Glutathione-Hemin/Hematin Adduct Formation to Disintegrate Cytotoxic Oxidant Hemin/Hematin in Human K562 Cells and Red Blood Cells' Hemolysates: Impact of Glutathione on the Hemolytic Disorders and Homeostasis

Sofia K Georgiou-Siafis; Martina K Samiotaki; Vassilis J Demopoulos; George Panayotou; Asterios S Tsiftsoglou

doi:10.3390/antiox11101959

Glutathione-Hemin/Hematin Adduct Formation to Disintegrate Cytotoxic Oxidant Hemin/Hematin in Human K562 Cells and Red Blood Cells' Hemolysates: Impact of Glutathione on the Hemolytic Disorders and Homeostasis

Antioxidants (Basel). 2022 Sep 30;11(10):1959. doi: 10.3390/antiox11101959.

Authors

Sofia K Georgiou-Siafis¹, Martina K Samiotaki², Vassilis J Demopoulos³, George Panayotou², Asterios S Tsiftsoglou¹

Affiliations

¹ Laboratory of Pharmacology, Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki (AUTh), 54124 Thessaloniki, Greece.
² B.S.R.C. "Alexander Fleming" Vari, 16672 Attiki, Greece.
³ Laboratory of Pharmaceutical Chemistry, Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki (AUTh), 54124 Thessaloniki, Greece.

Abstract

Hemin, an oxidized form of heme, acts as potent oxidant to regulate glutathione (GSH) content in pro-erythroid K562 nucleated cells, via activation of the KEAP1/NRF2 defensive signaling pathway. Moreover, GSH, as an essential metabolite, is involved in the regulation of cell-redox homeostasis and proposed to scavenge cytotoxic free heme, which is released from hemoglobin of damaged red blood cells (RBCs) during different hemolytic disorders. In the present study, we aimed to uncover the molecular mechanism by which GSH inhibits hemin-induced cytotoxicity (HIC) by affecting hemin's structural integrity in K562 cells and in RBC hemolysates. GSH, along with other thiols (cysteine, thioglycolic acid, and mercaptoethanol) altered the spectrum of hemin, while each of them co-added with hemin in cultures of K562 cells prevented HIC and growth arrest and markedly reduced the intracellular level of hemin. In addition, GSH endogenous levels served as a barrier to HIC in K562 cells, as shown by the depletion in GSH. LC-MS/MS analysis of the in vitro reaction between hemin and GSH revealed at least five different isomers of GSH-hemin adducts, as well as hydroxy derivatives as reaction products, which are characterized by unique mass spectra (MS). The latter allowed the detection of adducts in human RBC hemolysates. Based on these findings, we proposed a molecular mechanism via which GSH prevents HIC and structurally disintegrates heme. An analogous reaction was observed in RBC hemolysates via direct inter-reaction between hematin (ferric and hydroxide heme) released from hemoglobin and GSH. Overall, GSH-hematin adducts could be considered as novel entities of the human metabolome of RBCs in hemolytic disorders.

Keywords: GSH; GSH–hematin adducts; GSH–hemin adducts; K562 cells; N-acetyl cysteine; RBC hemolysates; hemin/hematin; hemolytic disorders; intracellular level of hemin; metabolites.

Grants and funding

This work was supported by grants from the Greek Secretariat of Science and Technology awarded to A.S.T., S.K.G-S. was funded by scholarships from the Bodossakis, the Onassis Foundations, as well as the Foundation of Education and European Culture (I.P.E.P.). Finally, the LC-MS analysis studies were supported by internal funds from Fleming Institute of Biological Research, Athens Greece.