**2. Absorption, distribution, metabolism, excretion and mechanisms of action of aflatoxins**

Aflatoxins are highly liposoluble compounds and are readily absorbed from the site of expo‐ sure usually through the gastrointestinal tract and respiratory tract into blood stream [22, 23]. Human and animals get exposed to aflatoxins by two major routes (a) direct ingestion of aflatoxin-contaminated foods or ingestion of aflatoxins carried over from feed into milk and milk products like cheese and powdered milk as well as other animal tissues mainly as AFM1 [22](b) by inhalation of dust particles of aflatoxins especially AFB1 in contaminated foods in industries and factories [24]. After entering the body, the aflatoxins are absorbed across the cell membranes where they reach the blood circulation. They are distributed in blood to different tissues and to the liver, the main organ of metabolism of xenobiotics. Afla‐ toxins are mainly metabolized by the liver to a reactive epoxide intermediate or hydroxylat‐ ed to become the less harmful aflatoxin M1 [25, 26]. In humans and susceptible animal species, aflatoxins especially AFB1 are metabolized by cytochrome P450 (CYP450) microso‐ mal enzymes to aflatoxin-8,9-epoxide, a reactive form that binds to DNA and to albumin in the blood serum, forming adducts and hence causing DNA damage [25, 26]. Various CYP450 enzymes isoforms occur in the liver and they metabolize aflatoxin into a reactive oxygen species (aflatoxin-8,9-epoxide), which may then bind to proteins and cause acute toxicity (aflatoxicosis) or to DNA and induce liver cancer [25, 26]. The predominant human CYP450 isoforms involved in human metabolism of AFB1 are CYP3A4 and CYP1A2. Both enzymes catalyze the biotransformation of AFB1 to the highly reactive *exo*-8,9-epoxide of AFB1[27]. CYP 1A2 is also capable of catalyzing the epoxidation of AFB1 to yield a high pro‐ portion of *endo-*epoxide and hydroxylation of AFB1 to form aflatoxin M1 (AFM1), which is a poor substrate for epoxidation [27] and less potent than AFB1 [28]. This is generally consid‐ ered as the major detoxification metabolic pathway for aflatoxins. The CYP3A4 is the major CYP450 enzyme responsible for activation of AFB1 into the epoxide form and also form AFQ1, a less toxic detoxification metabolite. The CYP3A5 metabolizes AFB1 mainly to the *exo-*epoxide and some AFQ1 [29]. However, polymorphism studies with CYP3A5 have indi‐ cated that, this enzyme isoform is not expressed by most people especially in Africans [28]. Studies in Gambian children showed that aflatoxin cross the placenta and transported to the fetus and the new born where they can cause detrimental effects [28]. The CYP3A7 is a ma‐ jor CYP450 enzyme isoform in human fetal liver and metabolizes AFB1 to the 8, 9- epoxide that may cause fetal defects to the developing fetus [30].

pounds. Aflatoxins B2 and G2 were established as the dihydroxy derivatives of B1 and G1, respectively. Whereas, aflatoxin M1 is 4-hydroxy aflatoxin B1 and aflatoxin M2 is 4-dihy‐ droxy aflatoxin B2. Of the four major aflatoxins (B1, B2, G1 and G2), G2 occurs in high quanti‐ ties though less toxic while AFB1 is the most toxic of all the aflatoxin. The World Health Organization (WHO) classifies AFB1 as a class 1 carcinogen [4, 6, 9, 18]. The aflatoxins dis‐ play potency of toxicity, carcinogenicity, mutagenicity in the order of AFB1> AFG1> AFB2> AFG2 [15-19]. The extent of toxicity depends on the organ affected especially the liver. The lethal toxicity of aflatoxin B1 varies in different animals from extremely susceptible (Sheep, Rat, Dog) to resistant species (Monkey, Chicken, Mouse). However, there are no toxicity in humans though epidemiological data from studies in Africa, South Africa, South East Asia and India implicate aflatoxins in the incidence of liver cancer especially the hepatobiliary carcinoma and death of children due to malnutrition, kwashiorkor and marasmus [20, 21]. Aflatoxins have been associated with various diseases like aflatoxicosis and other health

**2. Absorption, distribution, metabolism, excretion and mechanisms of**

Aflatoxins are highly liposoluble compounds and are readily absorbed from the site of expo‐ sure usually through the gastrointestinal tract and respiratory tract into blood stream [22, 23]. Human and animals get exposed to aflatoxins by two major routes (a) direct ingestion of aflatoxin-contaminated foods or ingestion of aflatoxins carried over from feed into milk and milk products like cheese and powdered milk as well as other animal tissues mainly as AFM1 [22](b) by inhalation of dust particles of aflatoxins especially AFB1 in contaminated foods in industries and factories [24]. After entering the body, the aflatoxins are absorbed across the cell membranes where they reach the blood circulation. They are distributed in blood to different tissues and to the liver, the main organ of metabolism of xenobiotics. Afla‐ toxins are mainly metabolized by the liver to a reactive epoxide intermediate or hydroxylat‐ ed to become the less harmful aflatoxin M1 [25, 26]. In humans and susceptible animal species, aflatoxins especially AFB1 are metabolized by cytochrome P450 (CYP450) microso‐ mal enzymes to aflatoxin-8,9-epoxide, a reactive form that binds to DNA and to albumin in the blood serum, forming adducts and hence causing DNA damage [25, 26]. Various CYP450 enzymes isoforms occur in the liver and they metabolize aflatoxin into a reactive oxygen species (aflatoxin-8,9-epoxide), which may then bind to proteins and cause acute toxicity (aflatoxicosis) or to DNA and induce liver cancer [25, 26]. The predominant human CYP450 isoforms involved in human metabolism of AFB1 are CYP3A4 and CYP1A2. Both enzymes catalyze the biotransformation of AFB1 to the highly reactive *exo*-8,9-epoxide of AFB1[27]. CYP 1A2 is also capable of catalyzing the epoxidation of AFB1 to yield a high pro‐ portion of *endo-*epoxide and hydroxylation of AFB1 to form aflatoxin M1 (AFM1), which is a poor substrate for epoxidation [27] and less potent than AFB1 [28]. This is generally consid‐ ered as the major detoxification metabolic pathway for aflatoxins. The CYP3A4 is the major CYP450 enzyme responsible for activation of AFB1 into the epoxide form and also form

problems in humans, livestock and domestic animals globally.

**action of aflatoxins**

242 Aflatoxins - Recent Advances and Future Prospects

The epoxidation of AFB1 to the exo-8, 9-epoxide is a critical step in the genotoxic pathway of this carcinogen. The binding of AFB1 to DNA and DNA adduction by AFB1 exo-8,9 epoxide has been reported to cause a functional changes of DNA conformation [31].The epoxide is highly unstable and binds with high affinity to guanine bases in DNA to form afltoxin-N7 guanine [32]. The aflatoxin-N7-guanine has been shown to be capable of forming guanine (purine) to thymine (pyrimidine) transversion mutations in DNA and hence affecting the p53 suppressor gene in the cell cycle [33, 34]. The p53 gene is important in preventing cell cycle progression when there are DNA mutations, or signaling apoptosis. The mutations have been reported to affect some base pair locations more than others especially in the third base of codon 249 of the p53 gene in the region corresponding to the DNA binding do‐ main of the corresponding protein [13, 34]and this appears to be more susceptible to aflatox‐ in-mediated mutations than nearby bases [35]. AFB1 induces the transversion of base G to base T in the third position of codon 249 and similar mutations have been observed in hepa‐ tocellular carcinoma (HCC) in high AFB1 contaminated food in regions in East Asia and Af‐ rica [34, 36, 37].

Epoxide hydrolase and glutathione-S-transferase (GST) are both involved in hepatic detoxi‐ fication of activated AFB1, but the GST-catalyzed conjugation of glutathione to AFB1-8,9-ep‐ oxides is thought to play the most important role in preventing epoxide binding to target macromolecules like DNA and various cell proteins [38]. Glutathione pathway is reported to play a vital role in the detoxification of AFB1 [39, 40]. The AFB1 8,9 *exo* and *endo*epoxides are conjugated by glutathione to form AFB-mercapturate and the reaction is catalyzed by gluta‐ thione S-transferase (GST) [39, 40]. The glutathione-aflatoxin conjugate is transported from the cells with an ATP-dependent multidrug-resistance protein through an accelerated proc‐ ess [39]. Despite a preference for conjugating the more mutagenic AFB1 exo-epoxide isomer, the relatively low capacity for GST-catalyzed detoxification of bio-activated AFB1 in lung may be an important factor in the susceptibility of the lung to AFB1 toxicity [4, 8, 41].The *exo* and *endo* epoxide can also be converted non-enzymatically to AFB1-8,9-dihydrodiol which in turn can slowly undergo a base-catalysed ring opening reaction to a dialdehyde phenolate ion [27]. AFB1 dialdehyde can form Schiff bases with lysine residues in serum albumin form‐ ing aflatoxin-albumin complex [42]. Also the aflatoxin dialdehyde are reduced to a dialcohol in a NADPH-dependent catalyzed reaction by aflatoxin aldehyde reductase (AFAR) [43]. However the guanine alkylation by aflatoxin B1 produces *exo*-8,9-epoxide which is the reac‐ tive form and a carcinogen to the liver and the reaction is more than 2000 times more effi‐ cient in DNA than in aqueous solution [44], (Figure 2).

compensate for the ATP shortages by triggering the replication of any nearby mitochondria but unfortunately, the response promotes replication of the very mitochondria that are caus‐ ing the local energy deficit hence aggravating the problem [46]. The AFB1 also binds to DNA and cause structural DNA alterations that lead to gene mutations as well as changes in the length of the telomeres and the check points in the cell cycle [47-49]. The binding of AFB1 to DNA at the guanine base in liver cells corrupt the genetic code that regulates cell growth, thereby leading to formation of tumors ([45-49]. The damage to mitDNA is caused by ad‐ duction and mutations of mitochondrial membranes leading to increased cell death (apopto‐ sis) as well as disruption of energy production (production of ATP) [46, 49, 50]. The reactive aflatoxin-8, 9-epoxide can affect the mitotic (M) phase, growth process (G1 and G2 phase) and DNA synthesis (S phase) in the cell cycle by disrupting the various check points that regulate the cell cycle development and proliferation leading to deregulation of the cell and

Review of the Biological and Health Effects of Aflatoxins on Body Organs and Body Systems

http://dx.doi.org/10.5772/51201

245

However in resistant rodents, their mitDNA is protected from aflatoxins from DNA adducts that effect mitochondrial transcription and translation [46-49]. The mycotoxin alters energylinked functions of ADP phosphorylation and FAD- and NAD-linked oxidizing substrates

AFB causes ultrastuctural changes in mitochondria [46-49]and also induces mitochondrial directed apoptosis thus reducing their function [20, 29, 48-51]. Also the aflatoxins may affect the telomere length and the various check point in the cell cycle causing further damage to the regulatory processes of the cell cycle [51]. Also the extent of aflatoxin binding to DNA and its damage, the level of different proteins changes from cell cycle and apoptotic path‐ ways such as c-Myc, p53, pRb, Ras, protein kinase A (PKA), protein kinase C (PKC), Bcl-2, NF-kB, CDK, cyclins and CKI contribute to the life or death decision making process that may contribute to the deregulation of the cell proliferation leading to cancer development

However like in hepatic detoxification of aflatoxins and other chemicals, GSH act as antioxi‐ dant and has many functions in membrane maintenance and stability as well as in reducing oxidative stress factors and the high reactive oxygen species (ROS) produced from the proc‐ ess of lipid peroxidation [38-41, 46, 52-56]. The increased depletion of GSH leads to abnor‐ mally high levels of ROS found in cells affected by aflatoxin due to uncoupling of metabolic processes resulting from the lack of GSH for GSH-peroxidase catalysis of O2 to H2O2 leading to lipid peroxidation and compromised cell membranes. Its reduction further enhances the damage to critical cellular components (DNA, lipids, proteins) by the 8,9 epoxides. However the most serious adverse effects of the AFB1-8,9-epoxide metabolite is that it reacts with amino acids in DNA and forms an adduct [38-41, 46, 52-55]. The adduct are fairly resistant to DNA repair processes and this causes gene mutation that leads to liver cancers especially

**2.3. Role of glutathione in detoxification of aflatoxins and their metabolites**

hence cancer development [47-49], (Figure 3).

and α-ketoglutarate-succinate cytochrome reductases [46-49].

**2.2. Effect of aflatoxins on mitochondrial structure**

the hepatocellular carcinomas [38-41, 46, 52-55].

[34, 48, 49](Figure 3).

**Figure 2.** Aflatoxin disease pathways in humans (Adopted from Wu, 2010; Wu, 2011)[10, 26]

**Figure 3.** Various check points that can be damaged by binding of aflatoxins and AF-8,9-epoxide causing the deregu‐ lation of the cell cycle; P –prophase, M-Metaphase, A- Anaphase, T- Telophase, S- Synthetic DNA phase, G1 and G2 – Gaps (growth phase) [47-49]

#### **2.1. Effect of aflatoxins on mitochondrial DNA**

The reactive aflatoxin-8,9-epoxide preferentially binds to mitochondrial DNA (mitDNA) during hepatocarcinogenesis as compared to nuclear DNA that hinder ATP production and FAD/NAD-linked enzymatic functions and this causes the disruption of mitochondrial func‐ tions in the various parts of the body that require production of energy in the form of ATP [45]. Aflatoxin damage to mitochondria can lead to mitochondrial diseases and may be re‐ sponsible for aging mechanisms [45]. It is reported that certain mitochondrial diseases result from the ability of the nucleus to detect energetic deficits in its area. The nucleus attempts to compensate for the ATP shortages by triggering the replication of any nearby mitochondria but unfortunately, the response promotes replication of the very mitochondria that are caus‐ ing the local energy deficit hence aggravating the problem [46]. The AFB1 also binds to DNA and cause structural DNA alterations that lead to gene mutations as well as changes in the length of the telomeres and the check points in the cell cycle [47-49]. The binding of AFB1 to DNA at the guanine base in liver cells corrupt the genetic code that regulates cell growth, thereby leading to formation of tumors ([45-49]. The damage to mitDNA is caused by ad‐ duction and mutations of mitochondrial membranes leading to increased cell death (apopto‐ sis) as well as disruption of energy production (production of ATP) [46, 49, 50]. The reactive aflatoxin-8, 9-epoxide can affect the mitotic (M) phase, growth process (G1 and G2 phase) and DNA synthesis (S phase) in the cell cycle by disrupting the various check points that regulate the cell cycle development and proliferation leading to deregulation of the cell and hence cancer development [47-49], (Figure 3).

However in resistant rodents, their mitDNA is protected from aflatoxins from DNA adducts that effect mitochondrial transcription and translation [46-49]. The mycotoxin alters energylinked functions of ADP phosphorylation and FAD- and NAD-linked oxidizing substrates and α-ketoglutarate-succinate cytochrome reductases [46-49].

#### **2.2. Effect of aflatoxins on mitochondrial structure**

**Figure 2.** Aflatoxin disease pathways in humans (Adopted from Wu, 2010; Wu, 2011)[10, 26]

**Figure 3.** Various check points that can be damaged by binding of aflatoxins and AF-8,9-epoxide causing the deregu‐ lation of the cell cycle; P –prophase, M-Metaphase, A- Anaphase, T- Telophase, S- Synthetic DNA phase, G1 and G2 –

The reactive aflatoxin-8,9-epoxide preferentially binds to mitochondrial DNA (mitDNA) during hepatocarcinogenesis as compared to nuclear DNA that hinder ATP production and FAD/NAD-linked enzymatic functions and this causes the disruption of mitochondrial func‐ tions in the various parts of the body that require production of energy in the form of ATP [45]. Aflatoxin damage to mitochondria can lead to mitochondrial diseases and may be re‐ sponsible for aging mechanisms [45]. It is reported that certain mitochondrial diseases result from the ability of the nucleus to detect energetic deficits in its area. The nucleus attempts to

Gaps (growth phase) [47-49]

244 Aflatoxins - Recent Advances and Future Prospects

**2.1. Effect of aflatoxins on mitochondrial DNA**

AFB causes ultrastuctural changes in mitochondria [46-49]and also induces mitochondrial directed apoptosis thus reducing their function [20, 29, 48-51]. Also the aflatoxins may affect the telomere length and the various check point in the cell cycle causing further damage to the regulatory processes of the cell cycle [51]. Also the extent of aflatoxin binding to DNA and its damage, the level of different proteins changes from cell cycle and apoptotic path‐ ways such as c-Myc, p53, pRb, Ras, protein kinase A (PKA), protein kinase C (PKC), Bcl-2, NF-kB, CDK, cyclins and CKI contribute to the life or death decision making process that may contribute to the deregulation of the cell proliferation leading to cancer development [34, 48, 49](Figure 3).

#### **2.3. Role of glutathione in detoxification of aflatoxins and their metabolites**

However like in hepatic detoxification of aflatoxins and other chemicals, GSH act as antioxi‐ dant and has many functions in membrane maintenance and stability as well as in reducing oxidative stress factors and the high reactive oxygen species (ROS) produced from the proc‐ ess of lipid peroxidation [38-41, 46, 52-56]. The increased depletion of GSH leads to abnor‐ mally high levels of ROS found in cells affected by aflatoxin due to uncoupling of metabolic processes resulting from the lack of GSH for GSH-peroxidase catalysis of O2 to H2O2 leading to lipid peroxidation and compromised cell membranes. Its reduction further enhances the damage to critical cellular components (DNA, lipids, proteins) by the 8,9 epoxides. However the most serious adverse effects of the AFB1-8,9-epoxide metabolite is that it reacts with amino acids in DNA and forms an adduct [38-41, 46, 52-55]. The adduct are fairly resistant to DNA repair processes and this causes gene mutation that leads to liver cancers especially the hepatocellular carcinomas [38-41, 46, 52-55].
