Oxygen consumption rate of Caenorhabditis elegans as a high-throughput endpoint of toxicity testing using the Seahorse XFe96 Extracellular Flux Analyzer

G Du Preez,M Daneel,H Fourie,S Höss,M Zouhar,G Engelbrecht,V Wepener,H Miller,C Ricci

doi:10.1038/s41598-020-61054-7

Abstract

Caenorhabditis elegans presents functioning, biologically relevant phenotypes and is frequently used as a bioindicator of toxicity. However, most C. elegans in vivo effect-assessment methods are laborious and time consuming. Therefore, we developed a novel method to measure the oxygen consumption rate of C. elegans as a sublethal endpoint of toxicity. This protocol was tested by exposing 50 larval stage one C. elegans individuals for 48 h (at 20 °C) to different concentrations of two toxicants i.e. benzylcetyldimethylammonium chloride (BAC-C16) and cadmium (Cd). Following exposures, the oxygen consumption rate of the C. elegans individuals were measured using the high-throughput functionality of the Seahorse XFe96 Extracellular Flux Analyzer. Dose-response curves for BAC-C16 (R2 = 0.93; P = 0.001) and Cd (R2 = 0.98; P = 0.001) were created. Furthermore, a strong, positive correlation was evidenced between C. elegans oxygen consumption rate and a commonly used, ecologically relevant endpoint of toxicity (growth inhibition) for BAC-C16 (R2 = 0.93; P = 0.0001) and Cd (R2 = 0.91; P = 0.0001). The data presented in this study show that C. elegans oxygen consumption rate can be used as a promising functional measurement of toxicity.

Highlights

Caenorhabditis elegans Maupas, 1900 has been extensively used to study the toxic effect of pollutants, drugs, and environmental samples[1]
Longer exposure periods would allow the measurement of a chronic response, which is facilitated by the short life cycle of C. elegans[3,18]
Nematode length was clearly inhibited by decreased food availability, a well-studied response often used to investigate the effect of dietary restrictions on C. elegans development[20,21]

Summary

Introduction

Caenorhabditis elegans Maupas, 1900 has been extensively used to study the toxic effect of pollutants, drugs, and environmental samples[1]. Studies have shown that C. elegans bioassays can be used to predict mammalian development at a fraction of the cost of traditional animal testing[2,5] These qualities and benefits complement C. elegans as a model organism for toxicity testing, as well as its use in high-throughput assessment protocols[3], as has been developed for drugs[6] and pollutants of environmental concern[7]. This has led to the development of acute response protocols that measure C. elegans OCR before and after the injection of pre-loaded compounds, typically oligomycin, FCCP, rotenone and antimycin A Such compounds facilitate the measurement of mitochondrial respiratory chain functionality in organisms by determining, for example, ATP production, proton leak, maximal respiration, spare respiratory capacity and non-mitochondrial respiration[15,16,17]. The aim of this research was to develop an environmentally relevant C. elegans OCR protocol for sublethal toxicity testing by utilizing the high-throughput capabilities of the Seahorse XFe96 Extracellular Flux Analyzer

Objectives

Methods

Results