[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 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.

[Quantitative assessment of breast cancer resistance protein during pregnancy in rabbits]. / Kolichestvennaia otsenka belka rezistentnosti raka molochnoi zhelezy pri beremennosti u krolikov.

Breast cancer resistance protein (BCRP,ABCG2) is an efflux transporter protein that transports various substrates from the cell to the extracellular space or organ cavities. The aim of this study was a complex assessment of the amount of BCRP during pregnancy in rabbits. The amount of BCRP in samples of the rabbit jejunum, liver, kidney, cerebral cortex, and placenta was determined by enzyme immunoassay, and in human hepatocellular carcinoma (HepG2) cells by the Western blot. To study the mechanisms involved in control of the dynamic BCRP levels during pregnancy, serum concentrations of sex hormones were investigated by radioimmunoassay and relative amounts of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in these organs were evaluated using the Western blot method. The putative role of CAR and PXR in regulation of the BCRP level by progesterone was evaluated in vitro experiments on HepG2 cells. It was found that amount of BCRP in the jejunum of pregnant rabbits was higher than in the placenta, liver, kidneys, and cerebral cortex. An increase in the amount of BCRP in the liver of rabbits was noted on the 21st day of pregnancy and a tendency to the increase was also detected on the 28th day; in the kidney and cerebral cortex increased BCRP levels were detected on the 28th day and 14th day of pregnancy, respectively, as compared with non-pregnant females. In vitro experiments with HepG2 cells have shown that the increase in the BCRP level is determined by the activating effect of progesterone on PXR.

Mechanisms of P-Glycoprotein Regulation Under Exogenous and Endogenous Oxidative Stress In Vitro.

We investigated the mechanisms of P-glycoprotein (P-gp) transporter regulation in Caco-2 cells under exogenous and endogenous oxidative stress (OS). Exogenous OS was modeled by exposure of the growth medium to hydrogen peroxide at concentrations of 0.1, 0.5, and 1 µM for 24 h or 10 µM for 72 h. Endogenous OS was modeled by incubating cells with DL-buthionine sulfoximine (BSO, gamma-glutamylcysteine synthetase inhibitor) at a concentration of 10, 50, and 100 µM for 24 h. The levels of intracellular reactive oxygen species (ROS) were assessed using MitoTracker Red CM-H2XRos fluorescent probes. Relative P-gp contents were analyzed using Western blot. Exogenous and endogenous OS was shown to increase relative to P-gp contents. An important role played by the Nrf2-Keap1 signaling pathway in increasing the P-gp contents under H2O2-induced exogenous OS was revealed using specific inhibitors. The transcription factor HIF1 is involved in the regulation of the P-gp levels under 24-hour exogenous OS, and the transcription factor CAR is involved in the regulation of transporter levels under 72-hour OS. All tested transcription factors and signaling pathways are involved in P-gp induction under endogenous OS. Most likely, this is associated with the bimodal effect of BSO on Pgp. On the one hand, BSO induces the development of OS; on the other, BSO, as a xenobiotic, is able to stimulate PXR and CAR, which, in turn, increase the P-gp contents.