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Pharmacological ascorbate (P-AscH - ) combined with standard of care (SOC) radiation and temozolomide is being evaluated in a phase 2 clinical trial (NCT02344355) in the treatment of glioblastoma (GBM). Previously published data demonstrated that paramagnetic iron (Fe 3+ ) catalyzes ascorbate's oxidation to form diamagnetic iron (Fe 2+ ). Because paramagnetic Fe 3+ may influence relaxation times observed in MR imaging, quantitative MR imaging of P-AscH - -induced changes in redox-active Fe was assessed as a biomarker for therapy response. Gel phantoms containing either Fe 3+ or Fe 2+ were imaged with T2* and quantitative susceptibility mapping (QSM). Fifteen subjects receiving P-AscH - plus SOC underwent T2* and QSM imaging four weeks into treatment. Subjects were scanned: pre-P-AscH - infusion, post-P-AscH - infusion, and post-radiation (3-4 h between scans). Changes in T2* and QSM relaxation times in tumor and normal tissue were calculated and compared to changes in Fe 3+ and Fe 2+ gel phantoms. A GBM mouse model was used to study the relationship between the imaging findings and the labile iron pool. Phantoms containing Fe 3+ demonstrated detectable changes in T2* and QSM relaxation times relative to Fe 2+ phantoms. Compared to pre-P-AscH - , GBM T2* and QSM imaging were significantly changed post-P-AscH - infusion consistent with conversion of Fe 3+ to Fe 2+ . No significant changes in T2* or QSM were observed in normal brain tissue. There was moderate concordance between T2* and QSM changes in both progression free survival and overall survival. The GBM mouse model showed similar results with P-AscH - inducing greater changes in tumor labile iron pools compared to the normal tissue. CONCLUSIONS: T2* and QSM MR-imaging responses are consistent with P-AscH - reducing Fe 3+ to Fe 2+ , selectively in GBM tumor volumes and represent a potential biomarker of response. This study is the first application using MR imaging in humans to measure P-AscH - -induced changes in redox-active iron., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Pharmacological doses (> 1mM) of ascorbate (a.k.a., vitamin C) have been shown to selectively kill cancer cells through a mechanism that is dependent on the generation of H 2 O 2 at doses that are safely achievable in humans using intravenous administration. The process by which ascorbate oxidizes to form H 2 O 2 is thought to be mediated catalytically by redox active metal ions such as iron (Fe). Because intravenous iron sucrose is often administered to colon cancer patients to help mitigate anemia, the current study assessed the ability of pharmacological ascorbate to kill colon cancer cells in the presence and absence of iron sucrose. In vitro survival assays showed that 10mM ascorbate exposure (2h) clonogenically inactivated 40-80% of exponentially growing colon cancer cell lines (HCT116 and HT29). When the H 2 O 2 scavenging enzyme, catalase, was added to the media, or conditionally over-expressed using a doxycycline inducible vector, the toxicity of pharmacological ascorbate was significantly blunted. When colon cancer cells were treated in the presence or absence of 250µM iron sucrose, then rinsed, and treated with 10mM ascorbate, the cells demonstrated increased levels of labile iron that resulted in significantly increased clonogenic cell killing, compared to pharmacological ascorbate alone. Interestingly, when colon cancer cells were treated with iron sucrose for 1h and then 10mM ascorbate was added to the media in the continued presence of iron sucrose, there was no enhancement of toxicity despite similar increases in intracellular labile iron. The combination of iron chelators, deferoxamine and diethylenetriaminepentaacetic acid, significantly inhibited the toxicity of either ascorbate alone or ascorbate following iron sucrose. These observations support the hypothesis that increasing intracellular labile iron pools, using iron sucrose, can be used to increase the toxicity of pharmacological ascorbate in human colon cancer cells by a mechanism involving increased generation of H 2 O 2 ., (Published by Elsevier B.V.)

Soft tissue sarcomas are a histologically heterogeneous group of rare mesenchymal cancers for which treatment options leading to increased overall survival have not improved in over two decades. The current study shows that pharmacological ascorbate (systemic high dose vitamin C achieving ≥ 20mM plasma levels) is a potentially efficacious and easily integrable addition to current standard of care treatment strategies in preclinical models of fibrosarcoma and liposarcoma both in vitro and in vivo. Furthermore, enhanced ascorbate-mediated toxicity and DNA damage in these sarcoma models were found to be dependent upon H 2 O 2 and intracellular labile iron. Together, these data support the hypothesis that pharmacological ascorbate may represent an easily implementable and non-toxic addition to conventional sarcoma therapies based on taking advantage of fundamental differences in cancer cell oxidative metabolism., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H 2 O 2 ; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O 2 ⋅- and H 2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H 2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H 2 O 2 . In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 2mM 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., Fe(3+)/Fe(2+), have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo., (Published by Elsevier Inc.)

Labile iron, i.e. iron that is weakly bound and is relatively unrestricted in its redox activity, has been implicated in both the pathogenesis as well as treatment of cancer. Two cancer treatments where labile iron may contribute to their mechanism of action are pharmacological ascorbate and ionizing radiation (IR). Pharmacological ascorbate has been shown to have tumor-specific toxic effects due to the formation of hydrogen peroxide. By catalyzing the oxidation of ascorbate, labile iron can enhance the rate of formation of hydrogen peroxide; labile iron can also react with hydrogen peroxide. Here we have investigated the magnitude of the labile iron pool in tumor and normal tissue. We also examined the ability of pharmacological ascorbate and IR to change the size of the labile iron pool. Although a significant amount of labile iron was seen in tumors (MIA PaCa-2 cells in athymic nude mice), higher levels were seen in murine tissues that were not susceptible to pharmacological ascorbate. Pharmacological ascorbate and irradiation were shown to increase the labile iron in tumor homogenates from this murine model of pancreatic cancer. As both IR and pharmacological ascorbate may rely on labile iron for their effects on tumor tissues, our data suggest that pharmacological ascorbate could be used as a radio-sensitizing agent for some radio-resistant tumors.

Environmental heat stress is associated with an age-related increase in hepatic oxidative damage and an exaggerated state of oxidative stress. The purpose of this investigation was to evaluate the regulation of hepatic iron after heat stress. A secondary aim was to determine a potential role for iron in heat stress-induced liver injury. Hyperthermia-induced alterations in hepatic iron were evaluated in young (6 mo) and old (24 mo) Fischer 344 rats by exposing them to a two-heat stress protocol. Livers were harvested at several time points after the second heating and assayed for labile and nonheme iron. In the control condition, there was no difference in labile iron between age groups. Both labile iron and storage iron were not altered by hyperthermia in young rats, but both were increased immediately after heating in old rats. To evaluate a role for iron in liver injury, hepatic iron content was manipulated in young and old rats, and then both groups were exposed to heat stress. Iron administration to young rats significantly increased hepatic iron content and ferritin but did not affect markers of lipid peroxidation under control conditions or after heat stress. In old rats, iron chelation with deferoxamine prevented the increase in nonheme iron, labile iron, ferritin, and lipid peroxidation after heat stress. These results suggest that iron may play a role in hepatic injury after hyperthermia. Thus, dysregulation of iron may contribute to the gradual decline in cellular and physiological function that occurs with aging.

Free radical injury is thought to play a significant role in the pathogenesis of several disease processes in low birth weight premature infants including retinopathy of prematurity and necrotizing enterocolitis. Because iron is a known catalyst in free radical-mediated oxidation reactions, the objectives of the present in vitro studies were to determine whether after exposure to air 1) iron present in infant formula, or that added to human milk or formula as medicinal iron or as iron contained in human milk fortifier, increases free radical and lipid peroxidation products; and 2) recombinant human lactoferrin added to formula or human milk attenuates iron-mediated free radical formation and lipid peroxidation. Before adding medicinal iron to formula and human milk, significantly more ascorbate and alpha-hydroxyethyl radical production and more lipid peroxidation products (i.e. thiobarbituric acid reactive substances, malondialdehyde, and ethane) were observed in formula. After the addition of medicinal iron to either formula or human milk, further increases were observed in free radical and lipid peroxidation products. When iron-containing human milk fortifier was added to human milk, free radicals also increased. In contrast, the addition of apo-recombinant human lactoferrin to formula or human milk decreased the levels of oxidative products when medicinal iron or human milk fortifier was present. We speculate that the presence of greater concentration of iron and the absence of lactoferrin in formula compared with human milk results in greater in vitro generation of free radicals and lipid peroxidation products. Whether iron-containing formula with lactoferrin administered enterally to preterm infants will result in less free radical generation in vivo has yet to be established.

Brain tissue being rich in polyunsaturated fatty acids, is very susceptible to lipid peroxidation. Iron is well known to be an important initiator of free radical oxidations. We propose that the principal route to iron-mediated lipid peroxidations is via iron-oxygen complexes rather than the reaction of iron with hydrogen peroxide, the Fenton reaction. To test this hypothesis, we enriched leukemia cells (K-562 and L1210 cells) with docosahexaenoic acid (DHA) as a model for brain tissue, increasing the amount of DHA from approximately 3 mole % to 32 mole %. These cells were then subjected to ferrous iron and dioxygen to initiate lipid peroxidation in the presence or absence of hydrogen peroxide. Lipid-derived radicals were detected using EPR spin trapping with alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN). As expected, lipid-derived radical formation increases with increasing cellular lipid unsaturation. Experiments with desferal demonstrate that iron is required for the formation of lipid radicals from these cells. Addition of iron to DHA-enriched L1210 cells resulted in significant amounts of radical formation; radical formation increased with increasing amount of iron. However, the exposure of cells to hydrogen peroxide before the addition of ferrous iron did not increase cellular radical formation, but actually decreased spin adduct formation. These data suggest that iron-oxygen complexes are the primary route to the initiation of biological free radical oxidations. This model proposes a mechanism to explain how catalytic iron in brain tissue can be so destructive.

The goal of our study was to investigate the mechanism by which changes in extracellular pH influence lipid peroxidation processes. Ferrous iron can react with hydroperoxides, via a Fenton-type reaction, to initiate free radical chain processes. Iron is more soluble at lower pH values, therefore we hypothesized that decreasing the environmental pH would lead to increased iron-mediated lipid peroxidation. We used Photofrin, a photosensitizer that produces singlet oxygen, to introduce lipid hydroperoxides into leukemia cells (HL-60, K-562, and L1210). Singlet oxygen reacts with the PUFA of cells producing lipid hydroperoxides. Using EPR spin trapping with POBN, free radical formation from HL-60 cells was only detected when Photofrin, light, and ferrous iron were present. Free radical formation increased with increasing iron concentration; in the absence of extracellular iron, radical formation was below the limit of detection and lipid hydroperoxides accumulated in the membrane. In the presence of iron, lipid-derived radical formation in cells is pH dependent; the lower the extracellular pH (7.5-5.5), the higher the free radical flux; the lower the pH, the greater the membrane permeability induced in K-562 cells, as determined by trypan blue dye exclusion. These data demonstrate that lipid peroxidation processes, mediated by iron, are enhanced with decreasing extracellular pH. Thus, acidic pH not only releases iron from "safe" sites, but this iron will also be more damaging.

Nitric oxide ((*)NO) can protect cells against the detrimental effects of reactive oxygen species. Using low-density lipoprotein as well as model systems, it has been demonstrated that (*)NO can serve as a chain-breaking antioxidant to blunt lipid peroxidation. To test the hypothesis that (*)NO can serve as a chain-breaking antioxidant in cell membranes, we examined the effect of (*)NO on iron-induced lipid peroxidation in human leukemia cells. We exposed HL-60 cells to an oxidative stress (20 microM Fe(2+)) and monitored the consumption of oxygen as a measure of lipid peroxidation. Oxygen consumption was arrested by the addition of (*)NO as a saturated aqueous solution. The duration of inhibition of oxygen consumption by (*)NO was concentration-dependent in the 0.4-1.8 microM range. The inhibition ended upon depletion of (*)NO. The addition of (*)NO prior to initiation of peroxidation delayed the onset of peroxidation; the nearer in time it was before Fe(2+) addition, the longer the inhibition. Depletion of cellular glutathione levels by d, l-buthionine-S,R-sulfoximine prior to Fe(2+) addition resulted in a more rapid initial rate of oxygen depletion and a shorter time for the (*)NO-induced inhibition of oxygen consumption. Complementary studies of this iron-induced lipid peroxidation, using thiobarbituric acid reactive substances as a marker, also demonstrated the protective effects of (*)NO. This protection of cells against lipid peroxidation also manifested itself as a reduction in trypan blue uptake, an observation demonstrating the protective effects of (*)NO on membrane integrity. We conclude that (*)NO protects HL-60 human leukemia cells from lipid peroxidation and that this protection ameliorates the toxicity of the oxidation processes initiated by Fe(2+) and dioxygen., (Copyright 1999 Academic Press.)

Iron can be a detrimental catalyst in biological free radical oxidations. Because of the high physiological ratio of [O2]/[H2O2] (> or = 10(3)), we hypothesize that the Fenton reaction with pre-existing H2O2 is only a minor initiator of free radical oxidations and that the major initiators of biological free radical oxidations are the oxidizing species formed by the reaction of Fe2+ with dioxygen. We have employed electron paramagnetic resonance spin trapping to examine this hypothesis. Free radical oxidation of: 1) chemical (ethanol, dimethyl sulfoxide); 2) biochemical (glucose, glyceraldehyde); and 3) cellular (L1210 murine leukemia cells) targets were examined when subjected to an aerobic Fenton (Fe2+ + H2O2 + O2) or an aerobic (Fe2+ + O2) system. As anticipated, the Fenton reaction initiates radical formation in all the above targets. Without pre-existing H2O2, however, Fe2+ and O2 also induce substantial target radical formation. Under various experimental ratios of [O2]/[H2O2] (1-100 with [O2] approximately 250 microM), we compared the radical yield from the Fenton reaction vs. the radical yield from Fe2+ + O2 reactions. When [O2]/[H2O2] < 10, the Fenton reaction dominates target molecule radical formation; however, production of target-molecule radicals via the Fenton reaction is minor when [O2]/[H2O2] > or = 100. Interestingly, when L1210 cells are the oxidation targets, Fe2+ + O2 is observed to be responsible for formation of nearly all of the cell-derived radicals detected, no matter the ratio of [O2]/[H2O2]. Our data demonstrate that when [O2]/[H2O2] > or = 100, Fe2+ + O2 chemistry is an important route to initiation of detrimental biological free radical oxidations.

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O2.- and H2O2. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H2O2 but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (.OH) as a consequence of the reaction of H2O2 with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H2O2 in the presence of an alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)-ethanol spin-trapping system, the 4-POBN-.CH(CH3)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating .OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H2O2 in a similar manner, the magnitude of .OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of .OH spin trapped. Exogenous SOD failed to inhibit .OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD.

We hypothesized that exposure of cells to H2O2 plus Fe2+ would increase formation of cell-derived lipid peroxides that would inactivate prostaglandin H synthase, resulting in decreased prostaglandin synthesis. Therefore, we treated human endothelial cells with 0-100 microM H2O2 followed immediately by addition of 0-200 microM Fe2+. After oxidant exposure, cells were stimulated with 20 microM arachidonic acid to induce prostaglandin I2 (PGI2) synthesis. Adding 100 microM H2O2 prior to arachidonic acid decreased PGI2 synthesis more than 80%. However, to our surprise, the addition of Fe2+, in increasing amounts, progressively protected PGI2 synthesis against the harmful effects of H2O2. A ratio of one part H2O2 to two parts Fe2+ offered almost complete protection, whereas Fe3+ did not protect PGI2 synthesis from H2O2. We found that 100 microM H2O2 was not cytolytic; however, 250 microM H2O2 was cytolytic; Fe2+ protected against this cytotoxicity. In addition, extracellular Fe2+ prevented the rise in intracellular calcium caused by H2O2 and extracellular Fe2+ preserved intracellular glutathione in H2O2-exposed cells. Electron paramagnetic resonance spin trapping demonstrated that extracellular Fe2+ generated the hydroxyl free radical, HO. outside the cell. We speculate that extracellular Fe2+ protects the intracellular space from H2O2 by initiating the Fenton reaction outside the cell. This reductive cleavage of H2O2 generates HO. in the extracellular space, where much of the HO. will react with noncellular components, thereby protecting the cell interior.

The reaction of superoxide with the spin trap DMPO (5,5-dimethylpyrroline-N-oxide) is widely used to study superoxide production as well as issues associated with the superoxide-related formation of hydroxyl radical. However, the interpretation of observed intensities of DMPO/.OOH and DMPO/.OH signals in electron paramagnetic resonance spin trapping experiments is not without its difficulties. In this paper, I report experiments that demonstrate: 1. That the flux of superoxide formation in a DMPO spin trapping experiment can alter the apparent importance of weak DMPO/.OH signals; 2. That iron can influence the DMPO/.OOH spin trapping results; 3. That there is very little spontaneous breakdown of DMPO/.OOH to form DMPO/.OH.

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.

Catalytic transition metals are an absolute requirement for the aerobic oxidation of ascorbate monoanion. Thus, for example, the concentration of iron can be determined by the metal-dependent rate of ascorbate oxidation in near-neutral solutions. The lower limit of detection of iron, as Fe(III)EDTA, by monitoring the decrease in absorbance at 265 nm of ascorbate is about 200 nM. However, by measuring the concentration of the ascorbyl radical by ESR spectroscopy the lower limit is about 10 nM. Using these assays, I have shown that the typical microliter laboratory syringe can introduce significant iron into solutions. Thus, for studies involving iron, these two tests can be used to determine the amount of contaminating iron in reagents as well as iron from other sources such as laboratory equipment.

Human synovial fluid, from a patient with synovitis disease, was examined by electron spin resonance spectroscopy for evidence of free radicals. The ascorbate free radical was observed and its intensity was affected by iron chelating agents, demonstrating that the iron in the synovial fluid is indeed available for oxidative catalysis.