Efekti heksabromociklododekana na steroidogeni potencijal Lejdigovih ćelija testisa i granuloza ćelija ovarijuma pacova

Abstract

<p>HBCDD&nbsp; je&nbsp; bromovani&nbsp; usporivač&nbsp; gorenja&nbsp; koji&nbsp; se&nbsp; u&nbsp; cilju&nbsp;za&scaron;tite&nbsp; od&nbsp; požara&nbsp; dodaje&nbsp; različitim&nbsp; materijalima&nbsp; kao&nbsp; &scaron;to&nbsp;su&nbsp; polistiren,&nbsp; tekstil,&nbsp; odnosno&nbsp; materijali&nbsp; koji&nbsp; ulaze&nbsp; u&nbsp;sastav&nbsp; električnih&nbsp; elektronskih&nbsp; uređaja.&nbsp; HBCDD&nbsp; se&nbsp;oslobađa iz tih materijala i danas ga detektujemo u svim&nbsp;matricama&nbsp; životne&nbsp; sredine.&nbsp; Predmet&nbsp; istraživanja&nbsp; ove&nbsp;<br />teze&nbsp; je&nbsp; bio&nbsp; moguć&nbsp; uticaj&nbsp; HBCDD&nbsp; na&nbsp; funkciju&nbsp; steroidprodukujućih&nbsp; ćelija&nbsp; gonada.&nbsp; Efekat&nbsp; HBCDD&nbsp; je&nbsp; ispitivan&nbsp;na&nbsp; tri&nbsp; modela:&nbsp; 1)&nbsp; primarna&nbsp; kultura&nbsp; Lejdigovih&nbsp; ćelija izolovanih&nbsp; iz&nbsp; testisa&nbsp; peripubertalnih&nbsp; mužjaka&nbsp; pacova;&nbsp; 2) primarna&nbsp; kultura&nbsp; nezrelih&nbsp; granuloza&nbsp; ćelija&nbsp; izolovanih&nbsp; iz ovarijuma&nbsp; estradiolom-pretretiranih &nbsp;nezrelih&nbsp; ženki pacova;&nbsp; i&nbsp; 3)&nbsp; primarna&nbsp; kultura&nbsp; preovulatornih&nbsp; granuloza ćelija&nbsp; izolovanih&nbsp; iz&nbsp; ovarijuma,&nbsp; konjskim&nbsp; serumskim gonadotropinom&nbsp; (PMSG)-pretretiranih&nbsp; nezrelih&nbsp; ženki pacova.&nbsp; Naglasak&nbsp; je&nbsp; bio&nbsp; na&nbsp; razja&scaron;njavanju&nbsp; mehanizma toksičnog&nbsp; dejstva&nbsp; HBCDD.&nbsp; Na&nbsp; nivou&nbsp; Lejdigovih&nbsp; ćelija, pokazali&nbsp; smo&nbsp; da&nbsp; HBCDD&nbsp; remeti&nbsp; sintezu&nbsp; androgena, inhibi&scaron;ući&nbsp; mitohondrijski&nbsp; membranski&nbsp; potencijal&nbsp; ( &Delta;&Psi;m). HBCDD-indukovana&nbsp; inhibicija&nbsp; &Delta;&Psi;m&nbsp; je&nbsp; dovela&nbsp; do inhibisane&nbsp; sinteze&nbsp; ATP,&nbsp; &scaron;to&nbsp; je&nbsp; rezultovalo&nbsp; u&nbsp; inhibisanoj sintezi&nbsp; cAMP.&nbsp; Inhibi&scaron;ući&nbsp; glavni&nbsp; signalni&nbsp; put&nbsp; koji&nbsp; reguli&scaron;e steroidogenezu cAMP/PKA, HBCDD je u prisustvu hCG inhibisao transport holestrola u mitohondrije&nbsp; i time akutno inhibisao steroidogenezu. U bazalnim uslovima, HBCDD je&nbsp; stimulisao&nbsp; steroidogenezu,&nbsp; olak&scaron;avajući&nbsp; transport holesterola&nbsp; u&nbsp; mitohondrije,&nbsp; efekat&nbsp; koji&nbsp; je&nbsp; najverovatnije rezultat naru&scaron;enog&nbsp; &Delta;&Psi;m. Duži HBCDD tretmani, praćeni redukovanim&nbsp; nivom&nbsp; cAMP&nbsp; su&nbsp; doveli&nbsp; do&nbsp; inhibisane ekspresije&nbsp; steroidogenih&nbsp; enzima&nbsp; i&nbsp; finalno&nbsp; inhibisali steroidogenezu&nbsp; i&nbsp; u&nbsp; bazalnim&nbsp; i&nbsp; u&nbsp; hCG-stimulisanim uslovima.&nbsp; Kod&nbsp; nezrelih&nbsp; granuloza&nbsp; ćelija&nbsp; HBCDD&nbsp; je inhibisao FSH-indukovanu diferencijaciju granuloza ćelja i&nbsp; time&nbsp; ugrozio&nbsp; proces&nbsp; ovulacije.&nbsp; Efekti&nbsp; se&nbsp; ogledaju&nbsp; u inhibisanoj&nbsp; ekspresiji&nbsp; LH&nbsp; receptora&nbsp; i&nbsp; inhibisanoj&nbsp; sintezi estradiola.&nbsp; HBCDD-indukovana&nbsp; inhibicija&nbsp; FSHindukovane ekspresije LHR je uzrokovala inhibiciju hCGindukovane&nbsp; ekspresije&nbsp; ovulatornih&nbsp; gena,&nbsp; sugeri&scaron;ući&nbsp; da HBCDD&nbsp; ima&nbsp; potencijal&nbsp; da&nbsp; remeti&nbsp; ovulaciju.&nbsp; Dobijeni rezultati&nbsp; ukazuju&nbsp; da&nbsp; je&nbsp; HBCDD&nbsp; potencirao&nbsp; FSHindukovanu&nbsp; aktivaciju&nbsp; ERK1/2&nbsp; i&nbsp; AKT,&nbsp; kao&nbsp; i&nbsp; njih ovog uzvodnog&nbsp; regulatora,&nbsp; takođe&nbsp; FSH-indukovanog,&nbsp; EGFR. Na&nbsp; osnovu&nbsp; rezultata&nbsp; na&nbsp; nivou&nbsp; ekspresije&nbsp; LHR, zaključujemo&nbsp; da&nbsp; su&nbsp; HBCDD-indukovani&nbsp; toksični&nbsp; efekti rezultat potenciranja aktivnosti&nbsp; EGFR/ERK1/2 signalnog puta.&nbsp; Kod&nbsp; preovulatornih&nbsp; granuloza&nbsp; ćelija,&nbsp; koje ekspresuju&nbsp; LHR,&nbsp; HBCDD&nbsp; nije&nbsp; uticao&nbsp; na&nbsp; ekspresiju ovulatornih gena, kao ni na ekspesiju steroidogenih gena i steroidogenezu. Generalno, pokazali smo da HBCDD u in&nbsp; vitro&nbsp; uslovima&nbsp; naru&scaron;ava&nbsp; funkciju&nbsp; i&nbsp; Lejdigovih&nbsp; ćelija testisa&nbsp; i&nbsp; granuloza&nbsp; ćelija&nbsp; ovarijuma,&nbsp; delujući&nbsp; na&nbsp; različite signalne&nbsp; puteve,&nbsp; cAMP/PKA&nbsp; kod&nbsp; Lejdigovih&nbsp; ćelija&nbsp; i EGFR/ERK1/2&nbsp; kod&nbsp; granuloza&nbsp; ćelija.&nbsp; Na&nbsp; osnovu celokupnih&nbsp; rezultata&nbsp; možemo&nbsp; zaključiti&nbsp; da&nbsp; HBCDD uticajem&nbsp; na&nbsp; odgovarajuće&nbsp; signalne&nbsp; puteve&nbsp; u&nbsp; steroidprodukujućim&nbsp; ćelijama&nbsp; gonada&nbsp; ispoljava &nbsp; inhibitorno dejstvo na reprodukciju.</p>

<p>HBCDD is brominated&nbsp; flame retardant routinely added to&nbsp;polystyrene&nbsp; plastics,&nbsp; textile&nbsp; and&nbsp; electric&nbsp; and&nbsp; electronic&nbsp;devices&nbsp; with&nbsp; the&nbsp; purpose&nbsp; of&nbsp; fire&nbsp; protection.&nbsp; Out&nbsp; of&nbsp; such&nbsp;materials&nbsp; HBCDD&nbsp; leaks,&nbsp; and&nbsp; today&nbsp; is&nbsp; detected&nbsp; in&nbsp; all&nbsp;environmental matrices. The subject of&nbsp; this study was&nbsp; to&nbsp;investigate&nbsp; toxic&nbsp; effects&nbsp; of&nbsp; HBCDD&nbsp; on&nbsp; steroid -producing&nbsp;cells of the testes and ovary. Three different test models&nbsp;were used: 1) primary culture of Leydig cells isolated from&nbsp;testes&nbsp; of&nbsp; peripubertal&nbsp; male&nbsp; rats;&nbsp; 2)&nbsp; primary&nbsp; culture&nbsp; of&nbsp;immature granulosa cells isolated from ovary of immature&nbsp;female&nbsp; rats&nbsp; pretreated&nbsp; with&nbsp; estradiol;&nbsp;&nbsp; and&nbsp; 3)&nbsp; primary&nbsp;culture of preovulatory granulosa cells &nbsp;isolated from ovary&nbsp;of&nbsp; immature&nbsp; female&nbsp; rats&nbsp; pretreated&nbsp; with&nbsp; pregnant&nbsp; mare&nbsp;serum&nbsp; gonadotropin&nbsp; (PMSG).&nbsp; The&nbsp; emphasis&nbsp; was&nbsp; on&nbsp;revealing&nbsp; mechanism&nbsp; of&nbsp; action&nbsp; of&nbsp; HBCDD.&nbsp; Here&nbsp; we&nbsp;showed that HBCDD disrupts Leydig cell steroidogenesis&nbsp;<br />via&nbsp; disruption&nbsp; of&nbsp; mitochondrial&nbsp; membrane&nbsp; potential&nbsp;(&Delta;&Psi;m). HBCDD-induced inhibition of&nbsp; &Delta;&Psi;m led to inhibited&nbsp;ATP&nbsp; synthesis&nbsp; and&nbsp; consequently&nbsp; to&nbsp; decreased&nbsp; cAMP&nbsp;production.&nbsp; By&nbsp; inhibiting&nbsp; main&nbsp; pathway&nbsp; regulating&nbsp;steroidogenesis&nbsp; -&nbsp; cAMP/PKA&nbsp; pathway,&nbsp; HBCDD&nbsp; acutely&nbsp;inhibited&nbsp; hCG-induced&nbsp; Leydig&nbsp; cell&nbsp; steroidogenesis&nbsp; by&nbsp;inhibititing cholesterol transport into mitochondria. In basal&nbsp;conditions&nbsp; HBCDD&nbsp; stimulated&nbsp; androgen&nbsp; production&nbsp; via&nbsp;facilitated&nbsp; transfer&nbsp; of&nbsp; cholesterol&nbsp; into&nbsp; mitochondria,&nbsp; an&nbsp;effect&nbsp; probably&nbsp; resulting&nbsp; out&nbsp; of&nbsp; disrupted&nbsp; &Delta;&Psi;m.&nbsp; Longer&nbsp;period of HBCDD exposure, due to reduced cAMP levels,&nbsp;caused&nbsp; inhibition&nbsp; of&nbsp; steroidogenic&nbsp; gene&nbsp; expression&nbsp; and&nbsp;androgenesis, both in basal and hCG-induced conditions.&nbsp;In&nbsp; immature&nbsp; granulosa&nbsp; cells&nbsp; HBCDD&nbsp; disrupted&nbsp; FSHinduced&nbsp; cell &nbsp; differentiation,&nbsp; and&nbsp; therefore&nbsp; ovulatory&nbsp;competence of granulosa cells.&nbsp; HBCDD inhibited both&nbsp; Lhr&nbsp;expression&nbsp; and&nbsp; estradiol&nbsp; production.&nbsp; HBCDD-induced&nbsp;inhibition&nbsp; of &nbsp; FSH-induced&nbsp; LHR&nbsp; expression&nbsp; resulted&nbsp; in&nbsp;inhibited&nbsp; hCG-induced&nbsp; expression&nbsp; of&nbsp; ovulatory&nbsp; genes,&nbsp;suggesting that HBCDD has potential to disrupt ovulation.&nbsp;The&nbsp; obtained&nbsp; results&nbsp; indicated&nbsp; to&nbsp; HBCDD&nbsp; induced&nbsp; overstimulation&nbsp; of&nbsp; FSH-driven &nbsp;extracellular-regulated&nbsp; kinase&nbsp;1/2 (ERK1/2) and protein kinase B (PKB, also known as&nbsp;AKT),&nbsp; as&nbsp; well&nbsp; as&nbsp; their&nbsp; upstream&nbsp; regulator,&nbsp; also&nbsp; FSH&nbsp;driven, EGFR. As observed &nbsp;at the level of&nbsp; Lhr&nbsp; expression,&nbsp;HBCDD&nbsp; induced&nbsp; toxic&nbsp; effects&nbsp; were&nbsp; mediated&nbsp; via&nbsp;overstimulation of EGFR/ERK1/2 pathway. In preovulatory&nbsp;granulosa&nbsp; cells,&nbsp; which&nbsp; express&nbsp; LHR,&nbsp; HBCDD&nbsp; had&nbsp; effect&nbsp;neither&nbsp; on&nbsp; ovulatory&nbsp; gene&nbsp; expression,&nbsp; nor&nbsp; on&nbsp;steroidogenic&nbsp; gene&nbsp; expression&nbsp; and&nbsp; steroidogenesis.&nbsp; In&nbsp;general,&nbsp; here&nbsp; we&nbsp; show&nbsp; that&nbsp; HBCDD&nbsp; in&nbsp; vitro&nbsp; disrupts&nbsp;function&nbsp; of&nbsp; both&nbsp; testicular&nbsp; Leydig&nbsp; cells&nbsp;&nbsp; and&nbsp; ovarian&nbsp;granulosa cells by disrupting different signaling pathways,&nbsp;cAMP/PKA in Lejdig cells and EGFR/ERK1/2 in granulosa&nbsp;cells,&nbsp; and&nbsp; therefore&nbsp; has&nbsp; potential&nbsp; to&nbsp; compromise&nbsp;reproduction.</p>