*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 func‐ tion 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), subfa‐ milies identified by a letter (e.g., 2A, 2B, 2D, and 2E), and specific members identified by an‐ other 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 xeno‐ biotics [50], [58], [84]. This superfamily of hemoproteins aids in the oxidation of various sub‐ strates such as steroids, eicosanoids, pharmaceuticals, pesticides, pollutants, and carcinogens [57]. As mentioned earlier, they bioactivate AFB1 to an electrophilic, highly reac‐ tive and unstable metabolite known as aflatoxin-8,9-epoxide, which binds to guanine resi‐ dues 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 nu‐ cleophiles 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, where‐ as much higher concentrations are catalyzed by CYP3A4 [30], [46], [79]. A recent study dem‐ onstrated 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 in‐ hibit AFBO production [29], while inducers of CYP3A4 activity such as 3-methylcholan‐ threne and rifampicin, increase AFB1 metabolism in cultured human hepatocytes [49].

CYP1A homologues also metabolize AFB1 to produce the detoxified metabolite AFM1, whereas CYP3A enzymes9 , produce another detoxified metabolite, aflatoxin Q1 (AFQ1), the major metabolite of AFB1 (Figure 2). [33]. Although both CYP1A and CYP3A isoforms oxi‐ dize 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.

**3.4. GST and aflatoxin**

274 Aflatoxins - Recent Advances and Future Prospects

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

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 func‐ tion 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), subfa‐ milies identified by a letter (e.g., 2A, 2B, 2D, and 2E), and specific members identified by an‐

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 xeno‐ biotics [50], [58], [84]. This superfamily of hemoproteins aids in the oxidation of various sub‐ strates such as steroids, eicosanoids, pharmaceuticals, pesticides, pollutants, and carcinogens [57]. As mentioned earlier, they bioactivate AFB1 to an electrophilic, highly reac‐ tive and unstable metabolite known as aflatoxin-8,9-epoxide, which binds to guanine resi‐ dues 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 nu‐

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, where‐ as much higher concentrations are catalyzed by CYP3A4 [30], [46], [79]. A recent study dem‐ onstrated 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 in‐

position of


cleophiles and can induce mutations by alkylating DNA, principally at the N7

*3.4.1. Introduction*

pathway [86].

*3.4.2. Primary metabolism of aflatoxin B1*

other number (e.g., CYP2E1 and CYP2A6) [47], [19].

guanine forming the 8,9-dihydro- 8-(N7

<sup>9 -</sup> P450 III AY and in the fetal liver P450 III A6

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].

GST to detoxify activated AFB1 has not been examined systematically. Three separate alphaclass 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 possess‐ es inducible GST and if so, whether these might be involved in AFB1 metabolism [34]. Nev‐ ertheless, 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 individu‐ als 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 differ‐ ent 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 car‐ cinoma compared to their adjacent normal tissues. Loss of GSTP1 has been suggested to in‐ crease 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 peroxida‐ tion. 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,

The Significance of Glutathione Conjugation in Aflatoxin Metabolism

http://dx.doi.org/10.5772/52096

277

with mammalian GSTs the most clearly characterized [Burns et al. 2005].

gated to AFB1 via the keto group in the cyclopentone ring [34].

Besides the formation of GSH conjugates, glucuronide and sulfate conjugates of AFB1 have al‐ so 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 impor‐ tance 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 me‐ tabolites 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 involv‐ ing the addition of water to the keto group in the cyclopentone ring, that result in the introduc‐ tion 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 conju‐

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 val‐ ues to form a dialdehydic phenolate ion. This AFB1-dialdehyde can undergo Schiff-base for‐ mation with primary amine groups in proteins and is therefore likely to be cytotoxic.

## *3.4.3. Secondary metabolism of aflatoxin B1*

Oxidative metabolism of AFB1 by cytochrome P450 results in the formation of several prod‐ ucts 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 nontumorous 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 macromole‐ cules, GSTs play a critical role in the protection of tissues from the deleterious effects of bio‐ activated 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-ep‐ oxide 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 con‐ jugated with reduced glutathione. Little is known about the identity of the GST which is re‐ sponsible 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 in‐ volved have not been characterized. In human liver, GST does not appear to play as impor‐ tant 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 pro‐ duced 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 detect‐ ed 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 alphaclass 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 possess‐ es inducible GST and if so, whether these might be involved in AFB1 metabolism [34]. Nev‐ ertheless, 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 individu‐ als 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 differ‐ ent 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 car‐ cinoma compared to their adjacent normal tissues. Loss of GSTP1 has been suggested to in‐ crease 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 peroxida‐ tion. 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].

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

Oxidative metabolism of AFB1 by cytochrome P450 results in the formation of several prod‐ ucts 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 nontumorous 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 macromole‐ cules, GSTs play a critical role in the protection of tissues from the deleterious effects of bio‐ activated 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-ep‐ oxide 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

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 con‐ jugated with reduced glutathione. Little is known about the identity of the GST which is re‐ sponsible 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 in‐ volved have not been characterized. In human liver, GST does not appear to play as impor‐ tant 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 pro‐ duced 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 detect‐ ed in the urine of marmoset monkeys treated with AFB1. The ability of human alpha-class

aflatoxicol (AFL), AFLH1, AFB2á and AFB1-2, 2-dihydrodiol [80].

*3.4.3. Secondary metabolism of aflatoxin B1*

276 Aflatoxins - Recent Advances and Future Prospects

endo stereoisomer. [43], [72].

Besides the formation of GSH conjugates, glucuronide and sulfate conjugates of AFB1 have al‐ so 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 impor‐ tance 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 me‐ tabolites 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 involv‐ ing the addition of water to the keto group in the cyclopentone ring, that result in the introduc‐ tion 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 conju‐ gated 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 val‐ ues to form a dialdehydic phenolate ion. This AFB1-dialdehyde can undergo Schiff-base for‐ mation 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)-treat‐ ed 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, de‐ termined 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 like‐ ly to be one of the key mechanisms for the protective action of this anti-carcinogen [56].

(AFB1- N7

from the cell [36].

following questions:

**Acknowledgements**

and his assistance.

**Author details**

*3.4.4. Conclusion and future directions*

toxin-GSH conjugate formation?

Tahereh Ziglari1\* and Abdolamir Allameh2

\*Address all correspondence to: Tz\_572@usc.edu

G) is the most abundant. It is chemically unstable and is lost spontaneously from

G is lost but at a much slower rate

http://dx.doi.org/10.5772/52096

279

The Significance of Glutathione Conjugation in Aflatoxin Metabolism

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-forma‐ mide-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 dis‐ tinct 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

Evidences presented in this review article clearly show that glutathione conjugation to afla‐ toxin metabolites which has been detected in aflatoxin-producing fungi as well as liver tis‐ sues 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

**1.** What is the relationship between the rate of aflatoxin metabolism and the level of afla‐

**2.** Which classes of glutathione S-transferases in each cellular system is directly responsi‐

**3.** What is the relationship between the efficiency of glutathione conjugation system and

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

ble for involvement of aflatoxin-GSH conjugate formation

toxic action of aflatoxins in different cell systems.

III adducts are no longer removed and only AFB1-N7

The transport of foreign compounds out of cells can be achieved by at least two distinct fam‐ ilies of efflux pump, both of which may provide protection against AFB1 by helping elimi‐ nate the mycotoxin from target cells. The best characterized of these two pumps is Pglycoprotein, 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 gluta‐ thione 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 sys‐ tems are ATP-dependent and are inhibited by vanadate but differ in that P-glycoprotein ap‐ pears to have specificity towards hydrophobic compounds whereas the glutathione Sconjugate 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 hy‐ pothesis 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 for‐ mation 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-forma‐ mide-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 dis‐ tinct 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*

Recently, a novel AFB1-aldehyde reductase (AFB1-AR) purified from ethoxyquin (EQ)-treat‐ ed 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, de‐ termined 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 like‐ ly to be one of the key mechanisms for the protective action of this anti-carcinogen [56].

278 Aflatoxins - Recent Advances and Future Prospects

The transport of foreign compounds out of cells can be achieved by at least two distinct fam‐ ilies of efflux pump, both of which may provide protection against AFB1 by helping elimi‐ nate the mycotoxin from target cells. The best characterized of these two pumps is Pglycoprotein, 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 gluta‐ thione 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 sys‐ tems are ATP-dependent and are inhibited by vanadate but differ in that P-glycoprotein ap‐ pears to have specificity towards hydrophobic compounds whereas the glutathione Sconjugate 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 hy‐ pothesis 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].

mation of three major adducts. Of these, trans-2,3-dihydro-2-(N7

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 for‐


Evidences presented in this review article clearly show that glutathione conjugation to afla‐ toxin metabolites which has been detected in aflatoxin-producing fungi as well as liver tis‐ sues 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:

