International Journal of Molecular Sciences Article Redox Interactions of Vitamin C and Iron: Inhibition of the Pro-Oxidant Activity by Deferiprone Viktor A. Timoshnikov 1,*, Tatyana V. Kobzeva 1, Nikolay E. Polyakov 1 and George J. Kontoghiorghes 2,* 1 Institute of Chemical Kinetics & Combustion, 630090 Novosibirsk, Russia; [email protected] (T.V.K.); [email protected] (N.E.P.) 2 Postgraduate Research Institute of Science, Technology, Environment and Medicine, CY-3021 Limassol, Cyprus * Correspondence: [email protected] (V.A.T.); [email protected] (G.J.K.); Tel./Fax: +7-383-3332947 (V.A.T.); +357-2627-2076 (G.J.K.) Received: 21 February 2020; Accepted: 28 May 2020; Published: 31 May 2020 Abstract: Ascorbic acid (AscH2) is one of the most important vitamins found in the human diet, with many biological functions including antioxidant, chelating, and coenzyme activities. Ascorbic acid is also widely used in medical practice especially for increasing iron absorption and as an adjuvant therapeutic in iron chelation therapy, but its mode of action and implications in iron metabolism and toxicity are not yet clear. In this study, we used UV–Vis spectrophotometry, NMR spectroscopy, and EPR spin trapping spectroscopy to investigate the antioxidant/pro-oxidant effects of ascorbic acid in reactions involving iron and the iron chelator deferiprone (L1). The experiments were carried out in a weak acidic (pH from 3 to 5) and neutral (pH 7.4) medium. Ascorbic acid exhibits predominantly pro-oxidant activity by reducing Fe3+ to Fe2+, followed by the formation of dehydroascorbic acid. As a result, ascorbic acid accelerates the redox cycle Fe3+ Fe2+ in the Fenton reaction, which leads $ to a significant increase in the yield of toxic hydroxyl radicals. The analysis of the experimental data suggests that despite a much lower stability constant of the iron–ascorbate complex compared to the FeL13 complex, ascorbic acid at high concentrations is able to substitute L1 in the FeL13 chelate complex resulting in the formation of mixed L12AscFe complex. This mixed chelate complex is redox stable at neutral pH = 7.4, but decomposes at pH = 4–5 during several minutes at sub-millimolar concentrations of ascorbic acid. The proposed mechanisms play a significant role in understanding the mechanism of action, pharmacological, therapeutic, and toxic effects of the interaction of ascorbic acid, iron, and L1. Keywords: vitamin C; ascorbic acid; iron toxicity; iron chelation; deferiprone; Fenton reaction; hydroxyl radical; ascorbyl radical; EPR spin trapping; TMIO spin trap 1. Introduction Vitamin C or ascorbic acid (AscH2, Scheme1) is a powerful water-soluble antioxidant, found in nature and present in fresh fruit and vegetables [1–3]. Ascorbic acid is also known as a reducing agent and a radical scavenger. As a radical scavenger AscH2 provides effective protection of membranes and proteins against oxidation by reactive oxygen species (ROS): superoxide (O2•−), hydrogen peroxide (H O ), hydroxyl radicals ( OH), peroxyl radicals ( OOH), and singlet oxygen 1O , especially at high 2 2 • • 2 concentrations [4,5]. Int. J. Mol. Sci. 2020, 21, 3967; doi:10.3390/ijms21113967 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3967 2 of 16 Int. J. Mol. Sci. 2020, 21, 3967 2 of 17 6 OH OH OH + OH 5 -H HO HO HO O O + HO OH - e- - H ,- e- O 4 3 2 O 1 O O O OH OH OH O O O O O - . AscH AscH AscH 2 DHA Scheme 1. The structure of ascorbic acid and the main products of redox reactions. Ascorbic Scheme 1. The structure of ascorbic acid and the main products of redox reactions. Ascorbic acid— acid—AscH2.; ascorbate anion—AscH−; ascorbyl radical—AscH•; dehydroascorbic acid—DHA. AscH2.; ascorbate anion—AscH−; ascorbyl radical—AscH•; dehydroascorbic acid—DHA. However, ascorbic acid is a multifaceted compound that can act as a chelator and also as a source of freeHowever, radicals [6 ascorbic–10]. The acid role is ofa multifaceted ascorbic acid compound as a chelator that incan iron act andas a chelator copper overloadand also as conditions a source is widelyof free discussed radicals [6–10]. [11–13 The]. In role this of regard, ascorbic the acid literature as a chelator intensively in iron discussesand copper the overload possibility conditions of using ascorbicis widely acid discussed to treat [11–13]. advanced In this cancer regard, in combination the literature withintensively anticancer discusses agents the[ possibility14–25]. In of particular, using ascorbic acid to treat advanced cancer in combination with anticancer agents [14–25]. In particular, some researchers have demonstrated biological validity and are ready to study the potential value of some researchers have demonstrated biological validity and are ready to study the potential value of ascorbic acid in the treatment of cancer in humans [17]. However, the evidence that ascorbic acid could ascorbic acid in the treatment of cancer in humans [17]. However, the evidence that ascorbic acid help human cancer patients is still thin and requires additional study. The results of published studies could help human cancer patients is still thin and requires additional study. The results of published fail to prove that there is a clinically relevant positive effect of ascorbic acid supplementation in cancer studies fail to prove that there is a clinically relevant positive effect of ascorbic acid supplementation patientsin cancer in general patients on in the general overall on survival, the overall clinical survival, status, clinical quality status, of life quality and performance of life and performance status [1,14,26 ]. statusIron [1,14,26]. is an essential element of life. It plays an important role in many cellular processes including energyIron production, is an essential oxygen element transport, of life.DNA It plays synthesis, an important as well role as in inmany many cellular other processes important including metabolic processesenergy production, involving iron-containing oxygen transport, enzymes DNA synthesis, [6,10]. The as catalyticwell as in activity many other of iron important stems from metabolic its redox 3+ 2+ properties,processes namelyinvolving the iron-containing ability to alternate enzymes between [6,10]. twoThe catalytic oxidation activity states, of Fe iron andstems Fe from, acting its redox as an electronproperties, donor namely or acceptor. the ability On to the alternate other hand,two oxidation an excess states, of iron Fe3+is and toxic Fe2+ because, acting as it canan electron cause the formationdonor or ofacceptor. an excessive On the amount other hand, of ROS, an excess which of caniron subsequently is toxic because cause it can biomolecular, cause the formation subcellular, of cellular,an excessive and tissue amount damage. of ROS, Excess which toxic can iron subsequently has been identifiedcause biomolecular, in many diseases, subcellular, such cellular, as age-related and maculartissue damage. degeneration, Excess age-related toxic iron ha Alzheimer’ss been identified and Parkinson’s in many diseas diseases,es, such as as well age-related as cancer macular [6,26–32 ]. degeneration,Iron chelation age-related therapy Alzheimer’s is widely used and to Parkinso preventn’s poisoning diseases, byas well removing as cancer excess [6,26–32]. iron caused from repeatedIron blood chelation transfusions therapy andis widely other used iron to overload prevent diseases.poisoning Itby includes removing the excess use of iron various caused chelating from agentsrepeated that blood bind andtransfusions remove excessand othe ironr iron from overload the body. diseases. [6,26,32 It –includes34]. In this the regard,use of various numerous chelating studies haveagents been that conducted bind and to remove elucidate excess the iron redox from activity the body. of iron [6,26,32–34]. in chelate complexesIn this regard, [35 –numerous39]. As an studies example, wehave recently been demonstrated conducted to theelucidate inhibition the redox of iron- activity and copper-induced of iron in chelate hydroxyl complexes radical [35–39]. production As an by example, we recently demonstrated the inhibition of iron- and copper-induced hydroxyl radical the water-soluble chelator deferiprone (L1, Figure1)[ 39,40]. L1 is an effective iron-chelating drug production by the water-soluble chelator deferiprone (L1, Figure 1) [39,40]. L1 is an effective iron- developed for the treatment of iron overload toxicity in thalassaemia and other iron-related toxicity chelating drug developed for the treatment of iron overload toxicity in thalassaemia and other iron- conditions. L1 is orally active and widespread in the body, thus binding toxic iron in many tissues related toxicity conditions. L1 is orally active and widespread in the body, thus binding toxic iron to and organs, including the brain [41–43]. Moreover, L1 is supposed to be an effective antioxidant that many tissues and organs, including the brain [41–43]. Moreover, L1 is supposed to be an effective prevents oxidative stress and biomolecular, subcellular, cellular, and tissue damage caused mainly by antioxidant that prevents oxidative stress and biomolecular, subcellular, cellular, and tissue damage ironcaused and copper-inducedmainly by iron freeand radicalcopper-induced formation free [43 ].radical The physicochemical formation [43]. The and physicochemical coordination properties and ofcoordination the L1 complex properties with iron of havethe L1 been complex previously with iron studied have inbeen detail previously by many studied investigators in detail [41 by,42 many,44–46 ]. investigatorsIn contrast, [41,42,44–46]. ascorbic acid is generally regarded as a weak chelating agent and cannot compete in iron binding with L1 and other stronger chelators. The coordination chemistry of ascorbic acid has been extensively studied in response to problems associated with its hydrolysis and redox reactions, as well as with the instability of its complexes [47–53]. As a weak dibasic acid (pKa1 = 4.25 and pKa2 = 11.79), at physiological pH ascorbic acid exists as monoanion (AscH−) with deprotonation of O(3)–H (see Scheme1).