Effetti dell'esposizione al BPA e ad altri plasticizzanti sulla proliferazione dei precursori neurali in vitro

Neurogenesis is the process by which neurons are generated from neural progenitor cells (NPCs). This process is strongly influenced by extrinsic factors derived from the micro- and macro-environment to which the NPs are exposed. In the last two decades, worldwide concerns have focused on the possible deleterious effects of exposure to environmental chemicals altering the endocrine system and brain development in humans and wildlife. These compounds are called ¿endocrine disrupting chemicals¿ (EDCs), and include synthetic hormones, pesticides, pharmaceuticals, compounds used in the plastics industry (plasticizers) and consumer products. Many EDCs can exert an estrogenic activity by binding to nuclear estrogen receptors (ERs), others can interfere with cell homeostasis and metabolism through activation of peroxisome proliferator-activated receptors (PPARs), retinoid X receptors (RXRs) and liver X receptors (LXRs). All these molecular targets belong to the superfamily of nuclear receptors (NRs), therefore their activation can affect the regulation of gene transcription at critical steps of neurogenesis. In the present work we used ST14A cells, an immortalised neural progenitor cell line derived from embryonic day 14 rat striatum primordia, to assess the effects of EDCs on cell proliferation and cell survival. As a first step we checked the expression of ERs, PPARs, RXRs and LXRs in proliferating ST14A cells by RT-PCR. We found that most molecular forms of these NRs were expressed, indicating that they could act as putative targets of EDCs with possible effects on cell proliferation. A number of plasticizers with computational affinity for the above mentioned NRs were selected as putative EDCs: Bisphenol A (BPA, a known xenoestrogenic compound), the phthalates Diisononyl Phthalate (DINP) and Diisodecyl Phthalate (DIDP), and the dibenzoate Diethylene Glycol Dibenzoate (DEGD). In parallel with BPA, we tested the effects of estradiol (endogenous estrogen, E2) and of the synthetic high-affinity estradiol derivative 17α-Ethinyl estradiol (EE2). All the chemical concentrations employed were previously tested to avoid cell death or suffering. The possible involvement of these plasticizers in the proliferative process was measured by crystal violet assays on ST14A cells treated for 24 and 48 hours. In order to minimize the content of estrogens in the medium, for BPA, E2 and EE2 treatments we used a phenol red-free DMEM supplemented with charcoal stripped (estrogen-free) fetal bovine serum (FBS). We found that BPA, as well as E2 and EE2, were able to increase cell proliferation in a broad range of concentrations following 24h exposure. However, this effect was no longer visible at 48h, with the exception of low doses (10 nM) of BPA. DINP, DIDP and DEGD at 200 nM concentration all induced a highly significant proliferation increase following 24h treatment; this effect was persistent at 48h for the last two chemicals. Lowering the concentration of DINP and DEGD to 100 nM still exerted significant effects. When DEGD/DINP-treated cells were cultured in estrogen-free medium, no proliferation increase was observed, implying that background estrogens are needed to exert their action on the proliferative process. Taken together, our data enforce the emerging awareness on alteration of neurogenesis following EDCs exposure in utero, especially in critical steps of neural development such as the early step of NP proliferation.