**2. Glutathione**

reticulum membrane, but others such as the dehydrogenases for example alcohol dehydro‐ genases and peroxidases located in the cytoplasm, while still others such as monoamine oxi‐ dase are localized in mitochondria. Monooxygenases are also known as mixed function oxidases because in a typical reaction, one molecule of oxygen is consumed (reduced) per substrate molecule: one oxygen atom appearing in the product and the other in a molecule of water. The reaction scope of monooxygenases includes heteroatom oxidation, aromatic and aliphatic hydroxylation, epoxidation, and Baeyer-Villiger oxidation. There are two ma‐ jor types of microsomal monooxygenase, both of which require NADPH as an external re‐ ductant: the cytochrome P450 (CYP) system and flavin-containing monooxygenases. The mechanism of CYP is a complex cascade of individual steps involving the interaction of pro‐ tein redox partners and consumption of reducing equivalents, usually in the form of NADPH. The iron heme containing enzyme, CYP, consists of two enzymes: NADPH–cyto‐ chrome P450 reductase and CYP. It is involved in the oxidative metabolism of many endog‐ enous substances such as steroids and bile acids, as well as the detoxication of a wide variety of xenobiotics. It can oxidize AFB1 to several products. Only one of these, the 8,9 exo-epoxide, appears to be mutagenic and the others are detoxification products. P4503A4, which can both activate and detoxicate AFB1, is found in the liver and the small intestine. [33], [52]. Flavincontaining monooxygenases catalyze an NADPH- and an oxygen-requiring oxidation of substances (primarily xenobiotics) bearing functional groups containing nitro‐ gen, sulfur, or phosphorus. The properties of the CYPs electron transport systems have also

In detoxification pathway, a series of enzyme-catalyzed processes with broad specificities convert the toxic substances into less toxic metabolites by chemical reactions within the body. Although biotransformation reactions take place within cytoplasm and mitochondria but they mostly happen within endoplasmic reticulum (E.R). Cell types also differ in their biotransforming potential for example cells located near the major points of xenobiotic entry into the body such as liver, lung, and intestine possess greater concentrations of biotrans‐

Phase II conjugation reactions which generally act follow phase I activation consists of reac‐ tions in which metabolites containing appropriate functional groups are conjugated with substances such as glucuronate, glutamate, sulfate, reduced glutathione or uridine diphos‐ phate (UDP)-glucuronic acid to finally discharge them through urine or bile. In general, con‐ jugation dramatically improves solubility, which then promotes rapid excretion. Among the several types of conjugation reactions which are present in the body, including glucuronida‐ tion, sulfation, and glutathione and amino acid conjugation, glutathione which is catalyzed by glutathione S-transferases, is the major phase II reaction in many species [52]. With the exception of acetylation, methylation and fatty acid conjugation, the strategy of phase II bio‐ transformation is to convert a xenobiotic to a more hydrophilic form via the attachment of a chemical moiety which is ionizable at physiological pH. This metabolic transformation also

In animals, elimination of the soluble compounds from cells and excretion of biotrans‐ formed molecules from the body referred to as phase III. It has been suggested that the

results in reduced affinity of the compound for its cellular target. [67], [23].

been reported [77].

forming enzymes than others [52].

268 Aflatoxins - Recent Advances and Future Prospects

Glutathione is a ubiquitous thiol-containing isotripeptide (γ-glu-cys-gly, FW 307.3), consist‐ ing of glycine, glutamic acid and cysteine molecules which was first discovered by Sir Fre‐ drick Gowland Hopkins in 1920s, synthesized de novo in mammalian cells (Figure 1). This water soluble antioxidant compound is an unusual peptide in that the peptide bond be‐ tween the glutamate residue and the cysteine residue is formed with the γ-carboxylate group of the former rather than the α-carboxylate group. Today along with β-carotene, as‐ corbic acid (vitamin C), α-tocopherol (vitamin E) and flavonoids *etc*., GSH6 is commonly re‐ ferred to as an antioxidant [17], which neutralizes free radicals due to the high electrondonating capacity of its sulfydryl (-SH) group, [13], and prevents damage to important cellular components, implicates in the cellular defense against xenobiotics. Glutathione sta‐ tus is a highly sensitive indicator of cell functionality and viability. Its levels in human tis‐ sues normally range from 0.1 to 10 mM, being most focused in liver (up to 10 mM) and in the spleen, kidney, lens, erythrocytes and leukocytes and its emptying be joined to a variety of diseases. Under normal conditions, glutathione is predominantly present in its reduced form, with only a small proportion present in its fully oxidized state [20].

Moreover, the GSH/GSSG7 pair with their high reduction potential participates in maintaining other cellular thiol in a reduced state. Finally, GSH tends to a substrate or cofactor in some of

<sup>6 -</sup> Glutathione, reduced form

<sup>7 -</sup> Glutathione, oxidized state

GSH linked enzymes. There are a number of GSH linked enzymes that are involved in cellular protection against toxic substances. The glyoxalase I and II which are responsible for catalyz‐ ing the conversion of methylglyoxal (a by-product in glycolysis) to lactic acid are among these enzymes [76]. Glutathione reductase (GR) which catalyzes the reduction of GSSG using NADPH as a reductant is also a glutathione-linked enzyme involved in cell protection. GR is important to keep the high cellular reductive potential. Selenium dependent glutathione perox‐ idase are other GSH-linked enzymes that catalyze the reduction of peroxides using GSH as the reducing agent [7]. Finally, last but not the least, glutathione transferases are also GSH depend‐ ent enzymes with many properties among which catalyzing the conjugation of GSH to vari‐ ous electrophilic compounds is one of the most investigated function [25].

bacterial fosfomycin-resistance proteins [39], [6], [69]. The cytosolic GSTs (cGSTs) have been subgrouped into numerous divergent classes on the basis of their chemical, physical and structural properties [39], [70]. The mitochondrial GSTs, also known as kappa class GSTs, are soluble enzymes that have been characterized in eukaryotes [65]. The third GST family comprises membrane-bound transferases called membrane-associated proteins involved in ecosanoid and glutathione metabolism, but these bear no similarity to soluble GSTs [44]. Representatives of all three families are also present in prokaryotes but the fourth family is found exclusively in bacteria [4]. The mammalian soluble GSTs are so far divided into eight classes based on their amino acid sequences including: Alpha (α), Kappa (κ), Mu (µ), Omega (ω), Pi (π), Sigma (σ), Theta (θ) and Zeta (ζ), [11], [78], [64], [40]. GSTs are named using a letter corresponding to their class membership and Arabic numerals after the subunit com‐ position (*e.g*. GST A1-1 is a homodimeric alpha class GST consisting of two subunit 1).

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For a long time, GST enzymes from microbial sources were neglected and were not system‐ atically studied. One of the reasons for this was the poor activity of microbial GSTs with

zymes are rare in unicellular organisms [81], [77]. The first evidence for the presence of GSTs in bacteria was reported more 30 years ago by Takashi Shishido who showed the presence of GST activity in a strain of *Escherichia coli* [71]. Since then, GSTs have been found to be broadly distributed in aerobic prokaryotes, but not in anaerobic bacteria [59]. The absence of the enzyme in these microorganisms is consistent with the lack of GSH [28]. Bacterial gluta‐ thione transferases are part of a superfamily of enzymes that play a key role in cellular de‐ toxification. Bacterial GSTs are implicated in a variety of distinct processes such as the biodegradation of xenobiotics, protection against chemical and oxidative stresses and anti‐ microbial drug resistance. In addition to their role in detoxification, bacterial GSTs are also involved in a variety of distinct metabolic processes such as the biotransformation of di‐ chloromethane, the degradation of lignin and atrazine, and the reductive dechlorination of

Until recently, relatively little was known about the presence and role of GST in fungi. How‐ ever, expression of GST has been reported in some fungal species such as *Issatchenkia orienta‐ lis*, [73], *Phanerochaete chrysosporium*, *Yarrowia lipolytica*, *Mucor circinelloides* [70] *Schizosaccharomyces pombe*, *Aspergillus nidulans*, *Aspergillus parasiticus, Aspergillus flavus, As‐ pergillus fumigates* [Burns et al., 2005] *Saccharomyces cerevisiae*, and *Cunninghamella elegans,* [70] [48] *etc*. However, the role of the enzyme in fungi, particularly toxigenic strains, is not well understood [2]. Although it has been shown that GST has a significant role in detoxifi‐

as a model substrate for GST activity, which led to the conclusion that these en‐

**3.2. Presence of GST in cells**

pentachlorophenol [4], [51].

*3.2.2. GSTs of fungi and yeasts*

8 - 1-chloro-2,4-dinitrobenzene

*3.2.1. Microbial GST*

CDNB8

**Figure 1.** Structure of reduced glutathione; glutamate is linked in an isopeptide bond (*via* its γ-carboxyl group) to cys‐ teine, which in turn forms a peptide linkage with glycine

## **3. Glutathione S-transferase**

Glutathione *S*-transferases (GST, EC 2.5.1.18), which first discovered as enzymes in 1961 [12], are abundant proteins encoded by a highly divergent, ancient gene family. These major cellular detoxification enzymes present mostly in liver and kidney as well as intestine. In spite of 40 years of research the exact function of this protein is more complex than ever, but it has been found that these intracellular dimeric proteins, play a major role in the intracellu‐ lar transport of endogenous compounds, metabolizes various electrophilic xenobiotics, li‐ gand transport and thus protects cells against toxic effects [31], [87], [85]. GST catalyzes the conjugation of glutathione on the sulfur atom of cysteine to various electrophiles and cataly‐ ses the conjugation of various electrophiles with GSH, detoxifying both exogenously and en‐ dogenously derived toxic compounds [13].

#### **3.1. Classification and structure**

The superfamily of the glutathione transferases are divided into at least four major families of proteins, namely cytosolic or soluble GSTs, mitochondrial GSTs, microsomal GSTs and bacterial fosfomycin-resistance proteins [39], [6], [69]. The cytosolic GSTs (cGSTs) have been subgrouped into numerous divergent classes on the basis of their chemical, physical and structural properties [39], [70]. The mitochondrial GSTs, also known as kappa class GSTs, are soluble enzymes that have been characterized in eukaryotes [65]. The third GST family comprises membrane-bound transferases called membrane-associated proteins involved in ecosanoid and glutathione metabolism, but these bear no similarity to soluble GSTs [44]. Representatives of all three families are also present in prokaryotes but the fourth family is found exclusively in bacteria [4]. The mammalian soluble GSTs are so far divided into eight classes based on their amino acid sequences including: Alpha (α), Kappa (κ), Mu (µ), Omega (ω), Pi (π), Sigma (σ), Theta (θ) and Zeta (ζ), [11], [78], [64], [40]. GSTs are named using a letter corresponding to their class membership and Arabic numerals after the subunit com‐ position (*e.g*. GST A1-1 is a homodimeric alpha class GST consisting of two subunit 1).

### **3.2. Presence of GST in cells**

#### *3.2.1. Microbial GST*

GSH linked enzymes. There are a number of GSH linked enzymes that are involved in cellular protection against toxic substances. The glyoxalase I and II which are responsible for catalyz‐ ing the conversion of methylglyoxal (a by-product in glycolysis) to lactic acid are among these enzymes [76]. Glutathione reductase (GR) which catalyzes the reduction of GSSG using NADPH as a reductant is also a glutathione-linked enzyme involved in cell protection. GR is important to keep the high cellular reductive potential. Selenium dependent glutathione perox‐ idase are other GSH-linked enzymes that catalyze the reduction of peroxides using GSH as the reducing agent [7]. Finally, last but not the least, glutathione transferases are also GSH depend‐ ent enzymes with many properties among which catalyzing the conjugation of GSH to vari‐

**Figure 1.** Structure of reduced glutathione; glutamate is linked in an isopeptide bond (*via* its γ-carboxyl group) to cys‐

Glutathione *S*-transferases (GST, EC 2.5.1.18), which first discovered as enzymes in 1961 [12], are abundant proteins encoded by a highly divergent, ancient gene family. These major cellular detoxification enzymes present mostly in liver and kidney as well as intestine. In spite of 40 years of research the exact function of this protein is more complex than ever, but it has been found that these intracellular dimeric proteins, play a major role in the intracellu‐ lar transport of endogenous compounds, metabolizes various electrophilic xenobiotics, li‐ gand transport and thus protects cells against toxic effects [31], [87], [85]. GST catalyzes the conjugation of glutathione on the sulfur atom of cysteine to various electrophiles and cataly‐ ses the conjugation of various electrophiles with GSH, detoxifying both exogenously and en‐

The superfamily of the glutathione transferases are divided into at least four major families of proteins, namely cytosolic or soluble GSTs, mitochondrial GSTs, microsomal GSTs and

ous electrophilic compounds is one of the most investigated function [25].

teine, which in turn forms a peptide linkage with glycine

dogenously derived toxic compounds [13].

**3.1. Classification and structure**

**3. Glutathione S-transferase**

270 Aflatoxins - Recent Advances and Future Prospects

For a long time, GST enzymes from microbial sources were neglected and were not system‐ atically studied. One of the reasons for this was the poor activity of microbial GSTs with CDNB8 as a model substrate for GST activity, which led to the conclusion that these en‐ zymes are rare in unicellular organisms [81], [77]. The first evidence for the presence of GSTs in bacteria was reported more 30 years ago by Takashi Shishido who showed the presence of GST activity in a strain of *Escherichia coli* [71]. Since then, GSTs have been found to be broadly distributed in aerobic prokaryotes, but not in anaerobic bacteria [59]. The absence of the enzyme in these microorganisms is consistent with the lack of GSH [28]. Bacterial gluta‐ thione transferases are part of a superfamily of enzymes that play a key role in cellular de‐ toxification. Bacterial GSTs are implicated in a variety of distinct processes such as the biodegradation of xenobiotics, protection against chemical and oxidative stresses and anti‐ microbial drug resistance. In addition to their role in detoxification, bacterial GSTs are also involved in a variety of distinct metabolic processes such as the biotransformation of di‐ chloromethane, the degradation of lignin and atrazine, and the reductive dechlorination of pentachlorophenol [4], [51].

#### *3.2.2. GSTs of fungi and yeasts*

Until recently, relatively little was known about the presence and role of GST in fungi. How‐ ever, expression of GST has been reported in some fungal species such as *Issatchenkia orienta‐ lis*, [73], *Phanerochaete chrysosporium*, *Yarrowia lipolytica*, *Mucor circinelloides* [70] *Schizosaccharomyces pombe*, *Aspergillus nidulans*, *Aspergillus parasiticus, Aspergillus flavus, As‐ pergillus fumigates* [Burns et al., 2005] *Saccharomyces cerevisiae*, and *Cunninghamella elegans,* [70] [48] *etc*. However, the role of the enzyme in fungi, particularly toxigenic strains, is not well understood [2]. Although it has been shown that GST has a significant role in detoxifi‐

<sup>8 - 1-</sup>chloro-2,4-dinitrobenzene

cation of aflatoxin and there is a possibility that this enzyme catalyses the conjugation of GSH to AFB1- epoxide to excrete its derivatives from the body, in 1988 and for the first time Saxena et al. reported that the relation of cytosolic GSH S-transferases from *A..flavus* to afla‐ toxin synthesis. In truth, they showed that in contrast to other cells that GST has a critical function to break down the aflatoxin, in aflatoxigenic *Aspergillus* spp., there is positive corre‐ lation between the GST activity and aflatoxin production [68], factors influencing aflatoxin formation such as growth period, medium etc., always enhanced GST activity in the toxi‐ genic strain. Since the non-toxigenic strain produces no aflatoxin, these factors have little ef‐ fect on its GST activity. Our experience with GSH-conjugation system using inducers/ inhibitors of aflatoxin metabolism in fungi also show a positive correlation of aflatoxin syn‐ thesis and GST activity in *Aspergillus* species [2], [88].

A3-3 catalyze the reduction of PGH2 to PGF2. The isomerization reaction of PGF2 to PGD2 is also catalyzed by sigma class of GST. PGD2, PGE2 and PGF2 act as hormones that bind to Gprotein coupled receptors which regulate other hormones and neurotransmittors. Prosta‐ glandin D2 and E2 are unstable and will easily be converted to prostaglandin J2 and A2, respectively and their derivatives inhibit NFκB, [66] a family of transcription factors that regulate the transcription of genes important for inflammatory processes. There are interest‐ ing speculations that GSTs might block other anti-inflammatory pathways by catalyzing the

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273

Because of exhibiting a ligand binding function, glutathione tranferases, have been known as ligandin, a function, which involves the noncovalent binding of nonsubstrate hydropho‐ bic ligands such as heme, bilirubin, various steroids, and conceivably some lipophilic anti‐ cancer drugs as well. Although GSTs are generally viewed as playing a protective role in foreign compound metabolism, they can also catalyze reactions that lead to toxification. Ex‐ amples include the GST dependent metabolism of 1,2-dibromoethane and related haloal‐ kanes and probably also metabolism of the 6-thiopurine prodrug azathioprin [60], [5]. Similarly, the cytotoxicity of the polypeptide antibiotic neocarzinostatin is greatly enhanced by thiols such as GSH, although in this case there is no apparent requirement for GST cataly‐

In addition to above functions, GSTs also are responsible for interacting the proteins and en‐ zymes. For example GST P1-1 interacts with c-Jun N-terminal kinase 1 (JNK1) suppressing the basal kinase activity. GST P1-1 also has a role in protection and cell survival after exposure to H2O2 but not against UV-induced apoptosis [1]. Whereas, mouse GST M1-1 protects cells against both UV-and H2O2-induced cell death and binds to apoptosis signal-regulating kinase 1 (ASK1), inhibits its kinase activity [16]. Moreover, mouse GST A4-4 has also been proposed to in‐

As enzymes, GSTs are involved in many different detoxification reactions. They are common‐ ly referred to as phase II enzymes. They catalyze the conjugation of GSH to a wide variety of endogenous and exogenous electrophilic toxic compounds. The GSH conjugates are excreted as mercapturic acids by the phase III metabolic pathway [41]. GST P1-1, GST M1-1 and GST A1-1 have been shown to catalyze the inactivation process of α, β unsaturated carbonyls like acrolein, (a cytotoxic compound present in tobacco smoke), propenals, (generated by oxida‐

teract with JNK and prevent cells from 4-hydroxynonenal induced apoptosis [15], [25].

tive damage to DNA) and alkenals, (formed by oxidative damage to lipids) [25], [70].

conjugation of GST to PGJ2 [38], [25]

*3.3.2. The ligandin function of GSTs*

*3.3.3. The regulatory function of GSTs*

*3.3.4. The detoxification function of GSTs*

sis [25], [18]. [82].

#### *3.2.3. Plant GSTs*

Plant GSTs are a family of multifunctional enzymes involved in the intracellular detoxifica‐ tion of xenobiotics and toxic compounds produced endogenously [54], [26]. Most of the en‐ zymes are stress-inducible and play a role in the protection of plants from adverse effects of stresses. However, the activities of different GSTs have been detected and characterized in many plants, including maize, wheat, tobacco, soybean, barley, chickpea, peanut, sorghum, and sugarcane [20], [21], [22], [75].

#### *3.2.4. Mammalian GST*

The isoenzymes of glutathione transferase have been most widely studied in rat liver. Six basic transferases in rat liver liver have been characterized. In rabbit, GST catalyzes the con‐ jugation of activated AFB1 with glutathione. In an experiment to assess the abilities of lung and liver GSTs to conjugate AFB1-8, 9-epoxide, it has been shown that alpha-class and muclass GSTs are of similar importance in catalyzing the reaction in the lung. The human gluta‐ thione S-transferase, possess both enzymatic and non-enzymatic functions and are involved in many important cellular processes, such as, phase II metabolism, stress response, cell pro‐ liferation, apoptosis, oncogenesis, tumor progression and drug resistance. The nonenzymat‐ ic functions of GSTs involve their interactions with cellular proteins, such as, Jun N-terminal kinase,(JNK), tumor necrosis factor receptor-associated factor-2 (TRAF2), apoptosis-signalregulating kinase 1 (ASK), serine/threonine kinases (PKA, PKC), and tissue transglutami‐ nase 2 (TGM2), during which, either the interacting protein partner undergoes functional alteration or the GST protein itself is post-translationally modified and/or functionally al‐ tered [53], [74].

#### **3.3. Different functions of GST**

#### *3.3.1. The metabolic function of GSTs*

GSTs have been reported to involve in steroid metabolism by catalyzing the isomerization of ∆5 -androstene-3, 17-dione to, ∆<sup>4</sup> -androstene-3, 17-dione, and biosynthesis of prostaglandins. GST M2-2 is a prostaglandin E synthase in the brain cortex [8] and rat GST A1-1 and GST A3-3 catalyze the reduction of PGH2 to PGF2. The isomerization reaction of PGF2 to PGD2 is also catalyzed by sigma class of GST. PGD2, PGE2 and PGF2 act as hormones that bind to Gprotein coupled receptors which regulate other hormones and neurotransmittors. Prosta‐ glandin D2 and E2 are unstable and will easily be converted to prostaglandin J2 and A2, respectively and their derivatives inhibit NFκB, [66] a family of transcription factors that regulate the transcription of genes important for inflammatory processes. There are interest‐ ing speculations that GSTs might block other anti-inflammatory pathways by catalyzing the conjugation of GST to PGJ2 [38], [25]

#### *3.3.2. The ligandin function of GSTs*

cation of aflatoxin and there is a possibility that this enzyme catalyses the conjugation of GSH to AFB1- epoxide to excrete its derivatives from the body, in 1988 and for the first time Saxena et al. reported that the relation of cytosolic GSH S-transferases from *A..flavus* to afla‐ toxin synthesis. In truth, they showed that in contrast to other cells that GST has a critical function to break down the aflatoxin, in aflatoxigenic *Aspergillus* spp., there is positive corre‐ lation between the GST activity and aflatoxin production [68], factors influencing aflatoxin formation such as growth period, medium etc., always enhanced GST activity in the toxi‐ genic strain. Since the non-toxigenic strain produces no aflatoxin, these factors have little ef‐ fect on its GST activity. Our experience with GSH-conjugation system using inducers/ inhibitors of aflatoxin metabolism in fungi also show a positive correlation of aflatoxin syn‐

Plant GSTs are a family of multifunctional enzymes involved in the intracellular detoxifica‐ tion of xenobiotics and toxic compounds produced endogenously [54], [26]. Most of the en‐ zymes are stress-inducible and play a role in the protection of plants from adverse effects of stresses. However, the activities of different GSTs have been detected and characterized in many plants, including maize, wheat, tobacco, soybean, barley, chickpea, peanut, sorghum,

The isoenzymes of glutathione transferase have been most widely studied in rat liver. Six basic transferases in rat liver liver have been characterized. In rabbit, GST catalyzes the con‐ jugation of activated AFB1 with glutathione. In an experiment to assess the abilities of lung and liver GSTs to conjugate AFB1-8, 9-epoxide, it has been shown that alpha-class and muclass GSTs are of similar importance in catalyzing the reaction in the lung. The human gluta‐ thione S-transferase, possess both enzymatic and non-enzymatic functions and are involved in many important cellular processes, such as, phase II metabolism, stress response, cell pro‐ liferation, apoptosis, oncogenesis, tumor progression and drug resistance. The nonenzymat‐ ic functions of GSTs involve their interactions with cellular proteins, such as, Jun N-terminal kinase,(JNK), tumor necrosis factor receptor-associated factor-2 (TRAF2), apoptosis-signalregulating kinase 1 (ASK), serine/threonine kinases (PKA, PKC), and tissue transglutami‐ nase 2 (TGM2), during which, either the interacting protein partner undergoes functional alteration or the GST protein itself is post-translationally modified and/or functionally al‐

GSTs have been reported to involve in steroid metabolism by catalyzing the isomerization of

GST M2-2 is a prostaglandin E synthase in the brain cortex [8] and rat GST A1-1 and GST


thesis and GST activity in *Aspergillus* species [2], [88].

*3.2.3. Plant GSTs*

and sugarcane [20], [21], [22], [75].

272 Aflatoxins - Recent Advances and Future Prospects

*3.2.4. Mammalian GST*

tered [53], [74].

∆5

**3.3. Different functions of GST**

*3.3.1. The metabolic function of GSTs*


Because of exhibiting a ligand binding function, glutathione tranferases, have been known as ligandin, a function, which involves the noncovalent binding of nonsubstrate hydropho‐ bic ligands such as heme, bilirubin, various steroids, and conceivably some lipophilic anti‐ cancer drugs as well. Although GSTs are generally viewed as playing a protective role in foreign compound metabolism, they can also catalyze reactions that lead to toxification. Ex‐ amples include the GST dependent metabolism of 1,2-dibromoethane and related haloal‐ kanes and probably also metabolism of the 6-thiopurine prodrug azathioprin [60], [5]. Similarly, the cytotoxicity of the polypeptide antibiotic neocarzinostatin is greatly enhanced by thiols such as GSH, although in this case there is no apparent requirement for GST cataly‐ sis [25], [18]. [82].

#### *3.3.3. The regulatory function of GSTs*

In addition to above functions, GSTs also are responsible for interacting the proteins and en‐ zymes. For example GST P1-1 interacts with c-Jun N-terminal kinase 1 (JNK1) suppressing the basal kinase activity. GST P1-1 also has a role in protection and cell survival after exposure to H2O2 but not against UV-induced apoptosis [1]. Whereas, mouse GST M1-1 protects cells against both UV-and H2O2-induced cell death and binds to apoptosis signal-regulating kinase 1 (ASK1), inhibits its kinase activity [16]. Moreover, mouse GST A4-4 has also been proposed to in‐ teract with JNK and prevent cells from 4-hydroxynonenal induced apoptosis [15], [25].

#### *3.3.4. The detoxification function of GSTs*

As enzymes, GSTs are involved in many different detoxification reactions. They are common‐ ly referred to as phase II enzymes. They catalyze the conjugation of GSH to a wide variety of endogenous and exogenous electrophilic toxic compounds. The GSH conjugates are excreted as mercapturic acids by the phase III metabolic pathway [41]. GST P1-1, GST M1-1 and GST A1-1 have been shown to catalyze the inactivation process of α, β unsaturated carbonyls like acrolein, (a cytotoxic compound present in tobacco smoke), propenals, (generated by oxida‐ tive damage to DNA) and alkenals, (formed by oxidative damage to lipids) [25], [70].

## **3.4. GST and aflatoxin**

#### *3.4.1. Introduction*

Study on GSTs of *Aspergillus flavus* stems from its ability to synthesize the aflatoxin. Aflatox‐ ins are one of the major causes of liver cancer in certain regions of Africa and Asia [83], [61]. These secondary metabolites which primarily produced by some *Aspergillus* spp. are ubiqui‐ tous, and under favorable conditions can grow on a wide variety of agricultural commodi‐ ties. Aflatoxins are major concern with to public health and the most important toxicological interest in aflatoxins has concentrated on aflatoxin B1, largely due to its acute toxicity and carcinogenicity in humans and animals. [3], [62], [88]. Genetic studies on aflatoxin biosyn‐ thesis in *A. flavus* and *A. parasiticus* has been led to the cloning of 25 clustered genes within a 70 kb DNA region responsible for the enzymatic conversions in the aflatoxin biosynthetic pathway [86].

hibit AFBO production [29], while inducers of CYP3A4 activity such as 3-methylcholan‐

CYP1A homologues also metabolize AFB1 to produce the detoxified metabolite AFM1,

major metabolite of AFB1 (Figure 2). [33]. Although both CYP1A and CYP3A isoforms oxi‐

**Figure 2.** Bioactivation of AFB1 to exo and endo-epoxides and subsequent GST-catalyzed conjugation with GSH.

9 - P450 III AY and in the fetal liver P450 III A6

, produce another detoxified metabolite, aflatoxin Q1 (AFQ1), the

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threne and rifampicin, increase AFB1 metabolism in cultured human hepatocytes [49].

dize AFB1, there are conflicting reports on their relative importance [63].

whereas CYP3A enzymes9
