In-Vitro Exposure to Endocrine Disruptors Alters Inflammatory Markers in Whole Hypothalami and Immortalized GnRH Neurons
Authors: Riaño Juan Manuel , Sorianello Eleonora Mariana , Libertun Carlos , Lux-Lantos Victoria Adela , Fernandez Marina Olga 
Published: May 2021

Abstract
Bisphenol A (BPA), a monomer of polycarbonate plastics, and Benzophenones (BPs), used as UV-filters, are endocrine disrupting chemicals (EDC) found in everyday products. Previously, we showed that the in-vitro exposure to BPA decreased Kiss-induced GnRH expression in GN11 cells (donated by Dr. Susan Wray, NIH), immature GnRH neurons, and that exposure to all the EDC decreased Kiss-induced GnRH gene expression in GT1-7 cells (donated by Dr. Pamela Mellon, UCSD), mature GnRH neurons. In this study, we analyzed the effect of in-vitro exposure to the same EDC (BPA, BP2 or BP3, Sigma, 1x10-9 M) or medium as control (C) in mature GnRH neurons (GT1-7 cells), and isolated hypothalami from adult Balb/c males on Glial fibrillary acidic protein (GFAP) and cytokine gene expression. Cells were exposed to the compounds for 12 or 24 h, in DMEM (high glucose) with charcoal-stripped FBS, and the hypothalami for 6 h to the EDC, in Krebs-Ringer buffer. After the incubations, RNA was extracted using Tri-Reagent (Molecular Research Center, OH, USA), 1-2 µg RNA was reverse transcribed and Real-Time PCR performed using specific primers. Results were expressed as Mean±SE and analyzed by T-test or ANOVA using Statistica v12 (StatSoft Inc, USA) Twenty-four hour BPA exposure increased il18 in GT1-7 cells (C=1.0±0.04, BPA=1.2±0.1, T-test p&lt;0.05, n=7), whereas neither BP2 nor BP3 had an effect on il18 expression (ANOVA: ns, n=7). When il6 was analyzed, 24-hour BPA decreased its expression relative to C and to 12-hour BPA, whereas BP3 had a dual effect depending on the time-point analyzed, increasing the expression at 12-hour stimulation and decreasing it after 24-hour stimulation (DMSO-12h=0.82±0.08, DMSO-24h= 1.01±0.13, BPA-12h=1.00±0.11, BPA-24h=0.68±0.06, BP2-12h=0.75±0.12, BP2-24h=0.82±0.18, BP3-12h=1.20±0.10, BP3-24h=0.83±0.05; Repeated Measures Two-way ANOVA: BPA-24h different from DMSO-24h and BPA-12h p&lt;0.05, BP3-12h different from DMSO-12h and from BP3-24h p&lt;0.05, n=4). In the hypothalami, 6-hour BPA exposure increased gfap gene expression (C=0.8±0.2, BPA=1.7±0.4; T-test p&lt;0.05, n=9), whereas neither BP2 nor BP3 had any significant effect (C=0.8±0.2, BP2=1.4±0.3, BP3=1.0±0.3, ANOVA ns, n=9). There was no significant change in il18, il6 or il1b gene expression in whole hypothalami with any of the EDC tested (ANOVA ns). Our results show that the EDC herein studied have the potential to alter the inflammatory state of mature GnRH neurons and to activate astrocytes in the hypothalamus. The pattern for cytokine expression in the whole tissue could be different from the one observed in GnRH neurons, as the hypothalamus contain multiple cell types, and effects can be different in the different cells. More experiments are needed to dissect the mechanisms involved in the effects observed. Funding: CONICET, ANPCyT, UBA, International Society for Neurochemistry, Asoc. ORT Arg., Fund. R. Barón, Fund. Williams.