**3. The biological role of glutathione and glutathione S-transferases in oxidative stress**

Numerous studies have shown that in order to avoid prolonged exposure to ROS produced during oxidative stress, the body has a very efficient antioxidant defense system. Glutathione S-transferases (GSTs) and glutathione (GSH) enzymes are part of this line of defense [21].

Glutathione (GSH) is a low molecular weight thiol found in all tissues, primarily in aerobic organisms. Also known as L-gamma-glutamyl-L-cysteinyl-glycine, GSH is a linear tripeptide consisting of three amino acids: glutamic acid, cysteine and glycine (**Figure 1**). Between the γ-glutamyl moiety and the free α-carboxylate group, there is a γ-peptide bond which, although unusual, prevents the hydrolysis of GSH by cellular peptidases [22].

In homeostasis conditions, GSH is the most efficient physiological reducing agent with the highest bioavailability (~ 10 mM) in the intracellular environment where it is synthesized,

**Figure 1.** Schematic representation of the GSSG reduction cycle by GR.

except in epithelial cells [23]. Its synthesis occurs in two phases and counts on the action of two enzymes: γ-glutamyl-cysteine-synthetase and glutathione-synthetase [24].

In the first phase, the γ-glutamyl-cysteine-synthetase enzyme favors the formation of the peptide bond between glutamic acid and cysteine, thus forming the dipeptide γ-L-glutamyl-L-cysteine [25]. In the second phase, the enzyme glutathione synthetase binds the newly formed dipeptide to glycine, giving rise to GSH which is distributed through the bloodstream and then brought to the tissues. In both phases, consumption of ATP and Mg+2 occurs. The regulation of the enzyme γ-glutamyl-cysteine-synthetase is done, by negative feedback, when the GSH itself begins to be formed. This regulatory mechanism ensures that, in normal conditions, the excessive production of GSH or the intermediate γ-L-glutamyl-L-cysteine does not occur (**Figure 2**) [22, 24].

An alternative route is activated in situations where conversion of γ-glutamyl-L-cysteine into GSH is insufficient. In this case, the enzyme γ-glutamylcyclotransferase catalyzes the conversion of γ-glutamyl-L-cysteine to 5-oxoproline, favoring the occurrence of 5-oxoprolinuria, chronic metabolic acidosis and neurological disorders (**Figure 2**) [22].

During the reaction catalyzed by γ-glutamylcysteine synthetase, activation of butionin sulfoximine (BSO), an inhibitor of GSH biosynthesis, may occur. Studies suggest that this suppression of GSH by BSO may be a rather efficient strategy in cancer therapy since, during this process, there is an increase in the sensitivity of cells to ionizing radiation and to cytostatic drugs, making them more susceptible to treatment. However, the disadvantage of this technique is that the toxic effect to normal cells has potency detrimental to the individual. An alternative to limit this toxicity would be the use of localized irradiation or the topical application of cytostatic drugs, but other studies are being carried out [26].

involved in the metabolism of eicosanoids and glutathione (GSH), thus being referred to as MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) [22]. It is important to note that other families of GSTs, absent in mammals, are also described in the literature. Cytosolic and mitochondrial GSTs are expressed in different isoforms and therefore divided into classes according to the amino acid and/or nucleotide sequence, immunological properties, enzymatic kinetic parameters and/or tertiary and quaternary structure

**Figure 2.** Scheme representing the biosynthesis and mechanism of regulation of glutathione (GSH). BSO, butionin

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Based on the similarity of the amino acid sequence, GSTS found in the cytosol are called α (GSTA), μ (GSTM), θ (GSTT), π (GSTP), σ (GSTS), omega (GSTO), and zeta (GSTZ) [28, 31]. The mitochondrial GST is known as kappa (GSTK) [31]. Mammalian cytosolic GSTs are all dimeric and contain 199–244 amino acid residues in their primary structures. Mitochondrial GSTs are also dimeric proteins and their subunits typically have 226 amino acid residues. Each of these enzymes differs in their functionality [22, 33]. It is suggested that in humans, the expression of these enzymes is uniform and independent of the cell type. GSTs have long been described as originating from mitochondria; however, recent studies indicate the presence of mitochondrial GSTs in peroxisomes. These findings reinforce their participation in the detoxification processes of by-products of β-oxidation of

During the detoxification process, the GSTs catalyze the conjugation of xenobiotics with endogenous substrates, mainly GSH. This conjugate is recognized by specific transporters and is carried to the intercellular medium where it undergoes action of γ-glutamyl transpeptidase (γGT) which removes the glutamic acid residue [32]. In sequencing, the dipeptidases remove the glycine residue, leaving only the cysteine residue associated with the xenobiotic. The

[22, 29, 30].

sulfoximine; Mg+2, magnesium; ATP, adenosine triphosphate.

fatty acids [22].

Glutathione can be found in the intracellular medium in its reduced (GSH) or oxidized form (GSSG, dimerized form of GSH) and the GSH/GSSG ratio determines the redox state of biological systems. This is because glutathione performs a cytotoxic and genotoxic inactivation of xenobiotics and consequently promotes detoxification and cellular protection against oxidative stress and additional damage [27].

The cellular detoxification process is divided into three distinct but related phases. In phases I and II, the xenobiotic is transformed into a more soluble and less toxic product and, in phase III, are transported for cellular excretion. It is noteworthy that the efficiency of phase II depends on the action of enzymes called glutathione S-transferases (GSTs) [22].

The GSTs belong to a superfamily of multigenic enzymes that catalyze the nucleophilic attack of the reduced form of Glutathione (GSH) to compounds that present a carbon, a nitrogen or an electrophilic sulfur atom [21]. Under natural conditions, GSTs are generally found in the biological environment as homo or heterodimers. Each dimer contains two active sites with independent activities. Each site has at least two binding regions: one specific for glutathione (GSH) and the other, with less specificity, for the electrophiles (alkyl halides, epoxides, quinones, iminoquinones, aldehydes, ketones, lactones and esters, halides of aryl and aromatic nitro) [22, 28].

Mammalian GSTs are divided into families according to their location: cytosolic, mitochondrial and microsomal. The cytosolic and mitochondrial GST enzymes are soluble, unlike the microsomal GSTs that are associated with the membrane [29]. This latter family is generally Impact of Oxidative Changes and Possible Effects of Genetics Polymorphisms of Glutathione… http://dx.doi.org/10.5772/intechopen.76222 51

except in epithelial cells [23]. Its synthesis occurs in two phases and counts on the action of

In the first phase, the γ-glutamyl-cysteine-synthetase enzyme favors the formation of the peptide bond between glutamic acid and cysteine, thus forming the dipeptide γ-L-glutamyl-L-cysteine [25]. In the second phase, the enzyme glutathione synthetase binds the newly formed dipeptide to glycine, giving rise to GSH which is distributed through the bloodstream and then brought to the tissues. In both phases, consumption of ATP and Mg+2 occurs. The regulation of the enzyme γ-glutamyl-cysteine-synthetase is done, by negative feedback, when the GSH itself begins to be formed. This regulatory mechanism ensures that, in normal conditions, the excessive production

An alternative route is activated in situations where conversion of γ-glutamyl-L-cysteine into GSH is insufficient. In this case, the enzyme γ-glutamylcyclotransferase catalyzes the conversion of γ-glutamyl-L-cysteine to 5-oxoproline, favoring the occurrence of 5-oxoprolinuria,

During the reaction catalyzed by γ-glutamylcysteine synthetase, activation of butionin sulfoximine (BSO), an inhibitor of GSH biosynthesis, may occur. Studies suggest that this suppression of GSH by BSO may be a rather efficient strategy in cancer therapy since, during this process, there is an increase in the sensitivity of cells to ionizing radiation and to cytostatic drugs, making them more susceptible to treatment. However, the disadvantage of this technique is that the toxic effect to normal cells has potency detrimental to the individual. An alternative to limit this toxicity would be the use of localized irradiation or the topical applica-

Glutathione can be found in the intracellular medium in its reduced (GSH) or oxidized form (GSSG, dimerized form of GSH) and the GSH/GSSG ratio determines the redox state of biological systems. This is because glutathione performs a cytotoxic and genotoxic inactivation of xenobiotics and consequently promotes detoxification and cellular protection against oxida-

The cellular detoxification process is divided into three distinct but related phases. In phases I and II, the xenobiotic is transformed into a more soluble and less toxic product and, in phase III, are transported for cellular excretion. It is noteworthy that the efficiency of phase II

The GSTs belong to a superfamily of multigenic enzymes that catalyze the nucleophilic attack of the reduced form of Glutathione (GSH) to compounds that present a carbon, a nitrogen or an electrophilic sulfur atom [21]. Under natural conditions, GSTs are generally found in the biological environment as homo or heterodimers. Each dimer contains two active sites with independent activities. Each site has at least two binding regions: one specific for glutathione (GSH) and the other, with less specificity, for the electrophiles (alkyl halides, epoxides, quinones, iminoquinones, aldehydes, ketones, lactones and esters, halides of aryl and aromatic nitro) [22, 28]. Mammalian GSTs are divided into families according to their location: cytosolic, mitochondrial and microsomal. The cytosolic and mitochondrial GST enzymes are soluble, unlike the microsomal GSTs that are associated with the membrane [29]. This latter family is generally

depends on the action of enzymes called glutathione S-transferases (GSTs) [22].

two enzymes: γ-glutamyl-cysteine-synthetase and glutathione-synthetase [24].

of GSH or the intermediate γ-L-glutamyl-L-cysteine does not occur (**Figure 2**) [22, 24].

chronic metabolic acidosis and neurological disorders (**Figure 2**) [22].

tion of cytostatic drugs, but other studies are being carried out [26].

tive stress and additional damage [27].

50 Glutathione in Health and Disease

**Figure 2.** Scheme representing the biosynthesis and mechanism of regulation of glutathione (GSH). BSO, butionin sulfoximine; Mg+2, magnesium; ATP, adenosine triphosphate.

involved in the metabolism of eicosanoids and glutathione (GSH), thus being referred to as MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) [22]. It is important to note that other families of GSTs, absent in mammals, are also described in the literature. Cytosolic and mitochondrial GSTs are expressed in different isoforms and therefore divided into classes according to the amino acid and/or nucleotide sequence, immunological properties, enzymatic kinetic parameters and/or tertiary and quaternary structure [22, 29, 30].

Based on the similarity of the amino acid sequence, GSTS found in the cytosol are called α (GSTA), μ (GSTM), θ (GSTT), π (GSTP), σ (GSTS), omega (GSTO), and zeta (GSTZ) [28, 31]. The mitochondrial GST is known as kappa (GSTK) [31]. Mammalian cytosolic GSTs are all dimeric and contain 199–244 amino acid residues in their primary structures. Mitochondrial GSTs are also dimeric proteins and their subunits typically have 226 amino acid residues. Each of these enzymes differs in their functionality [22, 33]. It is suggested that in humans, the expression of these enzymes is uniform and independent of the cell type. GSTs have long been described as originating from mitochondria; however, recent studies indicate the presence of mitochondrial GSTs in peroxisomes. These findings reinforce their participation in the detoxification processes of by-products of β-oxidation of fatty acids [22].

During the detoxification process, the GSTs catalyze the conjugation of xenobiotics with endogenous substrates, mainly GSH. This conjugate is recognized by specific transporters and is carried to the intercellular medium where it undergoes action of γ-glutamyl transpeptidase (γGT) which removes the glutamic acid residue [32]. In sequencing, the dipeptidases remove the glycine residue, leaving only the cysteine residue associated with the xenobiotic. The

It is important to highlight that this mechanism of detoxification via glutathione represents a fundamental biochemical evolution for the survival and guarantee of the perpetuation of many species and, although a co-transport mechanism without conjugate envelopment with glutathione has been proposed, there is no evidence experimental models that validate this

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**4. Oxidative alterations and the pathophysiology of diabetes and its** 

Many studies suggest that patients with diabetes present alterations in the levels of reactive oxygen species (ROS), a type of free radical whose electron is centered in the oxygen atoms [34]. This fact is justified by the toxic character of the persistent excess of glucose in the organism that ends up promoting glycation of proteins, hyperosmolarity and increase in the levels

Glucose is a vital source of energy for cells, and their serum levels are controlled by various organs such as intestine, liver, pancreas, skeletal muscle, adipose tissue and kidneys [36]. This regulation is facilitated by the action of hormones (glucagon and insulin), central and periph-

DM is defined as a heterogeneous group of metabolic disorders characterized by unusual hyperglycemia resulting from defects in insulin production and/or action [1]. In this situation, to revert the toxicity of excess glucose, this component undergoes auto-oxidation and, as

During auto-oxidation, excess glucose binds (protein glycation) [37] to lysine and valine residues in tissue and plasma proteins. This interaction results in the formation of Schiff's base, a labile or unstable compound that spontaneously transforms into ketoamine (glycated hemo-

These oxidation and rearrangement processes, followed by further dehydration and fragmentation of *Amadori* product, promote the formation of advanced glycation end products (AGEs) (**Figure 4**) and generate other compounds with chemically active carbonyl groups. These compounds favor the oxidative stress that affects β cells of the pancreas, responsible for

Accumulated AGEs bind to membrane receptors on endothelial cells and promote the onset of tissue damage and the activation of the proinflammatory pathway that involves the NF*κ*B transcription factor responsible for regulating the expression of other inflammatory cytokines

Moreover, the chronicity of this hostile environment causes the deactivation of the nitric oxide vasodilator (NO) formed by the endothelial cells [38]. This compromises the relaxation of vascular smooth muscle cells and has a degenerative effect on the vessels causing tissue death [34] and favoring the development of microvascular complications of diabetes, such as diabetic

eral nervous system, as well as metabolic requirements of the body [37].

model [22].

**complications**

of sorbitol inside the cells [35].

consequence, ROS are generated (**Figure 4**) [37].

globin) through the *Amadori* rearrangement [35].

synthesizing and secreting insulin [8, 38].

(**Figure 4**) [37].

nephropathy (DN) (**Figure 4**).

**Figure 3.** Schematic representation of main glutathiones S-Transferases correlated with oxidative stress in different biological conditions. A, normal intracellular environment; B, oxidative stress in an individual without polymorphism; C, oxidative stress in an individual with oxidative stress.

amino group of the cysteine residue is then acetylated by the intracellular N-acetyltransferase enzymes and thereby forms the mercapturic acid which, depending on its characteristics, is rapidly led to circulation, bile, urine or metabolized until it is eliminated (**Figure 3**) [22].

Once free, glutamate and glycine are reabsorbed by the cell and used in the regeneration of GSH through the catalytic cycle. In this stage of regeneration, three groups of enzymes are important: glutathione oxidase (GO) and glutathione peroxidase (GSH-Px), which catalyze the oxidation of GSH to GSSG, and the enzyme glutathione reductase (GR) that is responsible for the regeneration of GSH, from GSSG, in the presence of NADPH [33].

It is important to highlight that this mechanism of detoxification via glutathione represents a fundamental biochemical evolution for the survival and guarantee of the perpetuation of many species and, although a co-transport mechanism without conjugate envelopment with glutathione has been proposed, there is no evidence experimental models that validate this model [22].
