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Reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR) is a powerful and commonly used tool for gene expression analysis. It requires the right choice of stably expressed reference genes for accurate normalization. In this work, we aimed to select the optimal reference genes for qRT-PCR normalization within different brain areas during the first week following pentylenetetrazole-induced seizures in immature (P20–22) Wistar rats. We have tested the expression stability of a panel of nine housekeeping genes: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on geometric averaging of ranks obtained by four common algorithms (geNorm, NormFinder, BestKeeper, Comparative Delta-Ct), we found that the stability of tested reference genes varied significantly between different brain regions. The expression of the tested panel of genes was very stable within the medial prefrontal and temporal cortex, and the dorsal hippocampus. However, within the ventral hippocampus, the entorhinal cortex and amygdala expression levels of most of the tested genes were not steady. The data revealed that in the pentylenetetrazole-induced seizure model in juvenile rats, Pgk1, Ppia, and B2m expression are the most stable within the medial prefrontal cortex; Ppia, Rpl13a, and Sdha within the temporal cortex; Pgk1, Ppia, and Rpl13a within the entorhinal cortex; Gapdh, Ppia, and Pgk1 within the dorsal hippocampus; Rpl13a, Sdha, and Ppia within the ventral hippocampus; and Sdha, Pgk1, and Ppia within the amygdala. Our data indicate the need for a differential selection of reference genes across brain regions, including the dorsal and ventral hippocampus.

Natural killer (NK) cells are of special interest among a multitude of microvesicle (MV) source cells. NK cells are a lymphocyte subpopulation performing contact cytolysis of virus-infected cells and tumor cells. Each of the NK cell populations has a unique receptor repertoire on its surface and, thus, unique functions. During their contact with a target cell, the most common mechanism of cytolysis is an exocytosis of lytic granules. However, some indirect evidence suggests that MV with CD56 phenotype and leukocyte-derived MV with various phenotypes are present in the peripheral blood plasma.This research is aimed to study the phenotype, composition and cytotoxic activity of microvesicles produced by NK cells. The analysis of receptor expression showed that MV, as well as source cells of the NK-92 cell line, had a similar CD56 molecule expression profile. The expression profile in MV differs from the same in source cells by higher CD119 and CD11b expression and by lower CD18 expression. Culturing of NK-92 cells in the presence of PMA, IL-1β, TNFα, IFNγ resulted in alterations of cell phenotypes and MV. Immunoblots revealed a change of perforin and granzyme B (GrB) in MV. The analysis of the cytotoxic activity of NK-92 cells in a natural killer in vitro assay employing K562 target cells demonstrated that MV obtained from TNFα-activated cells of the NK-92 cell line increased the cytotoxicity of the same TNFα-activated NK-92 cells regarding cytotoxicity levels. This coincides with the previously revealed increased content of GrB in MV obtained from TNFα-activated cells of the NK-92 cell line. To sum up depending on the cytokine NK-92 cells produce MV that differ in their phenotype, composition and activity. Any changes in MV composition can result in changes in their functional activity: in particular, changes can increase the cytotoxic activity of NK cells of the NK-92 cell line. Thus, besides a well-known and proved way for GrB delivery to a target cell, we can suggest an additional way – the transportation of GrB within MV.

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Introduction. Benzo(a)pyrene metabolites are genotoxic compounds, the accumulation of which contributes to carcinogenesis. The main mechanism of metabolite formation is the benzo(a)pyrene oxidation by cytochromes P450 (CYP). Inhibitors of the main cytochromes can reduce the rate of metabolite formation and, as a result, to decrease the genotoxic effects of benzo(a)pyrene metabolites. In contrast, inducers of cytochromes contribute to the enhancement of genotoxicity. Objective. The aim of the work was to develop a cell model based on the HepaRG cells to study the role of cytochromes activity in the genotoxic effect of benzo (a) pyrene. Material and methods. To assess the effect of inhibitors of cytochromes CYP3A and CYP1A on the genotoxic effect of benzo(a)pyrene in HepaRG cells, the content of active forms of proteins of the DNA damage detection and repair system, phosphorylated forms of signaling cascade proteins was determined by immunoassay using Luminex xMAP technology. The cytotoxicity of benzo(a)pyrene was assessed by real-time cell analysis on xCelligence analyzer. Results. Inhibitors of CYP3A and CYP1A cytochromes, ketoconazole and α-naphthoflavone demonstrate the ability to diminish the toxic effects of benz (a) pyrene, reduce the activation of the DNA repair system, and have a multidirectional effect on the different tyrosine kinases phosphorylation in signaling pathways. Conclusion. HepaRG human hepatoma cells are a suitable cell model both to assess the contribution of cytochromes to the metabolism of xenobiotics and to study of the cell protection from the genotoxic effect of benzo (a) pyrene by cytochrome inhibitors. Limitations. The study was performed on a cell culture. To extrapolate the data to the organism, it is necessary to take into account the data of toxicodynamics and toxicokinetics.