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

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 IC₅₀ 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. LD₅₀ 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, IC₅₀ = 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 LD₅₀ 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.

1. Hajam Y.A., Rani R., Ganie S.Y., Sheikh T.A., Javaid D., Qadri S.S., Pramodh S., Alsulimani A., Alkhanani M.F., Harakeh S., Hussain A., Haque S., Reshi M.S. Oxidative stress in human pathology and aging: Molecular mechanisms and perspectives. Cells. 2022;11(3):552. doi: 10.3390/cells11030552

2. Mashkovsky M.D. Medicines. Moscow: Novaya volna, 2012. 1216 p.

3. Zhou G., Meng S., Li Y., Ghebre Y.T., Cooke J.P. Optimal ROS signaling is critical for nuclear reprogramming. Cell Rep. 2016;15(5):919–925. doi: 10.1016/j.celrep.2016.03.084

4. Cuadrado A., Rojo A.I., Wells G., Hayes J.D., Cousin S.P., Rumsey W.L., Attucks O.C., Franklin S., Levonen A.L., Kensler T.W., Dinkova-Kostova A.T. Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nat. Rev. Drug Discov. 2019;18(4):295–317. doi: 10.1038/s41573-018-0008-x

5. Blaner W.S., Shmarakov I.O., Traber M.G. Vitamin A and vitamin E: Will the real antioxidant please stand up? Annu. Rev. Nutr. 2021;41:105–131. doi: 10.1146/annurev-nutr-082018-124228

6. Speisky H., Shahidi F., Costa de Camargo A., Fuentes J. Revisiting the oxidation of flavonoids: Loss, conservation or enhancement of their antioxidant properties. Antioxidants. 2022;11(1):133. doi: 10.3390/antiox11010133

7. Diaz M., Mesa-Herrera F., Marin R. DHA and its elaborated modulation of antioxidant defenses of the brain: Implications in aging and AD neurodegeneration. Antioxidants. 2021;10(6):907. doi: 10.3390/antiox10060907

8. Matsumaru D., Motohashi H. The KEAP1-NRF2 system in healthy aging and longevity. Antioxidants. 2021;10(12):1929. doi: 10.3390/antiox10121929

9. Zenkov N.K., Kozhin P.M., Chechushkov A.V., Martinovich G.G., Kandalintseva N.V., Menshchikova E.B. Mazes of Nrf2 regulation. Biochemistry (Moscow). 2017;82(5):556–564. doi: 10.1134/s0006297917050030

10. Menshchikova E., Tkachev V., Lemza A., Sharkova T., Kandalintseva N., Vavilin V., Safronova O., Zenkov N. Water-soluble phenol TS-13 combats acute but not chronic inflammation. Inflamm. Res. 2014;63(9):729–740. doi: 10.1007/s00011-014-0746-0

11. Menshchikova E.B., Chechushkov A.V., Kozhin P.M., Kholshin S.V., Kandalintseva N.V., Martinovich G.G., Zenkov N.K. Activation of autophagy and Nrf2 signaling in human breast adenocarcinoma MCF-7 cells by novel monophenolic antioxidants. Cell Tissue Biol. 2019;13(2):85–92. doi: 10.1134/S1990519X1902007X

12. Gaynutdinov P.I., Kozhin P.M., Chechushkov A.V., Martinovich G.G., Kholshin S.V., Kandalintseva N.V., Zenkov N.K., Menshchikova E.B. Inverse relationship between the antioxidant activity of structurally related synthetic monophenols and their toxicity in tumor cells. Sibirskiy nauchnyy meditsinskiy zhurnal = Siberian Scientific Medical Journal. 2018;38(1):22–31. [In Russian]. doi: 10.15372/SSMJ20180104

13. Oleynik A.S., Kuprina T.S., Pevneva N.Y., Markov A.F., Kandalintseva N.V., Prosenko A.E., Grigoriev I.A. Synthesis and antioxidant properties of sodium S-[3-(hydroxyaryl)propyl] thiosulfates and [3-(hydroxyaryl)propane]-1-sulfonates. Russian Chemical Bulletin. 2007;58(6):1135–1143. doi: 10.1007/s11172-007-0172-3

14. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4

15. Fonseca N.A., Marioni J., Brazma A. RNA-Seq gene profiling – a systematic empirical comparison. PloS One. 2014;9(9):е107026. doi: 10.1371/journal.pone.0107026

16. Banerjee P., Eckert A.O., Schrey A.K., Preissner R. ProTox-II: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res. 2018;46(W1):W257–W263. doi: 10.1093/nar/gky318

17. Sukhachev V.S., Ivanov S.M., Filimonov D.A., Poroikov V.V. Alternative methods of studies. Computer-aided estimation of rodents acute toxicity. Laboratornyye zhivotnyye dlya nauchnykh issledovaniy = Laboratory Animals for Science. 2019;(4):4. doi: 10.29926/2618723X-2019-04-04

18. Nair A.B., Jacob S. A simple practice guide for dose conversion between animals and human. J. Basic. Clin. Pharm. 2016;7(2):27–31. doi: 10.4103/0976-0105.177703