Zhang Jian, Ali Hamed I, Bedi Yudhishtar Singh, Choudhury Mahua
Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, USA.
Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, USA.
Toxicology. 2015 Dec 2;338:130-41. doi: 10.1016/j.tox.2015.10.004. Epub 2015 Oct 28.
The plasticizer benzyl butyl phthalate (BBP) is a well-known endocrine disruptor. Widespread human exposure to phthalates has raised substantial public concern due to its detrimental health effects. However, molecular mechanisms of the phthalates effect require elucidation. In this study, we analyzed: 1) the binding interaction of several phthalates and persistent organic pollutants with epigenetic regulator sirtuins and 2) the effect of BBP on the sirtuins in HepG2 cells. AutoDock molecular docking analysis showed that BBP binds to Sirt1 and Sirt3 proteins similarly to the native ligands with shortest binding free energies (ΔGb) of -7.35 and -8.3 kcal/mol, respectively; and inhibition constants (Ki) of 4.07 μM and 0.82 μM, respectively. Furthermore, BBP was superimposed onto the co-crystallized ligands within the least root-mean-square deviation (RMSD) of 0.96Å and 1.55Å for Sirt1 and Sirt3, respectively, and bound into the sites with a sufficient number of hydrogen bonds, implying the best fit compared to other sirtuins. In HepG2 cells, BBP significantly down-regulated Sirt1 and Sirt3 (p<0.05) gene expression at a concentration as low as 10nM; other sirtuins remained unaffected. Consistent with decreased gene expression, Sirt1 and Sirt3 protein levels were significantly decreased at 48 h (p<0.05). In addition, mitochondrial biogenesis regulators PGC-1α, NRF-1, and NRF-2, were decreased (p<0.05). SiRNA studies showed that BBP did not regulate PGC-1α via sirtuin and BBP requires sirtuin's presence to regulate NRF-1 or NRF-2. BBP significantly increased ROS production (p<0.05) and ROS may be chiefly regulated by NRF-1 and NRF-2 in HepG2 cells under Sirt1 and Sirt3 silenced condition. This is the first report to demonstrate that BBP selectively disrupts specific sirtuins in HepG2 cells. In conclusion, our study suggests that BBP can impair two vital epigenetic regulators and mitochondrial biogenesis regulators in liver cells.
增塑剂邻苯二甲酸苄基丁酯（BBP）是一种著名的内分泌干扰物。由于其对健康的有害影响，人类广泛接触邻苯二甲酸盐引起了公众的极大关注。然而，邻苯二甲酸盐作用的分子机制仍需阐明。在本研究中，我们分析了：1）几种邻苯二甲酸盐和持久性有机污染物与表观遗传调节因子沉默调节蛋白的结合相互作用；2）BBP对HepG2细胞中沉默调节蛋白的影响。自动对接分子对接分析表明，BBP与Sirt1和Sirt3蛋白的结合方式与天然配体相似，结合自由能（ΔGb）分别为-7.35和-8.3 kcal/mol，抑制常数（Ki）分别为4.07 μM和0.82 μM。此外，BBP分别以0.96Å和1.55Å的最小均方根偏差（RMSD）叠加到Sirt1和Sirt3的共结晶配体上，并通过足够数量的氢键结合到位点，这意味着与其他沉默调节蛋白相比具有最佳匹配度。在HepG2细胞中，低至10nM的BBP浓度即可显著下调Sirt1和Sirt3（p<0.05）的基因表达；其他沉默调节蛋白不受影响。与基因表达降低一致，48小时时Sirt1和Sirt3蛋白水平显著降低（p<0.05）。此外，线粒体生物发生调节因子PGC-1α、NRF-1和NRF-2水平降低（p<0.05）。RNA干扰研究表明，BBP不通过沉默调节蛋白调节PGC-1α，且BBP需要沉默调节蛋白的存在才能调节NRF-1或NRF-2。BBP显著增加活性氧（ROS）的产生（p<0.05），在Sirt1和Sirt3沉默条件下，HepG2细胞中的ROS可能主要由NRF-1和NRF-2调节。这是首次证明BBP在HepG2细胞中选择性破坏特定沉默调节蛋白的报告。总之，我们的研究表明，BBP可损害肝细胞中的两种重要表观遗传调节因子和线粒体生物发生调节因子。
