**2. Aflatoxins**

Aflatoxins (AFs) are the best known and most widely studied mycotoxins. They were first isolated in the early 1960s when 100,000 turkey poults died after consuming aflatoxincontaminated peanut meal in the UK (the so-called Turkey X disease); this event was followed by proliferation in research on fungal toxins contaminating food and feeds. AFs were found to be the most potent naturally formed carcinogen, and researchers started their investigating on factors that influence this production (Blount, 1960; CAST, 1989).

AFs are highly toxic, mutagenic, and carcinogenic compounds (Wogan, 1999). They are secondary metabolites that are produced mainly by *Aspergillus parasiticus* and *Aspergillus flavus*; in fact, the name ''aflatoxin" is derived from the first letter in Aspergillus, and the first three letters in flavus. These fungi are found in many countries, especially in tropical and subtropical regions, where the temperature and humidity conditions are optimal for the growth of moulds and the production of toxin (Rustom, 1997).

Fig. 1. Principal aflatoxins and metabolites.

AFs are natural contaminants of several agricultural products, such as: corn, peanuts, cottonseed, and other grain crops (Gourama & Bullerman, 1995). Diet is the major way through which humans as well as animals are exposed to these mycotoxins. AFM1 is transformed at the hepatic level by means of cytochrome P450 enzymes and excreted into the milk in the mammary glands of both humans and lactating animals after the animals have ingested feeds contaminated with AFB1 (Oveisi et al., 2007; Prandini et al., 2009).

humans and animals are exposed to a mixture rather than to individual compounds. For example, the interactive (synergistic) cytotoxic effects of Ochratoxin A (OTA), Ochratoxin B (OTB), citrinin, and patulin, which are produced by a number of *Penicillium* and *Aspergillus* 

Aflatoxins (AFs) are the best known and most widely studied mycotoxins. They were first isolated in the early 1960s when 100,000 turkey poults died after consuming aflatoxincontaminated peanut meal in the UK (the so-called Turkey X disease); this event was followed by proliferation in research on fungal toxins contaminating food and feeds. AFs were found to be the most potent naturally formed carcinogen, and researchers started their

AFs are highly toxic, mutagenic, and carcinogenic compounds (Wogan, 1999). They are secondary metabolites that are produced mainly by *Aspergillus parasiticus* and *Aspergillus flavus*; in fact, the name ''aflatoxin" is derived from the first letter in Aspergillus, and the first three letters in flavus. These fungi are found in many countries, especially in tropical and subtropical regions, where the temperature and humidity conditions are optimal for the

O

O

O

O

O

O

O

O

O

OH AFM2

AFG2

O

AFB2

OCH3

O

O

OCH3

<sup>O</sup> <sup>O</sup>

OCH3

<sup>O</sup> <sup>O</sup>

AFs are natural contaminants of several agricultural products, such as: corn, peanuts, cottonseed, and other grain crops (Gourama & Bullerman, 1995). Diet is the major way through which humans as well as animals are exposed to these mycotoxins. AFM1 is transformed at the hepatic level by means of cytochrome P450 enzymes and excreted into the milk in the mammary glands of both humans and lactating animals after the animals have ingested feeds contaminated with AFB1 (Oveisi et al., 2007; Prandini et al., 2009).

O

investigating on factors that influence this production (Blount, 1960; CAST, 1989).

OCH3

O

OCH3

<sup>O</sup> <sup>O</sup>

OCH3

<sup>O</sup> <sup>O</sup>

species, have recently been evaluated by Heussner et al. (2006).

growth of moulds and the production of toxin (Rustom, 1997).

O

O

O

OH AFM1

AFG1

O

AFB1

Fig. 1. Principal aflatoxins and metabolites.

O

O

O

O

O

O

**2. Aflatoxins** 

Structurally, AFs are difurocoumarin derivatives that fluoresce under ultraviolet light. Depending upon colour of the fluorescence, AFs are divided into aflatoxin B1 and B2 (AFB1, AFB2) for blue, and G1 and G2 (AFG1, AFG2) for green (Dalvi, 1986) (Figure 1). Aflatoxin M1 and M2 (AFM1, AFM2), known as milk-AFs, are the metabolites of AFB1 and AFB2, respectively (Carnaghan et al., 1963). Other metabolites of AFB1 are aflatoxin Q1 (AFQ1) and aflatoxicol. Of the known AFs, AFB1 is the most common produced mycotoxin and the most potent; it has been reported to be the most powerful natural carcinogen in mammals (Creppy, 2002).

The biosynthesis of aflatoxins is a complex process, involving multi-enzymatic reactions. Genetic studies on the molecular mechanism of aflatoxin B1 biosynthesis have identified an aflatoxin pathway gene cluster of 70 kilobase pairs in length consisting of at least 24 identified structural genes including a positive regulatory gene as the transcription activator. The structural genes encode cytochrome P450 monooxygenases, dehydrogenases, oxidases, methyltransferases, a polyketide synthase and two unique fatty acid synthases (Yu et al., 2002).

#### **2.1 Fungi**

Aflatoxins are closely related to a group of aspergilli: *A. flavus*, *A. parasiticus*, and *A. nomius*; although one report also adds a sclerotium producing strain of *A. tamarii*, which is closely related to *A. flavus*, to the list (Goto et al., 1996). Earlier reports of the production of aflatoxins by other aspergilli, penicillia or even a species of *Rhizopus*, have not been adequately confirmed (Moss, 2002a).

*A. flavus* and *A. parasiticus*, which are found worldwide in the air and soil, usually infest both living and dead plants and animals, and as a consequence, aflatoxins in agricultural commodities are primarily produced by *A. flavus* and *A. parasiticus*. *A. flavus* produces only B aflatoxins, while *A. nomius* and *A. parasiticus* produce both B and G toxins (Rustom, 1997; Yu et al., 2002).

#### **2.2 Food**

Aflatoxin contamination of food and feeds is a serious problem worldwide. Studies focusing on AF contamination in foodstuffs have in fact been reported in many countries, especially those in tropical and subtropical regions, such as Asia and Africa (Bankole et al., 2010; Shundo et al., 2009; Soubra et al., 2009).

Aflatoxin contamination can develop both in the pre- and post-harvest periods, but the highest levels are usually associated with post-harvest spoilage of food commodities, stored under inappropriate high moisture content and high temperature conditions which facilitate the rapid growth of moulds; the level of contamination depends on the plant stress, temperature, water activity, genotype, culture and storage conditions, but appropriate postharvest treatments, under dry cool settings, should control this source of contamination (Moss, 2002a; Wilson & Payne, 1994).

As far as pre-harvest, is concerned, aflatoxigenic fungi have a complex ecology. The spores of *A. flavus* and *A. parasiticus* can germinate on the stigma surfaces of plants, and the germ tube can penetrate the developing embryo in a manner which mimics pollen germ tubes. The mycelium can establish an endotrophic relationship, which is not harmful to a healthy plant, while if the plant is stressed (e.g. drought), significant levels of aflatoxin may be produced during field growth. Under these circumstances food commodities may already be contaminated at harvesting and, even though the concentrations are never as high as

**Food commodity Country**  Soy beans Argentina Almonds; Brazil nuts USA

Nutmeg Japan Chilli Pakistan Herbs, spices UK Spices Sweden

Millet India

Coconut India Mustard seed India

**2.3 Toxicity** 

Sunflower oil China, Russia

about the occurrence of aflatoxins in food (Moss, 2002a).

primates, and rodents (Wogan, 1992).

hepatocarcinogenesis.

Dried figs Austria, Switzerland

Pistachio nuts Netherlands, USA, Turkey, Wheat Uruguay, China, Russia Rice Ecuador, China, India

Peanuts India, Sudan, Brazil, Egypt, South Africa Maize Argentina, India, China, Uganda, Nigeria, USA 169

Table 1. Presence of aflatoxins in food commodities (Moss, 2002a; Rustom, 1997).

Aflatoxins can be both acute and chronic toxins; acute poisoning is usually rare and exceptional, while chronic toxicity is of serious concern and it drives international concern

AFB1 is toxic for a wide range of animal species. AFB1 is principally a hepatotoxin and hepatocarcinogen (JECFA, 1998), but it can cause a myriad of other effects: immunosuppresion, reduced growth rate, lowered milk and egg production, reduced reproductivity, reduced feed utilization and efficiency and anemia. AFB1 has been shown to induce hepatocellular carcinoma in many animal species including fish, poultry, non-human

Species susceptibility to various acute toxic manifestations, as measured by TD50, is also variable (Gold et al., 1984). A wide variation exists in species susceptibility to AFB1

In humans, acute aflatoxicosis is manifested by vomiting, abdominal pain, pulmonary edema, coma, convulsions, and death with cerebral edema and fatty involvement of the liver, kidneys, and heart (Mwanda et al., 2005). Epidemiological studies have consistently demonstrated that AFB1 is a liver carcinogen in humans (Groopman et al., 1988; Van Rensburg et al., 1985). The International Agency for Research on Cancer has concluded that there is sufficient evidence for

AFB1 is not mutagenically active itself. It is primarily metabolized in the liver and has several metabolites, such as aflatoxicol and AFQ1. AFB1 is mainly activated by cytochrome P450 dependent monooxygenase; most of the metabolic products, such as AFM1 and AFQ1, are less toxic than the parent AFB1, but aflatoxin B1-8-9-expoxide (AFBO) is the most toxic metabolite (Hwan Do & Choi, 2007). The carcinogenic and mutagenic action of AFB1 might be the result of the affinity of the electrophilic and highly reactive AFBO for cellular nucleophiles, such as DNA (Coulombe, 1993). Thus, epoxidation is generally considered in metabolite activation, while hydroxylation, hydration, and demethylation are considered

the carcinogenicity of AFB1 in humans and hence placed this mycotoxin in group I.

those formed in stored commodities, they can be economically significant and this field contamination is much more difficult to control than post-harvest spoilage (Hill et al., 1983; Moss, 2002a).

Although a wide variety of foods are susceptible to aflatoxin contamination, it has most commonly been associated with peanuts, maize, pistachio, dried fruit, nuts, spices, figs, vegetable oils, cocoa beans, corn, rice and cotton seeds (JECFA, 1998; Reports on Carcinogens [ROC], 2003). Among the agricultural commodities usually infected by aflatoxigenic fungi (Table 1) some are food sources while others are used as animal feeds: the greatest difficulty is that aflatoxin affects the health of the humans and the livestock that consume these commodities and the related products. Speijers & Speijers (2004) reported that AFB1 and OTA are amongst the most frequently observed combinations of mycotoxins in different plant products. According to several other authors, cereals, olives and dried vines are other commodities which could support aflatoxigenic and ochratoxigenic mould growth and OTA and AFB1 production (Molinié et al., 2005; Zinedine et al., 2006).

While aflatoxin B1 is frequently found in contaminated feeds, aflatoxin M1, its hydroxylated metabolite, is normally not present in food, except though carry-over from animal feeds (Fallah, 2010; Kamkar, 2008): following the ingestion of contaminated feedstuffs by lactating dairy cows, AFB1 is biotransformed, by hepatic microsomal cytochrome P450 into AFM1, and is then excreted into the milk (Frobish et al., 1986). Moreover, the AFM1 content in milk is closely correlated to the level of AFB1 in the raw feedstuffs (Bakirci, 2001). AFM1 can be detected in milk 12–24 h after the first ingestion of AFB1; generally, it is deemed that approximately 1–3% of the aflatoxin B1 present in animal feeds appears as AFM1 in milk, depending on the animal, time of milking and many other factors. When the intake of the contaminated source is stopped, the concentration of the toxin in the milk decreases to an undetectable level within 72 h (Gurbay et al., 2006). Additionally, when specific conditions during feed storage are prevalent for the growth of aflatoxigenic species, an additional production and accumulation of AFB1 may occur; this in turn leads to the accumulation of additional AFM1 in the milk. Aflatoxin M1 can survive pasteurization and has even been reported in UHT milk (Unusan, 2006).

AFM1 binds to casein, has a high stability and concentrates in curd during cheese production, in different proportions according to the applied technology (Barbiroli et al., 2007; Brackett & Marth, 1982). In this way, it can also be present in dairy products, manufactured with contaminated milk, at higher concentrations than in the milk (Govaris et al., 2001; Lopez et al., 2001; Oruc et al., 2006). Cheese-making and the ripening period do not result in a reduction in the toxin (Dragacci et al., 1995; Yousef & Marth, 1985). This is why the risk remains, not only in commercially available milk, but also in other derived dairy products. The concentration of AFM1 in cheese varies according to the type of cheese, water content and production technologies (Bakirci, 2001; Lopez et al., 2001). Since the sources of aflatoxin contamination in animal feeds differ because they are location dependent and the incidence and occurrence of AFM1 contamination in animal feeds from different countries varies, there are many reports on AFM1 contamination in cheese and other dairy products from different countries: Slovenia, North Africa, Turkey, Brazil and Portugal (Bakirci, 2001; Elgerbi et al., 2004; Oliveira et al., 2006; Martins & Martins, 2000; Torkar & Vengus, 2008).

those formed in stored commodities, they can be economically significant and this field contamination is much more difficult to control than post-harvest spoilage (Hill et al., 1983;

Although a wide variety of foods are susceptible to aflatoxin contamination, it has most commonly been associated with peanuts, maize, pistachio, dried fruit, nuts, spices, figs, vegetable oils, cocoa beans, corn, rice and cotton seeds (JECFA, 1998; Reports on Carcinogens [ROC], 2003). Among the agricultural commodities usually infected by aflatoxigenic fungi (Table 1) some are food sources while others are used as animal feeds: the greatest difficulty is that aflatoxin affects the health of the humans and the livestock that consume these commodities and the related products. Speijers & Speijers (2004) reported that AFB1 and OTA are amongst the most frequently observed combinations of mycotoxins in different plant products. According to several other authors, cereals, olives and dried vines are other commodities which could support aflatoxigenic and ochratoxigenic mould growth and OTA and AFB1 production (Molinié et al., 2005;

While aflatoxin B1 is frequently found in contaminated feeds, aflatoxin M1, its hydroxylated metabolite, is normally not present in food, except though carry-over from animal feeds (Fallah, 2010; Kamkar, 2008): following the ingestion of contaminated feedstuffs by lactating dairy cows, AFB1 is biotransformed, by hepatic microsomal cytochrome P450 into AFM1, and is then excreted into the milk (Frobish et al., 1986). Moreover, the AFM1 content in milk is closely correlated to the level of AFB1 in the raw feedstuffs (Bakirci, 2001). AFM1 can be detected in milk 12–24 h after the first ingestion of AFB1; generally, it is deemed that approximately 1–3% of the aflatoxin B1 present in animal feeds appears as AFM1 in milk, depending on the animal, time of milking and many other factors. When the intake of the contaminated source is stopped, the concentration of the toxin in the milk decreases to an undetectable level within 72 h (Gurbay et al., 2006). Additionally, when specific conditions during feed storage are prevalent for the growth of aflatoxigenic species, an additional production and accumulation of AFB1 may occur; this in turn leads to the accumulation of additional AFM1 in the milk. Aflatoxin M1 can survive pasteurization and has even been

AFM1 binds to casein, has a high stability and concentrates in curd during cheese production, in different proportions according to the applied technology (Barbiroli et al., 2007; Brackett & Marth, 1982). In this way, it can also be present in dairy products, manufactured with contaminated milk, at higher concentrations than in the milk (Govaris et al., 2001; Lopez et al., 2001; Oruc et al., 2006). Cheese-making and the ripening period do not result in a reduction in the toxin (Dragacci et al., 1995; Yousef & Marth, 1985). This is why the risk remains, not only in commercially available milk, but also in other derived dairy products. The concentration of AFM1 in cheese varies according to the type of cheese, water content and production technologies (Bakirci, 2001; Lopez et al., 2001). Since the sources of aflatoxin contamination in animal feeds differ because they are location dependent and the incidence and occurrence of AFM1 contamination in animal feeds from different countries varies, there are many reports on AFM1 contamination in cheese and other dairy products from different countries: Slovenia, North Africa, Turkey, Brazil and Portugal (Bakirci, 2001; Elgerbi et al., 2004; Oliveira et al., 2006; Martins & Martins, 2000;

Moss, 2002a).

Zinedine et al., 2006).

reported in UHT milk (Unusan, 2006).

Torkar & Vengus, 2008).


Table 1. Presence of aflatoxins in food commodities (Moss, 2002a; Rustom, 1997).

#### **2.3 Toxicity**

Aflatoxins can be both acute and chronic toxins; acute poisoning is usually rare and exceptional, while chronic toxicity is of serious concern and it drives international concern about the occurrence of aflatoxins in food (Moss, 2002a).

AFB1 is toxic for a wide range of animal species. AFB1 is principally a hepatotoxin and hepatocarcinogen (JECFA, 1998), but it can cause a myriad of other effects: immunosuppresion, reduced growth rate, lowered milk and egg production, reduced reproductivity, reduced feed utilization and efficiency and anemia. AFB1 has been shown to induce hepatocellular carcinoma in many animal species including fish, poultry, non-human primates, and rodents (Wogan, 1992).

Species susceptibility to various acute toxic manifestations, as measured by TD50, is also variable (Gold et al., 1984). A wide variation exists in species susceptibility to AFB1 hepatocarcinogenesis.

In humans, acute aflatoxicosis is manifested by vomiting, abdominal pain, pulmonary edema, coma, convulsions, and death with cerebral edema and fatty involvement of the liver, kidneys, and heart (Mwanda et al., 2005). Epidemiological studies have consistently demonstrated that AFB1 is a liver carcinogen in humans (Groopman et al., 1988; Van Rensburg et al., 1985). The International Agency for Research on Cancer has concluded that there is sufficient evidence for the carcinogenicity of AFB1 in humans and hence placed this mycotoxin in group I.

AFB1 is not mutagenically active itself. It is primarily metabolized in the liver and has several metabolites, such as aflatoxicol and AFQ1. AFB1 is mainly activated by cytochrome P450 dependent monooxygenase; most of the metabolic products, such as AFM1 and AFQ1, are less toxic than the parent AFB1, but aflatoxin B1-8-9-expoxide (AFBO) is the most toxic metabolite (Hwan Do & Choi, 2007). The carcinogenic and mutagenic action of AFB1 might be the result of the affinity of the electrophilic and highly reactive AFBO for cellular nucleophiles, such as DNA (Coulombe, 1993). Thus, epoxidation is generally considered in metabolite activation, while hydroxylation, hydration, and demethylation are considered

**Name R1 R2 R3 R4 R5** 

171

OTA Phe\* Cl H H H OTB Phe H H H H OTC Ethyl-ester, Phe Cl H H H OTA methyl-ester Methyl-ester, Phe Cl H H H OTB methyl-ester Methyl-ester, Phe H H H H OTB-ethyl-ester Ethyl-ester, Phe H H H H OTα OH Cl H H H OTβ OH H H H H 4-R-hydroxy OTA Phe Cl H OH H 4-s-hydroxy OTA Phe Cl OH H H 10-hydroxy OTA Phe Cl H H OH Tyr\* analog of OTA Tyr Cl H H H Ser\* analog of OTA Ser Cl H H H Hyp\* analog of OTA Hyp Cl H H H Lys\* analog of OTA Lys Cl H H H Table 2. Radicals in OTA metabolites \*(Phenylalanine; Tyrosine; Serine; Hydroxyproline;

Ochratoxin A is produced by *Aspergillus* and *Penicillium* species listed in Table 3. These microorganisms differ according to the ecological conditions and commodities that characterize different geographical regions. In general, *Penicillium verrucosum* is responsible for OTA contamination in cool-temperate conditions, whereas *Aspergillus ochraceus* is particularly relevant in hot-tropical regions (Battaccone et al., 2010; Scudamore, 2005). The major *Aspergillus* producers in food and feeds are *A. alliaceus*, *A. carbonarius*, *A. ochraceus*, *A. steynii* and *A. westerdijkiae*. *A. melleus*, *A. ostianus*, *A. persii* and *A. petrakii* may produce trace amounts of OTA, but since the publication by Ciegler (1972) and Hesseltine et

In the genus *Penicillium*, *P. verrucosum* and *P. nordicum* are the only species that are able to produce OTA (Abruhnosa et al., 2010; El Khoury & Atoui, 2010). *P. chrysogenum*, *P. brevicompactum*, *P. crustosum*, *P. olsonii* and *P. oxalicum* have been claimed as OTA producers,

*Aspergillus section Circumdati A. cretensis; A. flocculosus; A. ochraceus ; A. pseudoelegans; A. roseoglobulosus; A. sclerotiorum ;* 

*Aspergillus section Flavi* 

*Aspergillus section Nigri* 

*Penicillium* 

*A. carbonarius; A. lacticoffeatus; A. niger; A. sclerotioniger; A. citricus ; A. fonsecaeus* 

Table 3. OTA producing fungi (Abrunhosa et al., 2010; El Khoury & Atoui, 2010; Moss,

Lysine) (El Khoury & Atoui, 2010).

*A. alliaceus; Petromyces albertensis* 

*P. nordicum; P. verrucosum*

2002b).

al. (1972) no further confirmation has been found.

but a confirmation of these findings is required (Paterson, 2006).

*A. steynii; A. sulphureus ; A. westerdijkiae; Neopetromyces muricatus* 

**3.1 Fungi** 

metabolic detoxications. The toxic and carcinogenic effects of aflatoxin B1 are intimately linked to both the rate of activation and the rate of detoxification at the primary and secondary levels of metabolism, in a similar way to chlorinated hydrocarbon (Olaniran et al., 2006).
