Akahoshi Eiichi, Yoshimura Seiko, Ishihara-Sugano Mitsuko
Environmental Technology Laboratory, Corporate Research & Development Center, Toshiba Corporation, 1 Komukai-Toshiba cho, Saiwai-ku, Kawasaki 212-8582, Japan.
Environ Health. 2006 Sep 7;5:24. doi: 10.1186/1476-069X-5-24.
Dioxins and related compounds are suspected of causing neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth. The molecular mechanism(s) of the actions in the brain, however, have not been fully investigated. A major participant in the process of the dioxin-toxicity is the dioxin receptor, namely the aryl hydrocarbon receptor (AhR). AhR regulates the transcription of diverse genes through binding to the xenobiotic-responsive element (XRE). Since the AhR has also been detected in various regions of the brain, the AhR may play a key role in the developmental neurotoxicity of dioxins. This study focused on the effect of AhR activation in the developing neuron.
The influence of the AhR on the developing neuron was assessed using the Neuro2a-AhR transfectant. The undifferentiated murine neuroblastoma Neuro2a cell line (ATCC) was stably transfected with AhR cDNA and the established cell line was named N2a-Ralpha. The activation of exogenous AhR in N2a-Ralpha cells was confirmed using RNAi, with si-AhR suppressing the expression of exogenous AhR. The neurological properties of N2a-Ralpha based on AhR activation were evaluated by immunohistochemical analysis of cytoskeletal molecules and by RT-PCR analysis of mRNA expression of neurotransmitter-production related molecules, such as tyrosine hydroxylase (TH).
N2a-Ralpha cells exhibited constant activation of the exogenous AhR. CYP1A1, a typical XRE-regulated gene, mRNA was induced without the application of ligand to the culture medium. N2a-Ralpha cells exhibited two significant functional features. Morphologically, N2a-Ralpha cells bore spontaneous neurites exhibiting axon-like properties with the localization of NF-H. In addition, cdc42 expression was increased in comparison to the control cell line. The other is the catecholaminergic neuron-like property. N2a-Ralpha cells expressed tyrosine hydroxylase (TH) mRNA as a functional marker of catecholaminergic neurotransmitter production. Thus, exogenous AhR induced catecholaminergic differentiation in N2a-Ralpha cells.
The excessive activation of AhR resulted in neural differentiation of Neuro2a cells. This result revealed that dioxins may affect the nervous system through the AhR-signaling pathway. Activated AhR may disrupt the strictly regulated brain formation with irregular differentiation occurring rather than cell death.
二噁英及相关化合物被怀疑会在发育和生长过程中围产期暴露后，导致人类和实验动物后代出现神经功能紊乱。然而，其在大脑中的作用分子机制尚未得到充分研究。二噁英毒性过程中的一个主要参与者是二噁英受体，即芳烃受体（AhR）。AhR通过与外源性反应元件（XRE）结合来调节多种基因的转录。由于在大脑的各个区域也检测到了AhR，AhR可能在二噁英的发育神经毒性中起关键作用。本研究聚焦于AhR激活对发育中神经元的影响。
使用Neuro2a-AhR转染细胞评估AhR对发育中神经元的影响。将未分化的小鼠神经母细胞瘤Neuro2a细胞系（ATCC）用AhR cDNA进行稳定转染，建立的细胞系命名为N2a-Ralpha。使用RNA干扰技术（RNAi），用si-AhR抑制外源性AhR的表达，从而在N2a-Ralpha细胞中证实外源性AhR的激活。通过对细胞骨架分子的免疫组织化学分析以及对神经递质产生相关分子（如酪氨酸羟化酶（TH））的mRNA表达的逆转录-聚合酶链反应（RT-PCR）分析，评估基于AhR激活的N2a-Ralpha细胞的神经学特性。
N2a-Ralpha细胞表现出外源性AhR的持续激活。典型的XRE调控基因CYP1A1的mRNA在未向培养基中添加配体的情况下被诱导。N2a-Ralpha细胞表现出两个显著的功能特征。在形态上，N2a-Ralpha细胞具有自发的神经突，表现出轴突样特性，且有NF-H的定位。此外，与对照细胞系相比，cdc42的表达增加。另一个是儿茶酚胺能神经元样特性。N2a-Ralpha细胞表达酪氨酸羟化酶（TH）mRNA，作为儿茶酚胺能神经递质产生的功能标志物。因此，外源性AhR诱导N2a-Ralpha细胞发生儿茶酚胺能分化。
AhR的过度激活导致Neuro2a细胞发生神经分化。这一结果表明，二噁英可能通过AhR信号通路影响神经系统。激活的AhR可能会破坏严格调控的大脑形成，导致出现不规则分化而非细胞死亡。
