A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure
A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure
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DOI：
10.1007/s00204-018-2353-2
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发表时间：
2019-02-01
影响因子：
6.1
通讯作者：
van de Water, Bob
中科院分区：
文献类型：
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作者：
Bischoff, Luc J. M.;Kuijper, Isoude A.;van de Water, Bob
氧化应激导致核因子-红细胞2相关因子2 （Nrf2）通路的激活。虽然大多数研究都集中在单一化学处理后Nrf2通路的激活上，但对重复暴露情景下Nrf2通路的动态调控知之甚少。在这里，我们利用两种HepG2荧光蛋白报告细胞系，分别表达GFP标记的Nrf2或Nrf2的直接下游靶点sulfiredoxin 1 (Srxn1)，利用单细胞活成像技术定量监测Nrf2在重复暴露过程中的动态变化。高通量实时共聚焦成像用于测量反复暴露于广泛浓度范围内的马来酸二乙酯（DEM）和叔丁基对苯二酚（tBHQ）后Nrf2通路的这两个组成部分的时间动态。单次用DEM或thbhq处理可诱导Nrf2和Srxn1随时间呈浓度依赖性。在相同浓度下，Nrf2对第二次处理的反应低于对第一次处理的反应，表明这种反应是适应性的。此外，在第二次治疗期间，个别细胞的有限部分致力于Nrf2反应。尽管Nrf2通路受到抑制，但与第一次处理相比，第二次处理导致Srxn1-GFP应答提高了三倍，所有细胞都参与了应答。在第一次处理后，Srxn1-GFP反应与Nrf2-GFP核易位呈线性相关，而在第二次处理时，这种线性关系就不那么明显了。sirna介导的敲低表明第二反应依赖于Nrf2的活性。其他几种临床相关的化合物（即，磺胺砜、呋喃醌和CDDO-Me）在连续两次重复暴露后也增强了Srxn1-GFP的诱导。综上所述，这些数据表明，对促氧化剂的适应降低了Nrf2的激活能力，但同时也为细胞增强抗氧化反应提供了条件，而抗氧化反应不仅取决于Nrf2，还取决于其他因素。这些数据为反复暴露后应激通路激活的整体动力学提供了进一步的见解，并强调了可能控制重复剂量毒性的反应的复杂性。
Oxidative stress leads to the activation of the Nuclear factor-erythroid-2-related factor 2 (Nrf2) pathway. While most studies have focused on the activation of the Nrf2 pathway after single chemical treatment, little is known about the dynamic regulation of the Nrf2 pathway in the context of repeated exposure scenarios. Here we employed single cell live imaging to quantitatively monitor the dynamics of the Nrf2 pathway during repeated exposure, making advantage of two HepG2 fluorescent protein reporter cell lines, expressing GFP tagged Nrf2 or sulfiredoxin 1 (Srxn1), a direct downstream target of Nrf2. High throughput live confocal imaging was used to measure the temporal dynamics of these two components of the Nrf2 pathway after repeated exposure to an extensive concentration range of diethyl maleate (DEM) and tert-butylhydroquinone (tBHQ). Single treatment with DEM or tBHQ induced Nrf2 and Srxn1 over time in a concentration-dependent manner. The Nrf2 response to a second treatment was lower than the response to the first exposure with the same concentration, indicating that the response is adaptive. Moreover, a limited fraction of individual cells committed themselves into the Nrf2 response during the second treatment. Despite the suppression of the Nrf2 pathway, the second treatment resulted in a three-fold higher Srxn1-GFP response compared to the first treatment, with all cells participating in the response. While after the first treatment Srxn1-GFP response was linearly related to Nrf2-GFP nuclear translocation, such a linear relationship was less clear for the second exposure. siRNA-mediated knockdown demonstrated that the second response is dependent on the activity of Nrf2. Several other, clinically relevant, compounds (i.e., sulphorophane, nitrofurantoin and CDDO-Me) also enhanced the induction of Srxn1-GFP upon two consecutive repeated exposure. Together the data indicate that adaptation towards pro-oxidants lowers the Nrf2 activation capacity, but simultaneously primes cells for the enhancement of an antioxidant response which depends on factors other than just Nrf2. These data provide further insight in the overall dynamics of stress pathway activation after repeated exposure and underscore the complexity of responses that may govern repeated dose toxicity.