SAT431 In-vitro Effects Of Benzophenones 2 And 3 On Cell Proliferation And Autophagy Factors Gene Expression In Lβt2 Cells

Abstract

Abstract Disclosure: J.M. Riaño Gómez: None. E. Sorianello: None. V.A. Lux-Lantos: None. N.J. Webster: None. M.O. Fernandez: None. Introduction: Humans and wildlife are exposed to Endocrine Disrupting Chemicals (EDC). Previously, we showed that the in-vitro exposure to Benzophenones 2 and 3 (BP2 and BP3, UV filters) increase cell proliferation in GN11 cells (Susan Wray, USA), and that BP2 and BP3 increase p62 gene expression after 12-h exposure in GT1-7 cells (Pam Mellon, UCSD, USA). In this study, we evaluated the in-vitro effects of BP2 and BP3 on cell proliferation and autophagy factors expression in LβT2 cells (pituitary gonadotropes, Pam Mellon). Materials and Methods: LβT2 cells were plated in 96-well plates (Proliferation Assay) or in 12-well plates (gene expression studies) in DMEM high glucose, with 10% FBS, sodium pyruvate and Penicillin/Streptomicin (complete medium). Before adding the stimuli, media were changed to DMEM without phenol red, with charcoal-stripped FBS (2% for proliferation and 10% for gene expression studies). Proliferation was evaluated using MTS (Promega, WI, USA), in response to BP2 and BP3 (1x10-7 y 1x10-9 M) after 12 or 24-h exposure. Ulk1, Lamp2 and p62 expression was evaluated after 6 or 24-h exposure to BP2 or BP3. RNA was extracted, 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). Results: After 12 or 24-h exposure, BP2 as well as BP3 increased cell proliferation in LβT2 cells [Control-12h:100.0±23.0; BP2-7-12h:201.6±64.0; BP2-9-12h:308.2±80.7; BP3-7-12h:325.2±98.1, BP3-9-12h:339.5±90.4; Control-24h:100.0±24.5; BP2-7-24h: 201.4±45.9; BP2-9-24h:318.2±97.1; BP3-7-24h:289.6±59.9, BP3-9-24h:272.3±99.0; ANOVA: Interaction ns, Main Effect Treatment: BP2-9, BP3-7, BP3-9 different from Control p<0.05, n=5]. After 6 or 24-h exposure, BP3 1x10-9 M increased p62 gene expression, whereas neither BP2 nor BP3 1x10-7 M had an effect on p62 expression [ANOVA: Interaction ns, Main Effect Treatment: BP3-9 different from Control, p<0.05, n=3]. There was no difference in Ulk1 and Lamp2 expression. Conclusions: BP2 and BP3, the EDC evaluated, increased cell proliferation in pituitary gonadotropes, LβT2 cells. Furthermore, BP3 increased expression of p62 at the lower dose used, but not at the higher dose. These results indicate that exposure to these chemicals alter normal physiology in the pituitary, what could promote pituitary pathologies. More results are underway to understand the mechanisms involved. Supported by CONICET, ANPCYT, VA SD Healthcare System, NIH, Fund. Williams, Fund. R. Barón. Presentation: Saturday, June 17, 2023