ToxSci Advance Access originally published online on June 19, 2008
Toxicological Sciences 2008 106(1):5-28; doi:10.1093/toxsci/kfn121
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Caenorhabditis elegans: An Emerging Model in Biomedical and Environmental Toxicology
* 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
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37240
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{at}duke.edu.
Received April 30, 2008; accepted June 10, 2008
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ABSTRACT |
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ABSTRACT
INTRODUCTIONThe 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.
Key Words: Caenorhabditis elegans; neurotoxicity; genotoxicity; environmental toxicology; high-throughput methods.
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INTRODUCTION |
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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.
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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 (300+) 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.
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Since reverse genetic and transgenic experiments are much easier and less expensive to conduct in C. elegans as compared 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 serotonergic 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 experiments 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.
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Caenorhabditis elegans AND NEUROTOXICITY |
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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 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 neuromuscular junctions (Chen et al., 2006). Additionally, the main neurotransmitter systems (cholinergic, 
-aminobutyric acid (GABA)ergic, glutamatergic, DAergic, and serotoninergic) and their genetic networks (from neurotransmitter metabolism to vesicle cycling and synaptic transmission) are phylogenetically 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 
1 and β (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 mechanosensory functions of glutamatergic neurons are evaluated by measuring the pharyngeal pumping rate and the response to touch. Mechanosensory functions of DAergic and serotoninergic 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 conducted 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 (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 transposons (discussed below under Caenorhabditis elegans and Genotoxicity). Hence, classical approaches to elucidate intracellular 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 morphology. 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 (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 (Ca2+) 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). Interestingly, 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 Ca2+ channels. In neurons, this blockage can induce spontaneous release of neurotransmitters (Atchison, 2003). In C. elegans, the Ca2+ channel blocker verapamil was found to protect against Hg exposure, suggesting that Ca2+ 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.
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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 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 overexpressing 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 neurotransmitter 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 LD50 values in C. elegans closely correlated with LD50 in both rats and mice. Pesticides (vapon, parathion, methyl parathion, methidathion, and funsulfothion) that showed cholinesterase inhibition were associated 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 disorders. 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, deposition of cytoplasmic Lewy bodies composed of aggregated protein, such as -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 neurotoxicants (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 (-SYNUCLEIN), PARK2 (PARKIN), PARK4 (-SYNUCLEIN), PARK5 (UCHL1), PARK6 (PINK1), PARK7 (DJ1), PARK8 (DARDARIN/LRRK2), and PARK9 (ATP13A2) (Wood-Kaczmar et al., 2006). All but -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 -SYNUCLEIN in DAergic neurons show differential levels of injury, including reduced DA content, DAergic neuron degeneration, motor deficits reversible by DA administration, intracellular -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 -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 -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 (MPP+) by the monoamine oxygenase B. MPP+ damages the DAergic nervous system, leading to a typical Parkinsonian syndrome (Kopin and Markey, 1988; Langston et al., 1984). Similarly, MPP+-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 identified 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 characterization 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 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 neurotransmission, such that DMD patients exhibit neurodegeneration associated with motor deficits and reduced cognitive performances (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 neurotransmission 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.
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Caenorhabditis elegans AND GENOTOXICITY |
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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 processes 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 biochemically 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, mycotoxins 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 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 developmental 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 developmental 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 developed 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.
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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., 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), methylmethanesulphonate (Holway et al., 2006), polycyclic aromatic hydrocarbons (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 |
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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