Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan.
Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea; Department of Biology, Kyung Hee University, Seoul 130-701, Republic of Korea.
Aquat Toxicol. 2014 Sep;154:39-47. doi: 10.1016/j.aquatox.2014.05.001. Epub 2014 May 10.
The toxicity of dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is mainly mediated by an aryl hydrocarbon receptor (AHR), which regulates the transcription of multiple target genes including cytochrome P450 1A (CYP1A). Our pervious study identified the presence of TCDD-induced defects of peripheral nerve projection in red seabream (Pagrus major) embryos. However, it remains unclear whether the TCDD-induced peripheral neurotoxicity is mediated by the AHR. To assess the contribution of the red seabream AHR (rsAHR) signaling pathway to the neuronal toxicity, red seabream embryos at 10h post-fertilization (hpf) were treated for 80 min with TCDD (0, 0.3, 5.3, and 37 nM in seawater) alone or in combination with CH223191 (500 nM in seawater), which is an AHR antagonist. A preliminary in vitro reporter gene assay confirmed that TCDD-induced transcriptional activity via rsAHR1 and rsAHR2 was suppressed by CH223191 treatment in a dose-dependent manner. CYP1A mRNA expression in embryos was determined by 2-step real time quantitative-polymerase chain reaction at 24 and 120 hpf and in situ hybridization at 48, 72, 96 and 120 hpf. The morphology of the peripheral nerve system (PNS) was also microscopically observed by fluorescent staining using an anti-acetylated tubulin antibody at 120 hpf. CYP1A mRNA expression was dose-dependently induced by TCDD at all of the examined developing stages. The suppression of TCDD-induced CYP1A expression by CH223191 treatment was observed in embryos at 24 and 48 hpf, while the effect of the rsAHR antagonist disappeared at 96 and 120 hpf. This phenomenon indicated the transient suppression of TCDD-induced rsAHR activation by CH223191 treatment. The immunostaining of peripheral nerves at 120 hpf demonstrated that the projections of the craniofacial nerve were altered in TCDD-treated embryos, and the frequency of TCDD-induced abnormal projections was not prevented by co-treatment with CH223191. These results indicate that the transient suppression of TCDD-induced rsAHR activation during the early developing stages of the red seabream does not influence the abnormal projection of peripheral nerves. In conclusion, transient rsAHR activation in the early stages of development is not involved in the neurotoxicity.
二恶英如 2,3,7,8-四氯二苯并对二恶英（TCDD）的毒性主要通过芳烃受体（AHR）介导，其调节细胞色素 P450 1A（CYP1A）等多种靶基因的转录。我们之前的研究发现，红鲷鱼胚胎外周神经投射存在 TCDD 诱导的缺陷。然而，TCDD 诱导的周围神经毒性是否通过 AHR 介导仍不清楚。为了评估红鲷鱼 AHR（rsAHR）信号通路对神经元毒性的贡献，在受精后 10 小时（hpf）的红鲷鱼胚胎中，单独或与 CH223191（海水中 500 nM）联合处理 80 分钟，CH223191 是一种 AHR 拮抗剂。初步的体外报告基因分析证实，TCDD 通过 rsAHR1 和 rsAHR2 诱导的转录活性被 CH223191 处理以剂量依赖的方式抑制。在 24 和 120 hpf 时通过两步实时定量聚合酶链反应和 48、72、96 和 120 hpf 时通过原位杂交测定胚胎中 CYP1A mRNA 的表达。在 120 hpf 时，通过抗乙酰化微管蛋白抗体的荧光染色也在显微镜下观察到周围神经系统（PNS）的形态。TCDD 在所有检查的发育阶段均呈剂量依赖性诱导 CYP1A mRNA 表达。在 24 和 48 hpf 的胚胎中观察到 CH223191 处理抑制 TCDD 诱导的 CYP1A 表达，而 rsAHR 拮抗剂的作用在 96 和 120 hpf 时消失。这种现象表明 CH223191 处理瞬时抑制 TCDD 诱导的 rsAHR 激活。120 hpf 时外周神经的免疫染色表明，颅神经的投射在 TCDD 处理的胚胎中发生改变，并且 CH223191 共同处理不能防止 TCDD 诱导的异常投射。这些结果表明，在红鲷鱼早期发育阶段瞬时抑制 TCDD 诱导的 rsAHR 激活不会影响周围神经的异常投射。总之，早期发育阶段瞬时 rsAHR 激活不参与神经毒性。
