8. Mode of action of aflatoxins

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

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

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

AF (AFB1, G1, B2 and G2) concentration, duration of dietary exposure, species, sex, breed, age and health status of animals are different factors that affect toxicity [42, 47]. Young animals are less resistant than older one presumably due to the lack of well-developed hepatic enzymatic systems required to degrade the toxins depending upon the specie [48]. Guinea-pig, duckling and rabbit represent a "fast metabolizing group" actually capable of handling LD50 dose in <12 minutes. Sheep, pig, mouse and chick fall into "intermediate group" metabolizing LD50 dose in few hours [49]. Currently, rat is the only example of a "slow metabolizing group" in which LD50 dose would probably disappear from the liver over a period of days (Hu et al.,

is restricted at less than 1% oxygen [17].

130 Mycotoxins - Impact and Management Strategies

AFs as high as 400,000 μg/kg AFs [42].

infection of crops [46].

7. Toxicity of aflatoxins

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 processes) [55].

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 carcinogenic and toxic properties than AFB1.

In liver cells, cytoplasmic reductase and microsomal mixed-function oxidase system metabolize AFB1 to aflatoxicol and aflatoxins M1, Q1, P1 and B1-epoxide (the most toxic and carcinogenic derivative), which are less toxic than AFB1. These are further conjugate with other molecules and rapidly eliminated from the body [3]. The metabolites (AFQ1, AFM1 and AFP1) being formed from AFB1 and other naturally occurring AFs e.g. G1, B2 and G2 are weaker for epoxidation, thus possess less carcinogenic and toxic properties than AFB1. The AFM1, AFQ1 and AFP1 are secreted as metabolites of AFB1 in the urine and can be used as biomarkers [62].

hepatocytes, about three folds mutations at the third base of codon 249) but neighboring guanines (247, 248 and 250) were also modified. About 20% of total AFB1 ingested remain in the body after a period of one week with a half-life in the plasma of 36.5 minutes, whereas M1 is almost excreted via urine within 48 hours [68]. Because there is a half-life of 20 days in serum albumin, the AFB1-albumin adduct can be used as an AF biomarker to check the chronic exposure within 1–2 months and is considered as an independent factor for advanced liver diseases in HCV-infected patients. The adduction levels of AFs with albumin by covalent bonding in the peripheral blood reflect AF exposure 2–3 months earlier depending on albumin

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A marked decrease in digestive enzymes (pancreatic ribonuclease, amylase, trypsin and lipase), hypocarotenoidaemia, steatorrhea and bile salts can be observed during aflatoxicosis in poultry. Protein requirements for growth were increased during aflatoxicosis which can be alleviated by dietary methionine fortification [43]. Fernandez et al. [69] conducted trials to investigate the hematological and serological changes on broilers from 21 to 42 days of age with oral administration of 2500 μg/kg AFB1. It was found that hematological (red blood cell, hemoglobin, leucocytes, eosinophils and basophils) and serological (serum protein, aspartate aminotransferase, alanine aminotransferase, urea, creatinine) parameters remained unchanged but caused hepatic and renal lesions which matches the findings of Bianchi et al. [39]. AFs are known to reduce protein synthesis that may lead to decreased blood protein levels. The AFs intoxications have been reported to decrease total protein, cholesterol, triglyceride and glucose

Mycotoxins including Aflatoxins are metabolized in the gastrointestinal tract, liver or kidneys according to their chemical structure. Their transfer to poultry meat and eggs leads to undesirable effects on human health [18]. Agag [3] examined the "carry-over" of AFB1 from layer feed to eggs was examined in laying hens at dietary levels of 100–400 μg/kg AFB1. This resulted in 0.2 to 3.3 μg/kg in eggs, and AFs ratios in feeds and tissues found to be are very low ranging from 500:1 to 14,000:1 excluding the liver, particularly when compared with milk (70:1). On the other hand, Zaghini et al. [55] showed no measurable residual AFB1 or its metabolites in eggs. These contrasting findings may be ascribed to mannan oligosaccharides in naturally AFs

In broilers and layer birds, the AFB1 residues have been reported to vary from no detection to 3.0 μg/kg in liver in birds fed 250–3310 μg/kg AFB1 over certain periods [71]. Fowler et al. [72] found no significant increase in AFs residues in liver until the 1800 μg/kg AF contaminated feed was fortified with AF at a concentration of 1200 μg/kg with no clay used as a binding agent. Younger birds were found to have significant increase in liver residues than those in

half-life [66].

levels significantly [70].

11. "Carry-over" of aflatoxins

contaminated feeds at different levels of toxicity [55].

10. Effect of aflatoxins on enzymes
