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

Summary

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.

Abstract

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.

Introduction

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 reproducibility^1,2,

Protocol

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

1. Maintain C. elegans N2 (var. Bristol) on an NGM agar plate fed with live E. coli OP50 at 20 °C^12,13.
2. Prepare age-synchronized eggs using either bleach solution treatm....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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