Mechanism of Bimodal Effect of DL-Butyonine Sulfoximine of Constitutive Androstane and Pregnane X Receptors <i>in vitro</i>

Abstract

The constitutive androstane receptor (CAR) and the pregnan X receptor (PXR) are nuclear receptors that are involved in the regulation of gene transcription of enzymes responsible for biotransformation and excretion of endo- and xenobiotics. The aim of the study was to study the effect of DL-butyonine sulfoximine (BSO, gamma-glutamylcysteine synthetase inhibitor) on the relative amount of CAR and PXR in Caco-2 cells and to evaluate its mechanisms. BSO was used in concentrations of 1‒500 μM and exposure duration of 24 and 72 h. The generation of reactive oxygen species (ROS) was determined using MitoTracker Red CM-H2 XRos fluorescent probes. Cytotoxicity was analyzed according to the results of the MTT test. The relative amount of CAR and PXR was estimated by the Western blot method. It was shown that BSO caused an increase in the formation of ROS at exposure duration of 24 h at concentrations of 10, 50 and 100 μM, at 72 h – at concentrations of 50, 100 μM. At the same time, at a concentration of 500 μM, BSO reduced the viability of cells during all periods of exposure. The relative amount of CAR increased at BSO concentrations of 50, 100 μM and exposure duration of 24 hours and 10, 50 μM at exposure of 72 h, and PXR – at concentrations of 100 and 500 μM and incubation of 24 h and 50, 100, 500 μM and incubation of 72 h. When used together with BSO, glutathione, the synthesis of which it blocks, it was found that at exposure time of 24 h and BSO concentrations of 50 μM and 72 h and concentrations of 10 and 50 μM, CAR induction was suppressed, and at 50 and 100 μM and exposure time of 72 h – PXR. The introduction of glutathione into the nutrient medium with BSO had no effect on PXR at xenobiotic concentrations of 100 and 500 μM (24 h) and 500 μM (72 h), on CAR – at concentrations of 100 μM (24 h) and 50 and 100 μM (72 h). Thus, BSO can induce CAR and PXR both by increasing the production of free radicals and the development of oxidative stress, and independently as a xenobiotic.