**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-de‐ rived carcinogen metabolite (e.g. N-acetoxy-PhIP), [52], plant toxins (such as *glycoalkaloids* in nightshades1 , *cyanogenic glucosides* <sup>2</sup> , or *pyrrolizidine alkaloids* in some herbs and herbal teas), xenobiotics3 producing during early human pregnancy, fungal and bacterial toxins such as aflatoxins4 ; and cyanotoxin5 ; 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 en‐ zymes that catalyzes oxidation, reduction or hydrolyze reactions, it usually converts sub‐ strates 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 abun‐ dantly in the liver, gastrointestinal tract, lung and kidney, consisting of families and subfa‐ milies 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

<sup>5 -</sup> A toxin producing by cyanobacteria of which microcystin-LR is predominant

© 2013 Ziglari and Allameh; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Ziglari and Allameh; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<sup>1 -</sup> Plants like potatoes, tomatoes, peppers, egg plant, tobacco, some spices.

<sup>2</sup>*- Like bitter almond, cassava root, sorghum root, lima bean, fruit seed, etc.*

<sup>3 -</sup> Chemical compounds foreign to the human organism without nutritional value

<sup>4 -</sup> A group of mycotoxins of which aflatoxin B1 is the most potent hepatocarcinogen

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 been reported [77].

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 nonmetabolized 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 circula‐ tion. 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 com‐ pounds, 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, gluta‐ thione conjugation pathway is the prominent route of AFB1 inactivation in liver of mamma‐ lians. Depending on the availability of cellular GSH and the activation of glutathione S-

The Significance of Glutathione Conjugation in Aflatoxin Metabolism

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

269

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

other cellular thiol in a reduced state. Finally, GSH tends to a substrate or cofactor in some of

pair with their high reduction potential participates in maintaining

transferase subclasses, detoxification of AFB1 is facilitated [24].

form, with only a small proportion present in its fully oxidized state [20].

**2. Glutathione**

Moreover, the GSH/GSSG7

6 - Glutathione, reduced form 7 - Glutathione, oxidized state

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‐ forming enzymes than others [52].

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 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 biotrans‐ formed 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 nonmetabolized 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 circula‐ tion. 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 com‐ pounds, 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, gluta‐ thione conjugation pathway is the prominent route of AFB1 inactivation in liver of mamma‐ lians. Depending on the availability of cellular GSH and the activation of glutathione Stransferase subclasses, detoxification of AFB1 is facilitated [24].
