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Agricultural and Biological Sciences » "Aflatoxins - Recent Advances and Future Prospects", book edited by Mehdi Razzaghi-Abyaneh, ISBN 978-953-51-0904-4, Published: January 23, 2013 under CC BY 3.0 license. © The Author(s).

Chapter 13

The Significance of Glutathione Conjugation in Aflatoxin Metabolism

By Tahereh Ziglari and Abdolamir Allameh
DOI: 10.5772/52096

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The Significance of Glutathione Conjugation in Aflatoxin Metabolism

Tahereh Ziglari1 and Abdolamir Allameh2

1. Introduction

We all are exposed through air, food, drinks and skin contacts to harmful compounds throughout the period of our lifetime, including, a variety of pharmaceuticals and food-derived carcinogen metabolite (e.g. N-acetoxy-PhIP), [52], plant toxins (such as glycoalkaloids in nightshades[1] - , cyanogenic glucosides [2] - , or pyrrolizidine alkaloids in some herbs and herbal teas), xenobiotics[3] - producing during early human pregnancy, fungal and bacterial toxins such as aflatoxins[4] - ; and cyanotoxin[5] - ; as well as free radicals and hydroperoxides. Many of these compounds are lipophilic and the organism can get rid of them only through metabolism.

Biotransformation has been conveniently categorized into three distinct phases, which act in a tightly integrated manner. Phases I and II enzymes catalyze the conversion of a lipophilic, non-polar xenobiotic into a more water-soluble and therefore less toxic metabolite, which can then be more easily excreted from the body. Phase I biotransformation seems to be enzymes that catalyzes oxidation, reduction or hydrolyze reactions, it usually converts substrates to more polar forms by introducing or unmasking a functional group (e.g., —OH, —NH2, or —SH). Phase I consist primarily of microsomal enzymes, which are found abundantly in the liver, gastrointestinal tract, lung and kidney, consisting of families and subfamilies of enzymes that are classified based on their amino acid sequence identities or similarities. [84]. Many of the enzymes like monooxygenases are found in the endoplasmic reticulum membrane, but others such as the dehydrogenases for example alcohol dehydrogenases and peroxidases located in the cytoplasm, while still others such as monoamine oxidase 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 major types of microsomal monooxygenase, both of which require NADPH as an external reductant: the cytochrome P450 (CYP) system and flavin-containing monooxygenases. The mechanism of CYP is a complex cascade of individual steps involving the interaction of protein redox partners and consumption of reducing equivalents, usually in the form of NADPH. The iron heme containing enzyme, CYP, consists of two enzymes: NADPH–cytochrome P450 reductase and CYP. It is involved in the oxidative metabolism of many endogenous 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 nitrogen, sulfur, or phosphorus. The properties of the CYPs electron transport systems have also been reported [77].

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 biotransforming enzymes than others [52].

Phase II conjugation reactions which generally act follow phase I activation consists of reactions in which metabolites containing appropriate functional groups are conjugated with substances such as glucuronate, glutamate, sulfate, reduced glutathione or uridine diphosphate (UDP)-glucuronic acid to finally discharge them through urine or bile. In general, conjugation dramatically improves solubility, which then promotes rapid excretion. Among the several types of conjugation reactions which are present in the body, including glucuronidation, 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 biotransformation 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 results in reduced affinity of the compound for its cellular target. [67], [23].

In animals, elimination of the soluble compounds from cells and excretion of biotransformed molecules from the body referred to as phase III. It has been suggested that the phase III of detoxification system to be called antiporter activity. Antiporter activity is an important factor in the first pass metabolism of pharmaceuticals and other xenobiotics. The antiporter is an energy-dependent efflux pump, which pumps xenobiotics out of a cell, thereby decreasing the intracellular concentration of xenobiotics. In eukaryotic organisms, they are actively excreted or compartmentalized in the vacuole by ATP-dependent GS-X pumps [42], [27]. Indeed, as the glutathionylated moiety is hydrophilic, the conjugate cannot usually simply re-diffuse back into the cell [77]. Antiporter activity in the intestine appears to be co-regulated with intestinal phase I CYP3A4 enzyme. This observation suggests the antiporter may support and promote detoxification. Possibly, its function of pumping non-metabolized xenobiotics out of the cell and back into the intestinal lumen, may allow more opportunities for phase I activity to metabolize the xenobiotic before it is taken into circulation. Although, most literature on detoxification refers to liver enzymes, as the liver is the site of the majority of detoxification activity for both endogenous and exogenous compounds, however, the first contact the body with the majority of xenobiotics take places in the gastrointestinal tract. Intestinal mucosa possesses enzyme systems capable of various types of biotransformation of xenobiotics [52]. Among the detoxification pathways, glutathione conjugation pathway is the prominent route of AFB1 inactivation in liver of mammalians. Depending on the availability of cellular GSH and the activation of glutathione S-transferase subclasses, detoxification of AFB1 is facilitated [24].

2. Glutathione

Glutathione is a ubiquitous thiol-containing isotripeptide (γ-glu-cys-gly, FW 307.3), consisting of glycine, glutamic acid and cysteine molecules which was first discovered by Sir Fredrick 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 between 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, ascorbic acid (vitamin C), α-tocopherol (vitamin E) and flavonoids etc., GSH[6] - is commonly referred to as an antioxidant [17], which neutralizes free radicals due to the high electron-donating capacity of its sulfydryl (-SH) group, [13], and prevents damage to important cellular components, implicates in the cellular defense against xenobiotics. Glutathione status is a highly sensitive indicator of cell functionality and viability. Its levels in human tissues 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/GSSG[7] - 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 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 catalyzing 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 peroxidase 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 dependent enzymes with many properties among which catalyzing the conjugation of GSH to various electrophilic compounds is one of the most investigated function [25].


Figure 1.

Structure of reduced glutathione; glutamate is linked in an isopeptide bond (via its γ-carboxyl group) to cysteine, which in turn forms a peptide linkage with glycine

3. Glutathione S-transferase

Glutathione S-transferases (GST, EC, 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 intracellular transport of endogenous compounds, metabolizes various electrophilic xenobiotics, ligand 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 catalyses the conjugation of various electrophiles with GSH, detoxifying both exogenously and endogenously 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 composition (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

For a long time, GST enzymes from microbial sources were neglected and were not systematically studied. One of the reasons for this was the poor activity of microbial GSTs with CDNB[8] - as a model substrate for GST activity, which led to the conclusion that these enzymes 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 glutathione transferases are part of a superfamily of enzymes that play a key role in cellular detoxification. Bacterial GSTs are implicated in a variety of distinct processes such as the biodegradation of xenobiotics, protection against chemical and oxidative stresses and antimicrobial 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 dichloromethane, 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. However, expression of GST has been reported in some fungal species such as Issatchenkia orientalis, [73], Phanerochaete chrysosporium, Yarrowia lipolytica, Mucor circinelloides [70] Schizosaccharomyces pombe, Aspergillus nidulans, Aspergillus parasiticus, Aspergillus flavus, Aspergillus 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 detoxification 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 aflatoxin 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 correlation between the GST activity and aflatoxin production [68], factors influencing aflatoxin formation such as growth period, medium etc., always enhanced GST activity in the toxigenic strain. Since the non-toxigenic strain produces no aflatoxin, these factors have little effect 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 synthesis and GST activity in Aspergillus species [2], [88].

3.2.3. Plant GSTs

Plant GSTs are a family of multifunctional enzymes involved in the intracellular detoxification of xenobiotics and toxic compounds produced endogenously [54], [26]. Most of the enzymes 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 conjugation 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 mu-class GSTs are of similar importance in catalyzing the reaction in the lung. The human glutathione 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 proliferation, apoptosis, oncogenesis, tumor progression and drug resistance. The nonenzymatic 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-signal-regulating kinase 1 (ASK), serine/threonine kinases (PKA, PKC), and tissue transglutaminase 2 (TGM2), during which, either the interacting protein partner undergoes functional alteration or the GST protein itself is post-translationally modified and/or functionally altered [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, ∆4-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 G-protein coupled receptors which regulate other hormones and neurotransmittors. Prostaglandin 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 interesting 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

Because of exhibiting a ligand binding function, glutathione tranferases, have been known as ligandin, a function, which involves the noncovalent binding of nonsubstrate hydrophobic ligands such as heme, bilirubin, various steroids, and conceivably some lipophilic anticancer 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. Examples include the GST dependent metabolism of 1,2-dibromoethane and related haloalkanes 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 catalysis [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 enzymes. 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 interact 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 commonly 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 oxidative 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. Aflatoxins 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 ubiquitous, and under favorable conditions can grow on a wide variety of agricultural commodities. 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 biosynthesis 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].

3.4.2. Primary metabolism of aflatoxin B1

Once inside the body and for toxicity to occur, AFB1 undergoes enzymatic conversion to electrophilic endo and exo stereoisomers of AFB1-8,9-epoxide by the action of mixed function mono-oxygenase enzyme systems, CYPs are an intensively studied family of enzymes with currently approximately 4,000 known members. They have been found in almost all branches of the “tree of life”, ranging from microorganisms over plants to mammalians. CYP enzymes are classified into families identified by a number (e.g., 1, 2, 3, and 4), subfamilies identified by a letter (e.g., 2A, 2B, 2D, and 2E), and specific members identified by another number (e.g., CYP2E1 and CYP2A6) [47], [19].

In human, five CYP gene families, namely; CYP1, CYP2, CYP3, CYP4 and CYP7 are believed to play crucial roles in hepatic as well as extra-hepatic metabolism and elimination of xenobiotics [50], [58], [84]. This superfamily of hemoproteins aids in the oxidation of various substrates such as steroids, eicosanoids, pharmaceuticals, pesticides, pollutants, and carcinogens [57]. As mentioned earlier, they bioactivate AFB1 to an electrophilic, highly reactive and unstable metabolite known as aflatoxin-8,9-epoxide, which binds to guanine residues in nucleic acids, leading to irreversible damage in DNA and causing hepatocarcinoma in humans, primates, and ducks [32], [84]. However, only AFB1 exo-epoxide (AFBO), binds appreciably to DNA (Figure 2). The AFBO is highly unstable, and it reacts with cellular nucleophiles and can induce mutations by alkylating DNA, principally at the N7 position of guanine forming the 8,9-dihydro- 8-(N7-guanyl)-9-hydroxy-AFB1. In addition, AFBO can bind to proteins and other critical cellular nucleophiles [43], [63]. Initial studies reported that concentrations of AFB1 which are likely to be achieved in the liver following ingestion of ‘‘real-world” concentrations of AFB1 are bioactivated to AFBO primarily by CYP1A2, whereas much higher concentrations are catalyzed by CYP3A4 [30], [46], [79]. A recent study demonstrated a dominant contribution of CYP3A4 homologues in AFBO production. AFB1 metabolism studies in human liver microsomal preparations indicate a predominant role for CYP3A4 and that its expression level was an important determinant of the AFB1 disposition in human liver [45]. Specific CYP3A4 inhibitors like troleandomycin have been shown to inhibit AFBO production [29], while inducers of CYP3A4 activity such as 3-methylcholanthrene and rifampicin, increase AFB1 metabolism in cultured human hepatocytes [49].

CYP1A homologues also metabolize AFB1 to produce the detoxified metabolite AFM1, whereas CYP3A enzymes[9] - , produce another detoxified metabolite, aflatoxin Q1 (AFQ1), the major metabolite of AFB1 (Figure 2). [33]. Although both CYP1A and CYP3A isoforms oxidize AFB1, there are conflicting reports on their relative importance [63].


Figure 2.

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

CYPs may also catalyze demethylation to aflatoxin P (AFP1) of the parent AFB1 molecule, resulting in products less toxic than AFB1. Other major metabolites in the human include aflatoxicol (AFL), AFLH1, AFB2á and AFB1-2, 2-dihydrodiol [80].

3.4.3. Secondary metabolism of aflatoxin B1

Oxidative metabolism of AFB1 by cytochrome P450 results in the formation of several products such as AFB1-epoxide which serve as substrates for phase II detoxification enzymes. Phase II enzymes such as GSTP1 and GSTA1, found in several mammalian species and non-tumorous liver tissues [14] are the first step in the mercapturic acid pathway, which leads to the excretion of the xenobiotics. Because conjugation of the electrophilic AFB1-8,9-epoxide with GSH is an alternative fate to binding to nucleophilic centers in cellular macromolecules, GSTs play a critical role in the protection of tissues from the deleterious effects of bioactivated AFB1, and tissues vary considerably in both GST concentration and distribution of specific GST isoforms. Two stereoisomers of AFB1-8,9-epoxide were identified: AFB1 exo-epoxide and AFB1 endo-epoxide, and their corresponding GSH conjugates; AFB1 exo-epoxide-GSH and AFB1 endo-epoxide-GSH. It has been reported that only the exo-epoxide effectively interacts with DNA and was at least 500-fold more potent as a mutagen than the endo stereoisomer. [43], [72].

Throughout the animal kingdom, significant variations exist in the susceptibility of different species to AFB1. Man and rats are sensitive to AFB1 but mice can tolerate this mycotoxin. [35]. In man and rat as well as many mammalian species, AFB1-8,9-epoxide is efficiently conjugated with reduced glutathione. Little is known about the identity of the GST which is responsible for detoxifying activated AFB1. To date, the catalytic conjugation of AFB1-8,9-epoxide has only been reported using rat and mouse GST as enzyme source and the ability of GST in other species to catalyze this reaction has not been described. In the investigation on hepatic rat GST responsible for catalyzing the conjugation of AFB1-8,9-epoxide with GSH, it has been shown that the alpha class but not mu-class of GST possess greatest ability to metabolize activated AFB1. Although the rat pi-class GST cannot catalyze this reaction it might be expected that the theta-class enzyme GST is active towards AFB1-8,9-epoxide. By contrast with the rat, the mouse exhibits high constitutive levels of GST activity towards AFB1-8,9-epoxide and alpha-class GST in Swiss-Webster mice possess high activity towards AFB1-8,9-epoxide and can protect against DNA-binding by AFB1 metabolites. Neither the murine mu-class nor pi-class GST can detoxify activated AFB1 and all the activity towards this substrate is contributed by the alpha-class GST. It can be concluded that in the mouse the theta-class enzymes do not play a major role in the detoxification of activated AFB1. Hamster liver contains significant levels of AFB1-GSH-conjugating activity but the GST involved have not been characterized. In human liver, GST does not appear to play as important a role in providing protection against AFB1 as the rodent GST. The in vitro studies have suggested that in comparison with rodents, relatively little AFB1-GSH conjugate is produced by human liver, but insufficient data exist to be certain that this reaction is not of physiological importance in man, particularly as an aflatoxin mercapturate has been detected in the urine of marmoset monkeys treated with AFB1. The ability of human alpha-class GST to detoxify activated AFB1 has not been examined systematically. Three separate alpha-class isoenzymes, which represent the dimeric combinations of two distinct subunits (B1 & B2) have been described in human liver. Furthermore, it is not known whether man possesses inducible GST and if so, whether these might be involved in AFB1 metabolism [34]. Nevertheless, it has been shown that in the humans, the GST with the highest activity toward AFB1 exo-epoxide is the polymorphic hGSTM1-1 which is absent in about 50% of individuals in most human populations. This suggests that AFB1-epoxide individuals lacking the beneficial effects of hGSTM1-1 may be at elevated risk. Indeed some reports suggest that the GSTM1 genetic polymorphism may affect AFB1 detoxification in human liver. In contrast to the liver, the lung is composed of many different cell types and expression of GSTs in different human lung cell types is heterogenous. Thus certain cell types with low levels of GSTs or lacking specific GST isoforms may be at higher risk of AFB1 toxicity [72]. GSTP was also demonstrated to significantly increase in early hepatocarcinogenesis and hepatocellular carcinoma compared to their adjacent normal tissues. Loss of GSTP1 has been suggested to increase the risk of DNA damage and mutation. Moreover, up-expression of GSTA was suggested to protect liver cells against oxidative stress via an extracellular signal-regulated kinases (ERKs) and p38 kinase (p38K)-related pathway, as well as through the inhibition of H2O2-induced apoptosis to inhibit reactive oxygen species (ROS)- induced lipid peroxidation. It was suggested that inactivated or down-regulated GSTP1 and GSTA1 genes could increase genomic damage when individuals were exposed to carcinogens. [14]. GSTs have also been shown to exhibit GSH-dependent peroxidase activity and thus may be involved in resistance to oxidative stress. Cytosolic GSTs have been identified in almost all organisms, with mammalian GSTs the most clearly characterized [Burns et al. 2005].

Besides the formation of GSH conjugates, glucuronide and sulfate conjugates of AFB1 have also been described in a variety of species including rat, mouse, monkey and trout. The ability to form these alternative secondary metabolites may be of considerable physiological importance in species, like the trout, that are unable to produce AFB1-GSH conjugates. Before AFB1 can form glucuronide and sulfate conjugates it requires to be hydroxylated. The primary metabolites AFM1, AFP1, and AFQ1 can readily form glucuronide or sulfate conjugates. Whilst such conjugation reactions may aid excretion of aflatoxin, their toxicological value is unclear as such hydroxylated metabolites are not particularly harmful because they are not subject to 8,9-epoxidation. However, it has been proposed that AFB1 is itself capable of forming glucuronide and sulfate conjugate; these reactions might entail a molecular rearrangement possibly involving the addition of water to the keto group in the cyclopentone ring, that result in the introduction of a hydroxyl group into the AFB1 structure. This proposal is of particular interest as it enables the direct detoxification of AFB1 through reactions that may not involve cytochrome P450. These workers have also proposed that amines, thiols and alcohols might also be conjugated to AFB1 via the keto group in the cyclopentone ring [34].

Alternatively, the AFB1-epoxide can hydrolyse spontaneously to AFB1-dihydrodiol. This is not a true detoxification process as the dihydrodiol product can rearrange at neutral pH values to form a dialdehydic phenolate ion. This AFB1-dialdehyde can undergo Schiff-base formation with primary amine groups in proteins and is therefore likely to be cytotoxic. Recently, a novel AFB1-aldehyde reductase (AFB1-AR) purified from ethoxyquin (EQ)-treated rat liver has been shown to metabolize the dialdehyde form of AFB1-dihydrodiol to an AFB1-dialcohol and its relative importance in AFB1 detoxification may be considerable [35]. The toxicity of AFB1 is selective towards certain species. In contrast with the mouse and hamster, the rat, guinea pig and man are susceptible to the hepatotoxic effects of AFB1 [34]. The toxicity of the mycotoxin is based on a balance between the rate of primary activation of AFB1 and the rate of detoxification of primary metabolites or repair of cellular damage, determined by the relative activity of enzymes responsible for these reactions; the differential toxicity of AFB1, between species is thought to be due mainly to different levels of activity of xenobiotic-metabolizing enzymes. In this regard, the livers of mice which are resistant to the hepatoxic effects of AFB1 contain high concentrations of a Yc-type GST subunit [55] that has considerable GSH conjugating activity towards AFB1-epoxide [34], [37], [10], [9]. By contrast, the Fischer rat, an inbred strain that is five times more susceptible to AFB1- induced liver cancer than the Wistar rat [34], possesses 20-fold less hepatic AFB1-GSH-conjugating activity than the mouse. Fischer rats can, however, be protected against AFB1 by treatment with the antioxidant EQ. It has shown that following EQ-treatment the livers of Fischer rats express a GST subunit that is immunochemically related to the mouse Yc subunit [35]. Moreover, this inducible polypeptide (Yc2, subunit 10) has high activity towards AFB1-epoxide [35]. Thus, the Yc2 subunit is thought to confer protection against AFB1, and its induction by EQ is likely to be one of the key mechanisms for the protective action of this anti-carcinogen [56].

The transport of foreign compounds out of cells can be achieved by at least two distinct families of efflux pump, both of which may provide protection against AFB1 by helping eliminate the mycotoxin from target cells. The best characterized of these two pumps is P-glycoprotein, the product of the mdr 1 gene which has been studied extensively because of its involvement in acquired resistance to anticancer drugs. The other pump is the glutathione S-conjugate carrier which is responsible for the transport of endogenous compounds such as oxidized glutathione and leukotriene C4 as well as glutathione conjugates of foreign compounds an example of which might be S-(2,4-dinitrophenyl)glutathione. Both pump systems are ATP-dependent and are inhibited by vanadate but differ in that P-glycoprotein appears to have specificity towards hydrophobic compounds whereas the glutathione S-conjugate carrier is as its name suggests specific for leukotrienes and drug-glutathione conjugates. Although it is not known whether P-glycoprotein is able to transport AFB1 the broad specificity of this efflux pump and its activity towards hydrophobic drugs suggests that this is likely. It also appears highly probable that the glutathione S-conjugate carrier is responsible for the transport of AFB1 conjugated with GSH. Both P-glycoprotein and the glutathione S-conjugate carrier are expressed in the liver which is compatible with the hypothesis that these pumps could be involved in the efflux of AFB1 and its metabolites. The involvement of P-glycoprotein in AFB1 transport is supported by the fact that aflatoxin has been shown to induce mRNA encoding this protein in mouse liver. [36].

Relatively little is known about the enzymes responsible for the removal of AFB1 that is bound covalently to DNA in mammalian cells. Exposure of cells to AFB1 results in the formation of three major adducts. Of these, trans-2,3-dihydro-2-(N7-guanyl)-3-hydroxy AFB1 (AFB1- N7 G) is the most abundant. It is chemically unstable and is lost spontaneously from DNA in vitro to yield apurinic sites. The other two adducts, 2,3-dihydro-2-(N-formyl-2,3,6-triamino-4-oxopyrimidine-N-yl)-3-hydroxy AFB1 and 8,9-dihydro-8-(2-amino-6-formamide-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy AFB1 (AFB1-FAPY and AFB1III respectively) are not spontaneously but appear to be removed catalytically by DNA repair enzymes. The loss of AFB1-DNA adducts in vivo is biphasic and this occurs through two distinct mechanisms. Following exposure to AFB1, all adduct species are removed rapidly until less than 1000 adducts per cell remain. Once this point is reached the AFB1-FAPY and AFB1 III adducts are no longer removed and only AFB1-N7 G is lost but at a much slower rate from the cell [36].

3.4.4. Conclusion and future directions

Evidences presented in this review article clearly show that glutathione conjugation to aflatoxin metabolites which has been detected in aflatoxin-producing fungi as well as liver tissues of mammalians play a crucial role in reducing the interaction of aflatoxins with cellular macromolecules. However further studies is needed to answer the main questions about the contribution of glutathione conjugation system in removing aflatoxin in different cellular systems. The future direction of this topic is to find out experimental-based answers to the following questions:

  1. What is the relationship between the rate of aflatoxin metabolism and the level of aflatoxin-GSH conjugate formation?

  2. Which classes of glutathione S-transferases in each cellular system is directly responsible for involvement of aflatoxin-GSH conjugate formation

  3. What is the relationship between the efficiency of glutathione conjugation system and toxic action of aflatoxins in different cell systems.


It is the time to express the deepest gratitude to Mr. Hamed Foroozesh for whole-hearted support and for his devoted care. Thanks for his vast knowledge and skill in many areas and his assistance.


1 - V. Adler, Z. Yin, S. Y. Fuchs, M. Benezra, L. Rosario, K. D. Tew, M. R. Pincus, M. Sardana, C. J. Henderson, C. R. Wolf, R. J. Davis, Z. Ronai, 1999 Regulation of JNK signaling by GSTp. EMBO J. 18 1321 34
2 - A. Allameh, M. Razzaghi-Abyaneh, M. Shams, M. B. Rezaee, K. Jaimand, 2002 Effect of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase, isocitrate dehydrogenase, and glutathione S-transferase in Aspergillus parasiticus. Mycopathologia 154 79 84
3 - A. A. Allameh, T. Ziglari, I. Rasooli, 2011 Phytoinhibition of growth and aflatoxin biosynthesis in toxigenic fungi Chapter 15 in the book: aflatoxin, detection, measurement and control. InTech publication, 978-9-53307-711-6
4 - N. Allocati, l. Federici, M. Masulli, C. Dillio, 2009 Glutathione transferases in bacteria. The FEBS Journal 276 58 75
5 - M. W. Anders, L. Lash, W. Dekant, A. A. Elfarra, D. R. Dohn, 1988 Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites. Crit. Rev. Toxicol 18 4 311 341
6 - R. N. Armstrong, 2000 Mechanistic diversity in a metalloenzyme superfamily. Biochemistry 39 13625 13632
7 - J. R. Arthur, 2000 The glutathione peroxidases. Cell. Mol. Life Sci. 57 1825 35
8 - C. T. Beuckmann, K. Fujimori, Y. Urade, O. Hayaishi, 2000 Identification of mu-class glutathione transferases M2-2 and M3-3 as cytosolic prostaglandin E synthases in the human brain. Neurochem. Res. 25 5 733 8
9 - T. M. Beutler, D. L. Eaton, 1992a Complementary DNA cloning, messenger RNA expression and induction of alpha-class glutathione S-transferases in mouse tissues. Cancer Res 52 314 318
10 - T. M. Beutler, D. Slone, D. L. Eaton, 1992b Comparison of the aflatoxin B1-8,9-epoxide conjugating activities of two bacterially expressed alpha class glutathione S-transferase isozymes from mouse and rat. Biochem. Biophys. Res. Commun 188 2 597 603
11 - P. G. Board, M. Coggan, G. Chelvanayagam, S. Easteal, L. S. Jermiin, G. K. Schulte, D. E. Danley, L. R. Hoth, M. C. Griffor, A. V. Kamath, M. H. Rosner, B. A. Chrunyk, D. E. Perregaux, C. A. Gabel, K. F. Geoghegan, J. Pandit, 2000 Identification, Characterization, and Crystal Structure of the Omega Class Glutathione Transferases. Journal of Biological Chemistry 275 32 24798 24806
12 - J. Booth, E. Boyland, P. Sims, 1961 An enzyme from rat liver catalyzing conjugations with glutathione. Biochem. J 79 516 24
13 - G. M. A. Cancado, V. E. De Rosa Jr, J. H. Fernandez, L. G. Maron, R. A. Jorge, M. Menossi, 2005 Glutathione S-transferase and aluminum toxicity in maize. Functional Plant Biology 32 1045 1055
14 - Y.l. Chen, H. S. Tseng, W. H. Kuo, S. F. Yang, D. R. Chen, H. T. Tsai, 2010 Glutathione S-Transferase P1 (GSTP1) gene polymorphism increases age-related susceptibility to hepatocellular carcinoma. BMC Medical Genetics 11 46
15 - J. Z. Cheng, S. S. Singhal, A. Sharma, M. Saini, Y. Yang, S. Awasthi, P. Zimniak, Y. C. Awasthi, 2001 Two distinct 4-hydroxynonenal metabolizing glutathione S-transferase isozymes are differentially expressed in human tissues. Biochem. Biophys. Res. Commun. 282 1268 74
16 - S. G. Cho, Y. H. Lee, H. S. Park, K. Ryoo, K. W. Kang, J. Park, S. J. Eom, M. J. Kim, T. S. Chang, S. Y. Choi, J. Shim, Y. Kim, M. S. Dong, M. J. Lee, S. G. Kim, H. Ichijo, E. J. Choi, 2001 Glutathione S-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1. J. Biol. Chem. 276 16 12749 55
17 - I. A. Cotgreave, R. G. Gerdes, 1998 Recent trends in glutathione biochemistry-glutathione-protein interactions: a molecular link between oxidative stress and cell proliferation? Biochem Biophys Res Commun 242 1 9
18 - W. G. De Graff, A. Russo, J. B. Mitchell, 1985 Glutathione depletion greatly reduces neocarzinostatin cytotoxicity in Chinese hamster V79 cells. J. Biol. Chem 260 8312 5
19 - G.j. Diaz, H. W. Murcia, S. M. Cepeda, 2010 Cytochrome P450 enzymes involved in the metabolism of aflatoxin B1 in chickens and quail. Poultry Science association Inc 89 2461 2469
20 - D. P. Dixon, L. Cummins, D. J. Cole, R. Edwards, 1998 Glutathione-mediated detoxification systems in plants. Current opinion in Plant Biology 258 266
21 - D. P. Dixon, A. Lapthorn, R. Edwards, 2002 Plant glutathione transferases Genome Biol 3 3 1 10
22 - D. P. Dixon, A. G. Mc Ewen, A. J. Lapthorn, R. Edwards, 2003 Forced evolution of a herbicide detoxifying glutathione transferase. J. Biol. Chem. 278 26 23930 23935
23 - M. Dusinska, M. Staruchova, A. Horska, B. Smolkova, A. Collins, S. Bonassi, K. Volkovova, 2012 Are glutathione S-transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. In press.
24 - D. L. Eaton, E. P. Gallagher, 1994 Mechanisms of aflatoxin carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 34 135 172
25 - M. Edalat, 2002 Multiple functions of glutathione transferase. Dissertation for the Degree of Doctor of Philosophy in Biochemistry presented at Uppsala University.
26 - R. Edwards, D. P. Dixon, V. Walbot, 2000 Plant glutathione Stransferase: enzymes with multiple functions in sickness and in health. Trends Plant Sci. 5 5 193 198
27 - R. Ernst, R. Klemm, L. Schmitt, K. Kuchler, Sies. L. P. Helmut, 2005 Yeast ATP-Binding Cassette Transporters: Cellular Cleaning Pumps. Methods in Enzymology 400 460 484
28 - R. C. Fahey, 2001 Novel thiols of prokaryotes. Annual Review of Microbiology 55 333 356
29 - E. P. Gallagher, L. C. Wienkers, P. L. Stapleton, K. L. Kunze, D. L. Eaton, 1994 Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1. Cancer Research 54 101 108
30 - E. P. Gallagher, K. L. Kunze, P. L. Stapleton, D. L. Eaton, 1996 The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4. Toxicology and Applied Pharmacology 141 595 606
31 - S. G. George, 1994 Enzymology and molecular biology of phase II xenobiotic conjugating enzymes in fish. Aquatic toxicology, molecular biochemical and cellular perspectives (Eds.: D.C. Malins and G.K.Ostrander). Lewis Publishers, Ann Arbor. 27 86
32 - F. P. Guengerich, E. M. Gillam, T. Shimada, 1996a New applications of bacterial systems to problems in toxicology. Critical Reviews in Toxicology 26 551 583
33 - F. P. Guengerich, W. W. Johnson, Y. F. Ueng, H. Yamazaki, T. Shimada, 1996b Involvement of Cytochrome 450Glutathione S-Transferase, and Epoxide Hydrolase in the Metabolism of Aflatoxin B1 and Relevance to Risk of Human Liver Cancer. Environment Health Perspectives 104 557 562
34 - J. D. Hayes, D. J. Judah, L. I. Mclellan, G. E. Neal, 1991a Contribution of the glutathione S-transferases to the mechanisms of resistance to aflatoxin B1. Pharmacol. Ther 50 443 472
35 - J. D. Hayes, D. J. Judah, L. I. Mc Lellan, L. A. Kerr, S. D. Peacock, G. E. Neal, 1991b Ethoxyquin-induced resistance to aflatoxin B1 in the rat is associated with the expression of a novel alpha-class glutathione S-transferase subunit, Yc2, which possesses high catalytic activity for aflatoxin B1-8,9-epoxide. Biochem. J 279 385 398
36 - J. D. Hayes, D. J. Judah, L. I. Mc Lellan, G. E. Neal, 1991c Contribution of the glutathione S-transferases to the mechanisms of persistence to aflatoxin B1. Pharmac. Ther 50 443 472
37 - J. D. Hayes, D. J. Judah, G. E. Neal, T. Nguyen, 1992 Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide. Biochem. J. 285 173 180
38 - J. D. Hayes, R. C. Strange, 2000 Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology 61 3 154 66
39 - J. D. Hayes, J. U. Flanagan, I. R. Jowsey, 2005 Glutathione transferases. Annu Rev Pharmacol Toxicol 45 51 88
40 - B. Hu, L. Deng, C. Wen, X. Yang, P. Pei, Y. Xie, S. Luo, 2012 Cloning, identification and functional characterization of a pi-class glutathione S-transferase from the freshwater mussel Cristaria plicata. Fish & Shellfish Immunology 32 1 51 60
41 - T. Ishikawa, 1992 ATP dependent glutathione S-conjugate transport pump. Trends Biochem. Sci 17 463 8
42 - T. Ishikawa, Z. S. Li, Y. P. Lu, P. A. Rea, 1997 The GS-X pump in plant, yeast, and animal cells: Structure, function, and gene expression. Bioscience Reports 17 189 207
43 - R. Iyer, B. Coles, K. D. Raney, R. Thier, F. P. Guengerich, T. M. Harris, 1994 DNA adduction by the potent carcinogen aflatoxin B1: mechanistic studies. Journal of the American Chemical Society 116 1603 1609
44 - P. J. Jakobsson, R. Morgenstern, J. Mancini, A. Ford-Hutchinson, B. Persson, 1999 Common structural features of MAPEG- a widespread superfamily of membrane-associated proteins with highly divergent functions in eicosanoid and glutathione metabolism. Protein Sci 8 689 692
45 - L. K. Kamdem, I. Meineke, U. Godtel-Armbrust, J. Brockmoller, L. Wojnowski, 2006 Dominant contribution of P450 3A4 to the hepatic carcinogenic activation of aflatoxin B1. Chemical Research in Toxicology 19 577 586
46 - J. D. Kelly, D. L. Eaton, F. P. Guengerich, R. A. Coulombe Jr, 1997 Aflatoxin B1 activation in human lung. Toxicology and Applied Pharmacology 144 88 95
47 - C. D. Klaassen, 2001 Casarett and Doull`s Toxicology: The Basic Science of Poisons. 6th ed. McGraw-Hill, New York, N.Y.
48 - M. P. Krajewski, B. Kanawati, A. Fekete, N. Kowalski, P. Schmitt-Kopplin, E. Grill, 2012 Analysis of Arabidopsis glutathione-transferases in yeast Phytochemistry In Press.
49 - S. Langouet, B. Coles, F. Morel, L. Becquemont, P. Beaune, F. P. Guengerich, B. Ketterer, A. Guillouzo, 1995 Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer Research 55 5574 5579
50 - D. E. Lewis, 2003 P450 structures and oxidative metabolism of xenobiotics. Pharmacogenomics 4 387 395
51 - C. Li, X. Su, Y. Li, T. Li, C. Sun, T. Zhou, H. Liu, 2012 Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum. Fish & Shellfish Immunology 32 1 219 222
52 - D. J. Liska, 1998 The detoxification enzyme system. Alternative Medicine Review 3 3 187 198
53 - H. W. Lo, F. Ali-Osman, 2007 Genetic polymorphism and function of glutathione S-transferases in tumor drug resistance Current Opinion in Pharmacology 7 367 374
54 - B. Mannervik, U. H. Danielson, 1988 Glutathione transferases structure and catalytic activity. Crit. Rev. Biochem. 23 2 283 337
55 - L. I. Mc Lellan, L. A. Kerr, A. D. Cronshaw, J. D. Hayes, 1991 Regulation of mouse glutathione S-transferases by chemoprotectors. Molecular evidence for the existence of three distinct alpha-class glutathione S-transferase subunits, Ya1, Ya2, and Ya3, in mouse liver. Biochem. J. 276 461 469
56 - L. Mc Lellan, D. J. Judah, G. E. Neal, J. D. Hayes, 1994 Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors. Biochem. J 300 117 124
57 - A. Parikh, E. M. Gillam, F. P. Guengerich, 1997 Drug metabolism by Escherichia coli expressing human cytochromes P450. Nature Biotechnology 15 784 788
58 - J. M. Pascussi, I. S. Gerba-Chaloin, L. Drocourt, P. Maurel, M. J. Vilarem, 2003 The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors. Biochim. Biophys. Acta 1619 243 253
59 - R. Piccolomini, A. Aceto, N. Allocati, A. Faraone, C. Di Ilio, 1991 Purification of a GSH-affinity binding protein from Bacteroides fragilis devoid of glutathione transferase activity. FEMS Microbiol Lett 82 101 106
60 - C. B. Pickett, 1989 Glutathione S-transferases. Gene structure, regulation, and biological function. Annu. Rev. Biochem 58 743 764
61 - M. Razzaghi-Abyaneh, A. Allameh, M. Shams, 2000 Screening of aflatoxin- producing mould isolates based on fluorescence production on a specific medium under ultraviolet light. Acta Medica Iranica. 38 67 73
62 - M. Razzaghi-Abyaneh, M. Shams-Ghahfarokhi, A. Allameh, M. Kazeroon-Shiri, S. Ranjbar-Bahadori, H. Mirzahoseini, M. B. Rezaee, 2006a A survey on distribution of Aspergillus section Flavi in corn field soils in Iran: population patterns based on aflatoxins, cyclopiazonic acid and sclerotia production. Mycopathologia 161 183 192
63 - S. Rawal, J. E. Kim, R. C. Jr , 2010 Aflatoxin B1 in poultry: Toxicology, metabolism and prevention. Research in Veterinary Science 89 325 331
64 - K. S. Revathy, N. Umasuthan, Y. Lee, C. Y. Choi, I. Whang, J. Lee, 2012 First molluscan theta-class glutathione S-transferase: Identification, cloning, characterization and transcriptional analysis post immune challenges. Biochemistry and Molecular Biology 162 1-3 10 23
65 - A. Robinson, G. A. Huttley, H. S. Booth, P. G. Board, 2004 Modelling and bioinformatics studies of the human kappa-class glutathione transferase predict a novel third glutathione transferase family with similarity to prokaryotic 2-hydroxychromene-2-carboxylate isomerases. Biochem J. 379 541 552
66 - A. Rossi, P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin, M. G. Santoro, 2000 Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase. Nature 403 103 8
67 - J. C. Sacco, 2006 Phase II biotransformation of xenobiotics in polar bear (Ursus maritimus) and channel catfish (Ictalurus punctatus). A dissertation presented to the graduate school of the University of Florida in partial fulfillment of the requirements for the degree of doctor of philosophy. Waxman
68 - M. Saxena, K. G. Mukerji, H. G. Raj, 1988 Positive correlation exists between glutathione S-transferase activity and aflatoxin formation in Aspergillus flavus. Biochem J 245 567 70
69 - S. Scarcella, P. Lamenza, G. Virkel, H. Solana, 2012 Expression differential of microsomal and cytosolic glutathione S-transferases in Fasciola hepatica resistant at triclabendazole. Molecular and Biochemical Parasitology 181 1 37 39
70 - D. Sheehan, G. Meade, V. M. Foley, CA Dowd, 2001 Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochemical Journal 360 1 16
71 - T. Shishido, 1981 Glutathione S-transferase from Escherichia coli. Agric Biol Chem 45 2951 2953
72 - R. K. Stewart, C. J. Serabjit-Singh, T. E. Massey, 1996 Glutathione S-Transferase-Catalyzed Conjugation of Bioactivated Aflatoxin B1 in Rabbit Lung and Liver. Toxicology and Applied Pharmacology 140 499 507
73 - H. Tamaki, K. Yamamoto, H. Kumagai, 1999 Expression of two glutathione S-transferase genes in the yeast Issatchenkia orientalis is induced by o-dinitrobenzene during cell growth arrest. J. Bacteriol. 181 2958 2962
74 - K. D. Tew, Y. Manevich, C. Grek, Y. Xiong, J. Uys, D. M. Townsend, 2011 The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer. Free Radical Biology and Medicine 15 July 2011 51 2 229 31
75 - R. Thom, I. Cummins, D. P. Dixon, R. Edwards, D. J. Cole, A. J. Lapthorn, 2002 Structure of a Tau class glutathione S-transferase from wheat active in herbicide detoxification. Structure of a Tau class glutathione S-transferase from wheat active in herbicide detoxification. Biochemistry 41 22 7008 7020
76 - PJ Thornalley, 1993 The glyoxalase system in health and disease Mol Aspects Med 14 287 371
77 - T. Todorova, 2007 Glutathione S-transferases and oxidative stress in Saccharomyces cerevisiae Thesis. A cotutelle project between the University Louis Pasteur, Strasbourg and the Sofia University St. Kliment Ohridski. France.
78 - N. Umasuthan, K. S. Revathy, Y. Lee, I. Whang, C. Y. Choi, J. Lee, 2012 A novel molluscan sigma-like glutathione S-transferase from Manila clam, Ruditapes philippinarum: Cloning, characterization and transcriptional profiling. Toxicology & Pharmacology 155 4 539 550
79 - T. R. Van Vleet, K. Mace, R. A. Coulombe Jr, 2002 Comparative aflatoxin B1 activation and cytotoxicity in human bronchial cells expressing cytochromes P450 1A2 and 3A4. Cancer Research 62 105 112
80 - R. J. Verma, 2004 Aflatoxin cause DNA damage Int j Hum Genet. 4 4 231 236
81 - S. Vuilleumier, 1997 Bacterial glutathione S-transferases: What are they good for? Journal of Bacteriology 179 1431 1441
82 - D. J. Waxman, 1990 Glutathione S-transferase: Role in Alkylating agent resistance and possible target for modulation chemotherapy. A review. Cancer 50 6449 6454
83 - World Health Organization International Agency for Research on Cancer (IARC), 1993 IARC Monographs on the Evaluation of Carcinogenic Risks to Humans
84 - C. Xu, C. Yong-Tao, A. T. Kong, 2005 Induction of Phase I, II and III Drug Metabolism/Transport by Xenobiotics. Arch Pharm Res 28 3 249 268
85 - A. Yamuna, Bhavan. P. Saravana, P. Geraldine, 2012 Glutathione S-transferase and metallothionein levels in the freshwater prawn Macrobrachium melcolmsonii exposed to mercury. J.Environ.Biol 33 133 137
86 - J. Yu, D. Bhatnagar, T. E. Cleveland, 2004 Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus. FEBS Lett 564 126 30
87 - S. J. Yu, 1996 Insect glutathione S-transferases- Review article. Zoological Studies 35 9 19
88 - T. Ziglari, A. Allameh, M. Razzaghi-Abyaneh, A. R. Khosravi, M. H. Yadegari, 2008 Comparison of glutathione S-transferase activity and concentration in aflatoxin-producing and their non-toxigenic counterpart isolates. Mycopathologia 166 219 226


[1] - - Plants like potatoes, tomatoes, peppers, egg plant, tobacco, some spices.

[2] - - Like bitter almond, cassava root, sorghum root, lima bean, fruit seed, etc.

[3] - - Chemical compounds foreign to the human organism without nutritional value

[4] - - A group of mycotoxins of which aflatoxin B1 is the most potent hepatocarcinogen

[5] - - A toxin producing by cyanobacteria of which microcystin-LR is predominant

[6] - - Glutathione, reduced form

[7] - - Glutathione, oxidized state

[8] - - 1-chloro-2,4-dinitrobenzene

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