5. Chemical nature and structural illustration

Due to recent advances in technology, modern methods and budding interests, more than 300– 500 mycotoxins have been discovered and characterized. Mycotoxins have very special chemical configurations [11, 18, 34]. However, only a relatively small number of toxins are of relevance in feed milling [11]. The AFs are difurocoumaro-lactones (difurocoumarin derivatives) in structure. These chemical structures comprise of a difuran ring with complex coumarin nucleus with a pentenone ring (in AFB and AFM)/a six membered lactone ring (AFG). The four compounds viz. AFB1, B2, G1 and G2 (Figure 1) can be differentiated by fluorescence under ultraviolet illumination (B = blue, G = green) [3]. AFs are indistinctly soluble in H2O and hydrocarbons, soluble in methanol, acetone and chloroform and insoluble in non-polar solvents. They appear to be unstable in air and light. These toxins are decomposed at their respective melting points which range between 237C (G) and 299C (M1) but not destroyed under normal cooking conditions. Rather these can be completely denatured by autoclaving in the presence of NH3 or by treatment with bleach [35].

6. Levels of toxin production

Figure 1. The chemical structure of aflatoxins [19].

According to Wayne [43], the amount of toxins produced depends on different factors that can be physical, chemical or biological. Physical factors include moisture, relative humidity, temperature and mechanical damage, while chemical factors include CO2, O2, substrate composition, pesticide and fungicide. Plant variety, stress (harsh weather), insects, and spore

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concentration collectively are biological factors that may affect toxin production.

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Figure 1. The chemical structure of aflatoxins [19].

Shareef [26] found AFs to be most prevalent mycotoxins group (91.1%) with average concentration of 179.1 μg/kg followed by ochratoxins (127 μg/kg) during a two-year survey (2005–2007) on different poultry feed samples in Pakistan. Anjum et al. [27] found AFB2 (10.80 2.16 to 39.20 3.67 μg/kg) in layer and broiler starter rations from ten different commercial feed mills in Punjab, Pakistan. Among them, 40% of samples were contained AFB2 at levels above 20 μg/kg (maximum tolerable levels for poultry). Bokhari [29] found 26.1% samples (seeds, oilseeds, spices, milk and milk products) contaminated with AFs principally poultry feed, cereal grains and oil seeds with AFB1 found as the most frequent contam-

Luttfullah and Hussain [29] found maximum incidence rate of AFs in walnuts with shell (40%), walnuts without shell (70%) and in peanuts with shell (40%) during a survey in Khyber Pakhtun and northern areas of Pakistan. Lutfullah and Arshad [30] found highest AFs incidence rate in corn (40%), sorghum (30%) and rice (25%) from different retail shops and local markets of different location in Pakistan. In Pakistan, A. flavus contamination occurs at the highest incidence

Borutova et al. [32] found a positive correlation between AFB1 and AFB2 prevalence on different feedstuffs i.e. corn, wheat, soybean meal, corn gluten meal, dried distiller grains, etc. in Asian-Oceania region in 2010 and concluded that the occurrence of single mycotoxins in any of the feedstuffs is rare. Mardani et al. [33] did not find via High Performance Liquid Chromatography (HPLC) any of the AFs at detectable levels in food samples from Kaskinen in Iran except for one sample that contained AFB1 (0.64 μg/kg). Basaran and Ozcan (2009) concluded AFB1 to be the most abundant in concentration (0.2–36.81 μg/kg) followed by four samples containing AFG1 (0.6–20.2 μg/kg) among 217 samples of hazelnuts, pistachio nuts and peanuts

Due to recent advances in technology, modern methods and budding interests, more than 300– 500 mycotoxins have been discovered and characterized. Mycotoxins have very special chemical configurations [11, 18, 34]. However, only a relatively small number of toxins are of relevance in feed milling [11]. The AFs are difurocoumaro-lactones (difurocoumarin derivatives) in structure. These chemical structures comprise of a difuran ring with complex coumarin nucleus with a pentenone ring (in AFB and AFM)/a six membered lactone ring (AFG). The four compounds viz. AFB1, B2, G1 and G2 (Figure 1) can be differentiated by fluorescence under ultraviolet illumination (B = blue, G = green) [3]. AFs are indistinctly soluble in H2O and hydrocarbons, soluble in methanol, acetone and chloroform and insoluble in non-polar solvents. They appear to be unstable in air and light. These toxins are decomposed at their respective melting points which range between 237C (G) and 299C (M1) but not destroyed under normal cooking conditions. Rather these can be completely denatured by autoclaving in

rate, being responsible for the production of AFB1 in the corn in Swat valley [31].

in the Turkey. About 87.09% of total samples were very low in AFB1.

5. Chemical nature and structural illustration

the presence of NH3 or by treatment with bleach [35].

inant especially in corn grains.

128 Mycotoxins - Impact and Management Strategies

#### 6. Levels of toxin production

According to Wayne [43], the amount of toxins produced depends on different factors that can be physical, chemical or biological. Physical factors include moisture, relative humidity, temperature and mechanical damage, while chemical factors include CO2, O2, substrate composition, pesticide and fungicide. Plant variety, stress (harsh weather), insects, and spore concentration collectively are biological factors that may affect toxin production.

Temperature, water activity (aw), oxygen and pH [1, 27, 36–39] play vital role in the production of mycotoxins by fungi. The aw range should be between 0.61 and 0.91 as most of storage fungi grow at aw <0.75. The ideal temperature for AFs production by A. flavus and A. parasiticus ranges between 12 and 41C with optimum production occurring at 25–32C. But the AF synthesis increases by temperature >27C, humidity >62% and moisture >14% [3]. Relative to AFG1, AFB1 production is stimulated by higher temperature [40]. Optimal production of AFB1 occurs between 24 and 28C, whereas 23C is optimal for AFG1 production. Low temperature (8–10C) induces the production of equal amounts of AFB and AFG. However, total AFs production is suppressed with more time is required [3]. At higher aw, fungi compete with bacteria as food spoilers [17]. Moreover, Aspergillus can tolerate lower aw than Fusarium [41]. Initially, fungal growth in grains produces adequate metabolic water for further expansion and mycotoxins production [42]. Oxygen is an essential factor for the fungal growth and its growth is restricted at less than 1% oxygen [17].

2011). AFB1 is classified by IARC [35], as a highly toxic compound (LD50, 1–50 mg/kg body weight) among most species, although it is extremely toxic (LD50 < 1 mg/kg) for some species

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Ducklings followed by turkey poults, broilers and laying hens are the most sensitive species to AFs as these showed 100% mortality at 1 mg/kg AFB1. Moreover, 0.11–0.2 mg/kg AFB1 decreased 230 and 163 g/bird feed intake and weight, approximately from 0 to 14 days of age, respectively [50]. Goslings, quails and pheasants are ranked at intermediate position regarding sensitivity while chickens appear to be the highly resistant. Ducklings are 5–15 times more sensitive than laying hens, but among layers, certain strains may be as much as 3 times more sensitive than others [38]. Broilers are more susceptible to AF than layers [36, 51]. Aflatoxincontaminated feed affect almost all systems in the body are affected, i.e. interference in bone metabolism resulting decreased bone strength, reduction in bone diameter, decrease in dressed

AFs are toxic to poultry at <1 mg/kg with liver as main target organ as the relative liver weight is altered by low levels of AFs [53, 54]. Respiratory exposure to AFB1 contaminated dust has been allied with increased incidence levels of tumor along the respiratory tract of animals and humans [3]. The AFs molecules are subjected through complex metabolic processes of different cytochrome P450 dependent pathways (bio-activation or detoxification

The carcinogenic and mutagenic effects of AFB1 [4], AFG1 and AFM1 occur after metabolic activation by microsomal mixed function oxidase system [3, 56]. AFs bind to both RNA and DNA and blocks transcription [17]. In the liver, cytochrome P450 activates AFB1 (procarcinogens) to form AFB1–8, 9-exo-epoxide (catalyzed by CYP3A4 leading to the formation of AFQ1) and endo-epoxide (catalyzed by CYP1A2) at 8, 9 position of the terminal furan ring and its subsequent covalent binding to nucleic acid but only exo-epoxide that is highly unstable binds with DNA resulting in the formation of 8,9-dihydro-8-(N7-guanyl)-9-hydro-AFB1 (AFB1-N7-Gua) adduct [18, 56, 57]. Toxin interaction with DNA and some enzymes to alter p53 gene results in GC to TA transversion, which results in mutagenic properties. This transversion is capable of binding to lysine in serum albumin [58] and also inhibits different activities on biological molecules e.g. synthesis of DNA adducts and conjugation with glutathione, and blocks of ribosomal translocase and RNA polymerase (inhibiting protein synthesis) and essential enzymes [59]. The RNA and DNA syntheses were inhibited in rats fed feed contaminated with 5 mg/kg AFs of over six weeks period [4]. AFB1-epoxide can covalently bind to different proteins which in turn, may affect structural and enzymatic protein function [3]. The structure of interaction between base pairs in DNA helix is determined by binding of exo-epoxide with guanine [60, 61]. The metabolites (AFQ1, AFM1 and AFP1) of AFB1 and other naturally occurring AFs such as AFG1, B2 and G2, are weaker for epoxide formation, thus they have less

such as cats, ducklings and rainbow trouts [3].

weight and breast yield etc. [52].

processes) [55].

8. Mode of action of aflatoxins

carcinogenic and toxic properties than AFB1.

The broken grains (by insects and birds) are often more susceptible to mycotoxins production. The grains with "musty" odor should be suspected and analyzed for mycotoxins [42]. Aflatoxins contamination is directly influenced by insects' attack to plants and is probably dominated by drought and high temperature [43]. These predisposing conditions allow "hot spots" to occur in stored grains. In severely affected crop of corn, the individual kernel may contain AFs as high as 400,000 μg/kg AFs [42].

The accrual of mycotoxins in the grains before and after harvest largely reflects the prevailing climatic conditions. For example, Fusarium toxins are produced in cereals with high moisture content during harvest, whereas pre-harvest AF contamination of crops like peanuts and maize is linked with high temperatures, insect damage and prolonged drought conditions [43].

Fungal geneticists have unraveled the pathways and genes for the synthesis and regulation of mycotoxins production, especially AFs and trichothecenes [37, 44], which assist in the breeding of plants resistant to toxin accumulation [45]. The transgenic Bt corn contains a gene isolated from the soil bacterium Bacillus thuringiensis, which encodes for a protein, being toxic to common lepidopteran corn pests. These hybrids offer a new tool for mycotoxins management as insect damage is often a major factor in facilitating toxigenic fungal infection of crops [46].
