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>
	<li>Blomme, E. A., Will, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+strategies+for+drug+discovery:+present+and+future.">Toxicology strategies for drug discovery: present and future.</a> Chem. Res. Toxicol. 29 (4), 473-504 (2016).</li><li>Lilienblum, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+methods+to+safety+studies+in+experimental+animals:+role+in+the+risk+assessment+of+chemicals+under+the+new+European+Chemicals+Legislation+(REACH).">Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH).</a> Arch. Toxicol. 82 (4), 211-236 (2008).</li><li>Honnen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+powerful+alternative+model+organism+to+promote+research+in+genetic+toxicology+and+biomedicine.">Caenorhabditis elegans as a powerful alternative model organism to promote research in genetic toxicology and biomedicine.</a> Arch. Toxicol. , (2017).</li><li>Hunt, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+model+in+toxicity+testing.">The C. elegans model in toxicity testing.</a> J. Appl. Toxicol. 37 (1), 50-59 (2017).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Lee, S. Y., Kim, J. Y., Jung, Y. J., Kang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+evaluation+of+the+topoisomerase+inhibitor,+etoposide,+in+the+model+animal+Caenorhabditis+elegans+and+3T3-L1+normal+murine+cells.">Toxicological evaluation of the topoisomerase inhibitor, etoposide, in the model animal Caenorhabditis elegans and 3T3-L1 normal murine cells.</a> Environ. Toxicol. 32 (6), 1836-1843 (2017).</li><li>Imanikia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+the+comet+assay+to+assess+the+genotoxicity+of+environmental+pollutants+in+the+nematode+Caenorhabditis+elegans.">The application of the comet assay to assess the genotoxicity of environmental pollutants in the nematode Caenorhabditis elegans.</a> Environ. Toxicol. Pharmacol. 45, 356-361 (2016).</li><li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorooctane+sulfonate+exposure+causes+gonadal+developmental+toxicity+in+Caenorhabditis+elegans+through+ROS-induced+DNA+damage.">Perfluorooctane sulfonate exposure causes gonadal developmental toxicity in Caenorhabditis elegans through ROS-induced DNA damage.</a> Chemosphere. 155, 115-126 (2016).</li><li>Allard, P., Kleinstreuer, N. C., Knudsen, T. B., Colaiacovo, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+elegans+screening+platform+for+the+rapid+assessment+of+chemical+disruption+of+germline+function.">A C. elegans screening platform for the rapid assessment of chemical disruption of germline function.</a> Environ. Health Perspect. 121 (6), 717-724 (2013).</li><li>Singh, S., Sharma, B., Kanwar, S. S., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lead+phytochemicals+for+anticancer+drug+development.">Lead phytochemicals for anticancer drug development.</a> Front. Plant Sci. 7, 1667 (2016).</li><li>Kang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+topoisomerase+inhibitor,+daurinol,+suppresses+growth+of+HCT116+cells+with+low+hematological+toxicity+compared+to+etoposide.">A novel topoisomerase inhibitor, daurinol, suppresses growth of HCT116 cells with low hematological toxicity compared to etoposide.</a> Neoplasia. 13 (11), 1043-1057 (2011).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> J. Vis. Exp. (47), e2293 (2011).</li><li>Zarse, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin/IGF1+signaling+extends+life+span+by+promoting+mitochondrial+L-proline+catabolism+to+induce+a+transient+ROS+signal.">Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal.</a> Cell Metab. 15 (4), 451-465 (2012).</li><li>Sutphin, G. L., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+on+solid+media.">Measuring Caenorhabditis elegans life span on solid media.</a> J. Vis. Exp. (27), e1152 (2009).</li><li>Weimer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D-Glucosamine+supplementation+extends+life+span+of+nematodes+and+of+ageing+mice.">D-Glucosamine supplementation extends life span of nematodes and of ageing mice.</a> Nat. Commun. 5, 3563 (2014).</li><li>Schmeisser, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+ROS+signaling+rather+than+AMPK/sirtuin-mediated+energy+sensing+links+dietary+restriction+to+lifespan+extension.">Neuronal ROS signaling rather than AMPK/sirtuin-mediated energy sensing links dietary restriction to lifespan extension.</a> Mol. Metab. 2 (2), 92-102 (2013).</li><li>Parodi, D. A., Damoiseaux, R., Allard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+assessment+of+germline+chemical+toxicity+using+the+nematode+Caenorhabditis+elegans.">Comprehensive assessment of germline chemical toxicity using the nematode Caenorhabditis elegans.</a> J. Vis. Exp. (96), e52445 (2015).</li><li>Hahm, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+maximum+velocity+correlates+with+healthspan+and+is+maintained+in+worms+with+an+insulin+receptor+mutation.">C. elegans maximum velocity correlates with healthspan and is maintained in worms with an insulin receptor mutation.</a> Nat. Commun. 6, 8919 (2015).</li><li>Nussbaum-Krammer, C. I., Neto, M. F., Brielmann, R. M., Pedersen, J. S., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+spreading+and+toxicity+of+prion-like+proteins+using+the+metazoan+model+organism+C.+elegans.">Investigating the spreading and toxicity of prion-like proteins using the metazoan model organism C. elegans.</a> J. Vis. Exp. (95), e52321 (2015).</li><li>Schmidt, B. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+acute+and+developmental+neurotoxicity+screening:+an+overview+of+cellular+platforms+and+high-throughput+technical+possibilities.">In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities.</a> Arch. Toxicol. 91 (1), 1-33 (2017).</li><li>Fey, S. J., Wrzesinski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+drug+toxicity+using+3D+spheroids+constructed+from+an+immortal+human+hepatocyte+cell+line.">Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell line.</a> Toxicol. Sci. 127 (2), 403-411 (2012).</li></ol>

The authors have nothing to disclose.

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 ...

The overall goal of this procedure is to measure the effect of chemicals, such as the topoisomerase inhibitor etoposide, on the growth and reproduction of the model animal Caenorhabditis elegans. This method can help answer key questions in the toxicology field, such as toxicological evaluation for the development of drugs and risk assessment of environmental toxicants. The main advantage of this technique is that the toxicity of various chemicals can be checked in non-mammalian systems, so this technique is convenient and cost-effective.Demonstrating the procedure will be So Young Lee, a graduate student from my laboratory. To begin, prepare two clean, 200-milliliter glass bottles by adding 0.6 grams of sodium chloride, 0.5 grams of peptone, 3.4 grams of agar, 195 milliliters of distilled water, and a magnetic stirrer to one bottle. Then add a magnetic stirrer to the other empty bottle.Autoclave the bottles for 15 minutes at 121 degrees Celsius, and then cool down the bottles in a water bath for 30 minutes at 55 degrees Celsius. Next, to the filled bottle, add 0.2 milliliters of one molar calcium chloride, 0.2 milliliters of five milligrams per milliliter cholesterol, 0.2 milliliters of one molar magnesium sulfate, and five milliliters of one molar potassium phosphate. Then mix the solution by magnetic stirring on a hot plate at 55 degrees Celsius.Pour half of the mixed NGM medium into the empty 200-milliliter bottle. Then add one milliliter of DMSO to one bottle, and use magnetic stirring to mix it again. Store the other bottle in a water bath at 55 degrees Celsius.Then aliquot three milliliters of the DMSO-containing medium to 35-by-10-millimeter Petri dishes. Remove the other bottle from the water bath, and use magnetic stirring to mix it. Then add one milliliter of 75-millimolar etoposide.And after mixing again, repeat the aliquoting procedure. Cool down the chemical-containing NGM plates by leaving them at room temperature for approximately three hours, and store them at four degrees Celsius until use. For the chemical test, spread 100 microliters of heat-inactivated E.coli OP50 on each NGM plate.Allow the plates to dry overnight in a C.elegans incubator at 20 degrees Celsius. Transfer the age-synchronized C.elegans eggs to an NGM plate supplemented with 1%DMSO or 750-micromolar etoposide. Place the C.elegans in an incubator at 20 degrees Celsius for four days.Observe and take microscopic images of C.elegans under stereo microscope every day. Also, take a microscopic image of the microscope stage micrometer for worm size calibration. To measure the body length of C.elegans treated with DMSO or etoposide at each time point, in ImageJ, open the microscope stage micrometer image.Then using the Straight Line tool, drag a line of 1, 000 micrometers on the image. Click Analyze and then Set Scale, and input 1, 000 and micrometers into the Known Distance box and Unit of Length box, respectively. Then check Global and click OK.Open the worm images using File and then Open.Then, using the Freehand Line tool, draw a line along the feature of each worm. Obtain the body length data by clicking Analyze and then Measure. Repeat these steps for 50 worms.Incubate the age-synchronized eggs on the NGM plate containing DMSO or etoposide at 20 degrees Celsius for 64 hours until the young adult stage. Transfer five adult worms to new NGM plates without chemicals or new NGM plates containing the same chemical pretreatment, and incubate them for 24 hours. Transfer the adults to new NGM plates, as just demonstrated, and count the number of eggs every day.In addition, count the worms that crawled off, died, or were internally hatched. Carry out calculations according to the text protocol. As shown here, the treatment of etoposide at 24 to 96 hours significantly retarded the growth of C.elegans.After 96 hours of incubation, etoposide-treated worms grew to 0.86 millimeters in body length while the vehicle-treated worms grew to 1.04 millimeters. Growth retardation was also observed under stereo microscope observation, as seen in these images of etoposide-treated worms, as compared to control-treated worms. After 72 hours of incubation, eggs from the vehicle-treated worms were observed.In contrast, eggs were observed after 96 hours of etoposide treatment, raising the possibility that etoposide treatment delayed the first birth time of C.Elegans. As depicted in these graphs, a decrease in total egg number by etoposide treatment was observed when the young adult worms were cultured, either in the presence or absence of continuous etoposide treatment. Once mastered, this technique can be done in one week if it is performed properly.After watching this video, you should have a good understanding of how to evaluate chemical toxicity in C.elegans, a model nematode.

measuring-effect-chemicals-on-growth-reproduction-caenorhabditis

Research

JoVE Journal

Medicine

16.5K Görüntüleme.  Korea Institute of Science and Technology. The overall goal of this procedure is to measure the effect of chemicals, such as the topoisomerase inhibitor etoposide, on the growth and reproduction of the model animal Caenorhabditis elegans. This method can help answer key questions in the toxicology field, such as toxicological evaluation for the development of drugs and risk assessment of environmental toxicants. The main advantage of this technique is that the toxicity of various chemicals can be checked in non-mammalian systems, so t...