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El pdf del artículo es la versión post-print., The organochlorine chemicals endosulfan, dieldrin, and γ-hexachlorocyclohexane (lindane) are persistent pesticides to which people are exposed mainly via diet. Their antagonism of the γ-aminobutyric acid-A (GABAA) receptor makes them convulsants. They are also endocrine disruptors because of their interaction with the estrogen receptor (ER). Here, we study the effects of dieldrin, endosulfan, and lindane on ERs in primary cultures of cortical neurons (CN) and cerebellar granule cells (CGC). All the compounds tested inhibited the binding of [3H]-estradiol to the ER in both CN and CGC, with dieldrin in CGC showing the highest affinity. We also determined the effects of the pesticides on protein kinase B (Akt) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Dieldrin and endosulfan increased Akt phosphorylation in CN, which was inhibited by the ERb antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-α]pyrimidin-3-yl]phenol. Instead, Akt and ERK1/2 phosphorylation induced by dieldrin in CGC was mediated by multiple activation of ERa, ERb, and G protein- coupled receptor 30. Lindane did not activate these pathways, but it inhibited estradiol-mediated Akt and ERK1/2 activation. In CN, all the chemicals activated ERK1/2 through a mechanism involving GABAA and glutamate receptors. Long-term exposure to these pesticides reduced the levels of ERα, but not of ERβ. Moreover, extracts of CN treated with endosulfan, dieldrin, or lindane induced cell proliferation in MCF-7 human breast cancer-derived cells, whereas only extracts of CGC treated with dieldrin induced MCF-7 cell proliferation. Overall, the observed alterations on ER-mediated signaling and ER levels in neurons might contribute to the neurotoxicity of these organochlorine pesticides. © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved., This research was supported by grants from Ministry of Health (FIS 061212, FIS10/0453), CIBERESP (AA08-001), Generalitat de Catalunya (2009/SGR/214) and from Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía (P09-CTS-5488).

Dieldrin is an endocrine disruptor that accumulates in mammalian adipose tissue and brain. It induces convulsions due to its antagonism of the γ-aminobutyric acid A receptor (GABA AR). We have previously reported that long-term exposure to dieldrin causes the internalization of the N-methyl-D-aspartate receptor (NMDAR) as a result of persistent GABAAR inhibition. Because the neurosteroids 17β-estradiol (E2) and allopregnanolone are known to modulate the function and trafficking of GABA AR and NMDAR, we examined the effects of E2 and allopregnanolone on dieldrin-induced GABA AR inhibition, NMDAR internalization, and neuronal death in cortical neurons. We found that 1 nM E2 increased the membrane expression of NR1/NR2B receptors and postsynaptic density 95 but did not induce their physical association. In contrast, 10 nM E2 had no effect on these proteins but reduced NR2A membrane expression. We also found that exposure to 60 nM dieldrin for 6 d in vitro caused the internalization of NR1 and NR2B but not NR2A. Treatment with either 1 nM E2 or 10 οM allopregnanolone prevented the dieldrin-induced reduction in membrane levels of the NR1/NR2B receptors. Furthermore, prolonged exposure to 200 nM dieldrin down-regulated the expression of NR2A; this was inhibited only by allopregnanolone. Although both hormones restored NMDAR function, as measured by the NMDA-induced rise in intracellular calcium, allopregnanolone (but not E2) reversed the inhibition of GABA AR and neuronal death caused by prolonged exposure to dieldrin. Our results indicate that allopregnanolone protects cortical neurons against the neurotoxicity caused by long-term exposure to dieldrin by maintaining GABA AR and NMDAR functionality. Copyright © 2012 by The Endocrine Society., This work was supported by the Ministry of Science and Innovation (FIS 10/0453) (to CS); Generalitat de Catalunya (2009/SGR/214) (to CS); the Agence Nationale pour la Recherche Grants ANR-07-NEURO-026-03 and ANR-09-BLAN-0267 (to VP); the European Society for Pediatric Endocrinology (ESPE) Research Unit Grant 2009-2011 coordinated by Dr. Sabine Heger (Children’s Hospital Bult, Hannover, Germany) (to V. P.).

Trabajo presentado en el XII International Congress of Toxicology, celebrado en Barcelona, España, del 19 al 23 de julio de 2010, The organochlorine chemicals endosulfan, dieldrin, and lindane are persistent pesticides, and endosulfan is frequently found in fruits and vegetables, contributing to diet exposure. These compounds are convulsants due to their antagonism on the GABAA receptor. They are also endocrine disruptors because of their interaction with the estrogen receptor (ER) in reporter cell lines. The aim of this study was to investigate the effects of dieldrin, endosulfan, and γ-hexachlorocyclohexane (γ-HCH, lindane) and its isomer β-HCH on ER binding as well as on Akt and MAPK pathways in primary cultures of either cortical neurons (CN) or cerebellar granule cells (CGC) and also in SH-SY5Y neuroblastoma cells. We observed that all the tested compounds, except β-HCH isomer, inhibited [3H]-17β-Estradiol binding in both primary cultures but with different IC50s, with dieldrin in CGC showing the highest potency. In addition, dieldrin and endosulfan enhanced Akt and ERK1/2 phosphorylation, whereas lindane inhibited the activation of both proteins mediated by estradiol. We further analyzed whether these pesticides could altered the intracellular estrogenic charge of the neuronal cultures. For this purpose we used the E-Screen assay, in which MCF-7 human breast cancer-derived cells were incubated with the intracellular content of the neuronal cultures that were previously exposed to these pollutants for a short- and long-term period of time. Prolonged exposure to the three compounds enhanced the basal estrogenic activity of CN whereas only exposure to dieldrin increases that of CGC. On the other hand, we have observed that long-term exposure to dieldrin reduced the levels of ER-alpha, but not ER-beta, in both primary cultures. In summary, we demonstrated that the three organochlorine pesticides tested are endocrine disruptors in neuronal cultures., Supported by FIS PI061212 and CIBERESP (AA08_001).

Trabajo presentado en el XII International Congress of Toxicology, celebrado en Barcelona, España, del 19 al 23 de julio de 2010, Dieldrin is a previously used pesticide that accumulates in the adipose tissue as well as in the brain of mammals. This organochlorine has convulsant activity because it blocks the GABAA receptor. However, little is known about the effects of a chronic exposure to this pollutant. We have previously reported that long-term exposure to dieldrin reduces the number of functional NMDA receptors (NMDARs) in neuronal cultures. On the other hand, estradiol increases synaptogenesis and promotes the delivery of NMDAR to the cell membrane. The aims of this study are: (i) elucidate which subunits of NMDAR are affected by dieldrin exposure and (ii) evaluate the effects of estradiol against dieldrin-induced NMDAR internalization. Long-term (6DIV) exposure to a subcitotoxic concentration (60 nM) of dieldrin caused the internalization of NR1 and NR2B, but not NR2A, subunits of NMDAR. Treatment with 1 nM (but not 10 nM) 17β-estradiol (E2) for 2DIV rescued dieldrin-induced decrease of NR1 and NR2B on the cell membrane but not their internalization, suggesting different mechanisms of action between the two compounds regarding NMDAR trafficking. E2 also restored NMDAR function, as measured by NMDA-induced [Ca2+]i increase. Furthermore, prolonged exposure to a higher concentration (200 nM) of dieldrin also reduced cell surface expression of NR2A, but this effect was not prevented by E2. In addition, we showed here by immunoprecipitation on membrane extraction that PSD-95 is highly physically associated with NR2A-containing (but not NR2B-containing) NMDARs in cortical neurons and moreover that this association is lost after prolonged exposure to 200 nM dieldrin. In the present study, we confirmed that the permanent blockade of the GABAA receptor by this persistent pesticide triggers adaptive neuronal changes consisting on NMDAR internalization, that might explain the learning and cognitive deficits observed in animals after chronic treatment with dieldrin. In addition, estradiol may exert beneficial effects against dieldrin-induced neurotoxicity., Supported by FIS PI061212.