A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay
Introduction
The National Research Council and government agencies including the National Institutes of Health and the U.S. Environmental Protection Agency have defined a need for reliable high-throughput screening (HTS) methods to evaluate the potential human health impacts of the large number of chemicals in production (National Research Council, 2000, Dix et al., 2007). Although rodent-based assays have been the traditional model for toxicological studies, many agencies are now recognizing the advantages of in vivo whole organism studies using invertebrate species. These advantages include rapid and inexpensive testing, as well as a lack of animal welfare issues. Although in vitro cell-based HTS assays are commonly used, whole organism testing allows researchers to observe phenotypes that are well characterized and biologically relevant.
The nematode Caenorhabditis elegans, a popular model organism for genetic and developmental biology research, is now being recognized as an attractive invertebrate model for high-throughput toxicological studies. C. elegans has a rapid and well-characterized life cycle and can be cultured in multi-well plates, making them amenable to HTS. There is also a high degree of conservation between C. elegans and mammalian species in processes controlling development, neurobiology, and stress responses (Kaletta and Hengartner, 2006). For these reasons, several pharmaceutical companies are using C. elegans as part of their drug discovery process (Artal-Sanz et al., 2006).
One easily quantifiable phenotype in C. elegans is reproduction. C. elegans develop from embryo to gravid adult through four distinct larval stages, termed L1–L4, in about three days at 20 °C (Wood, 1988). At the L4 stage, germ cells within C. elegans hermaphrodites mature to sperm, while in the adult stage germ cells mature to oocytes. Following internal fertilization, the developing embryos are released by muscle contractions of the vulva, which are regulated by specific neurons that release serotonin, acetylcholine, or neuropeptides (Trent et al., 1983, Weinshenker et al., 1995, Bany et al., 2003).
Reproduction and egg-laying are affected by a number of environmental conditions including salt concentration (Horvitz et al., 1982) and food availability (Trent et al., 1983). Exposure to ethanol (Dhawan et al., 1999), metals (Anderson et al., 2001), Enterobacteriaceae (Sicard et al., 2007), anthelmintic agents (Kim et al., 2001), and nicotinic agonists (Kim et al., 2001, Bull et al., 2007) also affect reproduction. Because such a wide variety of toxicants impact C. elegans reproduction, it is a promising endpoint for HTS. Egg-laying and reproduction have been measured in lowthroughput assays by placing one to several nematodes on the surface of an agar plate or in liquid media. After exposure, the number of offspring was manually counted with the aid of a microscope. Automated tracking systems and image analysis have also been used to monitor the frequency of egg-laying behavior on agar surfaces over several minutes to hours (Kim et al., 2001, Davies et al., 2003, Geng et al., 2005). A more high-throughput, but indirect approach to quantify reproduction, measures chitinase, which is released during embryo hatching (Kaletta and Hengartner, 2006).
In this report, an automated HTS method is described that directly quantifies the number of C. elegans offspring after exposure to potential toxicants. In this assay, a COPAS Biosort (Pulak, 2006) is used to load L4 hermaphrodite nematodes into each well of a 96-well plate containing test chemicals. Following a 48-h incubation, the number of offspring in each well is quantified using the Biosort. In the presence of the test chemicals, there were toxicant and concentration dependent decreases in the level of reproduction. These decreases could be the result of reducing the number of sperm or oocytes, disrupting germ cell maturation, affecting egg-laying behavior, or increasing embryonic or larval lethality after egg-laying. The strength of this assay was evaluated by comparing the toxicity of several different classes of chemicals in the C. elegans assay to toxicity measures in mice and rodents.
Section snippets
Nematode culture
The Bristol N2 (wild-type) and CB5584 mIs12[myo-2::GFP, pes-10::GFP, F22B7.9::GFP] (referred to as myo-2::GFP) strains of C. elegans were obtained from the Caenorhabditis Genetic Center (Minneapolis, MN) and maintained at 20 °C on K-agar plates (2% bacto-agar, 0.25% bacto-peptone, 51 mM sodium chloride, 32 mM potassium chloride, 13 µM cholesterol) seeded with Escherichia coli OP50 (Williams and Dusenbery, 1988). Age-synchronized adult nematodes were prepared as previously described (Khanna et al.,
Reproduction of untreated C. elegans
In the C. elegans reproduction assay, nematodes are exposed to chemicals from their last larval (L4) through the adult stage, and then their offspring are counted using a COPAS Biosort. Two strains of C. elegans that are routinely used in toxicological assays in our laboratory were examined: wild-type and myo-2::GFP. The myo-2::GFP strain, which has a fluorescently-labeled pharynx, is being used in the development of additional toxicological HTS assays, including motion tracking assays and
Discussion
The C. elegans reproduction assay described in this report used the COPAS Biosort to count the number of offspring produced between the L4 larval stage and adulthood. This period was chosen to coincide with the developmental stage when the number of germ cells reached its maximum, but before the embryonic membrane became impermeable (Anderson, 1995). Before testing chemical toxicity, preliminary experiments focused on optimizing the duration of the chemical exposure, the amount of bacterial
Conclusion
To assess the large number of chemicals that are lacking sufficient toxicity data, international agencies are exploring the use of high-throughput screening and testing using alternative model organisms. When using alternative model organisms it is necessary to consider the advantages and limitations of each system. The C. elegans reproduction assay is rapid, reproducible, and responses can be observed for a wide range of chemicals. In the future, C. elegans responses to additional toxicant
Acknowledgments
This work was supported in part by the National Toxicology Program, and by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (Z01ES102045 and Z01ES102046). Nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). The authors would like to thank Dr. Grace E. Kissling, Biostatistics Branch, NIEHS, for statistical advice.
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