Rat liver gene expression analysis as a specific test system for assessing the prooxidant activity of toxicants

Introduction. Prooxidants are substances capable of inducing oxidative stress through the generation of reactive oxygen species. Hydrogen peroxide belongs to the class of oxidizing agents, the biological effect of which is determined not only by the dose but also by the state of the cell’s antioxidant and detoxification systems. The liver, being the central organ for the biotransformation of xenobiotics, is of interest for assessing early molecular signs of adaptation to chronic peroxidative exposure. Developing specific approaches to assessing the prooxidant activity of chemical substances is an urgent task in preventive toxicology.

The study aims to develop a specific test system for assessing the pro oxidant activity of toxicants based on the method of analyzing gene expression in rat liver and to demonstrate its capabilities using the example of determining the threshold of chronic effect of hydrogen peroxide when administered orally.

Materials and methods. Female outbred rats received hydrogen peroxide by gavage for 6 months at doses of 0.05, 0.5, 5 and 50 mg/kg. Control animals received distilled water. Gene expression in liver samples was assessed by real-time quantitative RT-PCR and normalized to Gapdh. An integral GST-index was calculated as the mean expression of Gstp1, Gstt1 and Gstm.

Results. At exposure levels of 0.05 and 0.5 mg/kg, no statistically significant changes in the expression of GST genes were detected. At a dose of 5 mg/kg, a significant increase was observed in NFE2L2 to 1.51±0.28, Gstp1 to 1.97±0.07, Gstt1 to 2.04±0.12, and Gstm to 2.31±0.14 relative units. The GST index increased from 1.00±0.06 in the control group to 2.11±0.06 at 5 mg/kg and to 4.22±0.08 at 50 mg/kg. The calculation of the benchmark dose for a twofold increase in the GST index yielded BMD=4.06 mg/kg and BMDL=2.65 mg/kg.

Limitations. The study was conducted using a single experimental model and a single pro oxidant, which limits the direct extrapolation of the results to other compounds. The gene expression analysis reflected the mRNA level and did not include the determination of the corresponding proteins and the activity of GST enzymes. The obtained threshold values require confirmation in independent experimental series.

Conclusion. The obtained data indicate a stepwise activation of the NFE2L2/GST axis in the liver under chronic exposure to hydrogen peroxide, which makes it possible to use the analysis of gene expression in rat liver as a specific test system for assessing the pro oxidant activity of chemicals during their hygienic regulation. A dose of 5 mg/kg can be considered as the molecular threshold of a compensated adaptive response, at which the enzymes of the second phase of detoxification are activated without signs of total transcriptional decompensation.

Ethics. The experimental study was conducted in compliance with the necessary regulatory acts (the 2013 Helsinki Declaration, GOST 33044–2014 "Principles of Good Laboratory Practice"; Order of the Ministry of Health of the Russian Federation No. 188n dated April 1, 2016, "Rules of Good Laboratory Practice"). The study protocol was approved by the Ethics Commission of the F.F. Chernousov Federal Research Center for Hygiene. "Erisman" of Rospotrebnadzor (minutes of meeting No. 7 dated October 21, 2024).

Contributions:
Karimov D.O. — research concept and design, collection and processing of experimental data, writing the text;
Tonshin A.A. — research concept and design, text writing;
Bidevkina M.V. — research concept and design, conducting experiments, text writing;
Vinogradova A.I. — research concept and design, conducting experiments, collecting and processing experimental data;
Ryabova Yu.V. — collection and processing of experimental data;
Gizatullina A.A. — collecting and processing experimental data;
Akhmadeev A.R. — collecting and processing experimental data;
Ziatdinova M.M. — collecting and processing experimental data.

Funding. The study had no funding.

Conflict of interest. The authors declare no conflict of interest.

Received: 15.09.2026 / Accepted: 17.09.2026 / Published: 09.10.2026

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