**3. Glutathione S-transferases (GSTs, E.C. 2.5. 1.18) with diverse functions**

GSTs are found in almost every species, including plants [4] microorganisms [5,6], and animals [7]. GSTs are divided into classes based on their amino acid sequence, immunological, kinetic, and structural properties. In mammals, at least nine classes of GSTs have been identified, namely, Alpha, Mu, Pi, Theta, Omega, Sigma, Zeta, Kappa, and a microsomal class. Human GSTs have been reviewed in reference [8, 9], and [10]. The majority of GSTs are found mainly in the cytosol. Each class consists of one or more protein isoforms. The classes are defined such that the amino acid identity between two isoforms of the same class is more than 50% but more than 30% if they are in different classes [11]. Human cytosolic GSTs are not only in cytoplasm but may also be localized in the mitochondria or the nucleus [12]. The microsomal family of membrane-bound GSTs is also reported and is different from cytosolic GSTs in molecular weight, subunit structure, and immunological reactivity [13-15]. The microsomal GSTs are trimeric, membrane-bound proteins. Mitochondrial GST 13-13 previously purified from rat liver [16] has been later characterized as GSTK1-1 of a Kappa class GST [17]. The Kappa class GSTs are located in mammalian mitochondria and peroxisomes [18,19] and are structurally distinct from the microsomal and cytosolic GSTs [20].

GSTs have a broad and overlapping specificity. Among the reactions catalyzed by GSTs are the substitution of halogens in halogenohydrocarbon, the addition to double bonds, the cleavage of epoxides, and the reduction of organic peroxides. 1-Chloro-2,4-dinitrobenzene (CDNB) is the most common substrate used to assay GSTs in the laboratory as most, but not all, GSTs show catalytic activity with it. Other substrates that have been commonly used to characterize the enzymes are 1,2-dichloro-4-nitrobenzene (DCNB), trans-4-phenyl-3-butene-2one (PBO), ethacrynic acid (EA), 1,2-epoxy-3-nitrophenoxypropane (EPNP), *p*-nitrobenzyl chloride (NBC), and sulfobromophthalein (BSP) (Figure 1).

GSTs catalyze the nucleophilic attack by the thiol group of reduced glutathione (GSH) on a wide range of electrophilic substrates. GSTs play important roles in the development of resistance to a variety of exogenous xenobiotics, such as chemotherapeutic drugs [21], chemical carcinogens [22], herbicides [4], and insecticides [7].

#### **3.1. Conjugation of exogenous toxins (Biotransformation)**

crystalline solid with a melting point of 192–195°C and molecular weight of 307.33. It dissolves readily in water. There are two peptide bonds, two carboxylic acid groups (p*K*COOH = 3.53 and 2.12), one amino group (p*K*NH3+ = 8.66), and one thiol group (pHSH = 9.66). GSH is found intracellularly in all mammalian tissues and is the major nonprotein thiol compound present in the cell, with concentrations ranging from 0.1 to 10 mM. It is involved in a variety of metabolic processes, for instance, detoxication of xenobiotics, reduction of hydoperoxides, synthesis of leukotrienes and prostaglandins, maintenance of protein and membrane struc‐ tures, and regulation of numerous enzyme activities. This functional diversity is due to the properties of the thiol group that participates in redox transitions, thiol exchange reactions,

A variety of different enzymes utilize glutathione in a variety of biotransformations [1]. Glutathione reductase (GR) catalyses the reduction of GSSG (oxidized glutathione) using NADPH as a reductant. GR is important in maintaining the high cellular reduction potential. Selenium-dependent glutathione peroxidase (GPOX) is another type of GSH-requiring enzyme that catalyses the reduction of peroxides using GSH as the reducing agent. There are the glutathione S-transferases (GSTs) that are also GSH dependent enzymes with many

**3. Glutathione S-transferases (GSTs, E.C. 2.5. 1.18) with diverse functions**

GSTs are found in almost every species, including plants [4] microorganisms [5,6], and animals [7]. GSTs are divided into classes based on their amino acid sequence, immunological, kinetic, and structural properties. In mammals, at least nine classes of GSTs have been identified, namely, Alpha, Mu, Pi, Theta, Omega, Sigma, Zeta, Kappa, and a microsomal class. Human GSTs have been reviewed in reference [8, 9], and [10]. The majority of GSTs are found mainly in the cytosol. Each class consists of one or more protein isoforms. The classes are defined such that the amino acid identity between two isoforms of the same class is more than 50% but more than 30% if they are in different classes [11]. Human cytosolic GSTs are not only in cytoplasm but may also be localized in the mitochondria or the nucleus [12]. The microsomal family of membrane-bound GSTs is also reported and is different from cytosolic GSTs in molecular weight, subunit structure, and immunological reactivity [13-15]. The microsomal GSTs are trimeric, membrane-bound proteins. Mitochondrial GST 13-13 previously purified from rat liver [16] has been later characterized as GSTK1-1 of a Kappa class GST [17]. The Kappa class GSTs are located in mammalian mitochondria and peroxisomes [18,19] and are structurally

GSTs have a broad and overlapping specificity. Among the reactions catalyzed by GSTs are the substitution of halogens in halogenohydrocarbon, the addition to double bonds, the cleavage of epoxides, and the reduction of organic peroxides. 1-Chloro-2,4-dinitrobenzene (CDNB) is the most common substrate used to assay GSTs in the laboratory as most, but not all, GSTs show catalytic activity with it. Other substrates that have been commonly used to characterize the enzymes are 1,2-dichloro-4-nitrobenzene (DCNB), trans-4-phenyl-3-butene-2-

catalytic activities including the conjugation of GSH to xenobiotics [2,3].

distinct from the microsomal and cytosolic GSTs [20].

thioether formation, and radical scavenging.

316 Insecticides Resistance

GSTs play important roles in the protection of macromolecules from attack by reactive electrophiles. The enzymes generally exist in dimeric forms with a subunit molecular weight of approximately 26 kDa. GSTs occur both as homo- and heterodimers. The cytosolic isoen‐ zymes have two active sites per dimer that behave independently of one another [23]. Each active site consists of at least two ligand-binding regions, namely, the GSH binding site (hydrophilic G-site), which is specific for GSH, and the electrophile-binding site (hydrophobic H-site), which is less specific and thus enables GSTs to react with a wide variety of xenobiotics [24]. A review in reference [25] listed xenobiotics that could be conjugated by GSTs. These include halogenonitrobenzenes, organophosphorus compounds, steroids, αβ-unsaturated carbonyl compounds, aryl halides epoxides, quinones, isothiocyanates, and arylnitro com‐ pounds.

The conjugations catalyzed by the GSTs occur between the nucleophilic GSH and the com‐ pounds possessing a sufficiently electrophilic center [25]. The GSTs function by decreasing the p*K*a of GSH, thereby allowing its deprotonation and the formation of a more reactive thiolate anion. In most GST classes (Pi, Mu, Alpha, and Sigma), a tyrosine residue in the N-terminal region interacts with GSH to stabilize the thiolate anion. In Theta and Omega classes, this role is carried out by serine and cysteine residues, respectively [9].

This GSH conjugation has been shown to occur in mammals, birds, reptiles, amphibians, fish, insects, and other invertebrates [26], and it is the first step of mercapturic acid formation that is one of the metabolic pathways for detoxication of xenobiotics *in vivo*. The glutathione conjugates, which are water soluble and generally nontoxic, may be converted to the corre‐ sponding cysteine conjugate following sequential removal of glutamate and glycine. The cysteine conjugate is either N-acetylated to be excreted as a mercapturic acid or cleaved to a mercaptan by β-lyase. The thiol can be then further metabolized, for example, to be excreted as a glucuronide [26, 27].

Prostaglandin D-synthase, the enzyme involved in production of the D and J series of prosta‐ noids, was characterized as belonging to the Sigma class of GSTs [28]. GSTs also participate in the isomerization of biologically active molecules. A prostaglandin-H E-isomerase of *Ascaridia galli* [29] and prostaglandin-H D-isomerase of rat spleen [30] were identified as Sigma class GSTs. GSTs can also catalyze *cis–trans* isomerizations, for example, the isomerization of maleylacetoacetic acid to fumarylacetoacetic acid. The maleylacetoacetate isomerase activity of hGSTZ1-1 (human GST Zeta 1-1) has been investigated [31] using a spectrophotometric assay with (±)-2-bromo-3-(4-nitrophenyl)propanoic acid (BNPP) as substrate. Some GSTs

**Figure 1.** Enzymatic conjugation of common substrates by GST in the presence of GSH. (A) CDNB, (B) DCNB, (C) PBO, (D) EA, (E)EPNP, and (F) BSP.

possess keto-steroid isomerase activity and catalyze the conversion of Δ<sup>5</sup> -3-androstene-3,17 dione to Δ<sup>4</sup> -3-androstene-3,17-dione [21,32].

Human GST A3-3 was shown to efficiently catalyze the double-bond isomerization of Δ<sup>5</sup> androstene-3,17-dione and Δ<sup>5</sup> -pregnene-3,20-dione [12]. Human GSTs were reported to act as retinoic isomerases that catalyze the steric conversion of 13-*cis*-retinoic acid (13-cRA) to all*trans*-retinoic acid (t-RA) [33].

The Role of Glutathione Transferases in the Development of Insecticide Resistance http://dx.doi.org/10.5772/61972 319

**Figure 2.** Some examples of GSTs with isomerase and synthase functions. (adapted from [9])

#### **3.2. Participation in countering oxidative stress**

possess keto-steroid isomerase activity and catalyze the conversion of Δ<sup>5</sup>

Human GST A3-3 was shown to efficiently catalyze the double-bond isomerization of Δ<sup>5</sup>

**Figure 1.** Enzymatic conjugation of common substrates by GST in the presence of GSH. (A) CDNB, (B) DCNB, (C)

retinoic isomerases that catalyze the steric conversion of 13-*cis*-retinoic acid (13-cRA) to all-



dione to Δ<sup>4</sup>

318 Insecticides Resistance

androstene-3,17-dione and Δ<sup>5</sup>

PBO, (D) EA, (E)EPNP, and (F) BSP.

*trans*-retinoic acid (t-RA) [33].



Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and the hydroxyl radical are constantly produced during normal aerobic metabolism. The free radicals may attack the polyunsaturated fatty acyl moieties and lead to the peroxidation of lipid biomembranes. The cleavage of polyunsaturated fatty acids is known to be associated with formation of organic hydroperoxides and reactive aldehydes. These include alkanals, alkenals, and malondialdehyde. These may interfere with several biological processes such as DNA and protein synthesis by inhibition of specific enzyme reactions [34,35].

Peroxidized lipids, produced during oxidative stress, are substrates for GSTs. 4-Hydroxyal‐ kenals are derived from membrane-bound phospholipid hydroperoxides. They were shown to be efficiently conjugated to GSH by GST A4-4 [36,37]. A membrane-bound mouse GSTA4-4 [38]and a rat GSTA8-8 [39,40] were also shown efficiently conjugating 4-hydroxyalkenals. It was reported that transfected mGSTA4 protects HL 60 [41] and K562 human erythroleukemia cells [42] against 4-hydroxynonenal-induced apoptosis.

Free radicals can also cause DNA peroxidation. The toxicity of thymine propenal, which is generated by oxidative damage to DNA, was shown to be substantially reduced when HeLa cells received GSTP1-1 and GSH [43]. The rat GST6-6 had previously shown active toward thymine hydroperoxide [44].

Within the cells, peroxides occur either as hydrogen peroxide (H2O2) or organic hydroperox‐ ides, such as fatty acid and phospholipid hydroperoxides. GSTs protect tissues from endoge‐ nous organic hydroperoxides produced during oxidative stress [35,45]. Some GSTs have shown selenium-independent H2O2 and organic hydroperoxidase activity, which are involved in free radical reactions during oxidative stress [46]. A microsomal GST A1-1 of sheep liver exhibited peroxidase activity toward fatty acid hydroperoxides such as linoleic and arachi‐ donic acid hydroperoxides [47]. Human GSTs, such as hGSTA1-1 and hGSTA2-2 [48,49], also exhibited glutathione peroxidase activity toward phospholipid hydroperoxide [50]. Other workers observed elevated GSTs in *Nilaparvata lugens* when treated with pyrethroids, which induces oxidative stress and lipid peroxidation in insects [51].

#### **3.3. Involve in cells regulatory functions**

Recent studies of GSTs have demonstrated that a Pi class GST is involved in regulation of c-Jun N-terminal kinase (JNK) signaling in mammals. GSTP interacts with c-Jun N-terminal kinase 1 (JNK1) suppressing the basal kinase activity [52,53]. A model of GST inhibition of JNK signaling was proposed [52]. Under a nonstressed condition, GSTp can exist as free dimeric enzyme or complexed with Jun–JNK thus inhibiting JNK. Upon stress, GSTP forms larger aggregates, which are unable to associate with the Jun–JNK complex, thus enabling the JNK phosphorylation of c-Jun. Phosphorylated Jun can act as a stable and active transcription factor. The accumulation of ROS in response to oxidative stress results in the activation of multiple stress kinase cascades and an elevated level of GSTp expression [54].

Apoptosis signal-regulating kinase 1 (ASK 1) can activate the JNK and the p38 signaling pathways. It plays important role in stress-induced apoptosis. Mouse GSTM1-1 was shown to physically interact with ASK1 and repress ASK1-mediated signaling [55,56].

It has also been reported that human GST class Omega, GSTO1-1, modulates calcium channel (ryanodine receptors, RyRs) protecting mammalian cells from apoptosis induced by calcium (Ca2+) mobilization [57]. It was suggested that RyRs has two binding sites for GSTO1-1. The mammalian protein Bax (21 kDa) is an inducer of apoptosis. A study [58] has reported a plant GST (Theta class) as one of the Bax-inhibiting plant proteins, which prevent apoptosis in plants. GSTP1-1 was proposed [59] interacting with physiological nitric oxide (NO) carriers such as S-nitrosoglutathione (GSNO) and dinitrosyl-diglutathionyl iron complex (DNDGIC). In the absence of GSH, GSNO interacts with and modifies Cys47 and Cys101 residues of GSTP1-1 by an *S*-nitrosylation reaction. Thus, in the cellular depletion of GSH, GSTP1-1 acts directly as an NO carrier without losing its detoxication activity. The expression of 'tissue' transglutaminase (tTG) is induced in cells programmed to die such as in cells undergoing apoptosis. It was reported that [60] the overexpression of tTG in human neuroblastoma cells increases apoptosis. The study showed that tTG interacts with β-tubulin, histone H2B, and GSTP1-1 to form a protein complex. It was proposed that the interaction with tTG resulted in oligomerization of GSTP1-1. The formation of multimers of GSTP1-1 leads to inactivation of the enzyme toward ROS.
