilins PS1 and PS2. Study of the C. elegans presenilin
orthologues sel-12 (Baumeister et al., 1997; Levitan and
Greenwald, 1995) and hop-1 (Li and Greenwald, 1997;
Smialowska and Baumeister, 2006) linked AD to the apoptotic
pathway (Kitagawa et al., 2003) and Notch signaling, which
was later confirmed in vertebrates (Berezovska et al., 1998,
1999; Ray et al., 1999). Characterization of the C. elegans
betaAPP orthologue revealed a key role for microRNA in AD
gene regulation (Niwa et al., 2008). However, most of the
knowledge about AD acquired in C. elegans came from two
transgenic models: worms expressing the human betaAPP
(Boyd-Kimball et al., 2006; Drake et al., 2003; Gutierrez-
Zepeda and Luo, 2004; Wu and Luo, 2005; Wu et al., 2006) or
TAU (Brandt et al., in press; Kraemer et al., 2003). Studies on
betaAPP transgenic worms revealed toxicity mechanisms of
AD by identifying two new genes, aph-1 and pen-2, likely
involved in the progression of the disease (Boyd-Kimball et al.,
2006; Francis et al., 2002). They also allowed the character-
ization of oxidation processes preceding fibrillar deposition
(Drake et al., 2003) and the identification of genes activated upon
induction of betaAPP expression (Link et al., 2003). Furthermore,
protective mechanisms were identified (Florez-McClure et al.,
2007; Fonte et al., 2008) and potential therapeutic drugs for AD
(ginkgolides, Ginkgo biloba extract EGb 761, soy isoflavone
glycitein) were originally and successfully assayed in worms
(Gutierrez-Zepeda et al., 2005; Luo, 2006; Wu et al.,2006).
Caenorhabditis elegans overexpressing the human TAU or
a pseudohyperphosphorylated mutant TAU were found to exhibit
age-dependent motor neuron dysfunctions, neurodegeneration,
and locomotor defects due to impaired neurotransmission (Brandt
et al., 2007; Kraemer et al.,2003).
Likewise, while a few Huntingtin (Htt)-interacting genes
were identified in C. elegans (Chopra et al., 2000; Holbert
et al., 2003), most data came from transgenic worms expressing
polyQ variants of Htt. Several groups targeted different
neuronal subsets to study polyQHtt neurotoxicity in the worm.
They described behavioral defects prior to neurodegeneration
and protein aggregation and axonal defects and uncovered a role
for apoptosis in HD neurodegeneration (Bates et al., 2006; Faber
12
LEUNG ET AL.

et al., 1999; Holbert et al., 2003; Parker et al., 2001). Protective
mechanisms of the polyQ enhancer-1 and ubiquilin were
demonstrated (Faber et al. 2002; Wang et al., 2006), and
pharmacological screening using polyQHtt transgenic
C. elegans is ongoing (Faber et al. 2002; Wang et al., 2006).
A final illustration of the successful use of C. elegans in
elucidating the genetic basis of neurodegenerative disorder is
exemplified by the characterization of the genetic network
implicated in DMD. DMD is mainly characterized by
a progressive loss of muscular mass and function occurring
in males due to mutations in the DYSTROPHIN gene located
on the X chromosome, which commonly leads to paralysis and
death by the age of 30. DYSTROPHIN is both muscular and
neuronal, being required for brain architecture and neurotrans-
mission, such that DMD patients exhibit neurodegeneration
associated with motor deficits and reduced cognitive perform-
ances (average IQ is 85 in DMD boys) (Anderson et al., 2002;
Blake and Kroger, 2000; Poysky, 2007). DYSTROPHIN is
conserved in C. elegans, but its loss-of-function in the worm
results in hypercontractility due to impaired cholinergic activity
and does not affect muscle cells (Bessou et al., 1998; Gieseler
et al., 1999b). Nevertheless, the observation that double
mutants for Dystrophin/dys-1 and MyoD/hlh-1 display severe
and progressive muscle degeneration in the worm (as observed
in mice), set up the basis for a C. elegans model to study
dystrophin-dependent myopathies (Gieseler et al., 2000).
Using this model, several partners of DYSTROPHIN were
characterized, establishing their role in cholinergic neurotrans-
mission and muscle degeneration (Gieseler et al., 1999a,
1999b, 2001; Grisoni et al., 2002a, 2002b, 2003). Additionally,
it was shown that the overexpression of DYSTROBREVIN/
dyb-1 delays neurological and muscular defects (Gieseler et al.,
2002), and mutations in CHIP/chn-1, chemical inhibition of the
proteasome, and prednisone or serotonin treatments suppress
muscle degeneration in C. elegans (Carre-Pierrat et al., 2006;
Gaud et al., 2004; Nyamsuren et al., 2007).
Thus, though at first glance C. elegans appears quite
different from vertebrates, its nervous circuitry and the cellular
processes guiding neuronal development, neuronal death or
survival, neurotransmission, and signal integration rely on the
same neuronal and molecular networks as vertebrates.
Combined with the advantages of a small and fast-growing
organism, these properties make C. elegans a perfect system
for rapid genetic analysis of neurotoxicity mechanisms.
Caenorhabditis elegans AND GENOTOXICITY
As is the case for neurotoxicity, C. elegans provides a cost-
effective, in vivo, genetically manipulable and physiological
model for the study of the toxicological consequences of DNA
damage. As described below, the machinery that responds to
DNA damage in C. elegans is very similar genetically to the
corresponding machinery in higher eukaryotes. Many pro-
cesses related to DNA damage have been extensively studied in
C. elegans, providing an important biological context and clear
relevance to mechanistic studies. Finally, powerful tools for the
study of DNA damage, DNA repair, and mutations have been
developed in this organism.
DNA Damage Response Proteins Are Conserved
between C. elegans and Higher Eukaryotes
Genes and pathways involved in DNA repair in mammals
are generally well conserved in C. elegans (Boulton et al.,
2002; Hartman and Nelson, 1998; O’Neil and Rose, 2005).
Proteins involved in nucleotide excision repair, mismatch
repair, homologous recombination, and nonhomologous end
joining, for instance, are almost entirely conserved between
C. elegans, mouse, and human based on nucleotide sequence
homology (http://www.niehs.nih.gov/research/atniehs/labs/lmg/
dnarmd/docs/Cross-species-comparison-of-DNA-repair-genes.xls).
This is also true for proteins involved in many DNA repair–
related processes, such as translesion DNA polymerases,
helicases, and nucleases. Base excision repair proteins,
interestingly, show somewhat less conservation. While this
conservation is based in some cases only on sequence
homology, many of these proteins have now been biochemi-
cally or genetically characterized. Critically, proteins involved
in other DNA damage responses including apoptosis and cell
cycle arrest are also conserved in C. elegans and mammals
(Stergiou and Hengartner, 2004).
DNA Repair in C. elegans
Early studies on DNA repair in C. elegans were carried out
by Hartman and colleagues, who identified a series of
radiation-sensitive mutants (Hartman, 1985; Hartman and
Herman, 1982) and used an antibody-based assay to measure
induction and repair of ultraviolet (UV) radiation–induced
damage (Hartman et al., 1989). These and more recent studies
(Hyun et al., 2008; Meyer et al., 2007) have shown that
nucleotide excision repair is similar in C. elegans and humans
both in terms of conservation of genes and kinetics of repair.
Nucleotide excision repair is a critical pathway in the context
of exposure to environmental toxins since it recognizes and
repairs a wide variety of bulky, helix-distorting DNA lesions,
including polycyclic aromatic hydrocarbon metabolites, myco-
toxins such as aflatoxin B1, UV photoproducts, cisplatin
adducts, and others (Friedberg et al., 2006; Truglio et al.,
2006).
While nucleotide excision repair has been the best-studied
DNA repair pathway in C. elegans, significant progress has
been made in the study of genes involved in other DNA repair
pathways as well. The role of specific C. elegans gene products
in DNA repair has been studied both via high-throughput and
low-throughput methods. High-throughput methods including
CAENORHABDITIS ELEGANS IN TOXICOLOGY RESEARCH 13

RNAi knockdown and yeast two-hybrid analysis of protein-
protein interaction have been used to identify a large number of
genes coding for proteins involved in responding to DNA
damage (Boulton et al., 2002; van Haaften et al., 2004a,
2004b). Lower throughput studies involving biochemical
analyses of DNA repair activities (Dequen et al., 2005a;
Gagnon et al., 2002; Hevelone and Hartman, 1988; Kanugula
and Pegg, 2001; Munakata and Morohoshi, 1986; Shatilla
et al., 2005a, 2005b; Shatilla and Ramotar, 2002) as well
in vivo sensitivity to DNA damaging agents (Astin et al., 2008;
Boulton et al., 2004; Dequen et al., 2005b; Lee et al., 2002,
2004; Park et al. 2002, 2004; St-Laurent et al., 2007) or other
DNA damage–related phenotypes (Aoki et al., 2000; Kelly
et al., 2000; Sadaie and Sadaie, 1989; Takanami et al., 1998)
have supported the sequence similarity–based identification
of C. elegans homologues of DNA repair genes in higher
vertebrates, as well as in some cases permitting identification of
previously unknown genes involved in these pathways.
Apoptosis and Cell Cycle Checkpoints in C. elegans
DNA damage that is not repaired can trigger cell cycle arrest
and apoptosis, and these pathways are very well studied in
C. elegans. The great progress made in understanding them
mechanistically demonstrates the power of this model
organism. As mentioned, the cellular mechanisms regulating
apoptosis were discovered in C. elegans, and apoptosis and cell
cycle responses to DNA damage continue to be heavily studied
in C. elegans (Ahmed et al., 2001; Ahmed and Hodgkin, 2000;
Conradt and Xue, 2005; Gartner et al., 2000; Jagasia et al.,
2005; Kinchen and Hengartner, 2005; Lettre and Hengartner,
2006; Olsen et al., 2006; Schumacher et al., 2005; Stergiou
et al., 2007). The short life span of C. elegans has especially
lent itself to groundbreaking studies on the mechanisms of
germ line immortality (Ahmed, 2006; Ahmed and Hodgkin,
2000). Another important advantage of C. elegans is the ability
to easily study in vivo phenomena such as age- or de-
velopmental stage–related differences in DNA repair capacity.
For example, Clejan et al. (2006) showed that the error-prone
DNA repair pathway of nonhomologous end joining has little
or no role in the repair of DNA double-strand breaks in germ
cells but is functional in somatic cells. Holway et al. (2006)
showed that checkpoint silencing in response to DNA damage
occurs in developing embryos but not in the germ line. Both
these findings are important in our understanding develop-
mental exposure to genotoxins in that they suggest a special
protection for germ line cells.
DNA Damage–Related Pathological Processes
in C. elegans
DNA damage–related pathological processes including
carcinogenesis (He et al., 2007; Kroll, 2007; Pinkston-Gosse
and Kenyon, 2007; Poulin et al., 2004; Sherwood et al., 2005;
van Haaften et al., 2004a), aging (Antebi, 2007; Brys et al.,
2007; Hartman et al., 1988; Johnson, 2003; Kenyon, 2005;
Klass, 1977; Klass et al., 1983; Murakami, 2007; Rea et al.,
2007; Ventura et al., 2006), and neurodegenerative diseases
(described above) are also areas of active research in
C. elegans. This research has both established the relevance
of C. elegans as a model for the study of genotoxic agents (due
to conservation of the DNA damage response) and enormously
increased its utility in such studies by providing a wealth of
complementary and contextual biological information related
to the pathological responses to DNA damage in this organism.
Tools for the Study of DNA Damage, Repair, and
Mutation in C. elegans
Caenorhabditis elegans is an excellent model for studies of
genotoxicity due to the plethora of powerful tools available.
Genetic manipulation via RNAi and generation of KOs or other
mutants is relatively straightforward. If suitable mutants are not
already available, they can be generated by a variety of
approaches. These include untargeted and targeted methods,
including chemical mutagenesis, transposon insertion, and
biolistic transformation (Anderson, 1995; Barrett et al., 2004;
Berezikov et al., 2004; Plasterk, 1995; Plasterk and Groenen,
1992; Rushforth et al., 1993).
Assays for the measurement of mutagenesis, DNA damage
and repair, and transcriptional activity have also been de-
veloped for genotoxicity assessment in C. elegans (Table 5).
Some DNA damage and repair assays in C. elegans can be
carried out with as few as one or a few individual nematodes,
permitting studies of interindividual differences and permitting
high-throughput screening of DNA- damaging agents or genes
involved in DNA repair. It is also possible, using PCR- or
Southern blot–based methods, to distinguish damage and repair
in different genomic regions and genomes (i.e., mitochondrial
vs. nuclear DNA; (Hyun et al., 2008; Meyer et al., 2007)).
Mutagenesis has been studied by a variety of methods (Table 5)
including phenotype-based genetic mutation reversion
screens, an out-of-frame LacZ transgene reporter, and direct
sequencing.
Genotoxin Studies in C. elegans
Unlike the case of neurotoxicology, there have so far been
relatively few studies of genotoxicity per se using C. elegans.
One exception has been the study of UV radiation, typically
as a model genotoxin that introduces bulky DNA lesions (Astin
et al., 2008; Coohill et al., 1988; Hartman, 1984; Hartman
et al., 1988; Hyun et al., 2008; Jones and Hartman, 1996;
Keller et al., 1987; Meyer et al., 2007; Stergiou et al., 2007;
Stewart et al., 1991). However, other classes of genotoxins
have been studied, including ionizing radiation (Dequen et al.,
14
LEUNG ET AL.

2005a; Johnson and Hartman, 1988; Stergiou et al., 2007;
Weidhaas et al., 2006), heavy metals (Cui et al., 2007b; Neher
and Sturzenbaum, 2006; Wang et al., 2008), methylmethane-
sulphonate (Holway et al., 2006), polycyclic aromatic hydro-
carbons (Neher and Sturzenbaum, 2006), photosensitizers
(Fujita et al., 1984; Hartman and Marshall, 1992; Mills and
Hartman, 1998), and prooxidant compounds (Astin et al.,
2008; Hartman et al., 2004; Hyun et al., 2008; Salinas et al.,
2006). Studies have taken advantage of the utility of C. elegans
as an in vivo model; for example, it was shown that nucleotide
excision repair slowed in aging individuals (Meyer et al., 2007)
and that longer lived and stress-resistant strains have faster
nucleotide excision repair (Hyun et al., 2008) than do wild
type. It has been possible to identify cases in which UV
resistance was correlated to life span (Hyun et al., 2008;
Murakami and Johnson, 1996), and others in which it was not
(Hartman et al., 1988), so that theories about the relationship of
DNA damage and repair with aging can be directly tested.
Studies of aging populations or individuals are slow and
expensive in mammalian models and impossible in vitro.
High-Throughput Approaches with C. elegans
High-throughput screening has two specific definitions in
toxicology: (1) genome-wide screens for molecular targets or
mediators of toxicity and (2) rapid, high-content chemical
screens to detect potential toxicants. A genome-wide screen
can serve as a hypothesis-finding tool, providing a direction for
further mechanistic investigation. This approach is particularly
useful for studying any toxicant with a poorly understood
mechanism of action. Genome-wide screens can be done using
forward genetics, DNA microarrays, or genome-wide RNAi in
C. elegans.
High-throughput chemical screening, in comparison, has
been proposed as a quicker and less expensive method for
toxicity testing (Gibb, 2008). The conventional animal testing
used by companies or agencies is labor intensive and time
consuming, resulting in a large number of toxicants not being
tested at all. It is estimated, for instance, that there are more
than 10,000 environmental chemicals from several Environ-
mental Protection Agency programs that require further testing
(Dix et al., 2007). The objective of high-throughput chemical
screening is to shortlist chemicals showing high toxicity,
thereby setting priority for regulations as well as further
toxicity testing in mammalian models.
High-throughput screening is feasible with C. elegans due to
its experimental manipulability as well as several automation
technologies. Caenorhabditis elegans is easy to handle in the
laboratory; it can be cultivated on solid support or in liquid, in
Petri dishes, tubes, or 6-, 12-, 24-, 96-, or 384-well plates. It
can also be exposed to toxicants acutely or chronically by
injection, feeding, or soaking. Automated imaging methods
for absorbance, fluorescence, movement, or morphometric
TABLE 5
Genotoxicity Assays Available for the Caenorhabditis elegans Model
Endpoint Assay Principle References
A. Mutagenesis Direct sequencing The mutation rate of a given locus is calculated
using data from DNA sequencing.
Denver et al. (2000, 2004, 2006)
‘‘Big blue worms’’ Transgenic C. elegans carrying an out-of-frame
LacZ reporter gene expresses blue pigment upon
frameshift or insertion/deletion mutations.
Pothof et al. (2003);
Tijsterman et al. (2002)
Reversion assay Mutants with an easily scored phenotype (e.g.,
uncoordinated movement) are exposed to a
chemical of interest; the restoration of a normal
phenotype indicates mutagenesis.
Degtyareva et al. (2002);
Greenwald and Horvitz (1980);
Hartman et al. (1995)
Lethality assay The lethality of transgenic, mutation-sensitive
C. elegans was measured for mutagen detection
Rosenbluth et al. (1983);
Rosenbluth et al. (1985)
B. DNA damage and repair PCR-based assay The amount of PCR product is inversely proportional to
the amount of DNA damage on a given length of template
Meyer et al. (2007); Neher
and Sturzenbaum (2006)
Southern blot T4 endonuclease–sensitive sites in specific genes
(identified by genomic DNA sequence) indicate
the presence of UV photodimers
Hyun et al. (2008)
Immunoassay Antibodies to specific UV photoproducts are identified Hartman et al. (1989)
Enzymatic activity A diagnostic enzymatic activity is measured in vitro Shatilla and Ramotar (2002)
Reproduction/development
assay with KO mutants
Specific DNA damage (e.g., DNA adduct) can be
tested using simple reproduction/development
assays with mutants lacking a specific
DNA repair pathway (e.g., nucleotide excision repair)
Park et al. (2002, 2004)
C. Transcriptional activities RNA: DNA ratio A decrease in RNA: DNA ratio indicates the
inhibition of transcriptional activities
Ibiam and Grant (2005)
CAENORHABDITIS ELEGANS IN TOXICOLOGY RESEARCH
15

measurement have been developed since the late 1980s (Baek
et al., 2002; Bennett and Pax, 1986; Hoshi and Shingai, 2006;
Simonetta and Golombek, 2007; Tsibidis and Tavernarakis,
2007; Williams and Dusenbery, 1990b). Nowadays, cell sorters
adapted to sort worms based on morphometric parameters or
expression of fluorescent proteins combined with imaging
platforms have been successfully used for large-scale promoter
expression analyses and drug screening purposes (Burns et al.,
2006; Dupuy et al., 2007; Pulak, 2006). Recently, a micro-
fluidic C. elegans sorter with three dimensional subcellular
imaging capabilities was developed, allowing high-throughput
assays of higher complexity (Rohde et al., 2007).
While the simplicity and manipulability of the C. elegans
system enables high-throughput approaches, it also leads to several
potential disadvantages in toxicology studies. Caenorhabditis
elegans exhibits important metabolic differences compared
to vertebrates. For example, C. elegans is highly resistant to
benzo[a]pyrene (Miller and Hartman, 1998), likely because it
does not metabolize the chemical (M. Leung and J. Meyer,
unpublished data). This problem can be potentially solved,
however, by expressing the vertebrate cytochrome P450s in C.
elegans. The impermeable cuticle layer as well as selective
intestinal uptake, furthermore, may block the entry of chemicals,
thereby necessitating high exposure doses to impact the worm’s
physiology. A mutant strain (dal-1) has recently been isolated that
is healthy under laboratory conditions but exhibits altered
intestinal morphology and increased intestinal absorption of
a wide range of drugs (C. Paulson and J. Waddle, personal
communication). The resultant-increased vulnerability of this
strain to the toxic or pharmacological activities of tested
compounds has the potential to increase the sensitivity of the
C. elegans system.
Forward Genetics Screens in C. elegans
Forward genetics refers to the study of genes based on
a given phenotype. In a forward genetics screen, C. elegans are
treated with a mutagen, as described above. Mutant strains are
then exposed to a toxicant and are screened for increased
resistance or sensitivity. Once a resistant or hypersensitive
mutant is identified, the mutation is located using two-point
and three-point mapping and confirmed using single-gene rescue
or RNAi phenocopying (Hodgkin and Hope, 1999). Forward
genetics is efficient in C. elegans because the mutants can cover
genes expressed in a variety of tissues. Caenorhabditis elegans is
hermaphroditic, so homozygous mutant strains can be produced
in the F
2
generation via self-crossing.
Forward genetics screens are a useful method in mechanistic
toxicology. Griffitts et al. (2001, 2005), for instance,
discovered the role of glycolipid receptors and carbohydrate
metabolism in Bacillus thuringiensis (Bt) toxins using
C. elegans subjected to a forward genetics screen. The mutation
of glycolipid receptors prevents Bt toxin from entering intestinal
epithelium in C. elegans. Such a tissue-specific mechanism
would have been difficult to detect using in vitro cell cultures.
Gene Expression Analysis in C. elegans
Caenorhabditis elegans has several advantages over other
species in gene expression analysis. WormBase (Harris et al.,
2004), the information-rich central genomic database of
C. elegans, provides an intuitive interface into a well-annotated
genome. Caenorhabditis elegans also has a consistent system
of gene identification, thereby avoiding the confusion of gene
identification that is common in many species, including
human. The interactome modeling of C. elegans is also the
most developed among all animal species (Dupuy et al., 2007;
Li et al. 2004, 2008; Zhong and Sternberg, 2006) and along
with other genome-level bioinformatics tools (Kim et al., 2001)
greatly facilitates system-based analysis.
The results of gene expression analysis can be validated
in vivo using mutational or transgenic approaches in
C. elegans. For example, the gene expression of C. elegans
exposed to ethanol, atrazine, polychlorinated biphenyls,
endocrine disrupting chemicals, and polycyclic aromatic
hydrocarbons have been profiled (Custodia et al., 2001; Kwon
et al., 2004; Menzel et al., 2007; Reichert and Menzel, 2005).
Follow-up studies with transgenic C. elegans expressing
fluorescent markers were used to detect overexpression of
protein in specific tissues in vivo (Menzel et al., 2007; Reichert
and Menzel, 2005). Mutant C. elegans were also used to
confirm the role of specific molecular targets based on gene
expression analysis (Menzel et al., 2007).
Genome-Wide RNAi Screens in C. elegans
The discovery of RNAi mechanisms in C. elegans for which
the 2006 Nobel Prize was awarded (Fire et al., 1998) and the
complete sequencing of the nematode genome (C. elegans
Sequencing Consortium, 1998) led to the generation of
publically available RNAi libraries covering ~90% of its genes
(Fewell and Schmitt, 2006; Kamath and Ahringer, 2003).
Strategies to improve RNAi efficiency, especially in neurons,
were further developed (Esposito et al., 2007; Lee et al., 2006;
Simmer et al. 2002, 2003; Tabara et al., 2002; Tops et al.,
2005). RNAi can be triggered by injection of worms with
interfering double-strand RNA (dsRNA), by feeding them with
transgenic bacteria producing the dsRNA or by soaking them in
a solution of dsRNA. The latter allow timed RNAi exposure and
genome-wide screens in 96- or 384-well plates with liquid worm
cultures and have contributed to discoveries of mechanisms of
axon guidance as well as mitochondrial involvement in oxidative
stress and aging (Ayyadevara et al., 2007; Hamamichi et al.,
2008; Hamilton et al., 2005; Ichishita et al., 2008; Lee et al.,
2003; Schmitz et al., 2007; Zhang et al., 2006).
16
LEUNG ET AL.

A genome-wide RNAi screen typically assesses a number of
physiological parameters at the same time, such as viability,
movement, food intake, and development, thereby facilitating
the interpretation of screening results. While most RNAi screens
have been done in wild-type C. elegans, some are performed
using KO mutants to provide more sensitive or selective assays
(Kaletta and Hengartner, 2006). Genome-wide RNAi screens are
becoming a method of choice for discovering gene function. A
recent study by Kim and Sun (2007), for example, identified
anumberofdaf-2-dependent and nutrient-responsive genes that
are responsive to paraquat-induced oxidative stress.
High-Content Chemical Screens
The use of C. elegans as a predictive model for human
toxicity was first proposed in the context of heavy metals
(Williams and Dusenbery, 1988). The C. elegans assay was
validated as a predictor of mammalian acute lethality using
eight different metal salts, generating LC
50
values parallel to
the rat and mouse LD
50
values. A later study investigated the
acute behavioral toxicity of 15 OP pesticides in C. elegans
(Cole et al., 2004). The toxicity of these pesticides in
C. elegans was found to be significantly correlated to the
LD
50
acute lethality values in rats and mice. Several other
studies have also validated a number of C. elegans–based
assays for predicting neurological and developmental toxicity
in mammalian species (Anderson et al., 2004; Dhawan et al.,
1999; Tatara et al., 1998; Williams et al., 2000).
A C. elegans–based, high-throughput toxicity screen was first
published by the Freedman group at National Institute of
Environmental Health Sciences (Peterson et al., in press);
additional groups including industry and government groups in
the United States and elsewhere are also carrying out high-
throughput toxicity screening. Screens are typically conducted
on a 96-well plate with a robotic liquid handling workstation
(Biosort, Union Biometrica, Inc., Holliston, MA) to analyze the
length, optical density, motion, and fluorescence of C. elegans.
Caenorhabditis elegans is cultured in liquid from fertilized egg
to adult through four distinct larval stages. The development,
reproduction, and feeding behaviors of the C. elegans culture in
response to different chemical exposures are characterized. The
screen has been validated by the Freedman group with 60
chemicals including metals, pesticides, mutagens, and nontoxic
agents (Peterson et al., in press).
The high-throughput toxicity screen is being further
improved with additional genetics and automation techniques.
The generalized stress response of C. elegans, for instance, was
visualized with transgenic GFP constructs, providing a more
sensitive end point for toxicity screens (Dengg and van Meel,
2004; Roh et al., 2006). Nematode locomotion can be tracked
automatically, providing a more sensitive screen of neurotox-
icity (Cole et al., 2004; Williams and Dusenbery, 1990b).
Transgenic or mutant C. elegans can also be used in the high-
throughput screen to detect specific modes of action, including
metal response (Cioci et al., 2000), oxidative stress (Hasegawa
et al., 2008; Leiers et al., 2003), and DNA damage (Denver
et al., 2006). A microfluidic C. elegans sorter with three-
dimensional subcellular imaging capabilities was recently
reported, thereby allowing high-throughput assays of higher
complexity (Rohde et al., 2007).
Environmental Assessment of Chemical Exposure
Nematodes are the most abundant animal in soil ecosystems
and also found in aquatic and sediment environments. They
serve many important roles in nutrient cycling and in
maintaining environmental quality. These features have
supported their use in ecotoxicological studies and, from the
late 1970s, a variety of nematode species have been used to
study environmental issues. During the late 1990s, C. elegans
began to emerge as the nematode species of choice based on
the tremendous body of knowledge developed by basic
scientists using this model organism for biological studies.
Although generally considered a soil organism, C. elegans
lives in the interstitial water between soil particles and can be
easily cultured within the laboratory in aquatic medium. The
majority of environmental studies have been performed in an
aquatic medium, given its ease of use, and as toxicological end
points have been developed, the assessment tools have been
applied to sediment and soil medium which allows for a more
relevant direct environmental comparison.
The environmental toxicological literature using C. elegans
is extensive and Table 6 provides an overview of laboratory-
based studies where a toxicant of environmental interest has
been added to a medium (water, sediment, or soil) followed by
exposure to C. elegans and the assessment of an adverse effect.
In a limited number of situations, C. elegans testing has been
used to assess contamination in field settings (Table 7). Much
of the early work explored metal toxicity and used lethality as
an endpoint. Over time, a wider variety of toxicants have been
tested and more sophisticated sublethal end points have been
developed including the use of transgenic strains with specific
biomarkers (Candido and Jones, 1996; Chu et al., 2005; Dengg
and van Meel, 2004; Easton et al., 2001; Mutwakil et al., 1997;
Roh et al., 2006), growth and reproduction (Anderson et al.,
2001; Hoss and Weltje, 2007), feeding (Boyd et al., 2003), and
movement (Anderson et al., 2004). These types of end points
developed through environmental studies are directly applica-
ble to the use of the organism as an alternative for mammalian
testing.
Two of the principal limitations in using C. elegans in
environmental testing are concerns related to its comparison to
other nematodes and reliable and simple methods for extracting
them from soil and sediments. Given the almost countless
variety of nematodes, it is impossible for one species to be
representative of the entire Nematoda phylum. Limited studies
CAENORHABDITIS ELEGANS IN TOXICOLOGY RESEARCH 17

comparing the toxicological effects between nematodes species
indicate that C. elegans is as representative as any of the ones
commonly used and, in many cases, little difference in
response has been found between species (Boyd and Williams,
2003; Kammenga et al., 2000). Further, this organism is much
more thoroughly understood and benefits from its ease of use.
TABLE 7
Examples of Field Studies Using Caenorhabditis elegans to Assess Environmental Samples
Field site Environmental medium Overview References
Carnon River
system (England)
Water Transgenic strains of C. elegans that carry stress-inducible
lacZ reporter genes were used to assess metal
contamination of a river system.
Mutwakil et al. (1997)
Wastewater treatment
process (Georgia)
Water discharges from industrial
operations and a municipal
treatment plant
The contribution of several industrial operations to the waste
stream feeding a municipal wastewater treatment plant
and the treatment plant’s discharge were assessed to
identify sources of water contamination and effectiveness of
waste treatment. The 72-h mortality was used as end point.
Hitchcock et al. (1997)
Elbe River (Germany) Sediments Tested polluted sediments using growth and fertility as
end points.
Traunspurger et al. (1997)
Twelve freshwater
lakes (Germany)
Fresh water sediment Evaluated 26 sediment samples from unpolluted lakes
in southern Germany to determine the effect of
sediment size and organic content on growth and fertility.
Hoss et al. (1999)
Middle Tisza River flood
plain (Hungary)
Soil Following a major release of cyanide and heavy metals from a mine
waste lagoon in Romania, soil contamination was assessed
following a 100-year flood event using mortality as end point.
Black and Williams (2001)
Agricultural soil (Germany) Soil Assessed the toxicity of soil from fields cultivated with
transgenic corn (Bt corn; MON810) compared to
isogenic corn. Growth and reproduction used as end points.
Hoss et al. (2008)
TABLE 6
Representative Laboratory Studies Evaluating Environmentally Relevant Toxicants
Medium End point (test duration) Chemicals tested/comments References
A. Aquatic Lethality (24–96 h) Tested metallic salts of 14 metals (Ag, Hg, Be, Al, Cu, Zn, Pb, Cd,
Sr, Cr, As, Tl, Ni, Sb). Established initial aquatic testing procedures
and compared results to traditionally used aquatic invertebrates.
Williams and Dusenbery (1990a)
Lethality and stress reporter
gene induction (8–96 h)
Assessed the induction of hsp16-lacZ and lethality in C. elegans
exposed to water-soluble salts of Cd, Cu, Hg, As, and Pb.
Stringham and Candido 1994
Growth, behavior, feeding, and
reproduction (4–72 h)
Compared a number of sublethal end points and found feeding and
behavior to be the most sensitive. Tested metallic salts Cd, Cu, and Pb.
Anderson et al. (2001)
Feeding and movement (4–24 h) Determined changes in ingestion using microbeads and movement in
the presence of metals and varying availability of food
Boyd et al. (2003)
Behavior (4 h) Tested a variety of toxicants from several categories of chemicals including
metals, pesticides, and organic solvents. Established the use of a 4-h
exposure period for behavioral assessments.
Anderson et al. (2004)
Reproduction (96 h) Evaluated the effects on reproduction of several endocrine disruptors. Hoss and Weltje (2007)
B. Sediment Growth (72 h) CuSO
4
in spiked water added to whole sediments and refined
method for using organism in sediments.
Hoss et al. (1997)
Growth (72 h) Spiked natural sediments with CdCl
2
and extracted pore water to
determine effects.
Hoss et al. (2001)
C. Soil Lethality (24 h) Spiked soil with CuCl
2
and developed the recovery method used with
C. elegans exposed in soil.
Donkin and Dusenberry (1993)
Lethality (24 h) Tested metallic salts of five metals (Cu, Cd, Zn, Pb, Ni) in artificial
soil. Compared C. elegans data to earthworm data from same medium.
Determined that 24-h exposures for the nematode had similar effects
to 14-day exposures with earthworms.
Peredney and Williams (2000)
Lethality (24–48 h) Tested seven organic pollutants (four azarenes, one short-chain chlorinated
paraffin, and two organochlorinated pesticides) in soil, aquatic, and agar
and compared results across media.
Sochova et al. (2007)
18 LEUNG ET AL.

Much progress has been made to develop better methods to
extract the worm from soil and sediments. The initial method
developed by Donkin and Dusenbery (1993) has led to
a standardized soil toxicological testing method adopted in
2001 by the American Society for Testing and Materials
(ASTM, 2002) and recently the International Standards
Organization in Europe (ISO 2007). The initial extraction
method has been improved through the use of transgenic
strains of nematodes (Graves et al., 2005) which allows for
GFP-labeled worms to be used that distinguishes the worms
being tested in soils from the large numbers of indigenous
species that are similar in size and appearance. It also makes
easier removal from soil with high organic content. All this
work has led to more interest in using C. elegans in
environmental studies.
CONCLUSION: THE ROLE OF C. elegans IN
TOXICOLOGY RESEARCH
The unique features of C. elegans make it an excellent model
to complement mammalian models in toxicology research.
Experiments with C. elegans do not incur the same costs as
experiments with in vivo vertebrate models, while still
permitting testing of hypotheses in an intact metazoan
organism. The genetic tools available for C. elegans make it
an excellent model for studying the roles of specific genes in
toxicological processes and gene-environment interactions,
while the life history of this organism lends itself to high-
throughput analyses. Thus, C. elegans represents an excellent
complement to in vitro or cell culture–based systems and in
vivo vertebrate models.
FUNDING
National Institute of Environmental Health Sciences 10563;
Department of Defense W81XWH-05-1-0239; the Gray E.B.
Stahlman Chair of Neuroscience.
ACKNOWLEDGMENTS
We thank Windy A. Boyd, Richard T. Di Giulio, and
Jonathan H. Freedman for advice and assistance in the
preparation of the manuscript.
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