Of mice and men: mechanistic studies on developmental neurotoxicity of Polybrominated Diphenyl Ethers (PBDEs) in 3D in vitro models
Final Report Abstract
Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants used in many consumer products like carpets and computers. They rapidly bioaccumulate in the environment and thus in human tissue. PBDEs cause neurodevelopmental deficits like behavioral abnormalities in rodents and epidemiological studies suggest that they also impact human brain development. The molecular mechanisms underlying these neurodevelopmental effects are largely unknown. One of the postulated mechanisms of PBDE-induced developmental neurotoxicity (DNT) is endocrine disruption of thyroid hormone (TH) signaling. To study whether PBDE-induced TH disruption on the cellular level is responsible for their neurotoxicity, we use a three dimensional (3D) in vitro cell culture model based on primary neural progenitor cells (NPCs). This model mimics the basic processes of brain development: proliferation, migration and differentiation, offers the possibility to study species-specific differences in sensitivity towards chemicals by using NPCs from human, mouse and rat and allows their mechanistic evaluation. During the first year of the project the murine neurosphere culture had to be re-established due to high variability rates. Thereafter, we found that the PBDE congener BDE-99 reduced neurogenesis and oligodendrogenesis in a concentration-dependent manner in a species-specific way: human neurogenesis was less sensitive towards PBDEs (IC50: 23 µM BDE-99) than mouse neurogenesis (IC50: 10 µM BDE-99). In contrast to mouse, human oligodendrogenesis was over all the most sensitive endpoint (human IC50: 2 µM and mouse IC50: 14 µM BDE-99) with human NPCs being approx. 7x more sensitive than mouse NPCs. Next, we investigated whether we could antagonize those BDE-99 effects on differentiation by the thyroid hormones (TH) T3 or T4. Because only murine, not human oligodendrogenesis, was found to be TH-dependent, it is not quite clear at this point if the antagonizing effects of TH on BDE-99-dependent reduced oligodendrogenesis is antagonizing oligodendrocyte reduction due to TH disruption or an independent mechanism. Next, we studied by performing time course experiments if BDE-99 caused a reduction in oligodendrocyte formation/maturation or if the reduced numbers occurred via oligodendrocyte death. BDE-99 seemed to disrupt formation of oligodendrocytes in both species, which could partially be reverted by TH only in mouse NPCs. If the BDE-99-dependent inhibition of oligodendrocyte formation in mouse NPCs is due to TH disruption or provoked by an independent mechanism needs to be elucidated. In human NPCs, co-treatment with BDE-99 and vitamin C suggests that BDE-99 acts via induction of oxidative stress, which needs to be confirmed. Exposure of NPCs generated form TH receptor (THR)α and THRβ knockout mice revealed that none of the receptors is involved in the BDE-99-dependent reduction of oligodendrocytes, but that THRα is needed for the oligodendrocyte induction by T3. BDE-99 interfered with the T3-induced transcription of oligodendrocyte maturation markers MBP and MOG in human and mouse oligodendrocytes, respectively. The species specificity in BDE-99 effects is supported by expression analyses of the directly THRα regulated gene hairless: BDE-99 reduces T3 induced hairless expression in hNPCs, but not in mNPCs. So far these data show that i) TH regulates oligodendrogenesis in mouse, but not human NPCs, while oligodendrocyte maturation is TH-dependent in both species. ii) BDE-99 affects oligodendrogenesis in both species. In mouse NPCs, this happens independently of THRα and THRβ, in human NPCs involvement of THRα is also unlikely, because TH does not induce human oligodendrogenesis, but BDE-99 inhibits human NPC THRα-dependent transcription. This BDE-99-dependent reduction in human oligodendrogenesis seems to be caused by oxidative stress rather than TH disruption. iii) TH-dependent oligodendrocyte maturation is affected by BDE-99 in both species by a yet unknown mechanism. Mechanistic work on BDE-99 effects on oligodendrocyte formation and maturation will be completed before publication.
