An Untargeted Metabolomics Approach to Investigate the Metabolic Modulations of HepG2 Cells Exposed to Low Doses of Bisphenol A and 17β-Estradiol

Abstract

The model xeno-estrogen bisphenol A (BPA) has been extensively studied over the past two decades, contributing to major advances in the field of endocrine disrupting chemicals research. Besides its well documented adverse effects on reproduction and development observed in rodents, latest studies strongly suggest that BPA disrupts several endogenous metabolic pathways, with suspected steatogenic and obesogenic effects. BPA's adverse effects on reproduction are attributed to its ability to activate estrogen receptors (ERs), but its effects on metabolism and its mechanism(s) of action at low doses are so far only marginally understood. Metabolomics based approaches are increasingly used in toxicology to investigate the biological changes induced by model toxicants and chemical mixtures, to identify markers of toxicity and biological effects. In this study, we used proton nuclear magnetic resonance (1H-NMR) based untargeted metabolite profiling, followed by multivariate statistics and computational analysis of metabolic networks to examine the metabolic modulation induced in human hepatic cells (HepG2) by an exposure to low and very low doses of BPA (10−6M, 10−9M, and 10−12M), vs. the female reference hormone 17β-estradiol (E2, 10−9M, 10−12M, and 10−15M). Metabolomic analysis combined to metabolic network reconstruction highlighted different mechanisms at lower doses of exposure. At the highest dose, our results evidence that BPA shares with E2 the capability to modulate several major metabolic routes that ensure cellular functions and detoxification processes, although the effects of the model xeno-estrogen and of the natural hormone can still be distinguished.

The EDCMET Project: Metabolic Effects of Endocrine Disruptors

“…After the initial optimization and pre-validation of these assays [63,64], potential MDCs will be screened for their potency. These compounds have been preselected by using the in silico screening results (see also Sections 3 and 3.1) and knowledge from prior studies, e.g., [18,[65][66][67]. This information will be cross-referenced to the data available in public EU sources and ToxCast [68].…”

Important Considerations for Sample Collection in Metabolomics Studies with a Special Focus on Applications to Liver Functions

“…Regarding the hepatic HepG2 cells for instance, tests have been performed to optimize the number of cells needed to obtain NMR metabolic fingerprints allowing to detect and identify the metabolite content in the cells. The number of 10 6 cells was selected, as this seeding allowed the detection of the subtle modulations occurring after exposing the cells to xenobiotics such as estradiol or bisphenol A [18]. For cell extracts, it is usually recommended to work in very low volumes and concentrate the extract in a maximum of 50 uL.…”

“…Metabolomics has found numerous applications in the study of liver functions in health and disease. Among others, these include: Non-invasive biomarker investigations to discriminate between the different stages of progression of NAFLD using non-invasive biofluids (urine and plasma) [3,4]; investigation of mechanisms underlying hepatic disease progression such as acute-on-chronic liver failure using serum metabolic profiling [5] or fibrosis [6]; characterization of the gut microbiota metabotypes in urine of NAFLD patients [4,7]; nutrimetabolomics studies to unravel hepatic pathways dysregulated directly in liver samples upon various nutritional challenges [8,9]; discovery of new metabolic functions for nuclear receptors, that are important regulators of liver physiology using direct hepatic metabolomics or other informative fluids such as urine and bile [10][11][12][13]; identification of patients at risk for idiosyncratic drug-induced liver injury (IDILI) before drug administration, a concept named "pharmaco-metabolomics", that was first demonstrated in urine of animal models [14] and is now extended to human biofluids (urine and serum) [15,16]; study of mechanisms of action for pharmaceutical drugs in urine and fecal samples [17] and environmental contaminants in HepG2 cells and animal biofluids and tissues [18,19].…”

An Optimized Dual Extraction Method for the Simultaneous and Accurate Analysis of Polar Metabolites and Lipids Carried out on Single Biological Samples

“…Compared to other “omics” technologies (transcriptomics, proteomics…), metabolomics and lipidomics are considered to provide information that is closer to the actual phenotype and, therefore, more relevant for interpreting phenotypic changes [ 3 ]. These technologies are relevant and promising tools for studies related to metabolic disruption in many different contexts, including disease diagnosis [ 4 ], toxicology [ 5 , 6 ], nutrients’ effects [ 7 ] as well as in the field of plant sciences [ 8 ]. However, to explore changes occurring simultaneously at the level of many different metabolic pathways, it is essential to be able to perform unbiased measurement of a maximal number of metabolites on a given biological sample, which is often of limited size/volume [ 9 ].…”