Elsevier

Free Radical Biology and Medicine

Volume 84, July 2015, Pages 289-295
Free Radical Biology and Medicine

Original Contribution
Role of labile iron in the toxicity of pharmacological ascorbate

https://doi.org/10.1016/j.freeradbiomed.2015.03.033 Get rights and content

Highlights

  • Catalytic metals are central in determining the rate of oxidation of ascorbate and production of H2O2.

  • Extracellular catalytic iron is vital to the cellular toxicity of pharmacological ascorbate.

  • Increasing intracellular labile iron enhances the toxicity of pharmacological ascorbate.

Abstract

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H2O2 via the formation of ascorbate radical (Asc•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H2O2 generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H2O2 generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe3+/Fe2+, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.

Introduction

Pharmacological ascorbate has been shown to induce toxicity in cancer cells both in vitro and in vivo [1], [2], [3]. Pharmacological concentrations of ascorbate produce hydrogen peroxide via the formation of ascorbate radical (Asc•-) [4]. In in vitro environments, the rate of ascorbate oxidation is principally a function of the level of catalytically active iron and copper [5], [6]. For example, catalytic iron in cell culture media containing ascorbate contributes significantly to the rate of H2O2 generation; Dulbecco’s modification of Eagle’s MEM (DMEM) generates more H2O2 than RPMI 1640 during a 6-h incubation with increasing concentration of ascorbate [7] due to the fact that DMEM has an additional 0.25 μM Fe(NO3)3 in its formulation in addition to any adventitious iron.

Extracellular H2O2 readily diffuses into cells [8]; if not removed, it can lead to oxidative damage to proteins, lipids, and DNA [9]. These detrimental oxidations require that H2O2 be “activated” by appropriate redox-active transition metals, such as iron [10], [11]; labile iron, i.e., redox-active iron associated with macromolecules, will lead to production of hydroxyl radical (HO) causing site-specific damage [12], [13]. Intracellular labile iron can be coordinated by chelators such as deferoxamine (DFO), blunting their catalytically activity [14], and thereby protecting cells from H2O2-induced DNA damage [15]. Conversely, increasing intracellular iron can enhance H2O2-induced injury as seen with cardiomyocytes [16]. Preincubation of the epithelial cell line CNCMI221 with Fe3+/8-hydroxyquinoline enhanced the observed cytotoxic effects of H2O2 [17]. It is well known that the primary oxidant generated by the Fenton reaction (H2O2 + Fe2+) is the hydroxyl radical, HO [18]. HO is very reactive and as such has an extremely limited diffusion distance, only about 6 nm in cells [19]. In cell culture experiments, introduction of Fe2+ to media containing H2O2 can actually protect cells [20], [21] because the H2O2 in the media is removed; HO generated by the Fenton reaction in the media will have little consequence, as the majority will react with media components and not cells. Removal of extracellular H2O2 will minimize any possible site-specific oxidative damage to intracellular macromolecules, such as DNA.

We hypothesized that altering the concentration of iron would affect ascorbate-induced cytotoxicity: (1) by altering the rate of oxidation of ascorbate and thus the rate of production of H2O2; and (2) altering the rate of activation of H2O2 by intracellular labile iron, thereby altering the oxidative damage to cellular macromolecules, e.g., DNA, induced by site-specific production of HO. We found that treatment of cells with iron-chelators decreased H2O2 generation and reversed ascorbate-induced toxicity while increasing intracellular iron enhanced ascorbate-induced cytotoxicity. Our study indicates that redox-active metals, both inside and outside the cell, play an important role in ascorbate-induced cytotoxicity.

Section snippets

Chemicals

Iron (III) chloride (FeCl3), deferoxamine mesylate, 2,2′-dipyridyl (DPD), 8-hydroxyquinoline (8-HQ), and ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Asc-2 P) were from Sigma (St. Louis, MO). L-Ascorbic acid was purchased from Macron Chemicals (Center Valley, PA). Stock solutions of ascorbate (1.0 M) were made as previously described [3]. Phen Green SK diacetate (PG SK) was from Life Technologies (Grand Island, NY). Prior to use, DPD and PG SK were initially dissolved in a small amount

Iron chelators can protect cells from ascorbate-induced cytotoxicity

The oxidation of ascorbate in aqueous solution, in cell culture media, or in extracellular fluid is primarily dependent on the presence of catalytic metal ions [5], [6], [25]. To investigate the role of iron in ascorbate-induced cytotoxicity, cells were exposed to metal chelators DFO (75 µM) or DPD (50 µM) in HBSS for 1 h followed by addition of 2 mM ascorbate in DMEM–10% FBS for 1 h (Fig. 1A). There was no difference in clonogenic survival for cells treated with either DFO (41 ± 3%) or DPD (40 ±

Discussion

The effects of iron on ascorbate oxidation-induced cytotoxicity are not straightforward. Chelating the adventitious catalytic metals with DTPA or DFO slows ascorbate oxidation in phosphate buffer at neutral pH [5]. However, preincubation of ascorbate (500 μM) with apo-Tf (50 μg/mL) or 500 μM DFO, ferrozine, or DTPA and then incubating human dermal fibroblasts (HDFs) with these solutions did not prevent DNA damage induced by ascorbate oxidation [33]. In our experimental settings, we found that

Acknowledgments

This work was supported by NIH Grants U01 CA166800, R01 CA169046, R01 GM073929, and the Medical Research Service, Department of Veterans Affairs 1I01BX001318-01A2. Core facilities were supported in part by NIH P30 CA086862. The University of Iowa ESR Facility provided invaluable support. Data presented herein were obtained at the Flow Cytometry Facility, which is a Carver College of Medicine/Holden Comprehensive Cancer Center core research facility at the University of Iowa. The Facility is

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