Introductory Chapter: Reactive Oxygen Species – Origin and Significance

1. Introduction

In 1900, while serving as a faculty of chemistry at Michigan State University, Professor Moses Gomberg made a significant discovery that revolutionized the field of chemistry: the free radical. This concept was later recognized as a National Historic Chemical Landmark by the American Chemical Society in 2000, acknowledging the importance of his work. In 1954, Gershman proposed the “free radical theory of oxygen toxicity.” This theory suggests that oxygen is toxic because it has the ability to form free radicals. Another chemist, Fenton, discovered that the reaction between hydrogen peroxide and ferrous sulfate produces a violet color, which is the oxidation of tartaric acid upon adding alkali. This reaction is now known as the Fenton reaction and serves as the basis for producing the hydroxyl radical. These discoveries have paved the way for further exploration into the field of free radical science and have contributed greatly to our understanding of chemical reactions and their implications [1, 2, 3, 4].

Free radicals are molecules that contain one or more unpaired electrons and are capable of independent existence. They are highly reactive and play a crucial role in various metabolic processes such as oxidative reactions in mitochondria and oxidative bursts of phagocytes. However, in excess, free radicals can cause diseases such as autoimmune, cardiovascular, neurodegenerative conditions, and cancer. To minimize these pathological conditions, it is necessary to reduce the number of free radicals. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two types of free radicals that are formed in the body and consist of both radical and nonradical moieties [1].

It is important to note that the term “ROS” refers to a group of small molecules of oxidizing, nitrosating, nitrating, halogenating, and thiol-reactive species produced in biological systems. This group does not represent a single species, which makes it a bit nonspecific and ambiguous. In fact, some experts have criticized the use of ROS as an umbrella term for oxidants because of its lack of specificity. In 2014, Holmstrom and Finkel clarified the dual nature of reactive oxygen species (ROS) and their role in both cell damage and protection, as well as their involvement in various diseases. “Nonetheless, from a biological point of view, it is beginning to look as if ROS are neither cellular heroes nor villains—but instead something that occupies that always entertaining, captivating, and fertile middle ground” [5].

2. Sources of ROS

ROS is primarily expanded to include reactive oxygen-containing compounds or nonradical oxidizing agents such as singlet oxygen (¹O₂), ozone (O₃), hydrogen peroxide (H₂O₂), and hypochlorous acid (HOCl). ROS can originate from various sources such as the photolysis of gaseous ozone, materials-mediated catalytic reactions, and endogenous activities in biological systems [6]. In recent years, researchers have magnified their focus on studying ROS in living organisms because ROS plays an indisputable role in regulating various physiological functions. ROS sources in living organisms can be classified into exogenous and endogenous sources. Exogenous sources include exposure to engineered nanoparticles (NPs), radiation, chemotherapeutics, and microbial infection, whereas endogenous ROS can be produced from cellular respiration and normal metabolism [6, 7, 8, 9]. High-concentration ROS are extremely toxic to living organisms, but in normal physiological processes, ROS act as messengers in various cellular functions and can be identified as signal molecules or regulators in living systems [10]. It is believed that the effect of ROS on physiological processes is attributed to their capability to alter the activity of specific proteins [11].

ROS have been studied extensively in recent decades for their roles in normal physiological processes. These roles include blood vessel modulation, immune function, oxygen sensing, gene activation, and cellular growth [12, 13]. However, ROS are also implicated in the initiation and development of various pathological processes such as aging, cancer, insulin resistance, diabetes mellitus, cardiovascular diseases, Alzheimer’s disease, and more [7, 14]. Excessive ROS generation can lead to tissue dysfunction or cell death, while a stable concentration of ROS can effectively regulate physiological processes. Therefore, maintaining redox homeostasis is crucial to keeping normal physiological functions and reducing the incidence of diseases [7]. Antioxidants like vitamin E (tocopherols and tocotrienols), SOD, CAT, GPx, vitamin C (ascorbic acid), beta-carotene (β-carotene), and coenzyme Q10 (CoQ10) play a crucial role in neutralizing and eliminating ROS. They are an essential part of the body’s defense system against these toxic species. Polyphenolic compounds, another type of antioxidant, can also reduce the levels of harmful ROS by scavenging them, which helps prevent oxidative damage to macromolecules [4, 15].

3. Oxidative stress and diseases

Oxidative stress (OS) is the presence of reactive oxygen species in excess of the antioxidant buffering capacity. It can be described as an imbalance in ROS generation and antioxidant defense leading to the accumulation of ROS. It is an alteration in the balance of the prooxidant/antioxidant system in favor of the prooxidant with attendant lethal effects and resultant damage to the cellular macromolecules [16]. A wide range of methods have been developed and employed to measure the nature and extent of oxidative stress ranging from oxidation of lipids to free amino acids and proteins and DNA. Although diverse oxidative stress biomarkers are available as predictors of various diseases, the specificity of each seems to be yet established [17].

Autophagy is a remarkable catabolic process that efficiently delivers cytoplasmic macromolecules and organelles to the lysosomes for degradation [18]. Oxidative stress plays a crucial role in regulating the process of autophagic flux by influencing the transcription of autophagic genes, the activity of proteins, and the degradation of organelles. During periods of oxidative stress, ROS triggers macroautophagy (commonly known as autophagy), and the selective degradation of oxidized proteins occurs through CMA (chaperone-mediated autophagy) to ensure cellular viability. The susceptibility of CMA substrates to degradation increases because of oxidative modification, and LAMP2a (lysosome-associated membrane glycoprotein 2 alpha) upregulation enhances CMA in cells challenged with ROS. However, the effect of ROS on microautophagy remains unclear, and further research is needed to understand the roles of macroautophagy, microautophagy, and CMA in response to oxidative stress [18, 19].

The oversecretion of ROS in the brain leads to oxidative stress that if not suppressed or inhibited could lead to oxidative damage of essential components of the central nervous system. This can also initiate or enhance some reactions that may have detrimental effects on the physiological functions and health of the brain [20]. These reactions such as neuroinflammation and progressive neuronal cell loss via apoptosis if not abated can exacerbate protein misfolding and formation of protein aggregates resulting in neurodegeneration and associated neurobehavioral incompetence. Considering the pivotal roles of oxidative stress, neuroinflammation, protein misfolding, and apoptosis in neurodegenerative diseases, the manipulation of these major players in each of the pathological mechanisms may represent a promising treatment option to slow down neurodegeneration and alleviate associated symptoms [20].

Autoimmune diseases, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) are notable examples in which free radicals cause damage to cells and tissues. Arthritic inflammation is a medical condition where ROS acts as a potent inflammatory mediator, contributing significantly to the destruction of collagen tissues. The overproduction of ROS not only causes damage leading to the breakdown of cartilage but also triggers the apoptosis of the vital chondrocytes responsible for the structure and function of the cartilage. The reduced number of chondrocytes, in turn, results in an inability of the cartilage to self-repair, promoting the breakdown of the extracellular matrix in joints. Moreover, the H₂O₂ molecule causes chondrocyte lipid peroxidation, which significantly affects protein oxidation and degradation of the cartilage matrix. Scientific studies have established the crucial role of oxidative stress in joint pathology, which includes inflammatory infiltration, synovial proliferation, and angiogenesis These findings could pave the way for more targeted and effective treatments for these diseases in the future [21, 22, 23, 24].

Diabetes mellitus is a chronic condition that results in high levels of blood glucose known as hyperglycemia. This condition can be caused by a defect in insulin secretion (in Type I diabetes), resistance to insulin action (in Type II diabetes), or both [24]. Common symptoms of diabetes include thirst, hunger, weight loss, and weakness, which can eventually lead to coma. Diabetes is often linked to an increase in free radicals or a decrease in antioxidant systems, which leads to the development of oxidative stress [25]. DM is caused by both mitochondrial and nonmitochondrial-derived reactive oxygen species. Under normal conditions, the electron transport chain complexes I and III are the primary sites of superoxide production. However, in DM, increased glucose levels lead to an increase in glycolysis, resulting in elevated production of pyruvate. This causes an increase in the inner mitochondrial membrane potential, leading to mitochondrial dysfunction and an increase in ROS production at the electron transport chain complex II. By understanding these mechanisms, researchers can work toward developing effective strategies to mitigate the impact of oxidative stress on patients with DM [26, 27].

It is worth noting that DNA mutation, often caused by oxidative damage, plays an important role in the development of cancer. Cancer cells in particular, in comparison to normal cells, have higher levels of ROS and are more susceptible to mitochondrial dysfunction because of their higher metabolic rate [28]. Cancer cells display elevated levels of oxidative stress due to the activation of oncogenes and loss of tumor suppressors [29]. ROS by altering the growth signals and gene expression cause continuous proliferation of cancer cells [30]. ROS can damage DNA by inducing base modifications, deletions, strand breakage, chromosomal rearrangements, and hyper- and hypo-methylation of DNA. It is one of the initiators of carcinogenesis, wherein many tumors exhibit elevated levels of DNA damage. Epidemiological studies show an increased cancer risk with low antioxidant levels. Patients with various cancers exhibit increased total oxidant status levels and decreased total antioxidant status [31].

Cardiovascular diseases (CVDs) are a complex group of medical conditions that affect the heart and blood vessels. Atherosclerosis, also known as the hardening of the arteries, is a condition that can be caused by hyperlipidemia, which is a major risk factor. Patients with atherosclerosis, Type 2 diabetes, and obesity often have elevated levels of oxidized low-density lipoprotein (LDL), glucose, and free fatty acids [31, 32]. Oxidative stress caused by a significant imbalance of oxidants and antioxidants is observed in atherosclerosis. Free radicals are produced by endothelial cells, smooth muscle cells, and macrophages in the vessel wall. This results in endothelial dysfunction that leads to increased endothelial permeability, upregulation of endothelial adhesion molecules, and inflammatory cell infiltration into the arterial wall [32]. ROS are known to play a significant role in endothelial injury, dysfunction, and lesion progression. MMPs (matrix metalloproteinases) are activated by ROS, which leads to the degradation of intimal extracellular matrices and promotes smooth muscle cell migration. Cigarette smoking contains a large number of free radicals and may downregulate key exogenous and endogenous antioxidants such as vitamin D, carotenes, GPx, and SOD, leading to the dysfunction of monocytes and vascular smooth muscle cells. Proatherogenic agents such as oxidized lipids, high glucose, and cigarette constituents increase free radical production [33, 34].

Hypertension affects 40% of adults worldwide, making it a major health issue. Free radical-induced oxidative stress in part contributes to endothelial dysfunction and the development of hypertension [35]. One of the causes of hypertension is the increased generation of ROS, which leads to a decrease in the bioavailability of nitric oxide (NO•) by forming ONOO−, resulting in reduced endothelium-dependent vasodilation. It has been observed that there is a decrease in NO bioavailability and an increase in oxidative stress in patients with hypertension. The oxidation-induced impairment of NO also causes a reduction in its ability to counteract the vasoconstrictive and hypertensive effects of angiotensin II. Angiotensin II, in turn, promotes oxidative stress and decreases NO bioavailability [36, 37].

4. ROS as therapeutic agents

Over the past few decades, ROS have emerged as potential therapeutic products, finding applications in a wide range of areas, from wound healing and hair growth enhancement to cancer treatment, stem cell differentiation, and tissue engineering. Despite significant advances, there are still several challenges that need to be overcome for the clinical translation of ROS. Several studies have found that ROS can have a beneficial effect at low concentrations, and they may be used as a cost-effective and convenient way to induce tissue regeneration [38, 39].

Recent research has revealed that cancer cells are more sensitive to oxidative damage, which has led to the development of methods for selectively killing tumor cells using ROS. As a result, there is extensive investigation into creating sensors to monitor ROS production during cancer treatment. The high oxidative status of cancer cells led to the development of H₂O₂-responsive anticancer prodrugs. Additionally, various drugs inducing oxidative stress are being developed to exploit cancer cells’ susceptibility [38, 40].

The precise control of stem cell differentiation by ROS through ROS stimulation techniques such as cold atmospheric plasma and the use of biomaterials that release ROS has shown potential for tissue engineering applications. Some studies have demonstrated in vitro neurogenesis through cold atmospheric plasma stimulation techniques, whereas others are exploring the use of different biomaterials that release ROS. However, further research is required to fully understand the possible side effects of these techniques [41].

5. Conclusion

Reactive oxygen species (ROS) are a unique set of molecules that are produced naturally through metabolic reactions within cells. These molecules play a crucial role in cell function and survival by acting as signaling agents that regulate specific biochemical pathways. However, when there is an imbalance in ROS signaling or excessive ROS production, it can adversely impact disease pathophysiology. Therefore, an in-depth understanding of tissue-specific redox signaling complexities is essential to develop new and innovative therapies for cardiovascular and metabolic disease pathogenesis.

Despite the potential benefits of ROS in various fields of biology, such as cancer treatment, tissue engineering, wound healing, and developmental biology research, they possess a complex mechanism of action with possible side effects. It is crucial to develop new systems that can regulate the timing of ROS production and control their levels. Further research and advancements in this field will pave the way for a better understanding of ROS and their potential applications in various fields of biology.