Reactive Oxygen Species and Antioxidant Interactions in Erythrocytes

Abstract

There is a continuous generation of reactive oxygen species (ROS) in erythrocytes due to their microenvironment. Reactive oxygen species (ROS) and reactive nitrogen species are well known as both harmful and beneficial species. They help in activating the antioxidant enzymes. However, overproduction of ROS can cause fatal damage to cell structures, including lipids and membranes, proteins and cause oxidative stress. Erythrocytes have effective antioxidant defenses to maintain their structure and functions. They protect these cells from damage and maintain their activities. Studies have reported that antioxidant interventions in various situations have proved beneficial to erythrocytes. Therefore, they can be employed as in vitro models for antioxidant and free radical interactions and also are ideal cell models for translational studies.

1. Introduction

The erythrocyte [red blood cell (RBC)] is an ideal cell to study free-radical-mediated alterations. Approximately 25 trillion erythrocytes course through the human circulatory system. The main function of erythrocytes is the transport of oxygen (O₂) and the mediation of carbon dioxide (CO₂) production [1]. Reactive oxygen species (ROS) are continuously produced within the erythrocytes due to high O₂ tension in arterial blood and heme iron content [2].

The mature erythrocyte contains a variety of enzymes, proteins, carbohydrates, lipids, anions, and cations, to balance the cell’s metabolism and functions. An important consequence of erythrocyte imbalance is a reduced ability to deal with oxidative stress, which can lead to degenerative changes in hemoglobin, membrane, and enzymes [3].

Erythrocytes are exposed to circulating inflammatory mediators and related oxidative stress, which cause severe alterations in cellular membrane and functions in a variety of pathological conditions. These alterations have been defined as “erythropathy” [4] and can be observed in conditions of cardiovascular injury [5, 6]. The loss of lipid asymmetry, and thus the exposure of phosphatidyl serine (PS) on the outer monolayer, contributes to the premature destruction of thalassaemic and sickle red cells [7, 8]. Sickle cell disease is distinguished by a change in erythrocyte shape from biconcave discs to elongated and sickle-shaped erythrocytes, consequently leading to loss of function and anemia [9].

Chronic obstructive pulmonary disease (COPD) causes changes in erythrocyte shape, redistribution of microfilaments such as actin and spectrin, and elevations in membrane rigidity [10]. Alterations were also observed in terms of erythrocyte morphology (leptocytes and elliptocytes), elevated membrane F2-isoprostanes and 4-hydroxynonenal (4-HNE) protein adducts, and oxidative damage to actin proteins in Rett syndrome (RTT) and autism spectrum disorder (ASD) [11, 12].

The changes in erythrocyte morphology and stiffness have also been reported in pathologies (type 2 diabetes, obesity, hypertension, and hypercholesterolemia) characterized by consistent oxidative damage followed by reshaping of the lipid distribution and architecture [13]. Erythrocytes participate in physiological and pathological processes associated with oxidative stress, such as aging, Down syndrome, neurodegenerative diseases such as Alzheimer’s disease, erectile dysfunction, and cardiovascular disease [14].

The potential clinical application of these erythrocyte alterations as new biomarkers could be useful tools for monitoring a variety of oxidative-stress-related diseases.

2. Reactive oxygen species (ROS) in erythrocytes

Various physiological and pathological conditions, for example, aging, inflammation, and cell death develop through ROS generation. Several factors can lead to the generation of oxidizing radicals such as superoxide anion (O₂^•−), hydrogen peroxide (H₂O₂), and hydroxyl radical (HO^•) in erythrocytes [15].

Free radicals can be formed in three ways:

1. The cleavage of a covalent bond of a normal molecule, with each fragment retaining one of the paired electrons;

2. The loss of a single electron from a normal molecule;

3. The addition of a single electron to a normal molecule.

The latter, electron transfer, is a common process in biological systems [16].

Free radicals and ions are formed as illustrated below:

Radical formation by electron transfer: A + e⁻ → A^•−

Radical formation by homolytic fission: X: Y → X^• + Y^•

Ion formation by heterolytic fission: X: Y — > X: ⁻ + Y⁺

2.1 Nature of reactive oxygen species

ROS are defined as oxygen-containing species, which are highly reactive. O₂ undergoes one or two-electron reduction to form ROS, which reacts quickly with other compounds, attempting to capture the required electron in order to gain stability. ROS are oxygen-centered molecules that include hydrogen peroxide, singlet oxygen, superoxide anion, hydroxyl radical, and nitric oxide (NO) [16]. ROS are constantly produced in small quantities by normal metabolic processes. The addition of one electron to O₂ forms O₂^•−, whereas the addition of two electrons results in the production of H₂O₂.

There are two causes for O₂^•− generation in erythrocytes.

Firstly, Oxyhemoglobin (oxyHb) autoxidizes at a relatively slow rate to yield methemoglobin (metHb), and O₂^•−, which, further produces H₂O₂. Hemoglobin (Hb) is constantly exposed to an intracellular and extracellular flux of H₂O₂. When oxyHb is exposed to H₂O₂, it undergoes oxidative modifications that have been proposed as selective signals for proteolysis in erythrocytes [17]. Secondly, the oxidation state of trivalent iron (Fe³⁺) has lost an electron during its formation; consequently, O₂^•− has been generated from exogenous sources, such as drugs, etc. [18].

Hydrogen peroxide is hydrophilic; however, recent studies reported that aquaporins are not involved in facilitating H₂O₂ diffusion across RBC membranes; rather, diffusion occurs through the lipid fraction or an unidentified membrane protein [19]. While charged, O₂^•− can only cross membranes via transmembrane anion channels. MetHb, lipid peroxidation, and spectrin-Hb complexes increase with H₂O_2, which further generates a covalent complex of spectrin and Hb, leading to changes in cell shape, membrane deformability, phospholipid organization, and cell surface characteristics [20].

The Fenton reaction occurs when H₂O₂ reacts with ferrous iron to produce OH^•. H₂O₂ can react with O₂^•− to generate OH^•, the most active ROS that cannot travel far due to its short half-life of a few nanoseconds known as Haber-Weiss Reaction [21, 22].

$$F{e}^{++}+{\mathrm{H}}_2{\mathrm{O}}_2\to F{e}^{+++}+O{H}^{-}+O{H}^{•}\left(\mathrm{Fenton}\mathrm{Reaction}\right)$$E1

$${{\mathrm{O}}_2}^{•-}+{\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{O}}_2+O{H}^{-}+O{H}^{•}\left(\mathrm{Haber}­\mathrm{Weiss}\mathrm{Reaction}\right)$$E2

ROS have the ability to act as both oxidizing and reducing agents. ROS are capable of directly attacking the red cell membrane and causing changes in lipid and protein structure [23]. ROS also alter mechanical properties, increase rigidity, and RBC interactions with other cells and coagulation factors, as well as stimulate microparticle (MP) generation and phosphatidylserine (PS) exposure [24]. Human red cell aging could be attributed to oxidative damage. RBC deformability, membrane permeability, and surface antigenicity abnormalities, on the other hand, have been recognized as defects in cellular properties that contribute to RBC senescence [25].

Nitric oxide, along with O₂ and CO₂, is the third gas transported by erythrocytes. Erythrocytes are the primary NO scavengers in circulation due to their high Hb concentration. NO is taken up by heme prosthetic groups of Hb-chain cysteine residues. NO is converted to nitrate by oxyhemoglobin (HbFe⁺²O₂), whereas deoxyhemoglobin (HbFe²⁺) binds to NO to form iron-nitrosylhemoglobin (HbFe²⁺NO). NO consumption by erythrocytes can be regulated by HbFe²⁺NO formation under hypoxic conditions [26]. NO reaction with Hb greatly limits intravascular NO concentration. As a result, it is unlikely that NO is directly exported or produced by red blood cells as an intravascular signaling molecule. The rapid deoxygenation of NO by Hb results in the formation of nitrate and metHb, preventing NO diffusion from plasma to smooth muscle [15]. NO is produced in large amounts in inflammatory conditions and reacts with O₂^•− to generate peroxynitrite [27]. Peroxynitrite oxidizes plasma components, releasing secondary radicals that promote tyrosine nitration, leading to gain or loss of protein function [28].

3. Oxidative stress in erythrocytes

Erythrocytes are well endowed to combat oxidative stress due to their continuous contact with oxygen, as their inherent carrier function. When the concentration of ROS in cells or tissues exceeds the antioxidant protection, oxidative stress occurs [21]. RBC properties have been shown to change as a result of oxidative damage. Oxidative damage can also alter membrane permeability resulting in hemolysis [29, 30]. Oxidative cross-linking of spectrin can cause increased membrane rigidity and decreased erythrocyte deformability. Erythrocytes can be recognized by the immune system as a result of oxidative damage [31].

3.1 Extracellular hemoglobin as a source of oxidative stress

Extracellular Hb, which results from erythrocyte hemolysis or the infusion of cell-free Hb-based blood substitutes, can be a major source of oxidative stress. This potential source of oxidative stress is minimized under normal conditions by haptoglobin and hemopexin, which bind Hb and free heme, respectively. They inhibit the oxidative reactions of Hb and heme, allowing them to be removed from circulation. Elevated levels of free Hb and heme, which cannot be neutralized by haptoglobin and hemopexin, cause a variety of adverse clinical effects [32].

Autoxidation of Hb produces superoxide as well as methemoglobin. Hb is known to react with hydrogen peroxide to form ferrylhemoglobin, a strong oxidant [33]. Hb binds to RBC membrane proteins, especially under hypoxic conditions. The ROS produced by bound Hb may be inaccessible to cellular antioxidants, allowing the production of heme degradation products close to the membrane [34, 35, 36]. Extensive lipid peroxidation results in changes in fluidity such as a drop in membrane potential and an increase in permeability to different ions, which eventually leads to hemolysis. Thus, perturbations in erythrocyte function and structure can result in an increased flow of prooxidant generation that can lead to oxidative stress.

Oxidative stress and ROS accumulation in RBCs during aging may induce hemolysis. As a result, the plasma proteins haptoglobin and hemopexin can render free Hb and heme relatively inactive and deliver them safely to macrophages for phagocytosis [37, 38]. Oxidized Hb on the other hand exhibits impaired plasma clearance, due to its low affinity for haptoglobin protein.

Erythrocyte membranes exposed to oxidative stress undergo cellular component modifications such as oxidative denaturation of Hb, peroxidation of lipids, and high-molecular-weight cross-linked membrane proteins. The erythrocyte membrane is rich in sulfhydryl (∙SH) groups, which help to maintain cellular oxidative balance [39]. Changes in the membrane elasticity may occur due to oxidative damage to the membrane ∙SH groups [40]. Oxidative damage to erythrocytes can occur and manifest itself in a variety of ways, including potassium release, increase in malondialdehyde (MDA), phosphatidylserine externalization [41], decrements in glutathione, superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase as well as total antioxidant activity of plasma [42, 43, 44]. Oxidant stress is a key component to both normal RBC aging and pathological dysfunction [32].

Oxidative damage to a specific protein, particularly at the active site, can result in the progressive loss of a specific biochemical function [45]. Peroxidation causes globin cross-linking to proteins such as spectrin and band 3. These processes further lead to decreased phospholipid symmetry, formation of cross-linked spectrin and Hb, aggregation of band 3 protein, and increase in advanced glycation end products leading to deformability and morphologic and surface changes in the erythrocyte [15, 46, 47]. During erythrocyte aging, an irreversible oxidative complex is formed between the Hb globin chain and spectrin [20]. Iron release may be accompanied by the generation of senescent antigens (SCA) and oxidative alteration of membrane proteins [15, 48, 49]. Another event associated with red cell homeostasis disruption is an increased inflammatory state in the bloodstream, since erythrocytes are constantly exposed to inflammatory molecules transported in the vascular system, their membranes may be particularly vulnerable to their interaction. A large number of inflammatory mediators, including tumor necrosis factor-α, interleukins, interferons, and C-reactive protein (CRP), have been proposed as potential inflammatory response markers [50].

In general, erythrocytes respond to oxidative stress by activating tyrosine kinases [51, 52], resulting in tyrosine phosphorylation at the cytoplasmic domain of band 3 protein, which mediates interactions with ankyrin, leading to membrane destabilization. This also seen in β-thalassemia disease with the loss of stability between cytoskeleton and membrane complexes, such as band 3 protein [53]. Oxidative damage to band 3 has been linked to RBC aging including the exposure of senescent specific neo-antigens that bind autologous IgG triggering RBC removal [54].

Erythrocytes also contain NADH oxidases, which can generate endogenous ROS [55]. However, some forms of NADH oxidase were also detected in normal RBCs. L-isoaspartyl groups were discovered on erythrocyte membrane proteins in response to aging or pathological oxidative stress, such as glucose 6-phosphate dehydrogenase deficiency or Down syndrome [56, 57, 58, 59]. Thus, L-isoaspartyl group accumulation in RBC proteins correlates with RBC dysfunction and pathology.

The effects of oxidative stress observed in various pathological conditions in erythrocytes are depicted in Table 1.

Diseases	Oxidative stress markers	Reference
Sickle cell disease	Variations in Superoxide dismutase	[60, 61, 62, 63]
	Alterations in Catalase	[60, 62, 63]
	Glutathione—decreased	[64, 65]
	Changes in glutathione peroxidase	[61, 62]
	Vitamin E—decreased	[66, 67]
	Vitamin-C—decreased	[68]
	Lipid peroxidation—increased	[62, 63, 69, 70]
	Intracellular Ca^2+—increased	[71]
	Phosphatidylserine exposure—increased	[72, 73, 74]
	NADPH oxidase activity—increased	[55]
	ROS generation—increased	[55]
β-Thalassemia	Band 3 tyrosine phosphorylation (P)—increased	[53]
	Phosphatidylserine exposure—increased	[53]
	Release of microparticles	[53]
	Membrane thiols—decreased	[53]
	K^+, Cl^−, and water loss	[53]
Diabetes (T2DM)	Lipid peroxidation—increased	[75]
	GSH/GSSG ratio—decreased	[75]
	Glutathione—decreased	[75]
	Phosphatidylserine externalization—increased	[75]
	Annexin binding—increased	[75]
	Caspase-3 activation—increased	[76]
Malaria	Lipid peroxidation—increased	[77]
	Glutathione—decreased	[77]
	Catalase—decreased	[77]
	Membrane stiffness—increased	[78]
Autism	TBARS—increased	[79]
	Xanthine oxidase—increased	[79]
	Superoxide dismutase—increased	[79]
	Superoxide dismutase—decreased	[80]
	Glutathione peroxidase—decreased	[80]
	Modulations in catalase	[79, 81]
Chronic kidney disease	Membrane fluidity—increased	[82]
	Osmatic fragility—increased	[82]
	RBC life span—decreased	[82]
	Antioxidant enzymes—decreased	[83, 84, 85]
	NADPH oxidase—increased	[86]
	Hemoglobin leakage—increased	[86]
	Nitrite ions—increased	[86]
	Peroxynitrite—increased	[86]
	Hemoglobin—decreased	[86]
	Circulating RBCs—decreased	[86]
Systemic sclerosis (SSc)	Anion exchange capability via band 3—decreased	[87]
	Blood viscosity—increased	[87]
	Membrane protein structure destabilization	[87]
	SO4^2−—decreased	[87]
Corona virus disease-19 (Covid-19)	Hematocrit levels—decreased	[88]
	RBCs amplitude—increased	[88]
	Glutathione—decreased	[89, 90, 91]
	Arginase 1—increased	[92]
	ROS—increased	[92]
	Interferon-γ—increased	[92]
	NO bioactivity—decreased	[92]
	Oxidized glutathione (GSSG)—increased	[93]
	Glycolytic metabolites—increased	[93]
	Phosphoglucomutase—decreased	[93]
	Carboxylic acids—increased	[93]
	Glutathione peroxidase—decreased	[91]
	Catalase—decreased	[91]
	Superoxide dismutase—decreased	[91]

Table 1.

Effects of oxidative stress in erythrocytes during diseases.

4. Antioxidant defense in erythrocytes

The antioxidant defense system of erythrocytes prevents oxidative cell damage. This implies that the erythrocyte antioxidant defenses operate in a balanced manner. As a result, an appropriate redox state, balanced antioxidant action is required for ROS homeostasis.

Antioxidants are molecules that prevent or delay cellular damage by inhibiting or quenching ROS reactions [94]. Antioxidants can be synthesized in the body or obtained from the environment, such as through diet. Erythrocytes are well equipped to fight against oxidative stress, i.e., mechanisms to scavenge and detoxify ROS, prevent their production, and sequester transition metals [95]. Erythrocytes contain both enzymatic and non-enzymatic antioxidants to combat oxidative stress.

4.1 Enzymatic antioxidants

Enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and peroxiredoxin-2 (PRX-2). Their coordinated actions protect the erythrocytes from free-radical-mediated damage. Since there is no de novo synthesis of antioxidant enzymes in mature erythrocytes, their defense capacity is limited. Free radicals affect the capacities of antioxidative enzymes as well as the overall antioxidative system [96]. Under normal conditions, erythrocytes contain sufficient levels of scavenger enzymes such as Cu,Zn-SOD, CAT, and selenium-dependent GPX to protect from free radical injury.

Superoxide dismutase, a ubiquitous metal-containing enzyme, involves in the detoxification of O₂^•− into O₂ and H₂O₂.

$${\mathrm{O}}_2{{}^{•}}^{-}+{\mathrm{O}}_2{{}^{•}}^{-}+2{\mathrm{H}}^{+}\stackrel{\mathit{SOD}}{\to }{\mathrm{H}}_2{\mathrm{O}}_2+{\mathrm{O}}_2$$E3

SOD family comprises CuSOD, ZnSOD, MnSOD, and extracellular SOD, which protect from particularly O₂^•−. Cu,Zn-SOD catalyzes the dismutation of O₂^•− to H₂O₂, which is later converted to water by CAT or GPX [21, 27, 97]. The activity of these enzymes in erythrocytes is highest than that of other tissues in the body [98].

Erythrocytes are well protected against ROS due to the abundance of Cu,Zn-SOD, which scavenges free radicals and thus prevents metHb formation [43]. Cu, Zn-SOD synthesis is induced by O₂^•− formation through the activation of regulatory genes [15]. SOD scavenges O₂^•− and inhibits the formation of peroxynitrite, thereby preventing injury and regulating the bioavailability of NO [27]. Erythrocytes contain an abundant quantity of Cu,Zn-SOD, which maintains intra-erythrocyte O₂^•− levels at concentrations as low as 10⁻¹³ mol/L [96].

Catalases catalyze the direct decomposition of H₂O₂ to water and O₂ [21].

$$2{\mathrm{H}}_2{\mathrm{O}}_2\stackrel{\mathit{CAT}}{\to }2{\mathrm{H}}_2\mathrm{O}+{\mathrm{O}}_2$$E4

Catalase and SOD react synergistically to protect each other [99]. CAT and GPX are equally active in the detoxification of H₂O₂ in normal erythrocytes [100]. At physiological concentrations, GPX acts as a primary defense in H₂O₂ degradation by reducing H₂O₂ while also converting GSH to its oxidized form (GSSG). However, under H₂O₂ overproduction, CAT exhibits increased enzymatic activity, as measured by the Michaelis-Menten constant (Km). The Km for CAT (2.4 × 10⁻⁴ M) is significantly greater than the Km for GPX (1 × 10⁻⁶ M), which indicates that CAT scavenges H₂O₂ efficiently at higher concentrations [101, 102, 103]. GPX is important in dealing with endogenous H₂O₂ produced by Hb autoxidation, whereas CAT becomes increasingly important when erythrocytes are exposed to increased H₂O₂ flux [15]. H₂O₂ readily crosses erythrocyte membranes and can protect other tissues against extracellular H₂O₂ by “absorbing” and destroying it. Reduced glutathione (GSH) is used by GPX to detoxify hydrogen peroxide during normal antioxidant defense system function. Furthermore, glutathione reductase is required to convert H₂O₂ to GSH, which contributes to H₂O₂ detoxification [104].

Glutathione-S-transferases are important in the detoxification of electrophilic xenobiotics. This enzyme catalyzes the conjugation of GSH with exogenous and endogenous toxic compounds or their metabolites, making them more water-soluble, less toxic, and easier to excrete. In addition, they are responsible for various resistance mechanisms such as chemotherapeutic or antibiotic drug resistance [15].

Glucose-6-phosphate dehydrogenase (G6PD) is an important antioxidant enzyme in erythrocytes, which is the regulatory enzyme of the pentose-phosphate pathway (PPP). As erythrocytes lack mitochondria, the PPP pathway is the only source of NADPH, and it plays an important role in NADPH-dependent antioxidant defense [105, 106]. G6PD is required to protect erythrocytes from oxidative damage. The lack of this protection can lead to severe hemolysis [107].

In addition to primary antioxidant defense systems that prevent the generation of free radicals or radical chain reactions, secondary systems have been proposed. Proteases that preferentially degrade oxidatively damaged proteins are among them. In erythrocytes, a multicatalytic proteolytic complex appears to be responsible for the degradation of oxidized intracellular proteins [47]. The presence of an 80-kDa serine protease in the oxidized erythrocyte membranes preferentially degrades oxidized proteins specifically protein hydrolase. When cells are oxidized, this cytoplasmic protein becomes adherent to membranes, promoting membrane protein degradation. The protease is characterized by its inhibition by a serine protease inhibitor [108]. It is endogenously present in oxidized or aged erythrocyte membranes and plays a crucial role in the removal of the oxidation-induced membrane protein aggregates and in reducing the oxidation-induced anti-band 3 binding in aging. Oxidized protein hydrolase (OPH) acts as a secondary defense system by removing oxidized protein aggregates.

Peroxiredoxins (PRX), a class of thiol-containing enzymes, act as H₂O₂ and peroxynitrite scavengers in circulation. PRXs have a reductive capacity for hydroperoxides via a reductant thiol. Peroxiredoxins have been shown in studies to be catalytic peroxynitrite reductases. It has been also reported that PRX-II is present in the cytosol of erythrocytes. The catalytic cycle involves the reduction of oxidized PRX by thioredoxin and the reduction capacity of NADPH via NADPH-thioredoxin reductase [28, 109].

4.2 Non-enzymatic antioxidants

Endogenous non-enzymatic antioxidants are defined in two phases: lipophylic (vitamin E, carotenoids, ubiquinon, melatonin, etc.) and water-soluble (vitamin C, glutathione, uric acid, ceruloplasmin, transferin, haptoglobulin, etc.). Three antioxidant vitamins, A, C, and E, provide defense against oxidative damage. Vitamin C acts in the aqueous phase, whereas vitamin E acts in the lipid phase as a chain-breaking antioxidant. Vitamin C reduces O₂^•− and lipid peroxyl radical, but is also a well-known synergistic agent for vitamin E [2]. Uric acid is an endogenous antioxidant with metal-chelating properties and scavenges nitrogen radicals and superoxide in plasma, thereby blocking the generation of peroxynitrite. Uric acid in erythrocytes quenches the free radicals and ROS. Uric acid maintains the smooth membrane surface of RBCs, thus preventing echinocyte formation [110].

Additionally, erythrocytes have a plasma membrane redox system (PMRS) that transfers electrons from intracellular substrates to extracellular electron acceptors, which may be NAD+ or/and vitamin C [111].

Many studies have demonstrated the influence of different antioxidants on erythrocytes during oxidative stress (Table 2).

Sample	OS inducer	Antioxidant	Results	Reference
Human RBC membrane	Hydrogen peroxide (H2O2) & ozone	1,3-dimethyluric acid and 1,3,7-trimethyluric acid	Prevented lipid peroxidation	[112]
Human RBCs	Hydrogen peroxide	β-Carotene & resveratrol	Increased SOD & catalase. decreased conjugate dienes & TBARS	[113]
Human RBCs	Tert-butyl hydroperoxide	Resveratrol	Increase in GSH & membrane ∙SH	[114]
Human RBCs	tert-butyl hydroperoxide	Capsaicin or L-ascorbic acid	Decrease in MDA & protein carbonyls	[115]
Human RBCs	Hydrogen peroxide	Tea polyphenols (TPP)	Protected RBCs and membranes against lipid peroxidation	[116]
Sickle cell human RBCs	Tert-butyl hydroperoxide	Flavonoids (quercetin & rutin), Ascorbic acid	Protected against oxidative stress in and lipid peroxidation	[117]
Human RBCs	Hydrogen peroxide	Oak barrel-aged red wine extract (SD95)	Protected against hemolysis, ROS & maintained MDA	[118]
Human RBCs	AAPH or H2O2 or t-BOOH	Ether and water fraction of honey	Ether fraction inhibited hemolysis and t-BOOH-induced lipid peroxidation	[119]
Human RBCs	Bisphenol A (BPA)	Quercetin	Decreased MDA levels and increased antioxidant enzymes	[120]
Human RBCs	Hydrogen peroxide	Melatonin	Restored band 3 expression levels, cell shape alterations & lower TBARS.	[121]
Rat RBCs	Tert-butyl hydroperoxide	Flavonoids (quercetin, catechin and naringenin)	Inhibited membrane lipid peroxidation and decreased glutathione oxidation	[122]
Human RBCs	Sodium fluoride (NaF)	3,4-dihydroxy benzaldehyde (DHB)	Increased AOPP, lipid peroxidation, restored PMRS & AO enzyme activity	[123]
Human RBCs	Hydrogen peroxide	β-carotene (BC) and resveratrol (RSV)	TBARS and conjugate dienes decreased in BC and RSV groups. SOD increased in RSV.	[113]

Table 2.

The effects of antioxidants on erythrocytes during oxidative stress.

There are in vitro studies on oxidative stress in erythrocytes reporting the protective effects of antioxidants from plant extracts (Table 3).

Sample	OS inducer	Antioxidant	Results	Reference
Rat RBCs	H2O2	Potato peel extract (PPE)	Inhibited morphological alterations RBCs	[124]
Human RBCs	2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)	Olive leaf extract (OLE)	Inhibited hemolysis, TBARS formation, and hemoglobin oxidation	[125]
Goat RBCs	Cu^2+-ascorbate	Terminalia arjuna (TA) bark extract	Decreased lipid peroxidation and protein carbonyl content. Increased GSH	[126]
Human RBCs	2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)	Quince (Cydonia oblonga) fruit pulp and peel extracts	Protection of erythrocyte membrane from hemolysis	[127]
Human RBCs	Hypochlorous acid (HClO)	Ugni molinae Turcz aqueous extract	Reduced hemolysis	[128]
Rat RBCs	Cadmium	Salicornia arabica lipid extract	Ameliorated antioxidant status and inhibited MDA levels	[129]
Human RBCs	Hypochlorous acid (HClO)	Pitavia punctata extract	Protecting the membrane integrity and inhibiting the oxidation of the LDL lipoprotein	[130]
Human RBCs	H2O2	Orchis latifolia and Centratherum anthelminticum extract	Protected membrane integrity resulting in a reduction of RBC hemolysis and lipid peroxidation	[131]

Table 3.

The modulations of antioxidants from plant extracts in erythrocytes.

5. Conclusion

Many physiological and pathological circumstances have the potential to cause oxidative stress and are possibly harmful in susceptible individuals. Furthermore, this increased risk of adverse reactions is generally reflected in the erythrocytes. The administration of antioxidants has the prospects of diminishing oxidative damage. Therefore, erythrocytes act as unique cell models for translational studies on oxidant and antioxidant interactions. However, they may not be helpful to study these effects in relation to mitochondria (a major source of ROS), as the mature erythrocytes lack the cell organelles.

Acknowledgments

The authors acknowledge Dr. Leela Iyengar, Anusha BA, Ms. Magdaline Christina, and JAIN (Deemed-to-be University) for their support.

Conflict of interest

The authors have no conflict of interest to disclose.