Mohammadi-Bardbori Afshin, Vikström Bergander Linda, Rannug Ulf, Rannug Agneta
Institute of Environmental Medicine, Karolinska Institutet , SE-171 77 Stockholm, Sweden.
Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences , Shiraz, Fars 71345-1583, Iran.
Chem Res Toxicol. 2015 Dec 21;28(12):2278-86. doi: 10.1021/acs.chemrestox.5b00415. Epub 2015 Nov 16.
The mechanisms explaining arsenic toxicity are not well understood, but physiological consequences of stimulated aryl hydrocarbon receptor (AHR) signaling both directly and through cross-talk with other pathways have been indicated. The aim of this study was to establish how arsenic interacts with AHR-mediated transcription. The human hepatoma cell line (HepG2-XRE-Luc) carrying a luciferase reporter under the control of two AHR response elements (AHREs) and immortalized human keratinocytes (HaCaT) were exposed to sodium arsenite (NaAsO2; As(3+)), alone or in combination with the endogenous high affinity AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Luciferase activity, cytochrome P4501A1 (CYP1A1) activity, oxidative stress-related responses, metabolic clearance of FICZ, and NADPH oxidase (NOX) activity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent gene expression were measured. Arsenic inhibited CYP1A1 enzyme activity and reduced the metabolic clearance of FICZ. Arsenic also led to activated CYP1A1 transcription but only in cells grown in medium containing trace amounts of the endogenous ligand FICZ, pointing to an indirect mechanism of activation. Initially, arsenic caused dose-dependent inhibition of FICZ-activated AHR signaling, disturbed intracellular GSH status, and increased expression of oxidative stress-related genes. Silencing of NOX4, addition of N-acetylcystein, or pretreatment with arsenic itself attenuated the initial dose-dependent inhibition of AHR signaling. Arsenic pretreatment led to elevated GSH levels and sensitized the cells to ligand-dependent AHR signaling, while silencing of Nrf2 significantly reduced arsenic-mediated activation of the AHR. In addition, influence of NOX on AHR activation was also observed in cells treated with the SH-reactive metals cadmium, mercury, and nickel. Together, the results suggest that SH-reactive agents via a new and possibly general NOX/H2O2-dependent mechanism can interfere with the endogenous regulation of the AHR.
目前对解释砷毒性的机制尚未完全了解，但已表明刺激芳烃受体（AHR）信号传导直接或通过与其他途径的串扰所产生的生理后果。本研究的目的是确定砷如何与AHR介导的转录相互作用。将携带在两个AHR反应元件（AHREs）控制下的荧光素酶报告基因的人肝癌细胞系（HepG2-XRE-Luc）和永生化人角质形成细胞（HaCaT）单独或与内源性高亲和力AHR配体6-甲酰基吲哚并[3,2-b]咔唑（FICZ）联合暴露于亚砷酸钠（NaAsO2；As(3+)）。测量荧光素酶活性、细胞色素P4501A1（CYP1A1）活性、氧化应激相关反应、FICZ的代谢清除率、NADPH氧化酶（NOX）活性以及核因子（红细胞衍生2）样2（Nrf2）依赖性基因表达。砷抑制CYP1A1酶活性并降低FICZ的代谢清除率。砷还导致CYP1A1转录激活，但仅在含有微量内源性配体FICZ的培养基中生长的细胞中，这表明存在间接激活机制。最初，砷导致FICZ激活的AHR信号传导呈剂量依赖性抑制，扰乱细胞内谷胱甘肽（GSH）状态，并增加氧化应激相关基因的表达。沉默NOX4、添加N-乙酰半胱氨酸或用砷本身预处理可减弱AHR信号传导的初始剂量依赖性抑制。砷预处理导致GSH水平升高，并使细胞对配体依赖性AHR信号传导敏感，而沉默Nrf2可显著降低砷介导的AHR激活。此外，在用具有巯基反应性的金属镉、汞和镍处理的细胞中也观察到了NOX对AHR激活的影响。总之，结果表明具有巯基反应性的试剂可能通过一种新的、可能普遍的NOX/H2O2依赖性机制干扰AHR的内源性调节。
