A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.
Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.
Toxicological evaluation is essential for the development of pharmaceuticals, nutraceuticals, and cosmeceuticals, as well as the risk assessment of various environmental toxins. The rodent model is one of the most popular in vivo experimental systems for this toxicology study; alternatively, non-mammalian organisms such as C. elegans are also widely used. Non-mammalian toxicological evaluation models are beneficial because of not only animal ethical issues but also their convenience and usefulness considering cost-effectiveness, maintainability, speed, and reproducibility1,2,3,4.
C. elegans, a soil round worm, has been exploited as a model animal in various basic and applied biology and chemistry research. It is a 1 mm long, transparent nematode, which is simply maintained in solid or liquid Nematode Growth Media (NGM) fed with the bacterial strain Escherichia coli OP50. C. elegans has a short life cycle, and wild-type N2 C. elegans lays approximately 300 eggs. Therefore, it is easily propagated to be used as experimental materials3,4,5. C. elegans has also been widely used in the toxicological studies of many drugs and environmental pollutants6,7,8,9.
Because many anticancer drugs target rapidly dividing cancer cells, they can also damage rapidly dividing normal cells such as bone marrow, intestinal epithelium, and hair follicle cells. For example, topoisomerase inhibitory anticancer drugs target the DNA replication process of cancer cells; therefore, they also inhibit rapidly dividing normal cells. Every living organism has topoisomerases, and these topoisomerase inhibitors most likely impact environmental ecosystems6,10,11. Thus, a drug toxicological evaluation platform using a model animal is valuable for both the development of pharmaceuticals and environmental risk assessment.
In this article, we describe the detailed protocols to test the toxicity of etoposide, which is a clinical anticancer agent that targets topoisomerase II, as a model toxic chemical in C. elegans. For this purpose, we will describe the measurement method of body size and the total number of eggs laid in C. elegans treated with etoposide.
NOTE: The entire experiment must be performed in a clean isolated laboratory maintained at 20 °C with low dust and with minimization of contamination during worm and bacterial handling. For this purpose, experiments must be performed under the flame of an alcohol lamp or using a clean bench.
1. Maintenance of C. elegans and Egg Preparation for the Chemical Test
2. Preparation of Heat-inactivated E. coli OP50 to Feed C. elegans During the Toxicity Test
3. Preparation of NGM Plates Containing Either 1% DMSO or 750 µM Etoposide
4. Measurement of C. elegans Growth
5. C. elegans Egg Laying Assay
The treatment of etoposide (24-96 h) significantly retarded the growth of C. elegans. After 96 h of incubation, etoposide-treated worms grew to 0.86 mm in body length, while the vehicle-treated worms grew to 1.04 mm (Figure 1). Growth retardation was also apparently observed under stereo microscope observation (Figure 2). We started to see eggs from the vehicle-treated worms at 72 h of incubation. On the other hand, eggs were observed after 96 h of etoposide treatment. From these data, we speculated that etoposide treatment delayed the first birth time of C. elegans.
In addition to the delay of egg laying, etoposide treatment significantly decreased the total number of eggs laid per worm (Figure 3). The reproductive toxicity, the decrease of total egg number by etoposide treatment, was observed when the young adult worms were cultured in the presence (Figure 3B) or absence (Figure 3A) of continuous etoposide treatment. There were no significant differences between the control-treated worms under both experimental conditions. The total number of eggs from worms treated for a certain period of time (from eggs to the young adult stage) was not significantly different from that of worms continuously treated with etoposide. For this comparison, statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Tukey's multiple comparison test.
Figure 1: Measurement of growth in C. elegans treated with etoposide. Age-synchronized C. elegans eggs were grown on NGM plates supplemented with DMSO (1%, the vehicle control) or etoposide (750 µM) for 24-96 h. Photomicrographs (shown in Figure 2) were taken every day, and the body length was measured using ImageJ software. The data are expressed as the mean ± standard deviation (SD) (n = 50, 50 worms per group). *p <0.01, for significant differences between the vehicle control and etoposide treatment at each time point. Statistical analysis was performed using the Student's t-test. Please click here to view a larger version of this figure.
Figure 2: Stereo microscope images of C. elegans treated with etoposide. C. elegans were treated as described in Figure 1 (Scale bar = 1 mm). Please click here to view a larger version of this figure.
Figure 3: Total number of eggs laid from C. elegans treated with etoposide. Age-synchronized C. elegans eggs were incubated on NGM plates containing DMSO (1%, the vehicle control) or etoposide (750 µM) for 64 h. Next, the total number of eggs laid was observed in the absence (A) or presence (B) of continuous chemical treatment for 5 days. Worms were transferred every day to the normal NGM plate (A), or NGM plate supplemented with the same chemicals: 1% DMSO or etoposide (750 µM) (B). The number of eggs laid was counted and divided by the total number of worms every day to calculate the number of eggs laid per worm. All the numbers of eggs per worm were summed for 5 days. The data are expressed as the mean ± SD from quadruplicate experiments. *p <0.01, for significant differences between the vehicle control and etoposide treatment. Statistical analysis was performed using the Student's t-test. Please click here to view a larger version of this figure.
Normal NGM agar media – 1,000 mL for maintenance, and egg laying assay without continuous chemical treatment |
1. Add 2.5 g of peptone, 3 g of NaCl, 17 g of agar, and 975 mL of distilled water into a glass bottle. |
2. Autoclave for 15 min at 121 °C. |
3. Cool down in a water bath for 30 min at 55 °C, and then add 1 mL of 1 M CaCl2, 1 mL of 5 mg/mL cholesterol (dissolved in ethanol), 1 mL of 1 M MgSO4, 25 mL of KPO4. |
4. Mix with magnetic stirring, and then aliquot into Petri dishes (90 x 15 mm dishes for the maintenance; 35 x 10 mm2 dishes for the egg laying assay). |
5. Store the NGM plates at 4 °C until use. |
DYT media for the cultivation of E. coli OP50 – 500 mL |
1. Add 2.5 g of NaCl, 5 g of yeast extract, 8 g of peptone, and 485 mL of distilled water into an Erlenmeyer flask. |
2. Autoclave for 15 min at 121 °C. |
3. Cool down and store at room temperature until use. |
S-buffer – 1,000 mL |
1. Add 5.85 g of NaCl, 6 g of KH2PO4, 1 g of K2HPO4, and 987 mL of distilled water into a glass bottle. |
2. Adjust the pH of the solution to 6.0. |
3. Autoclave for 15 min at 121 °C. |
4. Cool down and store at room temperature until use. |
Table 1: Receipes of culture growth media and buffers.
In this article, we describe the toxicity evaluation of chemicals in C. elegans, a soil nematode, using etoposide as a toxicant example. For this purpose, we used two experimental conditions. In the first set, C. elegans were grown on etoposide containing plates from eggs to the young adult stage, and then the worms were allowed to lay eggs on normal NGM plates without chemicals. In the second experimental set, C. elegans were continuously treated with etoposide throughout the whole experimental period. By comparison of the egg numbers between the two experimental conditions, we can decide whether the reproductive toxicity of the tested compounds was reversible or irreversible. Etoposide significantly decreased the total number of eggs laid under both experimental conditions (Figure 3). These data implied that pretreatment of etoposide during the early growth stage was sufficient to induce the damage to reproductive organs, including gonad germ cells6; based on these data, we could speculate that etoposide treatment irreversibly induced reproductive toxicity in C. elegans.
In addition to the C. elegans experiments described in this method article, other toxicity tests such as the lifespan assay14,15, microscopic observation of gonad germ cells6,17, measurement of the pharyngeal pumping rate, and movement analysis18,19 can also be used for chemical toxicity evaluation in C. elegans. In vitro cultured human cell lines, primary cells, stem cells, and 3D spheroid culture are other possible options for evaluating the toxicity of various chemicals to augment the limitation of C. elegans experiments1,6,20,21.
Although there is evolutional conservation of essential genes in C. elegans, the non-mammalian organism is different from humans in terms of protein sequences, the digestive and intestinal systems, metabolism, as well as it lacks many genes. Therefore, mammalian animal experiments and clinical trials are needed for the full evaluation of the toxicity of chemicals. However, considering the advantages of the convenience and animal ethical issues, a C. elegans toxicity testing platform will be a useful and practical solution for the toxicological evaluation of various chemicals.
The authors have nothing to disclose.
This study was supported by the Korea Institute of Science and Technology intramural research grant (2E27513) and the High Value-added Food Technology Development Program (IPET) funded by the Ministry of Agriculture, Food and Rural Affairs (315067-03).
Agar | Affymetrix, USA | 10906 | |
Caenorhabditis elegans N2 | Caenorhabditis Genetics Center (CGC) | Wild type | |
Cholesterol | Sigma, USA | C3045 | |
Dimethyl sulfoxide | Sigma, USA | D2650 | |
Escherichia coli OP50 | Caenorhabditis Genetics Center (CGC) | ||
Etoposide | Sigma, USA | E1383 | |
Image J software (ver 1.4) | Natinoal Institute of Health, USA | https://imagej.nih.gov/ij/ | |
Microscope camera | Jenopitk, Progress Gryphax, Germany | ||
Peptone | Merck, USA | 107213 | |
35 × 10 mm Petri dish | SPL Life Sciences, South Korea | 10035 | |
90 × 15 mm Petri dish | SPL Life Sciences, South Korea | 10090 | |
Stereo microscope | Nikon, Japan | SMZ800N | |
Yeast extract | Becton Dickinson, USA | 212750 |