Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Summary

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Abstract

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Introduction

The application and waste management of chemicals is highly dependent upon the information of their effects at certain concentrations. Notably, time is another essential element between effects and concentrations. That is to say, chemicals, especially those at low concentrations in the actual environments, need time to provoke measurable effects¹. Therefore, researchers arrange different lengths of the exposure duration in animal experiments, and even cover the whole life cycle. For example, mice were exposed to nicotine for 30, 90 or 180 days to study its toxic effects ². Yet, such exposure durations are still not enoug....

Protocol

1. Culture E. coli OP50

1. Prepare 1 M sodium hydroxide solution by dissolving 4 g of sodium hydroxide in 100 mL water.
2. Prepare lysogeny broth (LB) medium by dissolving 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride with 1 L of ultrapure water in a 1 L conical flask. Adjust the pH to 7.0 with 1 M sodium hydroxide solution.
3. Aliquot the LB liquid medium from step1.2 into 20 conical flasks (maximum allowable volume: 100 mL) with 50 mL medium in each. Cover the conical flasks with kraft paper.
4. Sterilize the LB liquid medium at 121 °C and 0.105 MPa for 20 min. Cool the LB medium down to room temperatu....

Results

Here, we describe protocols for studying effects of chemicals over generations using C. elegans in trans-generational (TG), multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. Our own research results are presented as examples. One study presents the TG effects of heavy metals on locomotion behavior³. The other two studies present MGE and MGR effects of sulfomethoxazole and lindane on the reproduction and biochemical and.......

Discussion

In order to successfully conduct the described protocol, the following suggestions should be taken into consideration. Perform the overall experimental operations in a sterile environment. Improper operation may result in contamination of the E. coli strains, e.g., fungi and mites may hinder the normal growth of C. elegans and therefore affect the experimental results. In the section describing cultivating C. elegans, observe the growth scale of C. elegans on the NGM agars by naked eye.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
agar powder	OXOID, Thermo Fisher Scientific, UK	9002-18-0
79nnHT Fast Real-Time PCR System	Applied Biosystems
96-well sterile microplate	Costar?Corning?America
Autoclave sterilizer	Tomy, Tomy Digital Biology, Japan
Biosafety cabinet	LongYue, Shanghai longyue instrument equipment co. Ltd, China
calcium chloride	Sinopharm chemical reagent company Ltd, China	10043-52-4
centrifuge  5417R	Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Centrifuge tubes	Axygen, Aixjin biotechnology (Hangzhou) co. Ltd, America
cholesterol	Sinopharm chemical reagent company Ltd, China	57-88-5
Dimethyl sulfoxide	VETEC, Sigmar aldrich (Shanghai) trading co. Ltd, America	67-68-5
disodium hydrogen phosphate	Sinopharm chemical reagent company Ltd, China	7558-79-4
ethanol	Sinopharm chemical reagent company Ltd, China	64-17-5
Filter	Thermo, Thermo Fisher Scientific, America
incubator	YiHeng17, Shanghai yiheng scientific instrument co. Ltd, China
inoculating loop
K2HPO4•3H2O	Sinopharm chemical reagent company Ltd, China	16788-57-1
kraft paper
Mcroplate Reader	Boitek, Boten apparatus co. Ltd, America
MgSO4•7H2O	Sinopharm chemical reagent company Ltd, China	10034-99-8
Microscopes XTL-BM-9TD	BM, Shanghai BM optical instruments manufacturing co. Ltd, China
Petri dishes
Pipette	Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Potassium chloride	Sinopharm chemical reagent company Ltd, China	7447-40-7
potassium dihydrogen phosphate	Sinopharm chemical reagent company Ltd, China	7778-77-0
Qiagen RNeasy kits	Qiagen Inc., Valencia, CA, United States
QuantiTect SYBR Green RT-PCR kits	Qiagen Inc., Valencia, CA, United States
RevertAid First Strand cDNA Synthesis Kit	Thermo Scientific, Wilmington, DE, United States
sodium chloride	Sinopharm chemical reagent company Ltd, China	7647-14-5
sodium hydroxide	Sinopharm chemical reagent company Ltd, China	1310-73-2
sodium hypochlorite solution	Aladdin, Shanghai Aladdin biochemical technology co. Ltd, China	7681-52-9
tryptone	OXOID, Thermo Fisher Scientific, UK	73049-73-7
yeast extract	OXOID, Thermo Fisher Scientific, UK	119-44-8