Oxidative stress is characterized by an imbalance between the production of reactive species and the protective capacity of antioxidants. It represents a disruption in the pro-oxidant-antioxidant equilibrium, favoring the former and resulting in significant damage. This phenomenon has garnered significant attention from researchers worldwide due to its detrimental impact on the human body, being both essential for life and implicated in cellular demise. In various organisms, including humans, reactive oxygen species (ROS) and free radicals are generated during metabolic and immune system processes. Molecular oxygen (O2) possesses the ability to disassociate, leading to the formation of unstable and highly reactive free radicals, subsequently resulting in the generation of ROS. When the concentration of reactive oxygen species (ROS) exceeds a certain threshold, it can have beneficial effects on various biological functions, such as phagocytosis, apoptosis, necrosis, and pathogen protection. In oxidation reactions, certain enzymes like peroxidases utilize hydrogen peroxide (H2O2) as a substrate to facilitate the synthesis of complex organic molecules in organisms.

The human body possesses a defensive mechanism to counteract the effects of ROS, primarily relying on antioxidants and endogenous antioxidants such as Catalase, superoxide dismutase (SOD), thioredoxin, glutaredoxin, and glutathione. However, when ROS concentration surpasses a critical level, it can lead to damage to DNA, proteins, lipids, and carbohydrates, resulting in oxidative stress. Extensive research has linked oxidative stress to the development or exacerbation of various human diseases, including ulcerative colitis, nonulcer dyspepsia, Parkinson's disease, Alzheimer's disease, atherosclerosis, major depression, alcohol-induced liver disease, cancer, diabetic nephropathy, end-stage renal disease, cardiovascular disease, mild cognitive impairment, aging, and neural disorders. The human body maintains equilibrium among DNA, proteins, carbohydrates, and lipids. When ROS damage these essential biomolecules, it disrupts the metabolic state and growth and development of cells, leading to serious diseases collectively referred to as oxidative stress. For instance, ROS generation causes damage to nitrogenous bases and strand breaks in DNA, with various radicals like superoxide radical (O2), hydroxyl radical (.OH), and hydrogen peroxide (H2O2) being implicated in such damage. Among the radicals generated in our body, hydrogen peroxide is particularly noteworthy due to its ability to readily permeate membranes, longer longevity (approximately 1 minute), and lack of compartmentalization within the cell. Hydrogen peroxide, produced during oxidative stress, stands out as one of the most reactive ROS, leading to damage in proteins, nucleic acids, carbohydrates, and lipids, ultimately culminating in oxidative stress.

Free Radicals and Reactive Oxygen Species

Free radicals and reactive oxygen species (ROS) can be described as atoms or molecules with independent existence that possess one or more unpaired electrons in their outer valence shell. Reactive oxygen species specifically refer to free radicals that contain oxygen atoms and are commonly present in biological systems.

ROS can be categorized into two main types: radicals, which include Superoxide (O2.-), Hydroxyl (.OH), Peroxyl (RO2.), Alkoxyl (RO.), and Hydroperoxyl (HO2.), and non-radicals, which encompass Hydrogen peroxide (H2O2), Hypochlorous acid (HOCl-), Ozone (O3), Singlet oxygen (1O2), and Peroxynitrite (ONOO-).

There are various types of reactive oxygen species (ROS) that can be distinguished based on their chemical structure and reactivity. Some of the main types of ROS include:

Superoxide (O2.-):

Superoxide, although not highly reactive itself, exhibits the property of a reducing agent by facilitating the conversion of ferric (Fe+++) iron to its ferrous (Fe++) form. Due to its inability to penetrate lipid membranes, superoxide remains localized to the sites of its production. Notably, superoxide is spontaneously generated, particularly in the electron-rich aerobic environment of the inner mitochondrial membrane during respiratory chain activity. The endogenous formation of superoxide and hydrogen peroxide is facilitated by flavoenzymes, with Xanthine oxidase being a notable example, commonly activated during ischemia-reperfusion processes.

Cu⁺² / Fe⁺³ + O₂ →Cu+ / Fe+2 + O₂

Hydroxyl (.OH):

The hydroxyl radical exhibits higher reactivity compared to other ROS, making it particularly damaging to essential biomolecules such as DNA, proteins, carbohydrates, and lipids. The hydroxyl radical is formed through the Fenton reaction, wherein hydrogen peroxide (H2O2) reacts with proteins and other biomolecules containing transition metals (Fe+2 or Cu+). This reaction can lead to severe oxidative damage within the cellular environment.

H₂O₂ + Cu+ /Fe+2 →OH- + .OH + Cu⁺²/Fe⁺³ 

Hydrogen peroxide (H₂O₂):

Hydrogen peroxide (H₂O₂) is a pale-blue colored, covalent liquid that readily dissolves in water. It exhibits mild oxidizing and reducing properties, and while it can react with proteins and other molecules containing transition metals, it does not readily oxidize most biomolecules. In the human body, H₂O₂ serves as an essential defense mechanism against pathogens, playing a crucial role in activating and regulating the immune system. Neutrophils, a type of leukocyte, produce hydrogen peroxide as a primary line of defense against toxins, parasites, bacteria, viruses, and yeast, thus contributing to the body's ability to combat various threats effectively.

Hypochlorite (HOCl):

Upon reacting with chlorine, hydrogen peroxide (H₂O₂) gives rise to one of the most reactive oxygen species (ROS), known as hypochlorite.

 H + + Cl- + H₂O_{2 →}HOCl + H₂O

Exogenous sources of reactive oxygen species (ROS) encompass a variety of production mechanisms, including:

1. Radiation: Ultraviolet (UV) light, x-rays, and gamma rays
2. Chemicals that form peroxides: ozone and singlet oxygen.
3. Chemicals that promote superoxide formation: Quinones, nitro aromatics, and bi pyrimidiulium herbicides.
4. Chemicals that are metabolized to radicals: Poly halogenated alkanes, phenols, and aminophenols.
5. Chemicals that release iron: Ferritin and other transition metals.

The generation of ROS from these exogenous sources occurs primarily through Fenton's and Haber's reactions.

Fenton's reaction involves the reduction of molecular oxygen to produce superoxide, which can further generate more highly reactive ROS. Superoxide dismutates to form hydrogen peroxide: O₂- + O₂ + 2H → H₂O₂+ O₂

Hydrogen peroxide can then react with transition metals such as iron (Fe++) or copper (Cu+) to form highly reactive hydroxyl radicals: Fe²⁺ + H₂O₂+ → Fe³⁺ + OH + OH

The Haber-Weiss reaction involves the reaction of hydrogen peroxide with oxygen to produce superoxide and hydroxide radicals: O₂ + H₂O₂ → O₂ + OH- + .OH

Halogen atoms like Cl-, Br-, and I- can also react with hydrogen peroxide and be utilized by Myeloperoxidase to form more reactive hypochlorous acid or hypochlorite: H₂O₂ + Cl- → HOCl + OH

Endogenous sources of ROS within the human body involve various enzymes such as monoamine oxidase, lipoxygenase, cyclooxygenase, NADPH oxidase, cytochrome P450 monooxygenase, xanthine oxidoreductase, and nitric oxide synthase. These enzymes play crucial roles in generating ROS at the subcellular level.

NADPH Oxidase / Respiratory Burst Oxidase: The stimulation of reactive oxygen species (ROS) production in phagocytic cells was originally termed "the respiratory burst" due to the heightened oxygen consumption observed in these cells. This process is facilitated by NADPH oxidase, a multi-component, membrane-bound enzyme complex, and is essential for the bactericidal activity of phagocytes. While various enzymes are capable of producing ROS, NADPH oxidase holds particular significance. Its activity is regulated through a complex system involving the G-protein Rac.

Xanthine Oxidoreductase: This enzyme catalyzes the conversion of hypoxanthine into xanthine and further into uric acid. Xanthine Oxidoreductase (XOR) exists in two forms, Xanthine Dehydrogenase (XD) and Xanthine Oxidase (XO). XD can be converted into XO irreversibly through proteolysis and reversibly through the oxidation of sulfhydryl groups. XOR generates significant amounts of H₂O₂ and O₂- and is also involved in the transformation of nitrates into nitrites and nitric oxide (NO). Additionally, it catalyzes the reaction between NO and O₂- to form the highly reactive peroxynitrite.

Cytochrome P450 Oxidase: This haem-containing enzyme is present in mitochondria and participates in the metabolism of various compounds, such as cholesterol, hormones, steroids, bile acids, arachidonic acid, eicosanoids, vitamin D3, and retinoic acid, by facilitating intramolecular oxygen transfer. The enzyme transfers one electron bound to oxygen while the second electron is reduced to water.

Myeloperoxidase: Myeloperoxidase, a haem-containing enzyme found in neutrophils and eosinophils, catalyzes the reaction between H₂O₂ and various substrates to produce highly reactive hypochlorous acids. At low concentrations, ROS, including hypochlorous acids, play beneficial roles in processes such as phagocytosis, apoptosis, detoxification reactions, and elimination of precancerous cells and infections. ROS are also involved in signaling pathways that help maintain cellular homeostasis and regulate various metabolic and cellular processes, such as proliferation, immunity, gene expression, migration, and wound healing.

ROS Generation:

• Mitochondrial Production of ROS: ROS are generated within mitochondria through the release of electrons from the electron transport chain, leading to the reduction of oxygen molecules into superoxide (O₂-). Superoxide is subsequently converted into hydrogen peroxide with the assistance of superoxide dismutase (SOD). Hydrogen peroxide can react with biomolecules containing transition metals (Fe++, Cu+) and produce hydroxyl radicals through the Fenton's reaction.
• Endoplasmic Reticulum: Cytochrome P450 complexes in the endoplasmic reticulum are involved in detoxifying hydrophobic chemical compounds in the body, leading to the formation of superoxide anions. The enzyme Cytochrome P450 reductase facilitates the conversion of these compounds into hydrophilic forms.
• Peroxisomes: Peroxisomes contain enzymes like glycolate oxidase, urate oxidase, fattyacyl CoA oxidase, d-amino acid oxidase, and 1-α-hydroxyacid oxidase, which generate hydrogen peroxide. The enzyme catalase, found in peroxisomes, is involved in various peroxidative reactions and converts hydrogen peroxide into water and oxygen.
• ROS Generation by Lysosomes: This process leads to the reduction of oxygen and the formation of highly reactive hydroxyl radicals (OH-).
• Other Sources: Small molecules like epinephrine, dopamine, flavins, and hydroquinones can directly produce O₂- through autooxidation.

Viral Infections and ROS: Many viral infections are associated with ROS generation, particularly when intracellular and extracellular antioxidant levels decrease. ROS and reactive nitrogen intermediates possess antimicrobial and antitumor activities. For instance, viral infections like Sendai and influenza viruses induce respiratory bursts in phagocytic cells, elevating ROS/RNS levels. HIV increases oxidative stress by stimulating transcription factor NF-ĸB, cytokines, and TNF-α, leading to the release of H₂O₂ from T-cells. Additionally, hepatitis viruses directly affect the host genome, resulting in ROS production and increased cell proliferation, which may ultimately lead to cancer development.

Antioxidants

The term "antioxidant" is widely used but challenging to precisely define. In the context of food science, antioxidants are substances that inhibit lipid peroxidation, while in polymer science, they are employed to regulate polymerization in the manufacture of rubber, plastic, and paint. Essentially, antioxidants are substances present at low concentrations compared to oxidizable substrates (found in various molecules in vivo) that significantly delay or prevent the oxidation of those substrates.

Antioxidants can be classified into three categories:

1. Primary antioxidants: Involved in preventing the formation of oxidants.
2. Secondary antioxidants: Function as scavengers of reactive oxygen species (ROS).
3. Tertiary antioxidants: Engage in repairing oxidized molecules through dietary or consecutive antioxidants.

Antioxidants may be enzymatic or non-enzymatic in nature. Enzymatic systems directly or indirectly aid in defending against ROS. Examples include Superoxide dismutase (SODs), which remove superoxide by accelerating its conversion into hydrogen peroxide. SOD enzymes contain manganese (MnSOD) in mitochondria and copper and zinc (CuZnSOD) in the cytosol at their active sites. Other enzymes like Catalase convert hydrogen peroxide into water and oxygen, while glutathione peroxidase plays a crucial role in removing H2O2 from human cells, requiring selenium for its action. Glutathione reductase is a flavoprotein enzyme that regenerates reduced glutathione from oxidized glutathione, and thioredoxin also contributes to antioxidant defense.

Non-enzymatic antioxidants, on the other hand, function as scavengers of ROS and RNS. For example, Vitamin E inhibits lipid peroxidation by scavenging peroxyl radical intermediates. Vitamin C and Vitamin A, glutathione, uric acid, and melatonin react with ROS to form disulfide compounds, thus acting as effective non-enzymatic antioxidants.

Cell damage resulting from free radical-mediated reactions can be mitigated by enzymatic and non-enzymatic defense mechanisms. The antioxidant system comprises both endogenous antioxidants, produced within the body, and exogenous antioxidants obtained from dietary sources.

Endogenous antioxidants can be classified into primary and secondary antioxidants. Primary antioxidant enzymes, including Superoxide Dismutase (SOD), Catalase, and Glutathione Peroxidase, play a vital role in inactivating reactive oxygen species (ROS) into intermediates. In addition to these antioxidant enzymes, primary antioxidants also encompass water-soluble compounds such as Ascorbate, Glutathione, and Uric Acid, as well as lipid-soluble compounds like Tocopherols, Ubiquinols, and Carotenoids.

Secondary antioxidant enzymes, such as Glutathione Reductase, Glutathione-S-Transferase, Glucose-6-Phosphate Dehydrogenase, and Ubiquinone, assist in detoxifying ROS by reducing peroxide levels and continuously providing NADPH and Glutathione to maintain the proper functioning of primary antioxidant enzymes. Copper, iron, manganese, zinc, and selenium further enhance the activities of antioxidant enzymes.

Exogenous antioxidants are primarily derived from dietary sources, including various herbs, spices, vitamins, and vegetables, among others, that exhibit antioxidant activities.

Oxidative stress refers to a disturbance in the prooxidant and antioxidant balance, favoring prooxidants and leading to serious damage to biomolecules. This term describes the imbalance between the production of reactive species and the protective capacity of antioxidants. Oxidative stress has garnered significant attention from researchers worldwide due to its detrimental effects on important biomolecules such as DNA, Proteins, Lipids, Carbohydrates, and others within the human body.

Oxidative Stress and Its Impact on Disease

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and the body's antioxidant defense, resulting in numerous diseases in humans. Free radicals and other reactive species have been implicated in the pathology of over 100 human diseases, including atherosclerosis, cancer, AIDS, nonulcer dyspepsia, Parkinson's disease, Alzheimer's disease, major depression, diabetic nephropathy, end-stage renal disease, and cardiovascular disease, among others.

Various forms of stress, such as oxidative stress, heat stress, and denaturing stresses, disrupt the structure of proteins, carbohydrates, lipids, and DNA molecules. ROS, as a consequence of oxidative stress, play a key role in the development of human diseases, encompassing neurodegenerative diseases, immune disorders, arteriosclerosis, rheumatoid arthritis, diabetes, and cancer. Reactive oxygen species produced in the human body include superoxide, hydrogen peroxide, and hydroxyl radicals, with the hydroxyl radical being highly reactive and particularly prone to causing damage to biomolecules.

The process of oxidative stress involves hydrogen peroxide reacting with transition metals like iron and copper to generate highly reactive hydroxyl radicals, which can initiate lipid peroxidation in cell membranes and oxidize other macromolecules. Hemoglobin (Hb) and other heme proteins, with their higher oxidation state of iron (Fe+4), are also susceptible to ROS-induced damage. Hemoglobin is a fundamental molecule in living organisms, acting as a cofactor for various proteins and enzymes involved in essential cellular processes, including gas transport, redox reactions, and electron transport.

ROS, being highly reactive, can damage various biomolecules such as proteins, carbohydrates, lipids, and DNA. The substances that possess the capacity to scavenge ROS and protect biomolecules from injury are known as antioxidants. Extensive research has demonstrated that several enzymes, including SOD and catalase, vitamins such as A, C, and E, and amino acids like cysteine and methionine, exhibit antioxidant properties and play a crucial role in mitigating the detrimental effects of oxidative stress on human health.