Gas Chromatography- Mass Spectrometry Based Metabolomic Approach for Optimization and Toxicity Evaluation of Earthworm Sub-Lethal Responses to Carbofuran

Mohana Krishna Reddy Mudiam,Prem Narain Saxena,Ratnasekhar Ch,Jacob Guy Bundy

doi:10.1371/journal.pone.0081077

Abstract

Despite recent advances in understanding mechanism of toxicity, the development of biomarkers (biochemicals that vary significantly with exposure to chemicals) for pesticides and environmental contaminants exposure is still a challenging task. Carbofuran is one of the most commonly used pesticides in agriculture and said to be most toxic carbamate pesticide. It is necessary to identify the biochemicals that can vary significantly after carbofuran exposure on earthworms which will help to assess the soil ecotoxicity. Initially, we have optimized the extraction conditions which are suitable for high-throughput gas chromatography mass spectrometry (GC-MS) based metabolomics for the tissue of earthworm, Metaphire posthuma. Upon evaluation of five different extraction solvent systems, 80% methanol was found to have good extraction efficiency based on the yields of metabolites, multivariate analysis, total number of peaks and reproducibility of metabolites. Later the toxicity evaluation was performed to characterize the tissue specific metabolomic perturbation of earthworm, Metaphire posthuma after exposure to carbofuran at three different concentration levels (0.15, 0.3 and 0.6 mg/kg of soil). Seventeen metabolites, contributing to the best classification performance of highest dose dependent carbofuran exposed earthworms from healthy controls were identified. This study suggests that GC-MS based metabolomic approach was precise and sensitive to measure the earthworm responses to carbofuran exposure in soil, and can be used as a promising tool for environmental eco-toxicological studies.

Highlights

Metabolomics, an omics science of systems biology, is the untargeted profiling of endogenous metabolites within a biological system under various physiological conditions and offers a unique opportunity to look at genotype-phenotype relationships as well as genotype-environmental relationships [1], [2]
The workflow strategy was shown in fig 1 consisting of optimization of extraction conditions followed by biomarker evaluation of carbofuran exposure to earthworms
Environmental metabolomics is a rapidly developing and emerging sub-discipline of metabolomics and has the potential to relate between earthworm toxicity and bioavailability of soil contaminants

Summary

Introduction

Metabolomics, an omics science of systems biology, is the untargeted profiling of endogenous metabolites within a biological system under various physiological conditions and offers a unique opportunity to look at genotype-phenotype relationships as well as genotype-environmental relationships [1], [2]. Metabolomic profiling provides a powerful approach to identify and to quantitatively measure global changes in metabolites from biochemical pathways that are altered in response to disease, therapeutic intervention or toxicant. It has been widely employed in functional genomics, disease diagnosis, clinical [3], food and nutritional science [4], toxicology and pharmacology research [5]. Environmental pollutants, including organic chemicals and toxic metals may induce variety of adverse effects on ecosystems [14] These effects of organic pollutants and metals are monitored by taking the effects on earthworms as an illustration [15]. Varieties of endogenous metabolites have been identified as potential biomarkers of different environmental chemicals exposure to earthworms, for e.g. decrease in lactate and fatty acids for poly aromatic hydrocarbon (PAH) pyrene exposure [17], increase in alanine for pesticides DDT & endosulfan exposure [18], increase in histidine for copper exposure [19]

Methods

Results

Conclusion