Reactive oxygen species (ROS) are by-products of cellular oxidative metabolism, much of which occurs in the mitochondria of cells. Biologically relevant ROS include hydrogen peroxide (H2O2), superoxide, hydroxyl radicals, and singlet oxygen. In addition to cellular metabolism, there are several other biological reactions that can generate ROS in vivo. Transition metals such as copper and iron are essential dietary minerals that play important roles in enzyme activity and oxygen transport. However, they can also participate in one-electron oxidation-reduction reactions, leading to formation of ROS. They are therefore usually sequestered by protein ligands in vivo to limit their redox activity.
Reactive oxygen species play a dual role, being both beneficial and harmful.1–3
They play beneficial physiological roles in cellular signaling systems and induction of mitogenic responses.2,3
However, overproduction of ROS can induce oxidative stress which is associated with many age-related degenerative diseases.2,4–6
These degenerative diseases have major public health significance and include cardiovascular disease,7
The mechanisms underlying the involvement of ROS and oxidative stress in disease development may include oxidative modification of proteins,4
oxidation of lipids,13–15
DNA strand breaks and modification to nucleic acids,16
modulation of gene expression through activation of redox-sensitive transcription factors,17,18
and modulation of inflammatory responses through signal transduction.8
Enzymatic defenses have evolved to protect against these harmful biological oxidants. Superoxide dismutases, peroxidases, and catalases are some of the prominent and extensively studied antioxidant enzymes. Antioxidants also play an important role in preventing/limiting the damage caused by ROS.
The hydroxyl radical possesses the highest one-electron reduction potential of all the physiologically relevant ROS, and is extremely reactive with almost every type of biomolecule.19,20
The presence and pathological role of hydroxyl radicals in vivo
has been demonstrated. Targets for hydroxyl radicals include proteins and nucleic acids.16,21
Because of their reactivity and ability to damage biological targets, hydroxyl radicals can serve as a representative ROS for investigating dietary antioxidants for their potential to react directly with and to quench free radicals, as well as to protect important biomolecules from radical-mediated damage. Growing evidence suggests that dietary antioxidants may play an important role in limiting oxidative damage and reducing the risk of numerous chronic diseases related to advancing age.22,23
There is as yet no known enzymatic reaction that can detoxify the hydroxyl radical in vivo
. The only known defense against hydroxyl radicals is from antioxidants.
Many antioxidants are reducing agents; they participate in redox reactions by donating electrons or hydrogen atoms. There are several biologically relevant antioxidants that act to restore oxidative balance in the cellular environment. For example, vitamin C (ascorbate, AscH−
), can neutralize free radicals by donating a hydrogen atom, forming the ascorbyl radical, which readily reacts with NADH or NADPH-dependent reductases to regenerate ascorbate.24
Similar to ascorbate, reduced glutathione (GSH) can reduce free radicals by hydrogen atom donation.25
Similarly, because of the ability of thiols to undergo redox reactions, cysteine exhibits antioxidant properties by hydrogen atom donation. However, by virtue of their reducing ability, these beneficial antioxidant compounds can also activate transition metal ions (e.g. Fe3+
), making them behave as pro-oxidants. This may proceed in a cyclical manner (redox cycling), leading to continuous stream of ROS that can cause damage to DNA and other biomolecules.
Recent research has focused mostly on discovering antioxidant compounds for use in foods, cosmetics, and other products. There is less literature on the possible crossover effect from antioxidant to pro-oxidant activity that may be attributed to many biologically relevant antioxidants. A review of ingredients in marketed dietary supplements reveals a growing trend where antioxidants and redox active metals (e.g. chromium, cobalt, copper and iron) are present in the same formulation. These metals may participate in the formation of free radicals by a Fenton-like reaction mechanism (Mx+
, where M is a transition metal).20,26
The presence of antioxidant reducing agents together with redox active metals may lead to pro-oxidant activity. Most evidence for the pro-oxidant effect of antioxidants in the presence of redox active metals comes from in vitro
studies. However, there is some evidence that this effect may occur in vivo
. It has been shown that hydroxyl radicals can be detected in the bile of rats following intragastric administration of copper sulfate and ascorbic acid.27,28
Slivka and Kang have provided evidence that hydroxyl radicals are generated in the gastrointestinal tract following oral administration of ferrous sulfate and ascorbic acid to rats.29
Naito et al.30
have shown that injection of ferrous sulfate and ascorbic acid into the gastric wall of rats results in gastric ulcers. While it is generally accepted that antioxidants can act as pro-oxidants under certain conditions, there is no clear delineation of what these conditions are, how they differ among antioxidants, and how the adverse pro-oxidative effect can be avoided.
In the current study, we have focused on copper as a redox active metal which is present in many dietary supplements. Copper is an essential trace element having a recommended dietary allowance for adults of 900 μg/day.31
, most copper is bound securely to ceruloplasmin, which renders it inactive in Fenton-like reactions. However, about 5 to 15% is loosely bound to plasma albumin and other small molecules.32
This copper has been termed free copper and there is concern that free copper, in the presence of biological reducing agents, may increase the formation of free radicals. It has been previously shown that inorganic copper (e.g., copper present in drinking water and dietary supplements) is processed differently than organically complexed copper (i.e., copper present in food).32
Inorganic copper when ingested in large part bypasses the liver, ending up in the blood stream and contributing to the free copper pool.32
Dietary supplements contain primarily inorganic copper and, therefore, may increase free copper levels in the body, increasing the risk of free radical formation. The role of copper as a catalyst for free radical generation is well established. In fact, copper (Cu2+
) has been found to be a much more redox active metal than iron (Fe3+
) in many in vitro
Free radical generation involving copper is thought to be associated with development of some types of cancer and the acceleration of aging and age associated degenerative diseases.32,34–41
L-Ascorbic acid (Vit. C), L-cysteine, L-glutathione (GSH, reduced), and (−)-epigallocatechin gallate (EGCG) are biological antioxidants commonly present in dietary supplements. Many such dietary supplements also contain transition metals like iron and copper. This combination raises the question of the potential generation of free radicals in a Fenton-type reaction. In this study, we examined free radicals generated via a copper-based Fenton-type reaction. We determined if, and under what conditions, the selected antioxidants quench and/or promote radical formation. Conditions simulating the physiological pH of the stomach (pH =1.2) and of cells and tissues (pH = 7.4) were investigated. We also investigated whether albumin-bound copper can be redox activated by the presence of these antioxidants.