The Mechanism of Bimodal Effect of DL-Butyonine Sulfoximine on Constitutive Androstane and Pregnane X Receptors In Vitro

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 mu M 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 mu M for 24 h and at concentrations of 50 and 100 mu M for 72 h. However, 500 mu M 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 mu M and in 72 h at its concentrations of 10 and 50 mu M. The amount of PXR increased in 72 h during incubation with BSO at the concentration of 50 mu M and in 24 and 72 h at its concentrations of 100 and 500 mu M. The combined use of BSO (50 mu M, 24 h; 10 and 50 mu M, 72 h) and glutathione inhibited CAR induction, whereas 50 and 100 mu M BSO inhibited PXR formation for 72 h. The addition of 1 mM glutathione to the nutrient medium with BSO (100 and 500 mu M, 24 h; 500 mu M, 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 mu M, 24 h; 50 and 100 mu M, 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.

Automated summary

The effect of DL-butyonine sulfoximine (BSO) on the relative amounts of CAR and PXR in Caco-2 cells was studied. BSO increased ROS formation and acted as a xenobiotic to induce CAR and PXR formation.