Department of Pharmaceutical Science, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
Toxicol Appl Pharmacol. 2010 May 15;245(1):91-9. doi: 10.1016/j.taap.2010.02.007. Epub 2010 Feb 19.
Studies in our laboratory have demonstrated that subchronic 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure of adult mice results in hypertension, cardiac hypertrophy, and reduced nitric oxide (NO)-mediated vasodilation. Moreover, increased superoxide anion production was observed in cardiovascular organs of TCDD-exposed mice and this increase contributed to the reduced NO-mediated vasodilation. Since cytochrome P4501A1 (CYP1A1) can contribute to some TCDD-induced toxicity, we tested the hypothesis that TCDD increases reactive oxygen species (ROS) in endothelial cells by the induction of CYP1A1. A concentration-response to 24h TCDD exposure (10pM-10nM) was performed in confluent primary human aortic endothelial cells (HAECs). Oxidant-sensitive fluorescent probes dihydroethidium (DHE) and 2',7'-dichlorofluorescin diacetate (DCFH-DA), were used to measure superoxide anion, and hydrogen peroxide and hydroxyl radical, respectively. NO was also measured using the fluorescent probe diaminofluorescein-2 diacetate (DAF-2DA). These assessments were conducted in HAECs transfected with siRNA targeting the aryl hydrocarbon receptor (AhR), CYP1A1, or CYP1B1. TCDD concentration-dependently increased CYP1A1 and CYP1B1 mRNA, protein, and enzyme activity. Moreover, 1nM TCDD maximally increased DHE (Cont=1.0+/-0.3; TCDD=5.1+/-1.0; p=0.002) and DCFH-DA (Cont=1.0+/-0.2; TCDD=4.1+/-0.5; p=0.002) fluorescence and maximally decreased DAF-2DA fluorescence (Cont=1.0+/-0.4; TCDD=0.68+/-0.1). siRNA targeting AhR and CYP1A1 significantly decreased TCDD-induced DHE (siAhR: Cont=1.0+/-0.1; TCDD=1.3+/-0.2; p=0.093) (siCYP1A1: Cont=1.0+/-0.1; TCDD=1.1+/-0.1; p=0.454) and DCFH-DA (siAhR: Cont=1.0+/-0.2; TCDD=1.3+/-0.3; p=0.370) (siCYP1A1: Cont=1.0+/-0.1; TCDD=1.3+/-0.2; p=0.114) fluorescence and increased DAF-2DA fluorescence (siAhR: Cont=1.00+/-0.03; TCDD=0.97+/-0.03; p=0.481) (siCYP1A1: Cont=1.00+/-0.03; TCDD=0.92+/-0.03; p=0.034), while siRNA targeting CYP1B1 did not. These data suggest that TCDD-induced increase in ROS is AhR-dependent and may be mediated, in part, by CYP1A1 induction.
本实验室的研究表明，亚慢性 2,3,7,8-四氯二苯并对二恶英（TCDD）暴露于成年小鼠会导致高血压、心脏肥大和一氧化氮（NO）介导的血管舒张减少。此外，在 TCDD 暴露的小鼠的心血管器官中观察到超氧阴离子产生增加，并且这种增加导致 NO 介导的血管舒张减少。由于细胞色素 P4501A1（CYP1A1）可以导致一些 TCDD 诱导的毒性，我们测试了 TCDD 通过诱导 CYP1A1 在内皮细胞中增加活性氧物种（ROS）的假设。在汇合的原代人主动脉内皮细胞（HAEC）中进行了 24 小时 TCDD 暴露（10pM-10nM）的浓度反应。使用二氢乙啶（DHE）和 2'，7'-二氯荧光素二乙酸酯（DCFH-DA）等氧化敏感荧光探针分别测量超氧阴离子、过氧化氢和羟基自由基。还使用荧光探针二氨基荧光素-2 二乙酸酯（DAF-2DA）测量 NO。这些评估是在靶向芳香烃受体（AhR）、CYP1A1 或 CYP1B1 的 siRNA 转染的 HAEC 中进行的。TCDD 浓度依赖性地增加 CYP1A1 和 CYP1B1 mRNA、蛋白质和酶活性。此外，1nM TCDD 最大程度地增加了 DHE（Cont=1.0+/-0.3；TCDD=5.1+/-1.0；p=0.002）和 DCFH-DA（Cont=1.0+/-0.2；TCDD=4.1+/-0.5；p=0.002）荧光，并最大程度地降低了 DAF-2DA 荧光（Cont=1.0+/-0.4；TCDD=0.68+/-0.1）。靶向 AhR 和 CYP1A1 的 siRNA 显著降低了 TCDD 诱导的 DHE（siAhR：Cont=1.0+/-0.1；TCDD=1.3+/-0.2；p=0.093）（siCYP1A1：Cont=1.0+/-0.1；TCDD=1.1+/-0.1；p=0.454）和 DCFH-DA（siAhR：Cont=1.0+/-0.2；TCDD=1.3+/-0.3；p=0.370）（siCYP1A1：Cont=1.0+/-0.1；TCDD=1.3+/-0.2；p=0.114）荧光并增加了 DAF-2DA 荧光（siAhR：Cont=1.00+/-0.03；TCDD=0.97+/-0.03；p=0.481）（siCYP1A1：Cont=1.00+/-0.03；TCDD=0.92+/-0.03；p=0.034），而靶向 CYP1B1 的 siRNA 则没有。这些数据表明，TCDD 诱导的 ROS 增加是 AhR 依赖性的，并且可能部分由 CYP1A1 诱导介导。
