Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama, Japan.
Aquat Toxicol. 2013 Mar 15;128-129:193-202. doi: 10.1016/j.aquatox.2012.12.009. Epub 2012 Dec 21.
We investigated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced effects on the morphology of peripheral nervous system (PNS) in the developing red seabream (Pagrus major) embryos. The embryos at 10h post-fertilization (hpf) were treated with 0, 0.1, 0.4 or 1.7 μg/L of TCDD in seawater for 80 min. The morphology of PNS was microscopically observed with florescence staining using an anti-acetylated tubulin antibody at 48, 78, 120 and 136 hpf. Axon length of facial nerve (VII) was found to be shortened by TCDD exposure. Axon guidance in the glossopharyngeal nerve (IX) and vagus nerve (X) was altered at 120 and 136 hpf in a TCDD dose-dependent manner. Lowest observable effect level of TCDD (0.1 μg/L) that induced the morphological alteration of PNS was lower than those of other endpoints on morphological deformities so far reported. Given that the growth cone at the tip of growing nerve axons advances under the influence of its surrounding tissues, we hypothesized that TCDD exposure would affect (1) the nerve cell proliferation/differentiation, (2) the structure of muscle as an axon target and (3) the nerve guidance factor in the embryos. By the immunostaining of embryos with an antibody against the neuronal specific RNA-binding protein, HuD, and an antibody against the sarcomeric myosin, no morphological effects were observed on the neural proliferation/differentiation and the structure of facial muscles of TCDD-treated embryos. In contrast, whole mount in situ hybridization of semaphorin 3A (Sema3A), a secretory axon repulsion factor, revealed the altered expression pattern of its transcripts in TCDD-treated embryos. Our findings suggest that TCDD treatment affects the projection of PNS in the developing red seabream embryos through the effects on the axonal growth cone guidance molecule such as Sema3A, but not on the neuronal differentiation/proliferation and axon target. The PNS in developing embryos may be one of the most sensitive biomarkers to the exposure of dioxin-like compounds.
我们研究了 2,3,7,8-四氯二苯并对二恶英（TCDD）对发育中红鲷鱼胚胎外周神经系统（PNS）形态的诱导作用。在受精后 10 小时（hpf），将胚胎用 0、0.1、0.4 或 1.7μg/L 的 TCDD 在海水中处理 80 分钟。在 48、78、120 和 136 hpf 时，用抗乙酰化微管蛋白抗体进行荧光染色，观察 PNS 的形态。发现 TCDD 暴露会导致面神经（VII）轴突长度缩短。在 120 和 136 hpf 时，TCDD 暴露以剂量依赖的方式改变了舌咽神经（IX）和迷走神经（X）的轴突导向。TCDD 诱导 PNS 形态改变的最低可观察效应水平（0.1μg/L）低于迄今为止报道的其他形态畸形终点。鉴于生长锥在生长轴突的尖端在其周围组织的影响下前进，我们假设 TCDD 暴露会影响（1）神经细胞的增殖/分化，（2）肌肉作为轴突靶的结构和（3）胚胎中的神经导向因子。通过用神经元特异性 RNA 结合蛋白 HuD 的抗体和肌球蛋白的抗体对胚胎进行免疫染色，未观察到 TCDD 处理胚胎的神经增殖/分化和面部肌肉结构的形态效应。相比之下，轴突排斥因子 Sema3A 的全胚胎原位杂交显示其转录本在 TCDD 处理胚胎中的表达模式发生改变。我们的研究结果表明，TCDD 处理通过影响 Sema3A 等轴突生长锥导向分子来影响发育中红鲷鱼胚胎 PNS 的投射，而不是影响神经元分化/增殖和轴突靶。发育中的胚胎的 PNS 可能是对二恶英样化合物暴露最敏感的生物标志物之一。
