体外和计算机 AHR 测定评估鱼类中重油衍生多环芳烃的风险。
In vitro and in silico AHR assays for assessing the risk of heavy oil-derived polycyclic aromatic hydrocarbons in fish.
机构信息
Laboratory of Environmental Toxicology, Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama, 790-8577, Japan.
Faculty of the Advanced Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto, 860-8555, Japan.
出版信息
Ecotoxicol Environ Saf. 2019 Oct 15;181:214-223. doi: 10.1016/j.ecoenv.2019.06.008. Epub 2019 Jun 10.
In the aftermath of the Great East Japan Earthquake of March 11, 2011, marine fish in Kesennuma Bay, Japan, have been contaminated with heavy oil containing polycyclic aromatic hydrocarbons (PAHs). To estimate the risk of six PAHs (benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene), which have been detected at high levels in the tissues of fish from Kesennuma Bay, we attempted to evaluate the effects of these PAHs on the fish aryl hydrocarbon receptor (AHR) signaling pathway. We initially measured PAH concentrations and cytochrome P4501A catalytic activities (EROD: ethoxyresorufin-O-deethylase and MROD: methoxyresorufin-O-demethylase) as markers of AHR activation in greenlings (Hexagrammos otakii) collected from Kesennuma Bay in 2014. The results showed that alkylated PAH concentrations and EROD/MROD activities were higher in sites close to the oil-spilled sites than in the control site, suggesting AHR activation by spilled alkylated PAHs. We then investigated AHR-mediated responses to these PAHs in the in vitro reporter gene assay system where red seabream (Pagrus major) AHR1 (rsAHR1) or rsAHR2 expression plasmids were transiently transfected into COS-7 cells. The in vitro assay showed rsAHR isoform-, PAH-, and dose-dependent transactivation potencies. The relative effective concentrations of benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene that induce 20% of the maximum benzo[α]pyrene response (REC) for rsAHR1 activation were 0.052, 38, 79, 88, 270 nM, and no response, respectively, and those for rsAHR2 activation were 0.0049, 32, 53, 88, 60 nM, and no response, respectively. The results showed that the REC values of benzo[α]pyrene for both the rsAHR1 and rsAHR2 isoforms were lower than the concentrations (0.041-0.20 nM) detected in the muscle tissue of fish from Kesennuma Bay, while the REC values of other PAHs were higher than their tissue concentrations. In silico rsAHR homology modeling and subsequent ligand docking simulation analyses indicated that the rsAHR activation potencies of PAHs could be predicted from a rsAHR2 model. This study shows that in vitro and in silico rsAHR analyses may be a useful tool for assessing the risks to fish contaminated with PAHs.
在 2011 年 3 月 11 日东日本大地震之后，日本气仙沼湾的海洋鱼类受到了含有多环芳烃（PAHs）的重油污染。为了评估在气仙沼湾鱼类组织中高浓度检测到的六种 PAHs（苯并[a]芘、二苯并噻吩、菲、2,3,5-三甲基萘、苊和 1-甲基菲）的风险，我们试图评估这些 PAHs 对鱼类芳香烃受体（AHR）信号通路的影响。我们最初测量了 2014 年从气仙沼湾采集的绿鳍鱼（Hexagrammos otakii）中的 PAH 浓度和细胞色素 P4501A 催化活性（EROD：乙氧基Resorufin-O-脱乙基酶和 MROD：甲氧基Resorufin-O-去甲基酶）作为 AHR 激活的标志物。结果表明，靠近溢油点的地点的烷基化 PAH 浓度和 EROD/MROD 活性高于对照点，表明溢油的烷基化 PAHs 激活了 AHR。然后，我们在体外报告基因检测系统中研究了这些 PAHs 对 AHR 的介导反应，其中红色真鲷（Pagrus major）AHR1（rsAHR1）或 rsAHR2 表达质粒瞬时转染到 COS-7 细胞中。体外测定显示 rsAHR 同工型、PAH 和剂量依赖性的转激活能力。诱导 rsAHR1 激活的最大苯并[a]芘反应的 20%有效浓度（REC）的苯并[a]芘、二苯并噻吩、菲、2,3,5-三甲基萘、苊和 1-甲基菲分别为 0.052、38、79、88、270 nM 和无反应，而 rsAHR2 激活的 REC 值分别为 0.0049、32、53、88、60 nM 和无反应。结果表明，rsAHR1 和 rsAHR2 同工型的苯并[a]芘 REC 值均低于从气仙沼湾鱼类肌肉组织中检测到的浓度（0.041-0.20 nM），而其他 PAHs 的 REC 值均高于其组织浓度。rsAHR 同源建模和随后的配体对接模拟分析表明，PAHs 的 rsAHR 激活能力可以从 rsAHR2 模型中预测。本研究表明，体外和计算机 rsAHR 分析可能是评估受 PAHs 污染鱼类风险的有用工具。
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