Toxicity of new monophenolic synthetic activator of Keap1/Nrf2/ARE redox-sensitive signaling system <i>in vitro</i> and <i>in vivo</i>

One of the promising areas of modern pharmacology is the development of «indirect antioxidants» capable of activating redox-sensitive signaling systems, primarily the Keap1/Nrf2/ARE system. Among its chemical inductors is the hydrophilic monosubstituted monophenol (3’-tert-butyl-4’-hydroxyphenyl)sodium propylthiosulfonate (TS-13) in development. The aim of the study was to investigate TS-13 antiproliferative activity against tumor cell line BT-474 in vitro and acute oral toxicity in mice in vivo. Material and methods. The relationship between TS-13 concentration and proliferative activity of human breast ductal carcinoma cell line BT-474 was determined using the MTT test, the IC&lt;sub&gt;50&lt;/sub&gt; was calculated and compared to the previously obtained for MCF-7 line; results were correlated with the functional properties of cells based on gene expression (in silico GSEA). In vivo acute toxicity was studied in 50 female C57Bl/6J mice, who received a TS-13 solution in distilled water at various doses by intragastric gavage. LD&lt;sub&gt;50&lt;/sub&gt; obtained experimentally and predicted in silico using the GUSAR web service were compared. Results and discussion. TS-13 inhibited the proliferation of BT-474 cells in a concentration-dependent manner (exponential approximation, IC&lt;sub&gt;50&lt;/sub&gt; = 59.5 μM) and was 2.2 times more toxic than for MCF-7 cells. This may be due to functional differences between the BT-474 and MCF-7 lines, as evidenced by the GSEA results. The LD&lt;sub&gt;50&lt;/sub&gt; value established in the in vivo experiment was 936 mg/kg body weight, the obtained value satisfactorily corresponds to the predicted in silico (561 mg/kg), although in reality the compound turned out to be somewhat less toxic than could be expected based on its structure. Conclusions. A study of the acute toxicity of the new water-soluble monophenol TC-13 allows the classification of it as slightly toxic (toxicity rating level 4) according to the Hodge – Sterner scale) or as moderately hazardous (hazard class 3) according to GOST 12.1.007-76

Effect of new water-soluble phenolic antioxidants on the activity of Nrf2-driven enzymes, glutathione system, and Nrf2 translocation into the nucleus

Understanding the role of reactive oxygen and nitrogen species in eustress (redox balance) and distress (oxidative stress) development poses new challenges for biomedical scientists and pharmacologists in the search for compounds that can not only have a direct antioxidant (antiradical) effect, but also affect redox-sensitive signaling pathways, primarily Keap1/Nrf2/ARE system. Aim of the study was to investigate the influence of novel water-soluble structurally related monophenols on key elements of Keap1/Nrf2/ARE system induction (activity of Nrf2-driven enzymes, the state of the glutathione system, and intracellular redistribution of transcription factor Nrf2).Material and methods. Five original hydrophilic structurally related monophenols, differing in the number of tert-butyl ortho-substituents, the length of the para-alkyl substituent, and the presence of a divalent sulfur or selenium atom in it were investigated (phenoxane, the potassium salt of phenosan acid, was used as a reference compound). Cell lines U937 and J774 were cultured for 24 h in the presence of tested compounds, and comparative analysis was performed of its ability to induce the synthesis of Nrf2-driven enzymes of phase II xenobiotic detoxification pathway and antioxidant enzymes (NAD(P)H: quinone oxidoreductase 1 (NQO1), glutathione S-transferases (GST), glutathione peroxidases, glutathione reductase (biochemical spectrophotometric methods were used to study their activity), as well as to influence the state of glutathione system (spectrophotometry) and translocation of transcription factor Nrf2 into the nucleus (immunofluorescent staining, confocal microscopy) (key events of Keap1/Nrf2/ARE signaling system activation).Results and discussion. Monophenol TS-13 have found to be the most effective inducer of tested enzymes in U937 cells among the structural analogs, while the structure of the para-alkyl substituent and the degree of OH group hindrance are important for the implementation of this effect; TS-13 also effectively enhanced Nrf2 import into J774 cell nucleus. The NQO1- and GST-inducing abilities of structurally related monophenols are closely interrelated, which indicates the possibility of coordinated induction of these enzymes and the presence of a common regulatory system that ensures their activation in response to cell treatment with phenolic antioxidants