Abstract

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are nuclear receptors that are involved in the regulation of gene transcription of enzymes that are responsible for biotransformation and excretion of endo- and xenobiotics. The goal of the work was to study the effect of DL-butyonine sulfoximine (BSO, gamma-glutamylcysteine synthetase inhibitor) on the relative amounts of CAR and PXR in Caco-2 cells and to clarify its mechanisms. BSO was used at concentrations of 1‒500 μM for 24 and 72 h. The generation of reactive oxygen species (ROS) has been evaluated using the MitoTracker Red CM-H2 XRos fluorescent probes. Cytotoxicity was analyzed by the MTT test. The relative amount of CAR and PXR was assessed by the Western blot method. It has been shown that BSO caused an increase in ROS formation at concentrations of 10, 50, and 100 μM for 24 h and at concentrations of 50 and 100 μM for 72 h. However, 500 μM BSO reduced the viability of cells during all periods of exposure. The relative amount of CAR increased in 24 h at the BSO concentrations of 50 and 100 μM and in 72 h at its concentrations of 10 and 50 μM. The amount of PXR increased in 72 h during incubation with BSO at the concentration of 50 μM and in 24 and 72 h at its concentrations of 100 and 500 μM. The combined use of BSO (50 μM, 24 h; 10 and 50 μM, 72 h) and glutathione inhibited CAR induction, whereas 50 and 100 μM BSO inhibited PXR formation for 72 h. The addition of 1 mM glutathione to the nutrient medium with BSO (100 and 500 μM, 24 h; 500 μM, 72 h) did not affect the relative amount of PXR. No effect on CAR was observed when 1 mM glutathione was used together with BSO (100 μM, 24 h; 50 and 100 μM, 72 h). Thus, BSO can induce CAR and PXR formation by both increasing the production of free radicals, thus developing oxidative stress, and by acting independently as a xenobiotic.

中文翻译：

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DL-丁酰亚胺磺酰亚胺对雄甾烷和孕烷 X 受体的体外双峰作用机制

摘要

组成型雄甾烷受体 (CAR) 和孕烷 X 受体 (PXR) 是参与酶基因转录调节的核受体，这些酶负责内源性和外源性物质的生物转化和排泄。这项工作的目的是研究 DL-丁酰亚胺亚砜（BSO，γ-谷氨酰半胱氨酸合成酶抑制剂）对 Caco-2 细胞中 CAR 和 PXR 相对量的影响，并阐明其机制。BSO 以 1-500 μM 的浓度使用 24 和 72 小时。使用 MitoTracker Red CM-H2 XRos 荧光探针评估了活性氧 (ROS) 的产生。通过MTT试验分析细胞毒性。通过Western blot方法评估CAR和PXR的相对量。研究表明，BSO 在浓度为 10、50、和 100 μM 24 小时，以及 50 和 100 μM 浓度 72 小时。然而，500 μM BSO 在所有暴露期间都会降低细胞的活力。BSO浓度为50和100μM时，CAR的相对量在24小时内增加；在浓度为10和50μM时，CAR的相对量在72小时内增加。在与浓度为 50 μM 的 BSO 一起孵育期间，PXR 的量在 72 小时内增加；在浓度为 100 和 500 μM 时，在 24 小时和 72 小时内 PXR 的量增加。BSO（50 μM，24 小时；10 和 50 μM，72 小时）和谷胱甘肽的联合使用抑制 CAR 诱导，而 50 和 100 μM BSO 抑制 PXR 形成 72 小时。在含有 BSO 的营养培养基中添加 1 mM 谷胱甘肽（100 和 500 μM，24 小时；500 μM，72 小时）不会影响 PXR 的相对量。当 1 mM 谷胱甘肽与 BSO（100 μM，24 小时；50 和 100 μM，72 小时）。因此，BSO 可以通过增加自由基的产生（从而产生氧化应激）以及作为异生素独立发挥作用来诱导 CAR 和 PXR 形成。