PMON12 Effects of Endocrine Disruptors on Autophagy Markers Gene Expression in Immortalized GnRH Neurons and Hypothalami of Adult Male Mice

Abstract

Abstract Benzophenones (BP) and Bisphenol A (BPA) are endocrine disrupting chemicals (EDC). Previously, we showed that the in-vitro exposure to EDC inhibited Kiss-induced gene expression in GT1-7 and GN11 cells (donated by Pamela Mellon, UCSD), and altered expression of inflammatory markers in GT1-7 cells and hypothalami of adult male mice.In the present study, we evaluated the effects of the in-vivo exposure to BP2 and BP3 (250 µg/kg/day, orally, for 5 days) on the expression of autophagy markers in hypothalamic nuclei in C57Bl/6 adult male mice, and the in-vitro exposure to BPA, BP2 or BP3 in immortalized GnRH neurons. Animals were sacrificed, brains rapidly dissected and conserved -80°C until processed. Micropunches containing Anteroventral Periventricular (AVPV) or Arcuate (ARC) nucleus were obtained. Effects of the in-vitro exposure for 12 or 24 hours to BPA, BP2 or BP3 (1×10-9M, Sigma), or DMSO as control, were evaluated in GT1-7 and GN11 cell lines, mature and immature GnRH neurons. ARN was obtained using Tri-Reagent (Molecular Research Center, Inc), reverse-transcribed and gene expression analyzed by qPCR, using Ppib as control gene. Results were presented as Media±SE and analyzed by ANOVA (Statistica, StatSoft Inc, USA).In-vivo, five-day exposure to BP2 or BP3 did not alter Lamp2 or p62 gene expression in the AVPV or ARC in adult male mice (ANOVA: ns). In GT1-7 cells, Ulk1 was not significantly altered by any of the stimuli (ANOVA: ns; n=5), whereas in GN11 cells, BP3 significantly increased Ulk1 gene expression after 12 and 24 h exposure [DMSO-24h: 0.93±0.21, DMSO-12h: 0.73± 0.19, BPA-24h: 1.78±0.31, BPA-12h: 1.47±0.33, BP2-24h: 1.51±0.49, BP2-12h: 1.06±0.23, BP3-24h: 2.41±0.89, BP3-12h: 2.45±0.82, Repeated Measures ANOVA: Main effect "TREATMENT" p<0.05, BP3 different from DMSO, p<0.05, n=4]. Our results reinforce the notion that the exposure to EDC have different effects depending on the experimental model and time of exposure, with developing cells being more susceptible to disruption that mature cells. We need more experiments to fully understand if exposure to EDC alters other markers of the autophagy process in mouse hypothalamus and GnRH neurons.Funding: CONICET, ANPCYT, International Society for Neurochemistry, Fund. Williams, Fund. R. Barón. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.