Abstract
BackgroundIn vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively.ResultsThe study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.ConclusionsUntargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.
Highlights
In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing
Results were compared with data from previous studies concerning the effects of TCDD in non-metabolomics studies
The present study demonstrates that untargeted profiling of the polar and apolar extracts of in vitro cultured HepG2 cells using various analytical techniques is a feasible approach to studying the effect of a toxicant, TCDD, on the cell metabolome
Summary
In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Metabolomics has been defined as the quantitative measurement of the multi-parametric metabolic response of living systems to patho-physiological stimuli or genetic modification [1]. It encompasses the qualitative and quantitative measurement of metabolites interacting in a biological system; targeted and untargeted strategies for analysis of metabolites can be used. There has been an exponential growth in the number of published papers concerning metabolomics of a wide variety of systems [4,5,6,7]. Few toxicological studies have been published that concern the profiling of intracellular metabolites using in vitro cell culture systems [10,11]
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