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.

1. Niki E. Oxidative stress and antioxidants: Distress or eustress? Arch. Biochem. Biophys. 2016; 595: 19-24. doi: 10.1016/j.abb.2015.11.017

2. Sies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol. 2017; 11: 613-619. doi: 10.1016/j.redox.2016.12.035

3. Rolt A., Cox L.S. Structural basis of the antiageing effects of polyphenolics: mitigation of oxide tive stress. BMC Chem. 2020; 14 (1): 50. doi: 10.1186/s13065-020-00696-0

4. Sies H. Oxidative stress: concept and some practical aspects. Antioxidants (Basel). 2020; 9 (9). doi: 10.3390/antiox9090852

5. Tungmunnithum D., Thongboonyou A., Pholboon A., Yangsabai A. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview. Medicines (Basel). 2018; 5 (3). doi: 10.3390/medicines5030093

6. Jafari H., Bernaerts K.V., Dodi G., Shavandi A. Chitooligosaccharides for wound healing biomaterials engineering. Mater. Sci. Eng. C. Mater. Biol. Appl. 2020; 117: 111266. doi: 10.1016/j.msec.2020.111266

7. Zhou Y.X., Gong X.H., Zhang H., Peng C. A review on the pharmacokinetics of paeoniflorin and its anti-inflammatory and immunomodulatory effects. Biomed. Pharmacother. 2020; 130: 110505. doi: 10.1016/j.biopha.2020.110505

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

9. Oleynik A.S., Kuprina T.S., Pevneva N.Yu., Markov A.F., Kandalintseva N.V., Prosenko A.E., Grigorev I.A. Synthesis and antioxidant properties of sodium S-[3-(hydroxyaryl)propyl] thiosulfates and [3-(hydroxyaryl)propane]-1-sulfonates. Russ. Chem. Bull. 2007. 56 (6). 1135-1143.

10. Gainutdinov 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].

11. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 1976; 72: 248-254. doi: 10.1006/abio.1976.9999

12. Siegel D., Kepa J.K., Ross D. Biochemical and genetic analysis of NAD(P)H:quinone oxidoreductase 1 (NQO1). Curr. Protoc. Toxicol. 2007; Chapter 4: Unit4 22. doi: 10.1002/0471140856.tx0422s32

13. Mannervik B., Jemth P. Measurement of glutathione transferases. Curr. Protoc. Toxicol. 2001; Chapter 6: Unit6 4. doi: 10.1002/0471140856.tx0604s01

14. Li S., Yan T., Yang J.Q., Oberley T.D., Oberley L.W. The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res. 2000; 60 (14): 3927-3939.

15. Rahman I., Kode A., Biswas S.K. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 2006; 1 (6): 3159-3165. doi: 10.1038/nprot.2006.378

16. Satoh T., McKercher S.R., Lipton S.A. Nrf2/ ARE-mediated antioxidant actions of proelectrophilic drugs. Free Radic. Biol. Med. 2013; 65: 645-657. doi: 10.1016/j.freeradbiomed.2013.07.022

17. Lemza A.E., Tkachev V.O., Zenkov N.K., Kandalintseva N.V., Kholshin S.V., Men’shchikova E.B. Effect of novel water-soluble phenolic antioxidants on cell viability: structure-activity relationships. Si-birskiy nauchnyy meditsinskiy zhurnal = Siberian Scientific Medical Journal. 2015; (2): 16-22. [In Russian].

18. Kanner J. Polyphenols by generating H2O2, affect cell redox signaling, inhibit PTPs and activate Nrf2 axis for adaptation and cell surviving: in vitro, in vivo and human health. Antioxidants (Basel). 2020; 9 (9) doi: 10.3390/antiox9090797

19. Zenkov N.K., Menshchikova E.B., Kandalintseva N.V., Oleynik A.S., Prosenko A.E., Gusachenko O.N., Shklyaeva O.A., Vavilin V.A., Lyakhovich V.V. Antioxidant and antiinflammatory activity of new water-soluble sulfur-containing phenolic compounds. Biochemistry (Mosc.). 2007; 72 (6): 644-651.

20. 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 and Tissue Biology. 2019; 13 (2): 85-92. doi: 10.1134/s1990519x1902007x

21. 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