**Abstract**

Free radicals are highly reactive molecules that are unstable and have extremely short-short half-life. They are derived from either oxygen (reactive oxygen species, ROS) or nitrogen (reactive nitrogen species, RNS) in mitochondria, plasma membrane and endoplasmic reticulum due to oxidative stress and damage. ROS/RNS are physiologically useful at low concentrations and are responsible for the activation of redox-sensitive signaling pathways, phagocytosis of infected cells and removal of abnormal and aging cells. The endogenous sources of ROS are the electron transport chain, the respiratory burst of phagocytes and oxidation of lipids. These radicals react with biomolecules such as DNA, proteins and lipids and may cause pathophysiological conditions such as autoimmunity, carcinogenesis, and neurodegenerative diseases. The role of ROS in autoimmune response remains complex and they have been implicated in the initiation, generation and amplification of novel epitopes. ROS also appears to play a critical role in rheumatoid arthritis (RA), a systemic autoimmune disease of the joints also known as inflammatory arthritis (IA). ROS are involved in the initiation of various signaling pathways and have a significant role in the pathophysiology of RA.

**Keywords:** ROS, RNS, RA, autoimmunity, free radicals, rheumatoid arthritis

### **1. Introduction**

A free radical is a "molecule containing one or more unpaired electron(s) and which is capable of independent existence". Free radicals are highly reactive species and are involved in several metabolic processes including oxidative reactions in mitochondria, and 'oxidative burst' of phagocytes, etc. In excess, free radicals lead to diseases including autoimmune, cardiovascular, neurodegenerative, cancers and must be reduced to minimize these pathological conditions. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two types of free radicals formed in the body and consist of both radical and non-radical moieties [1]. Under normal conditions, living organisms utilize respiration to survive, they undergo a process of reduction of oxygen molecules through the addition of four electrons resulting in the formation of water. This process produces molecules such as superoxide anion (O2d), hydrogen peroxide (H2O2) and hydroxyl radical (OH) as a byproduct. During energy

transduction via electron transport of molecular oxygen, O2d is formed intracellularly within the mitochondria, which may potentially lead to the development of a variety of pathophysiological conditions. [2, 3] (**Figure 1**).

The O2**–** is converted into stable non-radical H2O2 by the enzyme superoxide dismutase (SOD). Catalase (CAT) and glutathione peroxidase (GPx) convert H2O2 to H2O and O2. Further, H2O2 is converted by myeloperoxidase (MPO) in the neutrophils to hypochlorous acid (HOCl), which is a strong oxidant acting as a bactericidal agent in phagocytic cells. The reaction of HOCl with H2O2 yields singlet oxygen (1 O2) and water. Finally, H2O2 is converted in a spontaneous reaction catalyzed by Fe2+ (Fenton reaction) to the highly reactive OH (**Table 1**). The OH causes oxidative damage to biological molecules such as lipid, protein and DNA leading to the etiopathogenesis of autoimmune disorders [5]. The excess generation of OH and peroxynitrite (ONOOd) causes oxidative damage to cell membranes and lipoproteins leading to lipid peroxidation resulting in the formation of harmful compounds such as malondialdehyde (MDA) and thiobarbituric acid reactive substances (TBARS) [6]. ROS/RNS damage nucleic acids leading to change in the function and conformation of DNA resulting in the formation of strand breaks, nitrogenous base damage-causing mutations [1]. The ROS are also produced from other sources such as NADPH oxidase (NOX) inactivated phagocytes and endothelial cells, macrophage and polymorphonuclear cells (PMN), lysosome and microsomes. ROS is also involved in inflammatory reactions through the activation of nuclear transcription factor kappa B (NF-κB), leading to upregulation of pro-inflammatory cytokines and leukocyte adhesion molecules (LAM) [5].

Oxidative stress results from reactive species from the biochemical reactions within the body including NADPH oxidases (NOXs), nitric oxide synthase (NOS), nitrate reductase (NR), mitochondrial electron transport chain (ETC) and the hydrogen sulphide (H2S) producing enzymes cystathionine-β synthase (CBS) and cystathionineγlyase (CSE). Superoxide undergoes a dismutation reaction to generate H2O2 which, in the presence of transition metal ions (e.g., ferrous ions), forms the OH. MPO, produces HOCl MPO is a heme-containing peroxidase expressed mainly in neutrophils and to a lesser degree in monocytes. MPO, in the presence of H2O2 and halides, catalyzes the

#### **Figure 1.**

*Pathways of ROS/RNS in the body. NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; GSH, glutathione; GSSG; glutathione disulfide; SOD, superoxide dismutase; NOS, nitric oxide synthases; MAO, monoamine oxidase; MPO, myeloperoxidase (*reprinted from *[3]).*


**Table 1.**

*Equations showing products generated by oxygen/nitrogen radicals (reprinted from [4]).*

formation of ROS such as HOCl. The MPO/HOCl system plays an important role in microbial killing by neutrophils. Moreover, it has also also been shown to be a local mediator of tissue damage and the inflammation in various inflammatory diseases. When ROS production exceeds the physiological antioxidant defense, oxidative stress occurs consequently leading to oxidative modification of proteins. This protein alteration may lead to the formation of neoepitopes resulting in the formation of autoantibodies [7, 8]. The pathogenesis of autoimmune diseases (ADs) such as RA is

characterized by the loss of peripheral tolerance to autoantigens, excessive activation of T and B cells, which leads to increased levels of cytokines and autoantibodies (rheumatoid factor, RF; anti-cyclic citrullinated peptide antibodies, ACPA; etc.) [9].

Studies have shown that anti-carbamylated proteins anti-CarP and anti-type II collagen antibodies can serve as a promising diagnostic such diagnostic tool in such ADs. The activation of endogenous cellular antioxidant defense systems (e.g., nuclear erythroid 2-related factor 2; Nrf2-dependent pathway), inhibition of ROS/RNS source (e.g., isoform-specific NOX inhibitors), etc., may become the potential future strategies for redox-based therapeutic compounds [7].
