Valeria Ceolin
Cristina Ghia
INTRODUCTION
More than eighty years since its discovery, the understanding of the functions of ascorbic acid has evolved from the prevention of scurvy to its potential use as a therapeutic drug for cancer treatment. Ascorbate maintains Fe2+ of collagen hydroxylases in an active state; therefore it plays a pivotal role in collagen synthesis; parallel reactions with a variety of dioxygenases affect the expression of a wide array of genes, for example via the HIF system, and possibly the epigenetic landscape of cells and tissues. The ability to donate one or two electrons makes ascorbate an excellent reducing agent and antioxidant. However, in the presence of catalytic metals, ascorbate also has pro-oxidant effects, where the redox-active metal is reduced by ascorbate and then in turn reacts with oxygen, producing superoxide that subsequently dismutes to produce H2O2.
THE DOUBLE FACED CHARACTER OF VITAMIN C
Ascorbic acid or Vitamin C is a potent dietary antioxidant with a double faced character, in that it exhibits a pro-oxidant activity arising from its routine antioxidant property that generates reactive free radicals. Vitamin C has antioxidant activity when it reduces oxidizing substances such as hydrogen peroxide, however, it can also reduce metal ions which leads to the generation of free radicals. The metal ion in this reaction can be reduced, oxidized, and then re-reduced, in a process called redox cycling that can generate reactive oxygen species.
[ Pro-oxydant - Wikipedia ]
So, although ascorbic acid has reported anti-oxidant properties, it plays a particular role in redox cycling metal ions and thus activates these ions to exacerbate oxidative stress.
Ascorbic acid has a number of known interactions with metal ions. These interactions involve redox reactions including the reduction reactions of Fe(III) to Fe(II), through the Fenton reaction.
THE FENTON REACTION
The oxidation of organic substrates by iron(II) and hydrogen peroxide is called the "Fenton chemistry" as it was first described by H.J.H. Fenton who first observed the oxidation of tartaric acid by H2O2 in the presence of ferrous iron ions. Alternatively, the name of “Fenton reaction” or “Fenton reagent” is often used, although Fenton never actually wrote it.
We know that the Fenton reagent defined as a mixture of hydrogen peroxide and ferrous iron is currently accepted as one of the most effective methods for the oxidation of organic pollutants.
More than 110 years after the Fenton reaction was discovered we know that this oxidation system is based on the formation of reactive oxidizing.
[ FENTON REACTION - CONTROVERSY CONCERNING THE CHEMISTRY. 2009 ]
Iron(II) is oxidized by hydrogen peroxide to iron(III), forming a hydroxyl radical and a hydroxide ion in the process. Iron(III) is then reduced back to iron(II) by another molecule of hydrogen peroxide, forming a peroxide radical and a proton. The net effect is a disproportionation of hydrogen peroxide to create two different oxygen-radical species, with water (H+ + OH–) as a byproduct.
- Fe2+ + H2O2 → Fe3+ + HO• + OH–
- Fe3+ + H2O2 → Fe2+ + HOO• + H+
Because of that the toxicity of iron, similarly as for other transition metals, may stem from Fenton reaction. It is commonly accepted that the oxidizing intermediates involved in Fenton reactions cause damage to biomolecules and play a major role in the aging process and a variety of diseases such as cancer . The Fenton reaction has been found to be the key reaction in the oxidation of membrane lipids, oxidation of amino acids and in the reactions where biological reduction agents are present, such as ascorbic acid. Its occurrence is also supposed in heart diseases, such as ischemia and reperfusion. Our knowledge of Fenton chemistry occurrence in biological systems is important also for its participation in pathological processes such as carcinogenesis, neurodegenerative diseases, atherosclerosis, etc. Finally, it could be said that the Fenton reaction has played an important role in biology for all of the time that life has existed on our planet, but its role in modern civilization diseases is new.
[ FENTON CHEMISTRY IN BIOLOGY AND MEDICINE. 2007 ]
ASCORBIC ACID AND THE FENTON REACTION
Ascorbic acid can recycle Fe(III) to Fe(II) facilitating further generation of reactive oxygen species by subsequent Fenton cycles.
- 2 Fe2+ + 2 H2O2 → 2 Fe3+ + 2 OH• + 2 OH−
- 2 Fe3+ + Ascorbate → 2 Fe2+ + Dehydroascorbate
It is speculated that in the presence of O2, complexation between Fe(II) and ascorbic acid results in the formation of an active oxygen species. The proposed mechanism shows the oxidation of ascorbic acid to dehydroascorbic acid, by electron transfer through Fe(II), and subsequent hydroxylation of an aromatic compound.
Iron and ascorbic acid form a potentially toxic cocktail. The chemical mechanisms given above have been established demonstrating the potential for these compounds to interact and oxidatively damage surrounding tissues.
VITAMIN C PRO-OXYDANT ACTIVITY DAMAGE
Ascorbic acid has been shown to exhibit both anti-oxidant and pro-oxidant effects in a dose related fashion.
So we can say that depending on concentrations, the effects of ascorbate can be pro- or antioxidant. There is considerable variability in the literature; this variability appears to be a result of the different concentrations and form of transition metal ions in the experiments.
The pro-oxidant effects of ascorbate may be important in vivo depending on the availability of catalytic metal ions. In healthy individuals, iron is largely sequestered by iron binding proteins such as transferrin and ferritin. This iron is essentially redox inactive. Instead, in pathological situations, such as thalassemia or hemochromatosis, non-transferrin-bound iron is present. Thus, supplemental ascorbate without administration of an iron chelator can lead to deleterious effects. Tissue damage resulting from ischemia/reperfusion is another example of increased availability of catalytic metal occurring in vivo. Intravenous ascorbate prior to vascular surgery increased concentrations of ascorbate radical and lipid hydroperoxides suggesting that catalytic iron released into the circulation during the ischemic phase of the surgery with ascorbate may promote iron-induced lipid peroxidation. Elevated levels of catalytic metal ions have also been demonstrated in chronic inflammatory diseases. There is an increased deposition of iron proteins in the synovial membranes in rheumatoid arthritis. Ascorbate radical has been detected in synovial fluid from patients with synovitis disease indicating that catalytic iron is in part responsible for the decreased levels of ascorbate and increased levels of DHA. In addition, ascorbate concentrations were decreased while levels of catalytic iron increased in patients with sepsis, compared to healthy subjects.
[ VITAMIN C CONTRIBUTES TO INFLAMMATION VIA RADICAL GENERATING MECHANISMS: A CAUTIONARY NOTE. 2003 - full text available on Biblioteca Biomedica Integrata Università A.S.O. S. Luigi ]
A study demonstrated that ferritin released by neuroblastoma cells enhanced pharmacologic ascorbate induced-cytotoxicity, indicating that ferritin with high iron-saturation could be a source of catalytic iron. Consistent with this, ascorbate has also been shown to be capable of releasing iron from cellular ferritin. Ferritin is only one candidate as a source of catalytic iron; extracellular iron chelates are present in tissue and seem to be increased under pathological conditions.
[ H(2)O(2)-MEDIATED CYTOTOXICITY OF PHARMACOLOGIC ASCORBATE CONCENTRATIONS TO NEUROBLASTOMA CELLS: POTENTIAL ROLE OF LACTATE AND FERRITIN. 2010 ]
Even in healthy subjects a positive or negative deviation from the optimal plasma ascorbic acid level results in oxidative damage.
A study published in Nature shows that vitamin C administered as a dietary supplement to healthy humans exhibits a pro-oxidant, as well as an antioxidant, effect in vivo.
Although the antioxidant nature in vivo of vitamin C has been questioned, it is nonetheless marketed as supplements in doses of 500 mg per day as an ‘antioxidant’. The discovery of an increase in a potentially mutagenic lesion following a typical vitamin C supplementation should therefore be of some concern, although at doses of less than 500 mg per day the antioxidant effect may predominate.
[ VITAMIN C EXHIBITS PRO-OXIDANT PROPERTIES. 1998 - full text available on Nature, vol. 392 ]
ASCORBIC ACID AND ITS PRO OXIDANT ACTIVITY AS A THERAPY
These many observations provide insights on the mechanism by which pharmacologic concentrations of ascorbate have potential in treating certain types of cancer. The inhibition effects of pharmacologic ascorbate on tumor growth have been confirmed in many laboratories.
It has been shown that ascorbate at pharmacologic concentrations was a pro-oxidant, generating hydrogen-peroxide-dependent cytotoxicity toward a variety of cancer cells in vitro without adversely affecting normal cells. In vitro, pharmacologic ascorbate concentrations mediated selective cancer cell toxicity via formation of Asc•− and H2O2 in cell culture media, with minimal Asc•− and no H2O2 detectable in blood . H2O2 in vitro were toxic to cancer cells.
Based on these data, many studies propose in vivo that pharmacologic ascorbate concentrations selectively generate Asc•− in extracellular fluid but not in blood. Pharmacokinetic data indicate that intravenous administration of ascorbate bypasses the tight control of the gut and renal excretion; thus, intravenous administration of ascorbate will produce highly elevated plasma levels; this ascorbate will autoxidize and the electron lost from ascorbate would reduce a protein-centered metal, resulting in a high flux of extracellular H2O2. This H2O2 will readily diffuse into cells challenging the intracellular peroxide-removal system, initiating oxidative cascades. These high fluxes of H2O2 appear to have little effect on normal cells but can be detrimental to certain tumor cells. In fact, in blood pharmacologic ascorbate concentrations would produce low Asc•− concentrations compared with extracellular fluid, whereas any H2O2 formed in blood would be immediately destroyed.
Knowledge and understanding of these mechanisms brings a rationale to the use of high-dose ascorbate to treat disease and thereby is reviving interest in the use of i.v. ascorbate in cancer treatment.
[ ASCORBATE IN PHARMACOLOGIC CONCENTRATIONS SELECTIVELY GENERATES ASCORBATE RADICAL AND HYDROGEN PEROXIDE IN EXTRACELLULAR FLUID IN VIVO. 2007 ]
The beneficial effects of ascorbate in cancer treatment reflect the ability of ascorbate to inhibit cancer cell proliferation. Ascorbate, acting as a pro-oxidant, inhibited cancer cell growth through other mechanisms, including induction of endoplasmic reticulum stress, suppression of insulin-like growth factor production, and inhibition of angiogenic factor production. Ascorbate can also inhibit the immune escape of cancer cells through suppression of IL-18 expression.
Thus, ascorbate may be a model antineoplastic agent, prolonging survival and improving the quality of life through selective inhibition of tumor growth.
[ THE PROSPECTS OF VITAMIN C IN CANCER THERAPY. 2009 ]
Real-time microdialysis sampling in mice bearing glioblastoma xenografts showed that a single pharmacologic dose of ascorbate produced sustained ascorbate radical and hydrogen peroxide formation selectively within interstitial fluids of tumors but not in blood.
Moreover, a regimen of daily pharmacologic ascorbate treatment significantly decreased growth rates of ovarian (P < 0.005), pancreatic (P < 0.05), and glioblastoma (P < 0.001) tumors established in mice. Similar pharmacologic concentrations were readily achieved in humans given ascorbate intravenously. These data suggest that ascorbate as a prodrug may have benefits in cancers with poor prognosis and limited therapeutic options.
[ PHARMACOLOGIC DOSES OF ASCORBATE ACT AS A PROOXIDANT AND DECREASE GROWTH OF AGGRESSIVE TUMOR XENOGRAFTS IN MICE. 2008 ]
A systematic review of the studies reported in literature that have studied the pro-oxidant activity of ascorbic acid as a therapeutic option for treatment of oral neoplasms and its effects on normal oral cells shows that the pro-oxidant activity of pharmacologic ascorbic acid is a part of its dose-dependent bimodal activity and is a result of the proposed Fenton mechanism. In vitro, animal and ex vivo studies of pharmacologic ascorbic acid (AA) have yielded meritorious results proving vitamin C as an effective cytotoxic agent against oral neoplastic cells with potentially no harming effects on normal cells. However, a shortage of clinical trials and in vivo human studies pertaining to evaluation of anti-tumour activity of vitamin C in tumours of oral cavity remains a lacuna in concluding ascorbic acid as a beneficial therapeutic option in treatment of oral neoplasms.
[ ASCORBIC ACID AND ITS PRO-OXIDANT ACTIVITY AS A THERAPY FOR TUMOURS OF ORAL CAVITY -- A SYSTEMATIC REVIEW. 2013 ]
Ascorbic acid just haven’t got a role in treating cancer: Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction. Vitamin C, driving the Fenton reaction, sterilizes cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis, the causative agent of tuberculosis. The bactericidal activity of vitamin C against M. tuberculosis is dependent on high ferrous ion levels and reactive oxygen species production, and causes a pleiotropic effect affecting several biological processes.
[ MYCOBACTERIUM TUBERCULOSIS IS EXTRAORDINARILY SENSITIVE TO KILLING BY A VITAMIN C-INDUCED FENTON REACTION. 2013 ]
CONCLUSION
The biological role of ascorbate is to act as a reducing agent, donating electrons to various enzymatic and a few non-enzymatic reactions.
Ascorbic acid behaves not only as an antioxidant but also as a pro-oxidant. However, usually in the body, free transition elements are unlikely to be present, while iron and copper are bound to diverse proteins and the use of a normal dose of vitamin C does not appear to increase pro-oxidant activity.
Instead, it has been studied the mechanistic basis for applying ascorbate as a pro-oxidant therapeutic agent, above all for cancer treatment.
