Use of omic approaches (proteomics and metabolomics) to elucidate bisphenols A and S mechanisms of toxicity in in vivo and in vitro models

Author(s)

Lara Ferreira Azevedo

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-04-01

Examining board members

Lusania Maria Greggi Antunes; Lucilene Delazari dos Santos

• Advisor: Fernando Barbosa Junior; Maria Fernanda Hornos Carneiro

Abstract

Bisphenol A (BPA) and bisphenol S (BPS) are endocrine disruptors that can lead to the development of several metabolic disorders such as obesity, diabetes and dyslipidemias. Nonetheless the mechanisms associated to the toxic effects are not yet fully understood. In this context, this work aimed at elucidating BPA and BPS mechanisms of toxicity in in vivo and in vitro models, through evaluation of biochemical-metabolic and pathophysiological alterations by the analysis of affected metabolic pathways and by means of omic tools (proteomics and metabolomics). To this end, this work was divided into 2 main studies: (I) Male Wistar rats were chronically exposed (20 weeks) to approximately 50 or 500 μg/kg/day of BPA and/or BPS through drinking water (ethical approval n. 15.1.979.60.7). Body mass gain was weekly determined and Insulin Tolerance Test (ITT) and Glucose Tolerance Test (GTT) were performed, respectively, at the 18th and 19th weeks of exposure. Following euthanasia, blood samples were collected to determine biochemical parameters in all experimental groups and liver and pancreas samples were obtained from animals exposed to 50 μg/kg/day of BFA or BFS to quantify changes in protein expression through nontargeted proteomic analyses by nanoUPLC-nanoESI-HDMSE (n=3) and gene expression by RT-qPCR (n=6). (II) HepG2 and INS-1E cells were exposed for 72 h to BPA or BPS (100; 10; 1; 0.1; 0.01 and 0.001 μM) to assess cellular proliferation and viability; and intracellular polar extracts from both strains were obtained following 24, 48 and 72 h of exposure to BPA or BPS (10 - 0.001 μM) for basal metabolic characterization through non-targeted metabolomic analyses by FIA-qTOF. Data from study I indicates that exposure to the lowest dose of the contaminants led to the development of glucose intolerance and animals exposed to 50 μg/kg/day of BPA+BPS and 500 μg/kg/day of BPS showed an increase in body mass. Exposure to 50 μg/kg/day of BPA caused hyperglycemia, 50 μg/kg/day of BPA and BPA+BPS caused an increase in total cholesterol and HDL-cholesterol and 500 μg/kg/day of BPA also increased this last parameter. Exposure to BPS (500 μg/kg/day) increased LDLcholesterol. Hepatic proteins differentially expressed in groups exposed to 50 μg/kg/day of BPA or BPS, compared to the control group, are related to triglyceride synthesis (glucokinase activity-related sequence 1, 1.8-fold change for BPA and 2.4- fold change for BPS) and with mevalonate biosynthesis (hydroxymethylglutaryl-CoA synthase cytoplasmic, 2-fold change for BPS). Proteins from electron transport chain as well as antioxidant enzymes also presented dysregulated expression following exposure to both contaminants. Proteomic analyses from pancreatic tissues evidenced that both compounds drastically reduced expression of several ribosomal proteins as well as of essential proteins for energy homeostasis such as insulin-1 (0.01-fold change for BPA), insulin-2 (0.01-fold change for BFS) and glucagon (0.01-fold change for BPS) compared to the control group. RT-qPCR assays confirmed the results obtained in proteomic analyses for the proteins analyzed. Results from study II showed antiproliferative and cytotoxic effects in HepG2 cells after exposure to the highest and lowest-concentrations of the contaminants (nonlinear dose-response curve), while the same exposure regimens produced milder effects on INS-1E proliferation and viability, except for the highest concentration studied of both contaminants. HepG2 and INS-1E viii metabolome analyses revealed time- and dose-dependent molecular changes and similar toxicity pathways for both compounds. This study shows the importance of conducting omic studies for the unveiling of mechanisms of toxicity and the need to reevaluate the use of BPS as a substitute for BPA, since the first produced significant metabolic changes in vivo and in vitro. (AU)

• FAPESP's process: 16/10456-0 - Evaluation of protein expression and biochemical parameters related to diabetes mellitus type II and dyslipidemias in animals exposed to the contaminants bisphenol A and S
• Grantee: Lara Ferreira Azevedo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)