In vitro exposure to Bisphenol A and benzophenones 2 and 3 alter GnRH gene expression in mature GnRH neurons.

MARIGLIANO CAMILA; FERNANDEZ, MARINA O.; LUX LANTOS V; LIBERTUN CARLOS

Congreso; XX Jornadas anuales de la Sociedad Argentina de Biologia y la XVII Jornada de la Sociedad Uruguaya de Biociencias.; 2018

IN-VITRO EXPOSURE TO BISPHENOL A AND BENZOPHENONES 2 AND 3 ALTER GNRH GENE EXPRESSION IN MATURE GNRH NEURONS Marigliano C1, Libertun C1,2, Lux-Lantos V1, Fernandez M1. 1-Laboratorio de Neuroendocrinología-Instituto de Biología y Medicina Experimental (IByME-CONICET) and 2-Facultad de Medicina-UBA. Email: mfernandez@dna.uba.ar.Bisphenol A (BPA), a monomer of polycarbonate plastics, and Benzophenones (BPs), UV-filters, are endocrine disrupting chemicals (EDC). Previously we showed that twenty-four-hour pre-treatment with BPA and BP2 (1x10-9M) inhibit Kisspeptin (Kiss)-induced GnRH gene expression in immature GnRH neurons, GN11 cells, whereas BP3 did not have this effect. In this study we investigated the in-vitro effects of BPA, BP2 and BP3 on GnRH gene expression in mature GnRH neurons; we used the GT1-7 cell line (Dr Pamela Mellon, UCSD, USA) and isolated hypothalami from adult Balb/c males. Hypothalami were incubated in Krebs-Ringer medium in the presence or absence of BPA, BP2 or BP3 (1x10-9M) or medium alone (C) for six hours. After the incubation, hypothalami were homogenized in Tri-Reagent (Molecular Research Center, OH, USA) and mRNA extracted for Gnrh analysis by qPCR. Media were removed and kept -20 ºC for GnRH determination (RIA). GT1-7 cells were cultured in 12-well plates (200000 cells/well) in DMEM supplemented with 10% FBS, pyruvate and Penicillin/Streptomicin (complete medium). After 24 hours, media were changed to DMEM without phenol red, supplemented with 10% charcolized-FBS, pyruvate and Penicillin/Streptomicin (stimulation medium). Cells were incubated with BPA, BP2, BP3 (1x10-9M) or vehicle (C) in stimulation media for 24 hours, and after this, media were changed, stimuli renewed and cells further stimulated with Kiss1 (1x10-9M) for 4 h to analyze gene expression. RNA was extracted using Tri-Reagent and Gnrh analyzed by qPCR. RNA (1ug) was reverse transcribed and Real-Time PCR performed using specific primers. Cyclophilin B (Ppib) was used as housekeeping gene and results were analyzed using the mathematical model of Pfaffl et al (Nucleic Acids Res 29: e45, 2001). Hypothalami exposed to BPA showed increased GnRH expression compared to C (Relative expression: C=4.6±0.9; BPA=10.7±2.2; ANOVA: BPA vs C p