Rhee Jae-Sung, Kim Bo-Mi, Lee Bo-Young, Hwang Un-Ki, Lee Yong Sung, Lee Jae-Seong
Department of Marine Science, College of Natural Sciences, Incheon National University, Incheon 406-772, South Korea.
Department of Biological Sciences, College of Science, Sungkyunkwan University, Suwon 440-746, South Korea.
Comp Biochem Physiol C Toxicol Pharmacol. 2014 Aug;164:11-20. doi: 10.1016/j.cbpc.2014.04.001. Epub 2014 Apr 12.
To investigate the effect of endocrine disrupting chemicals (EDCs) on the circadian rhythm pathway, we cloned clock and circadian rhythmic pathway-associated genes (e.g. Per2, Cry1, Cry2, and BMAL1) in the self-fertilizing mangrove killifish Kryptolebias marmoratus. The promoter region of Km-clock had 1 aryl hydrocarbon receptor element (AhRE, GTGCGTGACA) and 8 estrogen receptor (ER) half-sites, indicating that the AhRE and ER half sites would likely be associated with regulation of clock protein activity during EDCs-induced cellular stress. The Km-clock protein domains (bHLH, PAS1, PAS2) were highly conserved in five additional fish species (zebrafish, Japanese medaka, Southern platyfish, Nile tilapia, and spotted green pufferfish), suggesting that the fish clock protein may play an important role in controlling endogenous circadian rhythms. The promoter regions of Km-BMAL1, -Cry1, -Cry2, and -Per2 were found to contain several xenobiotic response elements (XREs), indicating that EDCs may be able to alter the expression of these genes. To analyze the endogenous circadian rhythm in K. marmoratus, we measured expression of Km-clock and other circadian rhythmic genes (e.g. Per2, Cry1, Cry2, and BMAL1) in different tissues, and found ubiquitous expression, although there were different patterns of transcript amplification during different developmental stages. In an estrogen (E2)-exposed group, Km-clock expression was down-regulated, however, a hydroxytamoxifen (TMX, nonsteroid estrogen antagonist)-exposed group showed an upregulated pattern of Km-clock expression, suggesting that the expression of Km-clock is closely associated with exposure to EDCs. In response to the exposure of bisphenol A (BPA) and 4-tert-octyphenol (OP), Km-clock expression was down-regulated in the pituitary/brain, muscle, and skin in both gender types (hermaphrodite and secondary male). In juvenile K. marmoratus liver tissue, expression of Km-clock and other circadian rhythmic pathway-associated genes showed a regular oscillation pattern over a period of approximately 24h during a 12L:12D cycle. However, the circadian rhythm of BPA-exposed juvenile K. marmoratus liver tissue was perturbed over a 12L:12D period. This study will aid in our understanding of how EDCs perturb endogenous circadian rhythms, particularly in BPA-exposed fish liver tissue.
为了研究内分泌干扰化学物质（EDCs）对昼夜节律途径的影响，我们在自体受精的红树林鳉鱼Kryptolebias marmoratus中克隆了生物钟及昼夜节律途径相关基因（如Per2、Cry1、Cry2和BMAL1）。Km-clock的启动子区域有1个芳烃受体元件（AhRE，GTGCGTGACA）和8个雌激素受体（ER）半位点，这表明AhRE和ER半位点可能与EDCs诱导的细胞应激过程中生物钟蛋白活性的调节有关。Km-clock蛋白结构域（bHLH、PAS1、PAS2）在另外5种鱼类（斑马鱼、日本青鳉、南方剑尾鱼、尼罗罗非鱼和斑点绿河豚）中高度保守，这表明鱼类生物钟蛋白可能在控制内源性昼夜节律中起重要作用。发现Km-BMAL1、-Cry1、-Cry2和-Per2的启动子区域含有多个外源性物质反应元件（XREs），这表明EDCs可能能够改变这些基因的表达。为了分析K. marmoratus的内源性昼夜节律，我们测量了Km-clock和其他昼夜节律基因（如Per2、Cry1、Cry2和BMAL1）在不同组织中的表达，发现它们普遍表达，尽管在不同发育阶段转录本扩增模式不同。在雌激素（E2）暴露组中，Km-clock表达下调，然而，羟他莫昔芬（TMX，非甾体雌激素拮抗剂）暴露组显示Km-clock表达上调，这表明Km-clock的表达与EDCs暴露密切相关。在双酚A（BPA）和4-叔辛基苯酚（OP）暴露后，两性类型（雌雄同体和次级雄性）的垂体/脑、肌肉和皮肤中的Km-clock表达均下调。在幼年K. marmoratus肝脏组织中，Km-clock和其他昼夜节律途径相关基因的表达在12L:12D周期内约24小时的时间段内呈现出规律的振荡模式。然而，BPA暴露的幼年K. marmoratus肝脏组织的昼夜节律在12L:12D周期内受到干扰。这项研究将有助于我们理解EDCs如何扰乱内源性昼夜节律，特别是在BPA暴露的鱼类肝脏组织中。
