Metabolomic analysis of conditioned media of HepaRG cells under the toxic action of benz[a]pyrene under the cytoprotective action of cytochrome CYP1A and CYP3A inhibitors and an arylhydrocarbon receptor agonist

Introduction. Benz[a]pyrene is one of the most dangerous carcinogens of group I according to the IARC classification; it is metabolically activated by enzymes of the cytochrome system P450 (CYP) with the formation of reactive metabolites capable of damaging cells and DNA, which leads to carcinogenesis. The HepaRG cell line, which exhibits active enzymes of phases I and II of biotransformation, serves as a model for studying polycyclic aromatic hydrocarbons (PAH) metabolism and evaluating the effect of CYP modulators ketoconazole, α-naphthoflavone and 6-formylindolo[3,2-b]carbazole (FICZ) on cell metabolism and the possibility of cytoprotection when exposed to benz[a]pyrene.

The aim of the study was to evaluate metabolic changes under the cytoprotective action of ketoconazole, α-naphthoflavone and FICZ against the background of benz[a]pyrene toxicity in the HepaRG cell line model.

Material and methods. To identify metabolomic biomarkers of PAH intoxication in HepaRG cells, changes in the metabolomic profile were analyzed using high-resolution chromatographic mass spectrometry. Potential biomarkers were identified through statistical data analysis methods.

Results. The possibility of using a metabolomic analysis of conditioned media to identify markers of benz[a]pyrene exposure and evaluate the effects of CYP modulators in vitro using the HepaRG cell line has been shown. Changes in the metabolomic profile of conditioned media after exposure to benz[a]pyrene on HepaRG cells in the presence of α-naphthoflavone indicate a decrease of the PAHs toxic effect on cells.

Limitations. The study was performed in vitro on a cell culture without analyzing the dose-dependent changes in the metabolome after exposure to the compounds studied and does not allow extrapolating the results obtained to a living organism.

Conclusion. According to the results of the metabolomic profiling of the HepaRG cellular media, a number of metabolomic markers were identified: 5-hydroxytryptophol, lysophosphatides, arginine and glutamic acid, which indicate the cytoprotective effect of α-naphthoflavone against the background of the toxic effect of benz[a]pyrene.

Compliance with ethical standards. The study does not require the submission of a biomedical ethics committee opinion or other documents.

Authors’ contribution:
Ilyushonok S.K. – collection and processing of material, statistical processing, writing a text;
Rogovskaya N.Yu. – collection of material, writing a text;
Gladchuk A.S. – editing;
Sharapov Ya.A. – writing a text;
Perevoznikov I.E. – writing a text;
Beltyukov P.P. – data processing and interpretation, text editing;
Podolskaya E.P. – research concept and design;
Babakov V.N. – the concept and design of the study, editing.
All co-authors are responsible for approving the final version of the article and ensuring the integrity of all parts of the article.

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

Funding. The work was carried out within the framework of the state contract (2224388100182000000000000/25.456.22.9), cipher “Monitoring-SOZ”.

Received: August 08, 2025 / Accepted: October 2, 2025 / Published: November 19, 2025

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