**6. Potential role of antioxidants in β-thalassemia**

Various antioxidant enzyme systems are activated by the oxidative stress to protect the body tissues from its damaging effects in β-thalassemia patients. These antioxidants include superoxide dismutase (SOD), catalase, glutathione (GSH), thioredoxin (Trx), and ferritin. Superoxide (O2 <sup>−</sup>) is the first reactive radical produced, and this radical can be neutralized by SOD. There are three distinct SODs: SOD1 (cu/Zn-SOD) is present in cytoplasm, whereas SOD2 (Mn-SOD) is present in the mitochondria, and SOD3 is almost exclusively extracellular [66, 67]. Each of these distinct SODs performs a specific function in human cells. In β-thalassemia, major patients higher levels of erythrocyte superoxide dismutase and glutathione peroxidase (GPx) as well as higher plasma malondialdehyde (MDA) were observed as compared to healthy controls [68]. Iron overload through repeated blood transfusions and subsequent oxidative stress produced by reactive oxygen species may be the cause of increased levels of MDA. The rise in SOD and glutathione peroxidase may occur as a result of compensatory mechanisms in response to oxidative stress [44]. Neutralization of O2 <sup>−</sup> produces H2O2, which can be metabolized into nontoxic products by a catalase and glutathione peroxidase (GPx) in conjunction with glutathione. Location of GPx depends on the subtype, whereas catalase is present in peroxisomes [67]. The stability of the cellular and subcellular membranes depends mainly on glutathione peroxidase, and the protective antioxidant effect of glutathione peroxidase depends on the presence of selenium. In patients with β-thalassemia, major was confirmed the peroxidative status generated by iron overload and the high increase in serum ferritin, iron, plasmatic thiobarbituric acid reactive substances (TBARS), SOD, and glutathione peroxidase activity, while the vitamin E and zinc concentration decreased in these patients [44, 69]. Glutathione (GSH) is present in nearly all cells in the body and is present in high levels in organs with high oxygen consumption and energy production, e.g., the brain [67, 70]. Glutathione, in conjunction with its oxidized form (GSSG), plays a major role in controlling cellular redox state. The ubiquitous thioredoxin system also plays an important role in maintaining the cell's redox state [67, 71]. Finally, ferritin is considered an endogenous antioxidant as it performs the important function of sequestering potentially toxic labile iron. When endogenous antioxidants are unable to neutralize oxidative stress, as in β-thalassemia, exogenous antioxidants can be used to augment the antioxidant system of the body. Iron metabolism underlies the dynamic interplay between oxidative stress and antioxidants in many pathophysiological processes. Iron overload can affect redox state, and not only this condition can be restored to physiological conditions using iron chelation, but also the addition of antioxidants to these treatment regimens can be a viable therapeutic approach for attenuating tissue damage induced by oxidative stress (**Table 1**), (**Figure 3**, [72–74]). Vitamin A (β-carotene), vitamin C, vitamin E (α-tocopherol), polyphenols, and other bioactive plant-derived compounds are effective exogenous antioxidants that also regulate iron metabolism. At the transcriptional level, antioxidant enzymes are regulated by the transcription factor Nrf2, which binds to the antioxidant response element (ARE) in the target gene's promoter region. Nrf2 is believed to be phosphorylated by protein kinase C (PKC), which causes the transcription factor to translocate to the nucleus, where it activates ARE-containing genes [67, 75], ultimately leading

**99**

**Table 1.**

*Mechanisms of iron regulation by antioxidants.*

*Oxidative Stress and Iron Overload in β-Thalassemia: An Overview*

**Antioxidant Mechanisms of iron regulation Sources of** 

• Attenuated lipopolysaccharide (LPS)-induced oxidative stress-related inflammation

• Activated hepatic IRPs and TfR1, repressed hepatic

• Increased BMP6, intranuclear SMAD4, SMAD4 binding to the HAMP promoter, and hepcidin

• Decreased ROS production and membrane lipid peroxidation by iron chelation in iron-overload A549 cells and activation of antioxidant catalase

• Increased HAMP promoter activity in both zebrafish and human hepatocytes via Stat3- and

**Silymarine** • Iron-chelating properties *Silybum marianum*

• Increased hepatic antioxidant and mitochondrial

cular cell function, low density lipoprotein (LDL)

**antioxidants**

Curcumin is a bright yellow chemical produced by *Curcuma longa* plants

Vegetables, leaves, grains, red onions, kale, red wine, and tea

Citrus fruits

extract

Lupin, fava beans, soy beans, kudzu, psoralea, *Maackia amurensis*, and *Flemingia vestita*

Vegetables, popcorn, bamboo shoots, cereals (bran, wheat, and barley grain)

Skin of grapes, blueberries, raspberries, mulberries, peanuts, and red wine

to the neutralization of free radicals and the attenuation of oxidative damage [76]. **Table 1** summarizes the flavonoids and other antioxidants that regulate both iron homeostasis and redox state, in some cases via independent mechanisms. Flavonoids are present in a wide variety of plants and represent the most common class of polyphenols, organic chemicals that protect the plant from ultraviolet radiation, pathogens, and effects of oxidative stress, making them suitable for therapeutic purposes [77, 78]. Examples of flavonoids include quercetin, cathechins, curcumin, and kaempferol, which are abundant in fruits, vegetables, legumes, red wine, and green tea. Curcumin is a potent flavonoid antioxidant that can chelate iron in addition to modulating redox state [79]. A flavonoid-rich extract of orange and bergamot juice has been shown to chelate iron in iron-overload A549 cells and to activate the antioxidant enzyme catalase, leading to a decrease in ROS production and membrane lipid peroxidation [80]. It is a promising candidate for regulating both oxidative stress and iron homeostasis. Quercetin can reduce hepatic iron deposition in mice

*DOI: http://dx.doi.org/10.5772/intechopen.90492*

**Curcumin** • Potent flavonoid antioxidant

**Quercetin** • Decreased hepatic iron levels

**Flavonoid-rich extract of orange and bergamot juice** expression

enzyme

**Genistein** • Reduced inflammation induced by ethanol and oxidative stress in mice

Smad4-dependent process

**Ferulic acid** • Decreased iron-induced oxidative stress, reduced liver injury, and ROS production

membrane potential

**Resveratrol** • Reduced myocardial damage by modulating vas-

oxidation, and platelet aggregation

• Iron chelator

• Redox state modulator • Decreased iron levels

hepcidin and ferritin synthesis

• Reduced iron-related damage


#### **Table 1.**

*Beta Thalassemia*

of which occur in β-thalassemia. In EPO-dependent fetal liver erythropoietic cells from β-thalassemic mice, the expression of HO-1 was augmented. The administration of tin protoporphyrin IX, an HO-1 inhibitor, improved ineffective erythropoi-

Various antioxidant enzyme systems are activated by the oxidative stress to protect the body tissues from its damaging effects in β-thalassemia patients. These antioxidants include superoxide dismutase (SOD), catalase, glutathione (GSH),

duced, and this radical can be neutralized by SOD. There are three distinct SODs: SOD1 (cu/Zn-SOD) is present in cytoplasm, whereas SOD2 (Mn-SOD) is present in the mitochondria, and SOD3 is almost exclusively extracellular [66, 67]. Each of these distinct SODs performs a specific function in human cells. In β-thalassemia, major patients higher levels of erythrocyte superoxide dismutase and glutathione peroxidase (GPx) as well as higher plasma malondialdehyde (MDA) were observed as compared to healthy controls [68]. Iron overload through repeated blood transfusions and subsequent oxidative stress produced by reactive oxygen species may be the cause of increased levels of MDA. The rise in SOD and glutathione peroxidase may occur as a result of compensatory mechanisms in response to oxidative stress

products by a catalase and glutathione peroxidase (GPx) in conjunction with glutathione. Location of GPx depends on the subtype, whereas catalase is present in peroxisomes [67]. The stability of the cellular and subcellular membranes depends mainly on glutathione peroxidase, and the protective antioxidant effect of glutathione peroxidase depends on the presence of selenium. In patients with β-thalassemia, major was confirmed the peroxidative status generated by iron overload and the high increase in serum ferritin, iron, plasmatic thiobarbituric acid reactive substances (TBARS), SOD, and glutathione peroxidase activity, while the vitamin E and zinc concentration decreased in these patients [44, 69]. Glutathione (GSH) is present in nearly all cells in the body and is present in high levels in organs with high oxygen consumption and energy production, e.g., the brain [67, 70]. Glutathione, in conjunction with its oxidized form (GSSG), plays a major role in controlling cellular redox state. The ubiquitous thioredoxin system also plays an important role in maintaining the cell's redox state [67, 71]. Finally, ferritin is considered an endogenous antioxidant as it performs the important function of sequestering potentially toxic labile iron. When endogenous antioxidants are unable to neutralize oxidative stress, as in β-thalassemia, exogenous antioxidants can be used to augment the antioxidant system of the body. Iron metabolism underlies the dynamic interplay between oxidative stress and antioxidants in many pathophysiological processes. Iron overload can affect redox state, and not only this condition can be restored to physiological conditions using iron chelation, but also the addition of antioxidants to these treatment regimens can be a viable therapeutic approach for attenuating tissue damage induced by oxidative stress (**Table 1**), (**Figure 3**, [72–74]). Vitamin A (β-carotene), vitamin C, vitamin E (α-tocopherol), polyphenols, and other bioactive plant-derived compounds are effective exogenous antioxidants that also regulate iron metabolism. At the transcriptional level, antioxidant enzymes are regulated by the transcription factor Nrf2, which binds to the antioxidant response element (ARE) in the target gene's promoter region. Nrf2 is believed to be phosphorylated by protein kinase C (PKC), which causes the transcription factor to translocate to the nucleus, where it activates ARE-containing genes [67, 75], ultimately leading

<sup>−</sup>) is the first reactive radical pro-

<sup>−</sup> produces H2O2, which can be metabolized into nontoxic

esis and Hb levels and decreased spleen size and liver iron [64, 65].

**6. Potential role of antioxidants in β-thalassemia**

thioredoxin (Trx), and ferritin. Superoxide (O2

[44]. Neutralization of O2

**98**

*Mechanisms of iron regulation by antioxidants.*

to the neutralization of free radicals and the attenuation of oxidative damage [76]. **Table 1** summarizes the flavonoids and other antioxidants that regulate both iron homeostasis and redox state, in some cases via independent mechanisms. Flavonoids are present in a wide variety of plants and represent the most common class of polyphenols, organic chemicals that protect the plant from ultraviolet radiation, pathogens, and effects of oxidative stress, making them suitable for therapeutic purposes [77, 78]. Examples of flavonoids include quercetin, cathechins, curcumin, and kaempferol, which are abundant in fruits, vegetables, legumes, red wine, and green tea. Curcumin is a potent flavonoid antioxidant that can chelate iron in addition to modulating redox state [79]. A flavonoid-rich extract of orange and bergamot juice has been shown to chelate iron in iron-overload A549 cells and to activate the antioxidant enzyme catalase, leading to a decrease in ROS production and membrane lipid peroxidation [80]. It is a promising candidate for regulating both oxidative stress and iron homeostasis. Quercetin can reduce hepatic iron deposition in mice

**Figure 3.**

*Summary of the mechanisms regulating iron and oxidative stress by antioxidants. BMP6-SMAD-HAMP: Bone morphogenetic factor-mothers against decapentaplegic homolog-hepcidin antimicrobial peptide; GPx: Glutathione peroxidase;Nrf2-ARE: Nuclear factor erythroid 2-related factor 2-antioxidant response element; SOD: Superoxide dismutase.*

that were exposed to either ethanol or excess iron and increase BMP6, intranuclear SMAD4, SMAD4 binding to the HAMP promoter, and hepcidin expression, leading to decreased hepatic iron levels and reduced iron-related damage [81]. Another potent antioxidant is genistein. It reduces inflammation induced by ethanol and oxidative stress in mice [82] and, similar to quercetin, increases HAMP promoter activity in both zebrafish and human hepatocytes via Stat3- and Smad4-dependent process [83]. Silymarin, another flavonoid, is present in milk thistle plant extract and may have iron-chelating properties [84]. It is safe, well tolerated, cost-effective alternative to currently available iron chelation therapies for treating patients with β-thalassemia [84]. Ferulic acid is present in a wide variety of plants, and the antioxidant effects are believed to be mediated via the neutralization of free radicals [85]. The antioxidant effects of resveratrol may prevent adverse changes that lead to cardiovascular disease by modulating vascular cell function, low density lipoprotein (LDL) oxidation, and platelet aggregation, thereby reducing myocardial damage [86, 87]. Both vitamin A and vitamin C have well-established antioxidant properties that are mediated via the attenuation of oxidative damage [88]. Vitamin A and β-carotene increase hepcidin and TfR expression and intestinal iron absorption, reduce inflammatory signaling and ferroportin expression, increase intracellular ferritin levels, and release intracellular trapped iron [89–91]. Vitamin C reduces Fe3+ to Fe2+ and inhibits hepcidin expression [92]. In recent years, research for new therapies based on plant-derived compounds has developed considerably. This is to maximize the benefits of plant phytochemicals and avoid the adverse effects often associated with synthetic pharmaceutical agents [93]. Several plant extracts, such as tucum-do-cerrado, astragalus, *Angelica sinensis*, *Caulis Spatholobi*, *Scutellaria baicalensis*, and others, have been studied for their putative effects on iron homeostasis and oxidative stress. The results obtained are very promising (for esaustive review, see Ref. [76]).

**101**

**Author details**

Nadia Maria Sposi

Center for Gender-Specific Medicine, Istituto Superiore di Sanità, Rome, Italy

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: nadia.sposi@iss.it

provided the original work is properly cited.

*Oxidative Stress and Iron Overload in β-Thalassemia: An Overview*

pharmaceutical agents are associated with adverse side effects.

Alteration in iron homeostasis is associated with oxidative stress and inflammation. Many bioactive antioxidants and plant-derived phytochemicals can regulate iron homeostasis, inflammation, and oxidative stress. Nevertheless, the majority of data collected to date are derived from in vitro and animal experiments, and further studies are needed in order to evaluate the efficacy of these phytochemicals as a natural substitute for pharmaceutical agents. This is very important because many

*DOI: http://dx.doi.org/10.5772/intechopen.90492*

**7. Conclusions**
