Name: measuring the effect of chemicals on the growth and reproduction of caenorhabditis elegans
Uploaded: 2017-10-05T15:00:00
Description: Watch this Scientific Journal Video about Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans at JoVE.com

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).

NOTE: The entire experiment must be performed in a clean isolated laboratory maintained at 20 &#176;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
<ol>
	<li>Maintain C. elegans N2 (var. Bristol) on an NGM agar plate fed with live E. coli OP50 at 2...

<ol>
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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 experimenta...

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.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Affymetrix, USA</td>
			<td>10906</td>
			<td></td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans N2</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Wild type</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma, USA</td>
			<td>C3045</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma, USA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli OP50</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Sigma, USA</td>
			<td>E1383</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software (ver 1.4)</td>
			<td>Natinoal Institute of Health, USA</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Jenopitk, Progress Gryphax, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck, USA</td>
			<td>107213</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#215; 10 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10035</td>
			<td></td>
		</tr>
		<tr>
			<td>90 &#215; 15 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Nikon, Japan</td>
			<td>SMZ800N</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Becton Dickinson, USA</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effect of chemicals on the growth and reproduction of caenorhabditis elegans

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.

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...

Watch this Scientific Journal Video about Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans at JoVE.com

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

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.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

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