Effetti cellulari provocati da plastificanti presenti negli involucri per alimenti su neuroni ipotalamici a GnRH immortalizzati (linea GT1-7)

Endocrine disruptor chemicals (EDCs) are exogenous substances that may interfere with the body endocrine system and produce adverse developmental, reproductive, neurological, and immune effects. They may be found in many everyday products, including plastic bottles, metal food cans, detergents, flame retardants, food, toys, cosmetics, and pesticides. EDCs have been shown to alter the pubertal process, having the reproductive system one of their main targets. EDCs may interfere with hormonal feedback regulation and neuroendocrine cell activity by binding to many receptor targets: classical nuclear receptors for estrogens ERα and ERβ, which regulate the expression of target genes, membrane-bound estrogen receptors GPR30, inducing rapid nongenomic responses through second messenger-triggered signal cascades, and the orphan nuclear receptor ERRγ. EDCs can also interfere with cell homeostasis and metabolism by binding to peroxisome proliferator-activated receptors (PPARs), retinoid X receptors (RXRs) and liver X receptors (LXRs). The aim of my thesis was to test the effects of a small set of putative EDCs on hypothalamic Gonadotropin Releasing Hormone (GnRH) neuron proliferation, neurite growth and calcium homeostasis, taking advantage of the GT1-7 murine cell line. Firstly, the expression of ERs, PPARs, RXRs and LXRs was checked in GT1-7 cells by RTPCR. We found that most receptors were expressed, suggesting that they could act as EDC targets. A set of plasticizers (plastic additives) with computational affinities for the above receptors were selected as putative EDCs: Bisphenol A (BPA, a known xenoestrogenic compound), the phthalates Diisononyl Phthalate (DINP) and Diisodecyl Phthalate (DIDP), the dibenzoate Diethylene Glycol Dibenzoate (DEGD), and the organophosphate tri-meta-cresyl phosphate (TMCP). The effects of these plasticizers on cell number was measured by crystal violet assays in high and low serum conditions (10% or 0.5% FBS), after 24 and 72 hours treatments at several different concentrations (from 10 nM to 100 μM). In proliferative conditions (10% FBS) a cell number increase was observed at 72 hours in the nM concentration range for all the selected molecules. Conversely, in 0.5% FBS, only BPA was able to positively affect cell number after 72 hours exposure at low nM and low μM concentrations. To assess if the increase in cell number observed with BPA was due to an enhancement of cell survival or cell proliferation, a BrdU assay was set; the results indicated that BPA is able to promote GT1-7 cells proliferation also in low serum condition. GT1-7 cells neurite growth was tested after 48 hours EDC exposure in low serum medium; none of the treatments was able to elicit a significant modulation of the measured parameters, indicating that this cellular activity is not affected. Finally, we tested BPA effects on GT1-7 cells in Ca2+ imaging-experiments. Acute exposure to BPA from 10 nM up to 5 μM was able to alter calcium homeostasis, inducing slow calcium influx from the extracellular medium, in a way dependent on voltage-gated calcium channels. Taken together, our data suggest that plasticizers present in everyday products may exert specific effects on development and cellular activity of GnRH neurons, possibly affecting the neuroendocrine control of puberty and reproduction.