# Reactive Oxygen Species (ROS) and Oxidative Stress

Oxygen is essential to all aerobic forms ranging from prokaryotes, protists, plants and fungi to animals for indispensable metabolic processes, but this oxygen dependence imposes universal toxicity to all aerobic life processes. The basis of this paradox is that one-electron reduction of oxygen generates the superoxide anion free radical (O2-). In all biological systems, O2- undergoes further reduction to $H_2O_2$ via Fenton reaction to the hydroxyl radical, (*OH). These (O2-, $H_2O_2$, *OH) and some other forms of activated $O_2$ i.e., singlet oxygen (1O2) constitutes Reactive Oxygen Species (ROS). In addition to these endogenously generated pro-oxidants, aerobic organisms are subjected to oxidative injury from pro-oxidants, originating in dietary products, environmental pollutants, therapeutic drugs, and agrochemicals.
In the living organisms, these pro-oxidants are counteracted by enzymatic and non-enzymatic antioxidants. Whenever there is an imbalance in favor of pro-oxidants, it results in oxidative stress and the oxidative damage. A pro-oxidant state may be either due to over-production of reactive substances or due to deficiency in the antioxidant defense system. The nature of pathological processes mediated by this stress includes leakage of cell membranes, dysfunction of mitochondria, depletion of glutathione, disturbed redox states of cells, and depletion of ATP. These processes, which affect cells and DNA, lead to cell apoptosis, aging, tumor promotion and cancer, inflammatory diseases, post-ischemic injury and numerous serious ailments.

# Biological sources of free radicals

Several reactions in biological systems contribute to maintain the steady state concentration of $H_2O_2$ and O2-. Mitochondrial respiration consumes 85-90% of the total oxygen used by cells and is a potential source of ROS within the cells. The electron-transfer chain of mitochondria is responsible for the reduction of $O_2$ to O2- . In addition to the mitochondrial electron transfer chain; other cellular sources of O2- and/or $H_2O_2$ in mammalian cells include the microsomal transfer chain and the NADP (H) oxidases in phagocytes. Xanthine oxidase, aldehyde oxidase and dioxygenase are additional sources of O2-. Two-electron reduction of $O_2$ to $H_2O_2$ occurs during the course of reactions catalyzed by a number of oxidases, such as monoamine oxidase, urate oxidase, acyl-CoA oxidase, and L-gulonolactone oxidase.

# Generation of ROS by electron transfer reactions

Oxygen Free Radical is a molecule or an atom with an unpaired electron in its outer orbit. They readily extract electrons from other molecules with which they collide, and this molecule in turn becomes a free radical capable of reacting. Thus a chain reaction is propagated. The generation of biologically important major oxygen free radicals is described below:

• Superoxide radical, the first member of the reactive oxygen species family, is generated when an electron is added to molecular oxygen. It is formed non-enzymatically in all tissues of vertebrates including that of testis as a consequence of autooxidation of constituents of the mitochondrial electron transport chain. In addition, superoxide anions are constantly generated enzymatically by a number of oxidases and oxygenases present in the cells.
                      $O_2$ + 2e- ----------------------------------> O2- (Superoxide radical)


Superoxide radical itself is not a very powerful oxidant; however, it can easily donate its electron to any nearby atom of Fe3+ and reduce it to Fe2+.

                     O2- + Fe3+ --------------------------------> Fe2+

• Hydrogen peroxide is generated by the spontaneous or SOD-catalyzed disproportionation of O2-. It is also produced, if oxygen accepts two electrons.
                    $O_2$ + 2e- + 2H+ --------------------------> $H_2O_2$ (hydrogen peroxide)
O2-  + O2- + 2 H+------------------------ > $H_2O_2$ + $O_2$


$H_2O_2$ is not a radical, but toxic oxygen intermediary which chelates with metal to form hydroxyl radical (HO*), more potent oxidant.

• Hydroxyl radical is the most reactive of biological free radicals and the most potent biological oxidizing agent known because of its extremely short half-life. There are two common and important biological sources of hydroxyl radical formation. First, Fenton Reaction- where hydroxyl radicals are produced by the metal-dependent decomposition of $H_2O_2$. In the presence of Fe2+, hydrogen peroxide produces hydroxyl radical (*OH)
                    $H_2O_2$ + Fe2+ ----------------------------> *OH + OH- + Fe3+


and, second, Haber-Weiss reaction- by the interaction of superoxide radical with hydrogen peroxide.

                    O2- + $H_2O_2$ -----------------------------> *OH + OH- + e


This reaction is called as Haber-Weiss reaction or the superoxide driven Fenton reaction.

• Singlet oxygen (1O2) is produced by energy transfer from photochemically excited donor molecules, called sensitizers, to ground-state molecular oxygen. Singlet oxygen has no unpaired electrons and can react both as a nucleophile and as an electrophile.

# Antioxidant defense system

To counteract the damage caused by the ROS, the living system is well equipped with antioxidant defense mechanism that can scavenge and convert ROS or free radicals into harmless species. The antioxidant defense systems responsible for cellular protection against oxidative stress are as diversified as free radicals themselves. To provide maximum protection, cells contain a variety of substances capable of scavenging many different species of free radicals. These scavengers are strategically compartmentalized in sub cellular organelles within the cell to provide maximum protection.
The cellular antioxidant defenses can be categorized into primary and secondary defense systems to designate the scavenging action of antioxidants. The primary defenses interact with free radicals generated directly from $O_2$ and secondary defenses scavenge radicals arising from dismutation of superoxide radical. Primary defenses consist of broadly studied antioxidant compounds, such as α-tocopherol, ascorbic acid, β-carotene, and uric acid, along with a variety of antioxidant enzymes. Secondary defenses include proteolytic and lipolytic enzymes, as well as the DNA-repair systems. The antioxidant defense system consists of enzymatic and non-enzymatic components. The enzymatic antioxidant defense system that assists in reducing the damage caused by ROS includes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-s-transferase. The non-enzymatic components are Glutathione, Vitamin-E (α-tocopherol), Vitamin-C (ascorbic acid), Uric acid, Ubiquinone, Carotenoids etc. The entire system works together to scavenge and eliminate the free radicals and ROS out of the body. Any imbalance in ROS/free radicals and antioxidants can results in oxidative injury and possible cell death.