**Abstract**

Cancer incidences and mortality in Kenya are increasing according to recent reports and now number among the top five causes of mortality in the country. The risk factors responsible for this increase in cancer incidences are assumed to be genetic and/or environmental in nature. The environmental factors include exposure to carcinogenic contaminants such aflatoxins (AFs). However, the exact causes of the increase in cancer incidences and prevalence in many developing countries are not fully known. Aflatoxins are known contaminants produced by the common fungi *Aspergillus flavus* and the closely related *Aspergillus parasiticus* which grow as moulds in human foods. Aflatoxin B1 (AFB1) is most common in food and is 1000 times more potent when compared with benzo(a)pyrene, the most potent carcinogenic polycyclic aromatic hydrocarbon (PAH). Aflatoxins have therefore drawn a lot of interest in research from food safety and human health point of view. In this chapter, the chemistry, synthesis, identification, toxicology and potential human health risks of AFB1 in Kenya are discussed.

**Keywords:** aflatoxin B1, chemistry, determination, toxicity, exposure, health risks, Kenya

### **1. AFB1 chemistry**

The aflatoxins were discovered in a toxic peanut meal after causing 'turkey X' disease, which killed large numbers of turkey poults, ducks, young pheasants and chicks in the UK in the early 1960s [1], and more than 100,000 young turkeys in poultry farms were killed [2]. The peanut meal was highly toxic, and the toxin-producing fungi was identified as *Aspergillus flavus* hence the name of the toxin, aflatoxin [2]. Extracts of the feed later induced the now known toxic symptoms in experimental animals, and purified metabolites with properties identical to aflatoxins B1 and G1 (AFB1 and AFG1) were later isolated from the *Aspergillus flavus* cultures [1, 3, 4].

Structural elucidation of aflatoxins was accomplished and confirmed by total synthesis in 1963 [4]. There are four major aflatoxins B1, B2, G1 and G2 plus two additional toxic metabolic products M1 and M2 that are of significance as direct contaminants of foods and feeds and whose structures have been elucidated [3, 4]. These toxins have similar structures and form a unique group of highly oxygenated, naturally occurring heterocyclic compounds [5]. Their structures and molecular formulae are shown in **Figure 1**. *Aspergillus flavus* typically produces aflatoxin B1, which is the most potent and the most frequently identified in aflatoxin contaminations, and aflatoxin B2, whereas *Aspergillus parasiticus* produces aflatoxin G1 and aflatoxin G2 as well as aflatoxin B1 and aflatoxin B2. Four other aflatoxins M1, M2, B2A and G2A [3, 6], which are produced in minor amounts, were subsequently isolated from cultures of *Aspergillus flavus* and *Aspergillus parasiticus*. Aflatoxins M1 and M2, which are found in milk of animals that have consumed feeds contaminated with AFB1, are the hydroxylated metabolites of aflatoxins B1 and B2, respectively [3, 7]. Aflatoxins are, in essence, known as a group of mycotoxins which are produced primarily by some strains of *Aspergillus flavus* and by most strains of *Aspergillus parasiticus*, plus related species of *Aspergillus niger*, among others [8].

Aflatoxins are just a subset of class of mycotoxins which are fungal metabolites rampant and invisible in the environment and have caused severe effects on food security and safety especially within sub-Saharan African (SSA) societies [9]. This class of mycotoxins include *Fusarium* mycotoxins which have been found in oesophageal cancer-prone areas of South Africa [10], aflatoxins, fumonisins and ochratoxin A which have all been found to be rampant across West, East and Central Africa [11, 12]. Aflatoxins have become the most common and ubiquitous food contaminants produced by the common fungi *Aspergillus flavus* and the closely related *Aspergillus parasiticus*.

**5**

**Table 1.**

*Aflatoxin B1: Chemistry, Environmental and Diet Sources and Potential Exposure in Human…*

Aflatoxin B1 (AFB1) is a secondary metabolite produced by *Aspergillus flavus* and *Aspergillus parasiticus* when environmental factors are favourable [13, 14]. It has also been characterized as a biological toxin. Biological toxins are defined as toxic substances produced by microorganisms, animals and plants that have the capability of causing harmful effects when inhaled, ingested, injected or absorbed (medical dictionary). Referring to **Figure 1**, all aflatoxins are heterocyclic compounds which have a common benzene ring, with slight variations only in terms of the presence of double bonds and ketonic groups and the metabolites having hydroxy groups, with hydroxylation positions varying from one metabolite to another. These structures indicate slight aqueous solubility and ease of epoxidation reaction, respectively, which are considered to influence both their excretion and toxicity. AFB1 which is the most prevalent and most potent, a human health hazard globally, has a peculiar double bond in the cyclic ring which is also observed in G1 and M1. For activation, AFB1 requires epoxidation to aflatoxin B1 2,3-epoxide. The microsomal cytochrome P450 (CYP450) monoxygenases biotransform the toxin to the less toxic metabolites aflatoxins M1 and G1 [5]. Aflatoxins are highly oxygenated and naturally occurring heterocyclic compounds [4] which have been separated based on their fluorescence under UV light and the presence or lack of a double bond at the 8, 9 carbons. Aflatoxins B1 and G1 have a double bond at the 8, 9 carbons, which allows for formation of an epoxide, a more toxic form of AFB1 and AFG1, while AFB2 and AFG2 do not. Aflatoxins B2 and G2 were established as the dihydroxy derivatives of B1 and G1, respectively. Whereas, aflatoxin M1 (AFM1) is 4-hydroxy aflatoxin B1, aflatoxin M2 is 4-dihydroxy

aflatoxin B2 [5]. Hydrogenation of B1 and G1 yields B2 and G2, respectively.

IUPAC name 2,3,6a,9a-Tetrahydro-4-methoxycyclopenta[c]

formamide)

polar solvents

Fluorescence emission Densely fluorescent blue (λmax = 450 nm)

Vapour pressure 2.65 × 10<sup>−</sup>10 mmHg at 25°C

Koc (soil) Ranges within 682–2.317 × 10<sup>−</sup><sup>4</sup>

UV absorption Absorbs at 223, 265 and 362 nm

Henry's law constant 1.4 × 10<sup>−</sup>13 atm m3

The important physico-chemical properties of AFB1 are shown in **Table 1**. It is odourless, tasteless and colourless. It is difficult to detect sensorically, and therefore it poses a real challenge to food handlers, consumers and regulators who are in a bid to control or eradicate it [15–17]. AFB1 exists as colourless to pale yellow crystals or white powder [18]. Aflatoxins are densely fluorescent; B refers to blue fluorescence, while

Furo[3′,2′:4,5]furo[2,3-*h*][l] benzopyran-1,11-dione

Water solubility 16.14 mg/l at 25°C; decreases at low temperature; generally soluble in water and

Stability Stable until melting point; decomposed by UV irradiation in water/chloroform

/mol at 25°C

Mass spectrum Identified by LC–MS; ionization ESI; precursor-type [M + H]+

−558 °/D at 25°C (0.1 M in chloroform) or −480 °/D at 25°C (0.1 M in dimethyl

; m/z 313.071

Physical state Colourless pale yellow crystalline to solid or white powder; odorless

*DOI: http://dx.doi.org/10.5772/intechopen.88773*

**Physico-chemical property**

Log Kow 1.23 BCF (fish) 3

*Physico-chemical properties of AFB1.*

Specific Optical rotation

MW 312.277 g/mol mp 268–269°C

#### *Aflatoxin B1: Chemistry, Environmental and Diet Sources and Potential Exposure in Human… DOI: http://dx.doi.org/10.5772/intechopen.88773*

Aflatoxin B1 (AFB1) is a secondary metabolite produced by *Aspergillus flavus* and *Aspergillus parasiticus* when environmental factors are favourable [13, 14]. It has also been characterized as a biological toxin. Biological toxins are defined as toxic substances produced by microorganisms, animals and plants that have the capability of causing harmful effects when inhaled, ingested, injected or absorbed (medical dictionary). Referring to **Figure 1**, all aflatoxins are heterocyclic compounds which have a common benzene ring, with slight variations only in terms of the presence of double bonds and ketonic groups and the metabolites having hydroxy groups, with hydroxylation positions varying from one metabolite to another. These structures indicate slight aqueous solubility and ease of epoxidation reaction, respectively, which are considered to influence both their excretion and toxicity. AFB1 which is the most prevalent and most potent, a human health hazard globally, has a peculiar double bond in the cyclic ring which is also observed in G1 and M1. For activation, AFB1 requires epoxidation to aflatoxin B1 2,3-epoxide. The microsomal cytochrome P450 (CYP450) monoxygenases biotransform the toxin to the less toxic metabolites aflatoxins M1 and G1 [5]. Aflatoxins are highly oxygenated and naturally occurring heterocyclic compounds [4] which have been separated based on their fluorescence under UV light and the presence or lack of a double bond at the 8, 9 carbons. Aflatoxins B1 and G1 have a double bond at the 8, 9 carbons, which allows for formation of an epoxide, a more toxic form of AFB1 and AFG1, while AFB2 and AFG2 do not. Aflatoxins B2 and G2 were established as the dihydroxy derivatives of B1 and G1, respectively. Whereas, aflatoxin M1 (AFM1) is 4-hydroxy aflatoxin B1, aflatoxin M2 is 4-dihydroxy aflatoxin B2 [5]. Hydrogenation of B1 and G1 yields B2 and G2, respectively.

The important physico-chemical properties of AFB1 are shown in **Table 1**. It is odourless, tasteless and colourless. It is difficult to detect sensorically, and therefore it poses a real challenge to food handlers, consumers and regulators who are in a bid to control or eradicate it [15–17]. AFB1 exists as colourless to pale yellow crystals or white powder [18]. Aflatoxins are densely fluorescent; B refers to blue fluorescence, while


#### **Table 1.**

*Physico-chemical properties of AFB1.*

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

related *Aspergillus parasiticus*.

These toxins have similar structures and form a unique group of highly oxygenated, naturally occurring heterocyclic compounds [5]. Their structures and molecular formulae are shown in **Figure 1**. *Aspergillus flavus* typically produces aflatoxin B1, which is the most potent and the most frequently identified in aflatoxin contaminations, and aflatoxin B2, whereas *Aspergillus parasiticus* produces aflatoxin G1 and aflatoxin G2 as well as aflatoxin B1 and aflatoxin B2. Four other aflatoxins M1, M2, B2A and G2A [3, 6], which are produced in minor amounts, were subsequently isolated from cultures of *Aspergillus flavus* and *Aspergillus parasiticus*. Aflatoxins M1 and M2, which are found in milk of animals that have consumed feeds contaminated with AFB1, are the hydroxylated metabolites of aflatoxins B1 and B2, respectively [3, 7]. Aflatoxins are, in essence, known as a group of mycotoxins which are produced primarily by some strains of *Aspergillus flavus* and by most strains of *Aspergillus parasiticus*, plus related species of *Aspergillus niger*, among others [8]. Aflatoxins are just a subset of class of mycotoxins which are fungal metabolites rampant and invisible in the environment and have caused severe effects on food security and safety especially within sub-Saharan African (SSA) societies [9]. This class of mycotoxins include *Fusarium* mycotoxins which have been found in oesophageal cancer-prone areas of South Africa [10], aflatoxins, fumonisins and ochratoxin A which have all been found to be rampant across West, East and Central Africa [11, 12]. Aflatoxins have become the most common and ubiquitous food contaminants produced by the common fungi *Aspergillus flavus* and the closely

**4**

**Figure 1.**

*Chemical structures of aflatoxin B1 and other related aflatoxin metabolites [3, 6].*

G signifies green fluorescence. AFB1 exhibits a blue fluorescence with a fluorescence emission spectrum maximum of 425 nm and has UV maximum absorbance values at 223, 265 and 362 nm (in ethanol). It strongly absorbs UV light and is decomposed by it when dissolved in water or chloroform or when it is in form of solid films. AFBI has a Henry's law constant value of 1.40 × 10<sup>−</sup>13 atm m3 /mol at 25°C and a vapour pressure of 2.65 × 10<sup>−</sup>10 mmHg at 25°C. These properties would enable it to be less volatile and therefore has become very ubiquitous in the environment, becoming distributed in air, water and soil [15, 18]. It therefore can spread easily on the farm or in stores causing heavy damage to agricultural food crops and stored grains, respectively.

The vapour pressure of AFB1 indicates that AFB1 will tend to exist solely in particulate phase in the atmosphere if released into air, according to a model of gas/ particle partitioning of semivolatile organic compounds [19]. The particulate bound AFB1 will then tend to be removed from the atmosphere by wet and dry deposition. Since it absorbs UV light, it is susceptible to direct photolysis by sunlight. If released to soil, AFB1 is expected to have low mobility based on its Koc value which ranges from 682 to 2.3 × 104 and Freundlich adsorption coefficients, ranging from 17 to 238 mg/kg in different soil types. Volatilization from moist soils or water surfaces is not expected to be an important fate process based on its Henry's law constant value of 1.4 × 10<sup>−</sup>13 atm-cm/mol. It is also not expected to volatilize much from dry soil surfaces based on its vapour pressure which is very low. The Koc of AFB1 indicates that it is expected to adsorb to soil and sediment. However, based on its Kow and BCF values, AFB1 would tend to have a relatively moderate potential for bioconcentration in aquatic organisms and animal adipose tissue. Perhaps this explains why it is rapidly absorbed in the stomach and intestines and why it is present in the blood, kidney and liver where it imparts its toxicity. In the water environment, AFB1 can undergo hydrolysis as it contains a cyclic ester functional group and the rates of hydrolysis are similar to those of non-cyclic esters, ranging from months to a year under normal environmental conditions (i.e. pH 5–9) [19]. However, ring strain and steric hindrance have been reported to prevent its ease of hydrolysis, and therefore the extent of hydrolysis is unexpectedly low [18]. AFB1 biodegradation in soil and water has been studied, and it has been found that biodegradation may not be a very important environmental fate process. For example, after incubation for 120 days in silt loam, clay loam and sandy loam soil types, respectively, only 8.1, 4.9 and 1.4% complete mineralization to CO2 was achieved [19]. Biodegradation in various soils with different pHs (ranging 5.8–7.3), organic carbon (OC) (ranging 0.46–2.82%) and cation exchange capacity (CEC) (ranging 11.7–18) showed very low concentrations of metabolites B2 and G2 after 1 day in a 20-day experiment, and the TLC results indicated that adsorption onto soil prevented AFB1 decomposition.

Biotransformation of aflatoxins has been studied and found to occur via four main routes [19–23]: (i) hydroxylation of carbon atom at junction of the two fused furan rings, aflatoxin B1 is converted into AFM1, and this occurs to some extent in the mammalian liver [19, 20]; (ii) oxidative o-demethylation of single aromatic methoxy-substituent gives aflatoxin P1 [19]; (iii) hydration of vinyl double bond would afford hemiacetals, and aflatoxin B1 has been converted to into hemiacetal AFB2A in pig, mouse and avian livers through this route [19, 22] and (iv) reduction of cyclopentenone ring, dihydroaflatoxicol, but this biotransformation seems to be confined to avian species and not mammals [19]. While the hydroxylated metabolite AFM1 is the product of metabolism of AFB1 and AFB2, G1 and G2 were established as dihydroxylated derivatives of B1 and B2, respectively. AFM1 is 4-hydroxy aflatoxin B1 and AFM2 is 4-hydroxy aflatoxin B2. The order of acute and chronic toxicity is B1 > FG1 > B2 > G2 [20].

Extensive studies on reactions of aflatoxins to various physico-chemical conditions and reagents have been conducted because of possible application of such

**7**

*Aflatoxin B1: Chemistry, Environmental and Diet Sources and Potential Exposure in Human…*

reactions in detoxification of materials contaminated with aflatoxins [24]. In dry state, aflatoxins are heat stable up to melting point. However, in the presence of moisture and elevated temperatures, aflatoxins are destroyed to certain extents over a period of time. Such destructions of aflatoxins have been found to occur in oil seeds, meals and roasted peanuts or in aqueous solution at pH 7 [15–17]. It is postulated that such treatments can lead to the opening of the lactose ring, with possible destruction of decarboxylation, at elevated temperature [21]. In alkaline solution, hydrolysis of the lactose ring occurs, but this hydrolysis appears reversible, since it has also been shown that recyclization occurs following acidification of basic solutions containing aflatoxin [21, 24]. At a temperature of 100°C, lactose ring opening can occur, followed by a decarboxylation reaction [21]; and this reaction can further lead to a loss of the methoxy group from the aromatic ring [22]. In the presence of mineral acids, aflatoxins B1 and G1 are converted to aflatoxins B2A and G2A, respectively, due to acid-catalyzed insertion of water molecules across the double bonds in the furan ring, leading to hydroxylation (see **Figure 1** chemical structures). In the presence of acetic and hydrochloric acids, the reaction of AFB1 and AFG1, respectively, gives the acetoxyl derivatives, with acetoxyl groups attached on the benzene rings [22]. Similar adducts of aflatoxins B1 and G1 are formed with methanoic acid-thionyl chloride, acetic acid-thionyl chloride and trifluoroacetic acid [22]. Reactions with oxidizing agents, such as sodium hypochlorite, potassium permanganate, chlorine, hydrogen peroxide, ozone and sodium perborate, change the aflatoxin molecule in some way as indicated by loss of fluorescences although the mechanisms of these reactions are still uncertain as the products remain unidentified in most cases [25]. Hydrogenation of aflatoxins B1 and G1 yields aflatoxins B2 and G2, respectively. If further reduced by 3 mol of hydrogen, aflatoxin B1 yields tetrahydroxyl aflatoxin, while reduction of aflatoxins B1 and B2 with sodium borohydride yields aflatoxins RB1 and RB2, respectively. The RB1 and RB2 arise because of the opening of the lactose ring followed by reduction of the acid group and the keto group in the cyclopentane ring. However, it should be noted that breakdown of aflatoxins by various means does not guarantee safety of the contaminated substance. At times this breakdown is reversible or may lead to another form of aflatoxin. Besides, reaction products have not been subjected to detailed examination, including length of time the reactions take place [25]. Researchers have just concluded that the

decomposition is not complete based on trials with food samples [26].

In general, the aflatoxins have been considered as difuranocoumarins, which are highly substituted coumarin derivatives containing a fused dihydrofurofuran moiety [1, 3, 4]. In particular, AFB1 is characterized by the fusion of a cyclopentenone ring to the lactone ring of the coumarin structure (**Figure 1**) and by strong fluorescence emission in the blue region (hence the designation B) when exposed to ultraviolet light [1, 3, 4]. Aflatoxins Bs strongly emit blue colour when they absorb UV light, and aflatoxins Gs strongly emit green colour when they absorb UV light. AFM1 is the principal hydroxylated metabolite of AFB1 and is produced upon the action of cytochrome P450 1A2 (CYP1A2) [27, 28]. It is strongly fluorescent, emitting blue-violet light. Specifically, AFB1 has similar chemical properties to other metabolites which include its slight solubility in water and polar organic solvents and less solubility in nonpolar solvents [23]. It has strong thermal stability, even at high temperature (>100°C), and this prevents it from being thermally degraded completely during food manufacturing, for example, when milk and dairy products are processed, since pasteurization and other thermal treatment methods alone are ineffective [29, 30]. Other chemical properties of AFB1, such as its instability to UV light or extreme pH conditions (<3 or >10) and reactivity of lactone moiety in the presence of ammonia or hypochlorite, have been useful in the development of methods for decontamination of feed and food [29, 30]. Several physical treatment

*DOI: http://dx.doi.org/10.5772/intechopen.88773*

#### *Aflatoxin B1: Chemistry, Environmental and Diet Sources and Potential Exposure in Human… DOI: http://dx.doi.org/10.5772/intechopen.88773*

reactions in detoxification of materials contaminated with aflatoxins [24]. In dry state, aflatoxins are heat stable up to melting point. However, in the presence of moisture and elevated temperatures, aflatoxins are destroyed to certain extents over a period of time. Such destructions of aflatoxins have been found to occur in oil seeds, meals and roasted peanuts or in aqueous solution at pH 7 [15–17]. It is postulated that such treatments can lead to the opening of the lactose ring, with possible destruction of decarboxylation, at elevated temperature [21]. In alkaline solution, hydrolysis of the lactose ring occurs, but this hydrolysis appears reversible, since it has also been shown that recyclization occurs following acidification of basic solutions containing aflatoxin [21, 24]. At a temperature of 100°C, lactose ring opening can occur, followed by a decarboxylation reaction [21]; and this reaction can further lead to a loss of the methoxy group from the aromatic ring [22]. In the presence of mineral acids, aflatoxins B1 and G1 are converted to aflatoxins B2A and G2A, respectively, due to acid-catalyzed insertion of water molecules across the double bonds in the furan ring, leading to hydroxylation (see **Figure 1** chemical structures). In the presence of acetic and hydrochloric acids, the reaction of AFB1 and AFG1, respectively, gives the acetoxyl derivatives, with acetoxyl groups attached on the benzene rings [22]. Similar adducts of aflatoxins B1 and G1 are formed with methanoic acid-thionyl chloride, acetic acid-thionyl chloride and trifluoroacetic acid [22]. Reactions with oxidizing agents, such as sodium hypochlorite, potassium permanganate, chlorine, hydrogen peroxide, ozone and sodium perborate, change the aflatoxin molecule in some way as indicated by loss of fluorescences although the mechanisms of these reactions are still uncertain as the products remain unidentified in most cases [25]. Hydrogenation of aflatoxins B1 and G1 yields aflatoxins B2 and G2, respectively. If further reduced by 3 mol of hydrogen, aflatoxin B1 yields tetrahydroxyl aflatoxin, while reduction of aflatoxins B1 and B2 with sodium borohydride yields aflatoxins RB1 and RB2, respectively. The RB1 and RB2 arise because of the opening of the lactose ring followed by reduction of the acid group and the keto group in the cyclopentane ring. However, it should be noted that breakdown of aflatoxins by various means does not guarantee safety of the contaminated substance. At times this breakdown is reversible or may lead to another form of aflatoxin. Besides, reaction products have not been subjected to detailed examination, including length of time the reactions take place [25]. Researchers have just concluded that the decomposition is not complete based on trials with food samples [26].

In general, the aflatoxins have been considered as difuranocoumarins, which are highly substituted coumarin derivatives containing a fused dihydrofurofuran moiety [1, 3, 4]. In particular, AFB1 is characterized by the fusion of a cyclopentenone ring to the lactone ring of the coumarin structure (**Figure 1**) and by strong fluorescence emission in the blue region (hence the designation B) when exposed to ultraviolet light [1, 3, 4]. Aflatoxins Bs strongly emit blue colour when they absorb UV light, and aflatoxins Gs strongly emit green colour when they absorb UV light. AFM1 is the principal hydroxylated metabolite of AFB1 and is produced upon the action of cytochrome P450 1A2 (CYP1A2) [27, 28]. It is strongly fluorescent, emitting blue-violet light. Specifically, AFB1 has similar chemical properties to other metabolites which include its slight solubility in water and polar organic solvents and less solubility in nonpolar solvents [23]. It has strong thermal stability, even at high temperature (>100°C), and this prevents it from being thermally degraded completely during food manufacturing, for example, when milk and dairy products are processed, since pasteurization and other thermal treatment methods alone are ineffective [29, 30]. Other chemical properties of AFB1, such as its instability to UV light or extreme pH conditions (<3 or >10) and reactivity of lactone moiety in the presence of ammonia or hypochlorite, have been useful in the development of methods for decontamination of feed and food [29, 30]. Several physical treatment

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

a Henry's law constant value of 1.40 × 10<sup>−</sup>13 atm m3

from 682 to 2.3 × 104

G signifies green fluorescence. AFB1 exhibits a blue fluorescence with a fluorescence emission spectrum maximum of 425 nm and has UV maximum absorbance values at 223, 265 and 362 nm (in ethanol). It strongly absorbs UV light and is decomposed by it when dissolved in water or chloroform or when it is in form of solid films. AFBI has

of 2.65 × 10<sup>−</sup>10 mmHg at 25°C. These properties would enable it to be less volatile and therefore has become very ubiquitous in the environment, becoming distributed in air, water and soil [15, 18]. It therefore can spread easily on the farm or in stores caus-

The vapour pressure of AFB1 indicates that AFB1 will tend to exist solely in particulate phase in the atmosphere if released into air, according to a model of gas/ particle partitioning of semivolatile organic compounds [19]. The particulate bound AFB1 will then tend to be removed from the atmosphere by wet and dry deposition. Since it absorbs UV light, it is susceptible to direct photolysis by sunlight. If released to soil, AFB1 is expected to have low mobility based on its Koc value which ranges

238 mg/kg in different soil types. Volatilization from moist soils or water surfaces is not expected to be an important fate process based on its Henry's law constant value of 1.4 × 10<sup>−</sup>13 atm-cm/mol. It is also not expected to volatilize much from dry soil surfaces based on its vapour pressure which is very low. The Koc of AFB1 indicates that it is expected to adsorb to soil and sediment. However, based on its Kow and BCF values, AFB1 would tend to have a relatively moderate potential for bioconcentration in aquatic organisms and animal adipose tissue. Perhaps this explains why it is rapidly absorbed in the stomach and intestines and why it is present in the blood, kidney and liver where it imparts its toxicity. In the water environment, AFB1 can undergo hydrolysis as it contains a cyclic ester functional group and the rates of hydrolysis are similar to those of non-cyclic esters, ranging from months to a year under normal environmental conditions (i.e. pH 5–9) [19]. However, ring strain and steric hindrance have been reported to prevent its ease of hydrolysis, and therefore the extent of hydrolysis is unexpectedly low [18]. AFB1 biodegradation in soil and water has been studied, and it has been found that biodegradation may not be a very important environmental fate process. For example, after incubation for 120 days in silt loam, clay loam and sandy loam soil types, respectively, only 8.1, 4.9 and 1.4% complete mineralization to CO2 was achieved [19]. Biodegradation in various soils with different pHs (ranging 5.8–7.3), organic carbon (OC) (ranging 0.46–2.82%) and cation exchange capacity (CEC) (ranging 11.7–18) showed very low concentrations of metabolites B2 and G2 after 1 day in a 20-day experiment, and the TLC results indicated that adsorption onto soil prevented AFB1 decomposition.

Biotransformation of aflatoxins has been studied and found to occur via four main routes [19–23]: (i) hydroxylation of carbon atom at junction of the two fused furan rings, aflatoxin B1 is converted into AFM1, and this occurs to some extent in the mammalian liver [19, 20]; (ii) oxidative o-demethylation of single aromatic methoxy-substituent gives aflatoxin P1 [19]; (iii) hydration of vinyl double bond would afford hemiacetals, and aflatoxin B1 has been converted to into hemiacetal AFB2A in pig, mouse and avian livers through this route [19, 22] and (iv) reduction of cyclopentenone ring, dihydroaflatoxicol, but this biotransformation seems to be confined to avian species and not mammals [19]. While the hydroxylated metabolite AFM1 is the product of metabolism of AFB1 and AFB2, G1 and G2 were established as dihydroxylated derivatives of B1 and B2, respectively. AFM1 is 4-hydroxy aflatoxin B1 and AFM2 is 4-hydroxy aflatoxin B2. The order of acute and chronic

Extensive studies on reactions of aflatoxins to various physico-chemical conditions and reagents have been conducted because of possible application of such

and Freundlich adsorption coefficients, ranging from 17 to

ing heavy damage to agricultural food crops and stored grains, respectively.

/mol at 25°C and a vapour pressure

**6**

toxicity is B1 > FG1 > B2 > G2 [20].

methods like exposure to microwaves, gamma rays, X-rays and ultraviolet light have been investigated, but inconsistency of the results has discouraged their use, especially for heavily contaminated samples [31]. At present, ammoniation [32] and adsorption on clays or organic adsorbents [29] have commonly been used to achieve a good level of decontamination without disruption of the nutritional properties or safety of feed.

Biological methods of detoxification of mycotoxins are of two different types: the first being via enzymatic degradation and the second via sorption. In enzymatic biochemical processes, live microorganisms can biodegrade and mineralize the mycotoxins completely to CO2 or absorb them by attaching them to their cells by active interaction and accumulation and thereby reducing them from the media. Dead organisms can adsorb mycotoxins, and they can be used to make biofilters for fluid decontamination of products, where the aflatoxins are left on the filter and the products become subsequently decontaminated, or as probiotics to bind and remove mycotoxins from the human intestine [15, 33]. Enzymatic degradation can be complete mineralization to CO2, in which either extracellular or intracellular enzymes and various species of bacteria have been identified including *Pseudomonas*, *Bacillus* and *Lactobacillus* and used to inhibit toxicity or production of aflatoxins by *Aspergillus*. A large number of microorganisms (approximately 1000) have been screened for this purpose, but only *Lactobacillus* have been adopted [34, 35]. AFB1 and AFM1 have been shown to have a strong binding ability to other molecules, and recently research has been focusing on the AFB1-binding capacity to certain metabolites, for example, different strains of *Lactobacillus* in milk for aflatoxin decontamination in different products such as yoghurt [34, 36, 37].

Various chemical treatment processes have been tried, including sodium hypochlorite (NaOCl), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), sodium bicarbonate, sodium chloride and sodium borohydride (NaHBO3) a wellknown reducing agent, to detoxify or decompose aflatoxins in various foods [16, 38, 39]. These reagents can be used, and, for example, formaldehyde and NH3 were found to neutralize AFB1, while NaSO4 was found to be less efficient in neutralizing AFB1 [38]. However, these reactions have to be optimized in terms of quantities needed and reaction time as well as temperature and pressure conditions required. Different cooking methods have also been tried to remove aflatoxins from foods [16, 17, 38, 40]. Normal cooking of rice was found to destroy only 49% AFB1 [16, 17]. In other experiments to study the reduction of aflatoxins in various products, boiling of maize in traditional cooking used in Kenya destroys 11–17.6% AFB1 and AFG2 [40], while in beer making 18–27% AFB1 still remain [38] and in bread making 25% still remain [26]. Kirui [39], in assessing the levels of aflatoxins that were left after various treatments following physico-chemical and traditional cooking methods for maize and maize products, found that boiling maize reduced total aflatoxin level from 83 to 7 ppb, dry decortication reduced the level from 51.3 to 9.6 ppb, boiling with Magadi soda (food softener) reduced the level from 59 to 13.4 ppb, solar irradiation (18 h) reduced the level from 60.8 to 13.7 ppb and UV irradiation (18 h) reduced the level from 81.7 to 61.4 ppb. He found that only dry decortication method, which involves boiling with Magadi soda followed by washing with water and boiling, respectively, reduced the levels significantly but not completely below the maximum limits. Alkali treatment with inorganic (e.g. boiling with NaCl) and organic bases were reported to be effective and economically feasible [17]. Occupational exposure to AFB1 has been reported to occur through inhalation and dermal contact at work places where commodities such as peanuts, grains, linseed oil or animal feeds are produced, stored or used. An average AFB1 exposure of 64 ng/d-kg body weight was reported for Danish workers in the animal feed production industry. General population may most likely be exposed to AFB1 via ingestion of contaminated food [18].

**9**

*Aflatoxin B1: Chemistry, Environmental and Diet Sources and Potential Exposure in Human…*

The biosynthetic pathway of AFB1 has been explained by researchers. It is derived from both a dedicated fatty acid synthase (FAS) and a polyketide synthase (PKS) which occur in the mould, together known as norsolorinic acid synthases. The biosynthetic pathway has been described by Singh and Hsich [41], Yu et al. [42] and Dewick [43], among others, and, an outline of the method can be found in Wikipedia. The process begins with a FAS-aided synthesis of hexanoic acid, which is the starter unit for the iterative type I PKS. A PKS catalyzes addition of seven malonyl-CoA molecules to the hexanoic acid to form a C20 polyketide compound. The polyketide folds through a cyclization process induced by a PKS to form an anthraquinone norsolorinic acid, and a reductase enzyme then catalyzes the reduction of the ketone on the norsolorinic acid side chain to yield an intermediate, an averantin [41–43]. From here, various processes which are assisted with different enzymes including hydroxylases, dehydrogenases (for oxygenation and cyclization), CYP450 oxidases, esterases, reductases, methyl transferases and oxidoreductases occur, leading to different intermediates. The pathway for AFB1 biosynthesis is very complicated, and some of the enzymes and

intermediates involved continue to be elucidated and characterized [43].

Under favorable moulding conditions, *Aspergillus flavus* spores germinate by attaching their mycelium in a food substrate and secreting enzymes which break down nutrients into simpler compounds capable of digestion. During digestion, *Aspergillus flavus* then produces, as described in the foregoing paragraph, secondary metabolites, including AFB1, meant to give the fungi a competitive edge against

For research and other purposes, aflatoxins can be produced in small quantities by fermentation of *Aspergillus flavus* or *Aspergillus parasiticus* on solid substrates or media [45]. It is extracted by solvents and purified by chromatography [45]. AFB1 and other aflatoxins have been produced through this method by many chemical companies including Sigma-Aldrich, among others. While doing the purification, it is important to note that *Aspergillus flavus* produces only B aflatoxins and sometimes the mycotoxin cyclopiazonic acid (CPA), while *Aspergillus parasiticus* produces both B and G aflatoxins but not CPA. Various mutants of *Aspergillus flavus* have varying relative stability ratios of B2/B1 [45, 46]. Ada and Matcha [46] described a method for aflatoxin production by fermentation in which an *Aspergillus flavus* strain isolated from groundnut, referred to as *Aspergillus flavus* strain AJ, was used. The *Aspergillus flavus* strain AJ was found to be very stable and consistently yielded higher levels of aflatoxins, especially AFB1, after transfers [46]. In their preparation Ada and Matcha [46] used inoculums prepared by inoculating tubes (1.5 × 15 cm) of potato-dextroseagar with spores of the AJ. This strain was used to produce an aflatoxin stain AJ2010. The potato-dextrose-agar gel was prepared by adding 20 g dextrose, 20 mg NaCl and 1 g of agar in 100 ml distilled in a conical flask, adjusting and maintaining the mixture at pH 7. The mixture was kept momentarily at 121°C in an autoclave and then platted in a laminar flow [46, 47]. The inoculated slants were then incubated for 7–21 days at 28°C after which the cultures had a heavy crop of green *conidia*, and the spores were scraped loose with a loop. The slants were shaken to give a uniform suspension of spores, and the spore suspension (0.5 ml) was used to inoculate each of 100 g of the substrate (groundnut), a fish feed. Fermentation which involved the growth of *A. flavus* on the feed (100 g) at high moisture levels to produce a pale green aflatoxin substrate was carried out by mixing 25 ml distilled water with 50 g of fish feed in an Erlenmeyer flask. The mixture was allowed to stand for 1 h with frequent shaking, and then the flasks were autoclaved at 15 psi for 15 min before cooling and inoculation, keeping the flasks at 28°C and blending on a shaker at 188 rev/min. The flasks were removed, and the feed was prevented from binding with

*DOI: http://dx.doi.org/10.5772/intechopen.88773*

**2. Synthesis of aflatoxin B1**

other microorganisms [44].

*Aflatoxin B1: Chemistry, Environmental and Diet Sources and Potential Exposure in Human… DOI: http://dx.doi.org/10.5772/intechopen.88773*
