N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

Department of Anatomy Faculty of Medicine and Dentistry Palacky University Olomouc Hnevotinska 3 77715 Olomouc Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc Hnevotinska 3 77715 Olomouc Czech Republic

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Flanagan R.J., Meredith T. Use of N-acetylcysteine in clinical toxicology. Am. J. Med. 1991;91:S131–S139. doi: 10.1016/0002-9343(91)90296-A. PubMed DOI

Kelly G.S. Clinical applications of N-acetylcysteine. Altern. Med. Rev. J. Clin. Ther. 1998;3:114–127. PubMed

Millea P.J. N-acetylcysteine: Multiple clinical applications. Am. Fam. Physician. 2009;80:265–269. PubMed

Rushworth G.F., Megson I.L. Existing and potential therapeutic uses for N-acetylcysteine: The need for conversion to intracellular glutathione for antioxidant benefits. Pharmacol. Ther. 2014;141:150–159. doi: 10.1016/j.pharmthera.2013.09.006. PubMed DOI

Samuni Y., Goldstein S., Dean O.M., Berk M. The chemistry and biological activities of N-acetylcysteine. Biochim. Biophys. Acta (BBA)-Gen. Subj. 2013;1830:4117–4129. doi: 10.1016/j.bbagen.2013.04.016. PubMed DOI

Aruoma O.I., Halliwell B., Hoey B.M., Butler J. The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic. Biol. Med. 1989;6:593–597. doi: 10.1016/0891-5849(89)90066-X. PubMed DOI

Winterbourn C.C., Metodiewa D. Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radic. Biol. Med. 1999;27:322–328. doi: 10.1016/S0891-5849(99)00051-9. PubMed DOI

de Flora S., Cesarone C.F., Balansky R.M., Albini A., D’Agostini F., Bennicelli C., Bagnasco M., Camoirano A., Scatolini L., Rovida A., et al. Chemopreventive properties and mechanisms of N-acetylcysteine. The experimental background. J. Cell. Biochem. 1995;59:33–41. doi: 10.1002/jcb.240590806. PubMed DOI

Mlejnek P., Dolezel P. N-acetylcysteine prevents the geldanamycin cytotoxicity by forming geldanamycin–N-acetylcysteine adduct. Chem.-Biol. Interact. 2014;220:248–254. doi: 10.1016/j.cbi.2014.06.025. PubMed DOI

Mlejnek P., Dolezel P. Loss of mitochondrial transmembrane potential and glutathione depletion are not sufficient to account for induction of apoptosis by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone in human leukemia K562 cells. Chem.-Biol. Interact. 2015;239:100–110. doi: 10.1016/j.cbi.2015.06.033. PubMed DOI

Skoupa N., Dolezel P., Ruzickova E., Mlejnek P. Apoptosis Induced by the Curcumin Analogue EF-24 Is Neither Mediated by Oxidative Stress-Related Mechanisms nor Affected by Expression of Main Drug Transporters ABCB1 and ABCG2 in Human Leukemia Cells. Int. J. Mol. Sci. 2017;18:2289. doi: 10.3390/ijms18112289. PubMed DOI PMC

Georgiou-Siafis S.K., Samiotaki M., Demopoulos V.J., Panayotou G., Tsiftsoglou A.S. Formation of novel N-acetylcysteine-hemin adducts abrogates hemin-induced cytotoxicity and suppresses the NRF2-driven stress response in human pro-erythroid K562 cells. Eur. J. Pharmacol. 2020;880:173077. doi: 10.1016/j.ejphar.2020.173077. PubMed DOI

Rakshit S., Bagchi J., Mandal L., Paul K., Ganguly D., Bhattacharjee S., Ghosh M., Biswas N., Chaudhuri U., Bandyopadhyay S. N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide. Apoptosis. 2009;14:298–308. doi: 10.1007/s10495-008-0305-7. PubMed DOI

Wu M.-S., Lien G.-S., Shen S.-C., Yang L.-Y., Chen Y.-C. N-acetyl-L-cysteine enhances fisetin-induced cytotoxicity via induction of ROS-independent apoptosis in human colonic cancer cells. Mol. Carcinog. 2014;53:E119–E129. doi: 10.1002/mc.22053. PubMed DOI

Zheng J., Lou J.R., Zhang X.-X., Benbrook D.M., Hanigan M.H., Lind S.E., Ding W.-Q. N-acetylcysteine interacts with copper to generate hydrogen peroxide and selectively induce cancer cell death. Cancer Lett. 2010;298:186–194. doi: 10.1016/j.canlet.2010.07.003. PubMed DOI PMC

Mlejnek P., Dolezel P., Maier V., Kikalova K., Skoupa N. N-acetylcysteine dual and antagonistic effect on cadmium cytotoxicity in human leukemia cells. Environ. Toxicol. Pharmacol. 2019;71:103213. doi: 10.1016/j.etap.2019.103213. PubMed DOI

Kelner M.J., Bagnell R., Welch K.J. Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat. J. Biol. Chem. 1990;265:1306–1311. doi: 10.1016/S0021-9258(19)40014-8. PubMed DOI

Freyhaus H.T., Huntgeburth M., Wingler K., Schnitker J., Bäumer A.T., Vantler M., Bekhite M.M., Wartenberg M., Sauer H., Rosenkranz S. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. Cardiovasc. Res. 2006;71:331–341. doi: 10.1016/j.cardiores.2006.01.022. PubMed DOI

Mlejnek P. Caspase inhibition and N6-benzyladenosine-induced apoptosis in HL-60 cells. J. Cell. Biochem. 2001;83:678–689. doi: 10.1002/jcb.1262. PubMed DOI

Mlejnek P. Caspase-3 Activity and Carbonyl Cyanide m-Chlorophenylhydrazone-induced Apoptosis in HL-60 cells. Altern. Lab. Anim. 2001;29:243–249. doi: 10.1177/026119290102900313. PubMed DOI

Hayyan M., Hashim M.A., AlNashef I.M. Superoxide Ion: Generation and Chemical Implications. Chem. Rev. 2016;116:3029–3085. doi: 10.1021/acs.chemrev.5b00407. PubMed DOI

Bedard K., Krause K.H. The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology. Physiol. Rev. 2007;87:245–313. doi: 10.1152/physrev.00044.2005. PubMed DOI

Sumimoto H. Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J. 2008;275:3249–3277. doi: 10.1111/j.1742-4658.2008.06488.x. PubMed DOI

Azad M., Chen Y., Gibson S.B. Regulation of Autophagy by Reactive Oxygen Species (ROS): Implications for Cancer Progression and Treatment. Antioxid. Redox Signal. 2009;11:777–790. doi: 10.1089/ars.2008.2270. PubMed DOI

Redza-Dutordoir M., Averill-Bates D.A. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim. Biophys. Acta (BBA)-Mol. Cell Res. 2016;1863:2977–2992. doi: 10.1016/j.bbamcr.2016.09.012. PubMed DOI

Valko M., Rhodes C.J., Moncol J., Izakovic M., Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 2006;160:1–40. doi: 10.1016/j.cbi.2005.12.009. PubMed DOI

Prasad A., Sedlářová M., Balukova A., Ovsii A., Rác M., Křupka M., Kasai S., Pospíšil P. Reactive Oxygen Species Imaging in U937 Cells. Front. Physiol. 2020;11:552569. doi: 10.3389/fphys.2020.552569. PubMed DOI PMC

Borutaite V., Brown G.C. Caspases are reversibly inactivated by hydrogen peroxide. FEBS Lett. 2001;500:114–118. doi: 10.1016/S0014-5793(01)02593-5. PubMed DOI

Phillips H., Terryberry J. Counting actively metabolizing tissue cultured cells. Exp. Cell Res. 1957;13:341–347. doi: 10.1016/0014-4827(57)90013-7. PubMed DOI

LeBel C.P., Ischiropoulos H., Bondy S.C. Evaluation of the probe 2’,7’-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 1992;5:227–231. doi: 10.1021/tx00026a012. PubMed DOI

Dyskova T., Fillerova R., Novosad T., Kudelka M., Zurkova M., Gajdos P., Kolek V., Kriegova E. Correlation Network Analysis Reveals Relationships between MicroRNAs, Transcription FactorT-bet, and Deregulated Cytokine/Chemokine-Receptor Network in Pulmonary Sarcoidosis. Mediat. Inflamm. 2015;2015:121378. doi: 10.1155/2015/121378. PubMed DOI PMC

Petrackova A., Horak P., Radvansky M., Fillerova R., Kraiczova V.S., Kudelka M., Mrazek F., Skacelova M., Smrzova A., Kriegova E. Revealed heterogeneity in rheumatoid arthritis based on multivariate innate signature analysis. Clin. Exp. Rheumatol. 2019;38:289–298. PubMed

Kriegova E., Arakelyan A., Fillerova R., Zatloukal J., Mrazek F., Navratilova Z., Kolek V., Du Bois R.M., Petrek M. PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells. BMC Mol. Biol. 2008;9:69. doi: 10.1186/1471-2199-9-69. PubMed DOI PMC

Nicoletti I., Migliorati G., Pagliacci M., Grignani F., Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods. 1991;139:271–279. doi: 10.1016/0022-1759(91)90198-O. PubMed DOI

Darzynkiewicz Z., Bruno S., del Bino G., Gorczyca W., Hotz M.A., Lassota P., Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry. 1992;13:795–808. doi: 10.1002/cyto.990130802. PubMed DOI

Mlejnek P., Kuglík P. Induction of apoptosis in HL-60 cells by N(6)-benzyladenosine. J. Cell. Biochem. 2000;77:6–17. doi: 10.1002/(SICI)1097-4644(20000401)77:1<6::AID-JCB2>3.0.CO;2-3. PubMed DOI

Frydrych I., Mlejnek P. Serine protease inhibitors N-α-Tosyl-L-Lysinyl-Chloromethylketone (TLCK) and N-Tosyl-L-Phenylalaninyl-Chloromethylketone (TPCK) are potent inhibitors of activated caspase proteases. J. Cell. Biochem. 2008;103:1646–1656. doi: 10.1002/jcb.21550. PubMed DOI

Direct Interaction between N-Acetylcysteine and Cytotoxic Electrophile-An Overlooked In Vitro Mechanism of Protection