**2. Aflatoxin biosynthesis, metabolism, and toxification**

#### **2.1. Aflatoxin biosynthesis**

The biosynthesis of aflatoxins has been fully summarized in several previous reviews [7, 8]. In brief, aflatoxins are an important type of mycotoxins, which were the most early identified in the *Aspergillus flavus* (*A. flavus*) and regarded as causative agents of "turkey X" disease in the late 1950s and early 1960s. Thus, these toxins were named as "aflatoxins (namely *A. flavus* toxins)" according to their origin fungus [9]. Until now, 17 related aflatoxin isoforms and aflatoxin metabolites have been identified, and 4 of them often contaminated a number of agricultural commodities [10]. According to the amounts and fluorescent reactions, four aflatoxins primarily identified in foodstuffs are named as AFB1, aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). Among these four known aflatoxins, AFB1 and AFB2 are named as B-type aflatoxins because they are attached to a pentanone and can produce blue-color fluorescent under UV light, whereas AFG1 and AFG2 are termed as G-type aflatoxins because of their attachment to a 6-membered lactone and producing green fluorescent color feature. These aflatoxins are mainly produced by *A. flavus*, *Aspergillus parasiticus* (*A. parasiticus*), *Aspergillus nidulans* (*A. nidulans*), *Aspergillus pseudotamarii* (*A. pseudotamarii*), and *Aspergillus bombycis* (*A. bombycis*) [7, 8].

reaction: R09–R12) (**Figure 3**) and third stages (biosynthesis reaction: R13–R18) (**Figure 4**) refer from hydroxyversicolorone to versicolorin B and from versicolorin B (VB) to the formation of ultimate products, respectively. These two stages involve in the formation of hydroxy- and non–hydroxy-versicolorone, and toxins. During the aflatoxin synthesis, more than 10 nicotinamide-adenine dinucleotide phosphate reduced form (NAPDH), one nicotinamide-adenine dinucleotide (NAD), and 2*S*-adenosylmethionine (SAM) are required. These cofactors may play

**Figure 1.** The aflatoxin gene cluster and their expression productions and functions. In the fungus-producing aflatoxins including *A. nidulans*, *A. parasiticus,* and *A. flavus*, genes encoding the enzymes and the transcription factors involving in aflatoxin biosynthesis commonly locate within a huge gene cluster of about 60–70 kb in the genomes. These genes, except for aflR and aflJ, involve in the 18 enzyme reaction steps (R01–R18) of aflatoxin biosynthesis, whereas aflR and aflJ expressing proteins are two important transcription factors and can regulate enzyme-related gene expression. "?" shows that the function of the corresponding gene is unknown (Note: adapted from Yabe and Nakajima [7]). *Abbreviations*. MCA, malonyl CoA; HAS, hexanoate synthase (also termed fatty acid synthase); PKS, polyketide synthase; NAS, Norsolorinic acid (NA) synthase; NAR, norsolorinic acid (NA) reductase; AVN, averantin; AVNM, averantin (AVN) monooxygenase; HAVN, 5′-hydroxyaverantin; HAVNR, 5′-hydroxyaverantin reductase; OVENC, 5′-oxoaverantin (OAVN) cyclase; AVRM, averufin (AVR) monooxygenase; VHAS, versiconal hemiacetal acetate (VHA) synthase; VHOHC, versiconal (VHOH) cyclase (also called versicolorin B synthase); VHAR, versiconal hemiacetal acetate (VHA) reductase; VBD, versicolorin B (VB) desaturase; DMSTSS, demethylsterigmatocystin (DMST) synthase system; OMTI,

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Aflatoxins synthesized in the mycelia are finally excreted into such mediums as cereals (maize, wheat, sorghum, rice, and millet), nuts (peanuts, pistachios, walnuts, Brazil nut, and coconut), spices (chili, turmeric, paprika, black pepper, and ginger), and seeds. Epidemiological studies have exhibited that AFB1 is the most common in contaminated human foods [8, 10]. Once this aflatoxin in the mediums is taken into body, it is metabolized via two-stage reactions in the liver. The first-stage metabolisms include reduction reaction (ketoreduction to aflatoxicol), oxidative reaction (O-dealkylation to aflatoxin P1), and hydrolytic reactions (hydroxylation to aflatoxin M1, aflatoxin Q1, and aflatoxin B2). This stage reaction involves numerous enzymes such as cytochromes P450 (CYP450), monooxygenases, amino-oxidases,

a critical role in the control of aflatoxin biosynthesis [7–10].

*O*-methyltransferase I; OMTII, *O*-methyltransferase II; OAE, OrdA enzyme.

**2.2. The metabolism of aflatoxins in liver**

Toxigenic strains of *A. flavus* produce only B-type aflatoxins, but do not synthesize G-type aflatoxins due to the deletion of an unstable microsome enzyme and a-220 kDa cytosolic protein. The other aflatoxigenic species including *A. parasiticus*, *A. nidulans*, *A. pseudotamarii*, and *A. bombycis* can produce all four aflatoxins [8].

Numeral synthetical genes, such as aflatoxin regulatory protein gene (aflR), are required for aflatoxin biosynthesis and act as a huge neighbor gene cluster consisting of about 60–70 kb in original fungi (**Figure 1**) [8–10]. All corresponding gene-encoding enzymes and transcription factors produce aflatoxin production and regulate biosynthesis. Increasing evidence has proved that aflatoxin biosynthesis involves in at least 3 stages and 18 enzyme steps (**Figures 2**–**4**). The first stage, including the first (R01) to eighth reaction (R08) of biosynthesis, refers from acetyl CoA to hydroxyversicolorone. The primary product hydroxyversicolorone will be formed and regulated by transcription factors aflR and aflJ (**Figure 2**) [8, 10]. The second (biosynthesis

**Figure 1.** The aflatoxin gene cluster and their expression productions and functions. In the fungus-producing aflatoxins including *A. nidulans*, *A. parasiticus,* and *A. flavus*, genes encoding the enzymes and the transcription factors involving in aflatoxin biosynthesis commonly locate within a huge gene cluster of about 60–70 kb in the genomes. These genes, except for aflR and aflJ, involve in the 18 enzyme reaction steps (R01–R18) of aflatoxin biosynthesis, whereas aflR and aflJ expressing proteins are two important transcription factors and can regulate enzyme-related gene expression. "?" shows that the function of the corresponding gene is unknown (Note: adapted from Yabe and Nakajima [7]). *Abbreviations*. MCA, malonyl CoA; HAS, hexanoate synthase (also termed fatty acid synthase); PKS, polyketide synthase; NAS, Norsolorinic acid (NA) synthase; NAR, norsolorinic acid (NA) reductase; AVN, averantin; AVNM, averantin (AVN) monooxygenase; HAVN, 5′-hydroxyaverantin; HAVNR, 5′-hydroxyaverantin reductase; OVENC, 5′-oxoaverantin (OAVN) cyclase; AVRM, averufin (AVR) monooxygenase; VHAS, versiconal hemiacetal acetate (VHA) synthase; VHOHC, versiconal (VHOH) cyclase (also called versicolorin B synthase); VHAR, versiconal hemiacetal acetate (VHA) reductase; VBD, versicolorin B (VB) desaturase; DMSTSS, demethylsterigmatocystin (DMST) synthase system; OMTI, *O*-methyltransferase I; OMTII, *O*-methyltransferase II; OAE, OrdA enzyme.

reaction: R09–R12) (**Figure 3**) and third stages (biosynthesis reaction: R13–R18) (**Figure 4**) refer from hydroxyversicolorone to versicolorin B and from versicolorin B (VB) to the formation of ultimate products, respectively. These two stages involve in the formation of hydroxy- and non–hydroxy-versicolorone, and toxins. During the aflatoxin synthesis, more than 10 nicotinamide-adenine dinucleotide phosphate reduced form (NAPDH), one nicotinamide-adenine dinucleotide (NAD), and 2*S*-adenosylmethionine (SAM) are required. These cofactors may play a critical role in the control of aflatoxin biosynthesis [7–10].

#### **2.2. The metabolism of aflatoxins in liver**

Increasing evidence has exhibited that several mechanisms, including the toxic production from metabolism, the accumulation of DNA damage and genic mutation–induced aflatoxins, the decreasing DNA repair capacity, and dysregulation of signal pathways may play a central role in the tumorigenesis of aflatoxin-induced hepatocarcinoma [4–6]. In this review, we discuss the biogenesis, metabolism, and genic toxification of aflatoxins. We also discuss the molecular mechanisms of aflatoxin-induced hepatocarcinoma, involving in aflatoxin toxification, abnormal change of tumor relative genes, the interaction of aflatoxins and genetic factors, and signal pathway for tumorigenesis. The roles of some important genes related to cell apoptosis, DNA repair, drug metabolism, and tumor metastasis in hepatocarcinogenesis

The biosynthesis of aflatoxins has been fully summarized in several previous reviews [7, 8]. In brief, aflatoxins are an important type of mycotoxins, which were the most early identified in the *Aspergillus flavus* (*A. flavus*) and regarded as causative agents of "turkey X" disease in the late 1950s and early 1960s. Thus, these toxins were named as "aflatoxins (namely *A. flavus* toxins)" according to their origin fungus [9]. Until now, 17 related aflatoxin isoforms and aflatoxin metabolites have been identified, and 4 of them often contaminated a number of agricultural commodities [10]. According to the amounts and fluorescent reactions, four aflatoxins primarily identified in foodstuffs are named as AFB1, aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). Among these four known aflatoxins, AFB1 and AFB2 are named as B-type aflatoxins because they are attached to a pentanone and can produce blue-color fluorescent under UV light, whereas AFG1 and AFG2 are termed as G-type aflatoxins because of their attachment to a 6-membered lactone and producing green fluorescent color feature. These aflatoxins are mainly produced by *A. flavus*, *Aspergillus parasiticus* (*A. parasiticus*), *Aspergillus nidulans* (*A. nidulans*), *Aspergillus pseudota-*

Toxigenic strains of *A. flavus* produce only B-type aflatoxins, but do not synthesize G-type aflatoxins due to the deletion of an unstable microsome enzyme and a-220 kDa cytosolic protein. The other aflatoxigenic species including *A. parasiticus*, *A. nidulans*, *A. pseudotamarii*, and

Numeral synthetical genes, such as aflatoxin regulatory protein gene (aflR), are required for aflatoxin biosynthesis and act as a huge neighbor gene cluster consisting of about 60–70 kb in original fungi (**Figure 1**) [8–10]. All corresponding gene-encoding enzymes and transcription factors produce aflatoxin production and regulate biosynthesis. Increasing evidence has proved that aflatoxin biosynthesis involves in at least 3 stages and 18 enzyme steps (**Figures 2**–**4**). The first stage, including the first (R01) to eighth reaction (R08) of biosynthesis, refers from acetyl CoA to hydroxyversicolorone. The primary product hydroxyversicolorone will be formed and regulated by transcription factors aflR and aflJ (**Figure 2**) [8, 10]. The second (biosynthesis

related to aflatoxins are further emphasized.

**2.1. Aflatoxin biosynthesis**

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**2. Aflatoxin biosynthesis, metabolism, and toxification**

*marii* (*A. pseudotamarii*), and *Aspergillus bombycis* (*A. bombycis*) [7, 8].

*A. bombycis* can produce all four aflatoxins [8].

Aflatoxins synthesized in the mycelia are finally excreted into such mediums as cereals (maize, wheat, sorghum, rice, and millet), nuts (peanuts, pistachios, walnuts, Brazil nut, and coconut), spices (chili, turmeric, paprika, black pepper, and ginger), and seeds. Epidemiological studies have exhibited that AFB1 is the most common in contaminated human foods [8, 10]. Once this aflatoxin in the mediums is taken into body, it is metabolized via two-stage reactions in the liver. The first-stage metabolisms include reduction reaction (ketoreduction to aflatoxicol), oxidative reaction (O-dealkylation to aflatoxin P1), and hydrolytic reactions (hydroxylation to aflatoxin M1, aflatoxin Q1, and aflatoxin B2). This stage reaction involves numerous enzymes such as cytochromes P450 (CYP450), monooxygenases, amino-oxidases, alcohol dehydrogenases, epoxide-hydrolases, aldehyde-reductases, and ketone-reductases. The second-stage reaction mainly comprises covalent binding reaction (toxic produces) and conjugation reaction (excretion and detoxification). Through these metabolites, aflatoxins ultimately transform into nontoxic secretions and toxic products [10, 11].

illness or death. This kind of acute effect is mainly associated with malfunction of the liver induced by toxic metabolic products. For chronic toxic effects, chronic exposure of aflatoxins can induce DNA damage and produce genotoxicity and carcinogenicity. In the past decades, increasing evidence has proved that AFB1 as aflatoxins often induce genic mutations such as TP53 and are among the most carcinogenic substances known and the major cancerous

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**Figure 3.** The second stage of aflatoxin biosynthesis. The second stage of aflatoxin biosynthesis, including the ninth (R09) to twelfth reaction (R12) of biosynthesis, refers from hydroxyversicolorone to versicolorin B (VB). *Abbreviations*. VHAS, versiconal hemiacetal acetate (VHA) synthase; VHOHC, versiconal (VHOH) cyclase (also called versicolorin B synthase); VHAR, versiconal hemiacetal acetate (VHA) reductase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH,

nicotinamide-adenine dinucleotide phosphate (reduced form). *Noted:* adapted from Yabe and Nakajima [7].

hepatocarcinoma risk factor.

#### **2.3. The toxification of aflatoxins in liver**

Toxification of aflatoxins in liver is mainly divided into acute and chronic toxic effects. Data from epidemiological, experimental, and clinical studies have shown that above 6000 mg exposure of aflatoxin through digestion will cause acute severe liver damage and subsequent

**Figure 2.** The first stage of aflatoxin biosynthesis. The first stage of aflatoxin biosynthesis, including the first (R01) to eighth reaction (R08) of biosynthesis, refers from acetyl CoA to hydroxyversicolorone. *Abbreviations*. MCA, malonyl CoA; HAS, hexanoate synthase (also termed fatty acid synthase); PKS, polyketide synthase; NAS, norsolorinic acid (NA) synthase; NAR, norsolorinic acid (NA) reductase; AVN, averantin; AVNM, averantin (AVN) monooxygenase; HAVN, 5′-hydroxyaverantin; HAVNR, 5′-hydroxyaverantin reductase; OVENC, 5′-oxoaverantin (OAVN) cyclase; AVRM, averufin (AVR) monooxygenase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide-adenine dinucleotide phosphate (reduced form); CoA, coenzyme A. *Noted:* adapted from Yabe and Nakajima [7].

illness or death. This kind of acute effect is mainly associated with malfunction of the liver induced by toxic metabolic products. For chronic toxic effects, chronic exposure of aflatoxins can induce DNA damage and produce genotoxicity and carcinogenicity. In the past decades, increasing evidence has proved that AFB1 as aflatoxins often induce genic mutations such as TP53 and are among the most carcinogenic substances known and the major cancerous hepatocarcinoma risk factor.

alcohol dehydrogenases, epoxide-hydrolases, aldehyde-reductases, and ketone-reductases. The second-stage reaction mainly comprises covalent binding reaction (toxic produces) and conjugation reaction (excretion and detoxification). Through these metabolites, aflatoxins

Toxification of aflatoxins in liver is mainly divided into acute and chronic toxic effects. Data from epidemiological, experimental, and clinical studies have shown that above 6000 mg exposure of aflatoxin through digestion will cause acute severe liver damage and subsequent

**Figure 2.** The first stage of aflatoxin biosynthesis. The first stage of aflatoxin biosynthesis, including the first (R01) to eighth reaction (R08) of biosynthesis, refers from acetyl CoA to hydroxyversicolorone. *Abbreviations*. MCA, malonyl CoA; HAS, hexanoate synthase (also termed fatty acid synthase); PKS, polyketide synthase; NAS, norsolorinic acid (NA) synthase; NAR, norsolorinic acid (NA) reductase; AVN, averantin; AVNM, averantin (AVN) monooxygenase; HAVN, 5′-hydroxyaverantin; HAVNR, 5′-hydroxyaverantin reductase; OVENC, 5′-oxoaverantin (OAVN) cyclase; AVRM, averufin (AVR) monooxygenase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide-adenine

dinucleotide phosphate (reduced form); CoA, coenzyme A. *Noted:* adapted from Yabe and Nakajima [7].

ultimately transform into nontoxic secretions and toxic products [10, 11].

**2.3. The toxification of aflatoxins in liver**

116 Liver Research and Clinical Management

**Figure 3.** The second stage of aflatoxin biosynthesis. The second stage of aflatoxin biosynthesis, including the ninth (R09) to twelfth reaction (R12) of biosynthesis, refers from hydroxyversicolorone to versicolorin B (VB). *Abbreviations*. VHAS, versiconal hemiacetal acetate (VHA) synthase; VHOHC, versiconal (VHOH) cyclase (also called versicolorin B synthase); VHAR, versiconal hemiacetal acetate (VHA) reductase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide-adenine dinucleotide phosphate (reduced form). *Noted:* adapted from Yabe and Nakajima [7].

**3. The molecular mechanisms of aflatoxin-induced hepatocarcinoma**

genic mutations, abnormal immunoreaction, and inheritance alterations.

dehydrogenase; AFB1-R, aflatoxin B1 reductase. *Noted:* adapted from Wu and Jezkova [10].

**3.1. Aflatoxin-induced DNA damage**

As described earlier, the main chronic toxification of aflatoxins is chronic liver damage and induced tumorigenesis of hepatocarcinoma. AFB1 has been proved as an I-type chemical carcinogen. Mechanisms of AFB1-induced hepatocarcinoma mainly involve in DNA damage and repair, the inactivation of tumor suppressor genes and the activation of oncogenes from

**Figure 5.** The metabolite of aflatoxins in the liver. Aflatoxins are metabolized via four metabolic pathways: O-dealkylation to aflatoxin P1 (AFP1), ketoreduction to aflatoxicol (AFL), epoxidation to AFB1-8,9-epoxide (AFBO, highly toxic, mutagenic, and carcinogenic), and hydroxylation to aflatoxin M1 (AFM1, highly toxic), AFP1, aflatoxin Q1 (AFQ1), or aflatoxin B2a (AFB2a). *Abbreviations*. AFM2, aflatoxin M2; AFP2, aflatoxin P2; AFQ2, aflatoxin Q2; AFL-D, aflatoxicol

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Increasing evidence has shown that the carcinogenicity of aflatoxins results from aflatoxininduced DNA damage, including the formation of DNA adducts, DNA single strand breaks (SSBs) or double strand breaks (DSBs), chromosomal aberration damage (CAD), unscheduled DNA synthesis (USDS), abnormal chromatid exchange (ACE), the formation of micronuclei and macronuclei, and oxidation DNA damage. Of these DNA damages, AFB1-DNA adducts

**Figure 4.** The third stage of aflatoxin biosynthesis. The third stage of aflatoxin biosynthesis, including the 13th (R13) to 18th reaction (R18) of biosynthesis, refers from versicolorin B (VB) to the formation of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). *Abbreviations*. VBD, versicolorin B (VB) desaturase; DMSTSS, demethylsterigmatocystin (DMST) synthase system; OMTI, *O*-methyltransferase I; OMTII, *O*-methyltransferase II; OAE, OrdA enzyme; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide-adenine dinucleotide phosphate (reduced form). *Noted:* adapted from Yabe and Nakajima [7].

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**Figure 5.** The metabolite of aflatoxins in the liver. Aflatoxins are metabolized via four metabolic pathways: O-dealkylation to aflatoxin P1 (AFP1), ketoreduction to aflatoxicol (AFL), epoxidation to AFB1-8,9-epoxide (AFBO, highly toxic, mutagenic, and carcinogenic), and hydroxylation to aflatoxin M1 (AFM1, highly toxic), AFP1, aflatoxin Q1 (AFQ1), or aflatoxin B2a (AFB2a). *Abbreviations*. AFM2, aflatoxin M2; AFP2, aflatoxin P2; AFQ2, aflatoxin Q2; AFL-D, aflatoxicol dehydrogenase; AFB1-R, aflatoxin B1 reductase. *Noted:* adapted from Wu and Jezkova [10].
