The Study of the Effectiveness of Ethylmethylhydroxypyridine Succinate in Acute Alcohol Intoxication.

The effectiveness of ethylmethylhydroxypyridine succinate (EMHPS) in acute alcohol intoxication was tested in a study on SPF male outbred ICR mice. Ethanol (concentration 40%) was administered to animals once intraperitoneally at a dose of 4 g/kg. Control animals were injected with saline in an equivalent volume. In 15 min after the administration of alcohol, the animals were injected intravenously or intramuscularly with EMHPS at a dose of 50 or 100 mg/kg or with saline via the same route in an equivalent volume. Animal behavior was tested 3 and 24 h later after administration of the substances. After 3 and 24 h, mice in the pathological control groups developed semiptosis, the gait and the turning over reflex were impaired, the strength of the hind limbs decreased and the distance between the hind limbs increased when landing; in the open-field test, the latency of the first movement increased, and the number of rearing postures decreased. Intravenous and intramuscular administration of EMHPS in doses of 50 and 100 mg/kg had a pronounced antitoxic and neuroprotective effect in acute alcohol intoxication: all studied parameters did not differ significantly from the control.

The effect of lactoferin on the free radical and cytokine status of cornea in the experimental thermal burn.

The free radical and cytokine statuses of the cornea during its thermal burn and the possibility of its correction by lactoferrin have been studied in Soviet Chinchilla rabbits. The development of a corneal thermal burn was accompanied by the development of oxidative stress (increased levels of TBA-reactive substances and carbonyl derivatives of proteins, decreased activity of SOD and GPx enzymes) and a pronounced inflammatory reaction with increased levels of TNF-1α, IL-10, TGF-1ß. The use of lactoferrin had a pronounced therapeutic effect, which was manifested by accelerated healing, prevention of the development of complications (corneal perforations), a decrease in the severity of oxidative stress, an increase in the concentrations of TNF-1α (in the early stages), IL-10 (in the later stages), TGF-1ß (throughout the experiment). At the same time, by the end of regeneration more severe corneal opacification was recognized compared to the control group. This may be associated with an increased level of anti-inflammatory cytokines, especially TGF-1ß.

[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.

The Effect of Original Russian Neurotropic Drugs on Organic Anion Transporting Polypeptides OATP1B1 and OATP1B3.

In experiments on HepG2 cells, we studied the effect of the original domestic neurotropic drugs omberacetam, fabomotizole, and ethylmethylhydroxypyridine succinate (EMHPS) (1-500 µM) on the activity and content of organic anion transporting polypeptides OATP1B1 and OATP1B3. It was shown that omberacetam (500 µM) increased the content of OATP1B1 and OATP1B3, fabomotizole did not affect the level of both transporters, and EMHPS (500 µM) increased the content of OATP1B1 compared to the control and did not affect the level of OATP1B3. The tested substances also reduced the OATP1B1/OATP1B3 ratio, as evidenced by a decrease in the penetration of atorvastatin, a substrate of the transporters, into HepG2 cells in the presence of omberacetam (100-500 µM), fabomotizole (500 µM), and EMHPS (10-500 µM). Evaluation of clinical significance of the obtained results, according to the FDA approach based on the calculation of the Cmax/IC50 ratio, showed that the effect of the tested substances on OATP1B1/OATP1B3 is clinically insignificant.

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.

[Mechanism of regulation of the constitutive androstane receptor under conditions of modeling oxidative stress in vitro]. / Reguliatsiia konstitutivnogo androstanovogo retseptora v kletkakh linii Caso-2 pri modelirovanii okislitel'nogo stressa in vitro.

The constitutive androstane receptor (CAR) is a nuclear receptor that participates in the regulation of biotransformation of toxic substances and metabolic processes. The mechanisms of expression changes of CAR under conditions of oxidative stress (OS) have not been studied yet and this was the purpose of the study. OS was modeled by incubating Caco2 cells with hydrogen peroxide 10-100 µM for 72 h. The amount of CAR was determined by the Western blot, nuclear factor erythroid 2-related factor 2 (Nrf2) was evaluated by a heterogeneous enzyme immunoassay malondialdehyde (MDA), the lipid peroxidation products (LPP) was assayed by a photometric method. Incubation of cells with 10 µM and 50 µM H2O2 led to an increase in the amount of CAR and Nrf2, while incubation with 100 µM H2O2 caused their decrease. Nrf2 inhibition did not influence the CAR content under OS conditions. 10 µM MDA increased the CAR content, 100 µM MDA had no effect, while 150 µM reduced the amount of CAR.

Regulation and Role of Hypoxia-Induced Factor 1α (HIF-1α) under Conditions of Endogenous Oxidative Stress In Vitro.

The effect of endogenous oxidative stress induced by γ-glutamyl cysteinesynthetase inhibitor D,L-buthionine sulfoximine (BSO) on the functioning of hypoxia-induced factor 1α (HIF-1α) was studied on Caco-2 cells. BSO was added for 24 h in concentrations of 5, 10, 50, 100, and 500 µM. It was shown that BSO in concentrations of 10, 50, and 100 µM induced endogenous oxidative stress and increased the content of HIF-1α; this effect was regulated through nuclear factor of erythroid origin 2 (Nrf2). Activation of HIF-1α had an independent protective effect, as evidenced by the decrease in cell viability after HIF-1α inhibition under these conditions. When the concentration of BSO was increased to 500 µM the content of HIF-1α did not change, and cell viability decreased.

Effect of Nitric Oxide on the Functioning of the P-Glycoprotein Transporter.

We studied the effect of nitric oxide (NO) on the functioning of P-glycoprotein transporter (Pgp) in Caco-2 cells. NO donor S-nitrosoglutathione (GSNO) was used in concentrations of 1, 10, 50, 100, and 500 µM; the duration of exposure was 24 h. The content of Pgp was analyzed by the Western blotting, activity of the transport protein was analyzed by the transport of its substrate fexofenadine. It was shown that GSNO in concentrations of 10 and 50 µM increased the content and activity of Pgp. Increasing the GSNO concentration to 500 µM led to the development of nitrosative stress and a decrease in the content and activity of the transporter protein.

Assessment of Malondialdehyde Belonging to Modulators and Substrates of the P-Glycoprotein Transporter Protein.

In the study on cells of the Caco-2 line, the affiliation of malondialdehyde (MDA) to modulators and substrates of P-glycoprotein (Pgp) was assessed, and the biological role of Pgp in conditions of oxidative stress (OS) was studied. MDA was used at concentrations of 10, 50, 100, and 150 µM; OS was simulated by incubation with hydrogen peroxide (H2O2) at concentrations of 0.1-100 µM for 24 h. The relative amount of Pgp was evaluated by the Western blot hybridization, and the activity was estimated by the transport of its substrate fexofenadine (HPLC with UV detection, HPLC MS/MS). In this study, it was shown that MDA at concentrations of 10 and 50 µM and exposure duration of 24 h increases the relative amount and activity of Pgp by acting through CAR and PXR, and MDA can be transported by Pgp. The induction of Pgp under the action of MDA during the development of OS can have a protective significance, ensuring the removal of the peroxidation product from cells into the extracellular space and thereby increasing the viability of cells.

Induction of Constitutive Androstane Receptor during the Development of Oxidative Stress.

We studied the effect of 3-, 24-, and 72-h exposure to H2O2 in concentrations of 0.1-100.0 µM on the level of constitutive androstane receptor in Caco-2 cells. It was shown that 3- and 24-h incubation with Н2О2 in all concentrations had no effect on the level of constitutive androstane receptors. Increasing the incubation time to 72 h led to an increase in the level of constitutive androstane receptor at H2O2 concentrations of 5, 10, and 50 µM and to a decrease at a concentration of 100 µM. Antioxidant glutathione (1 mM) in parallel to the prooxidant neutralized these changes.