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.

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.

Introduction. Aflatoxins are highly toxic metabolites produced by Aspergillus fungi, with strong carcinogenic and immunosuppressive properties. Their genotoxicity arises from the formation of reactive epoxides during metabolic activation by CYP1A2 and CYP3A4 cytochrome enzymes. The aryl hydrocarbon receptor (AhR) plays a critical role in regulating aflatoxin metabolism and cellular response to toxicity.This study investigates the genotoxic effects of aflatoxin G1 (AFG1) with a focus on its interaction with CYP450 enzymes and AhR, and its impact on DNA repair and apoptosis in human hepatoma HepaRG cells.Material and methods. Cellular toxicity of AFG1 was assessed using xCELLigence RTCA impedance analysis, which revealed an LC50 value of 9.79 µM. DNA repair activity was analyzed using the Luminex xMAP multiplex technology. The study also evaluated the effects of AhR agonists (FICZ, ITE), an antagonist (CH223191), and CYP inhibitors (α-naphthoflavone for CYP1A2, ketoconazole for CYP3A4) on AFG1-induced genotoxicity.Results. AhR ligands exhibited moderate cytoprotective effects, increasing cell viability under AFG1 exposure. In contrast, CYP inhibitors more effectively reduced activation of DNA repair proteins. The protective role of AhR ligands may involve competitive binding with AFG₁ and modulation of downstream signaling.Limitations. The study was conducted using a cell culture model; extrapolation of the findings to the whole organism requires consideration of toxicodynamic and toxicokinetic data.Conclusion. These findings highlight the pivotal role of CYP enzymes and AhR in shaping the genotoxic profile of AFG₁.Compliance with ethical standards. The study does not require approval from a biomedical ethics committee or any other related documentation.Authors’ contribution: Perevoznikov I.E. – collection and processing of material, statistical processing, writing a text; Rogovskaya N.Yu. – collection and processing of material; Malygina D.A. – collection and processing of material; Beltyukov P.P. – the concept and design of the study, editing; Babakov V.N. – the concept and design of the study, editing. All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final version.Conflict of interest. The authors declare no conflict of interest.Funding. The study had no sponsorship.Received: August 21, 2025 / Accepted: October 2, 2025 / Published: November 19, 2025