Aflatoxin Biosynthetic Pathway and Pathway Genes
Among the over 185 known species within the genus Aspergillus, Aspergillus flavus is the most economically important because it produces the toxic and carcinogenic aflatoxins. Its non-aflatoxigenic relative A. oryzae is used extensively for food fermentations (Jelinek et al., 1989). It is one of the most abundant soil-borne molds on earth. A. flavus fungus is a saprobe mold that is capable of surviving on many organic nutrient sources like plant debris, tree leaves, decaying wood, animal fodder, cotton, compost piles, dead insects and animal carcasses, stored grains, and even immunocompromised humans and animals (Klich, 1998). It has the ability to survive temperatures ranging from 12°C to 48°C, but the optimal growth temperature ranges from 28°C to 37°C. Its ability to grow at relatively high temperatures contributes to its pathogenicity toward humans and other warm blooded animals. For most of its lifecycle, the fungus exists in the form of mycelium or asexual spores known as conidia. Under adverse conditions such as lack of adequated nutrients or water, the fungal mycelium will transform to resistant structures called sclerotia which can survive extremely harsh environmental conditions. The fungus overwinters either as spores, sclerotia, or as mycelium in debris. When conditions become favorable the sclerotia germinate directly to produce new colonies or conidiophores with conidia (Bennett et al., 1986; Cotty, 1988; Chang et al., 2002). Aflatoxins were first identified as the cause of a severe animal poisoning incident in England in 1960 called the Turkey X disease (Allcroft et al., 1961; Lancaster et al., 1961). A. flavus produces aflatoxin B1 and B2 whereas A. parasiticus, produces aflatoxins B1, B2, G1, and G2. These four major aflatoxins are named based on their blue (B) or green (G) fluorescence under ultraviolet light, and their relative mobility by thin-layer chromatography on silica gel. Aflatoxin M1 is a hydroxylated derivative metabolized from aflatoxin B1 by cows and secreted in milk (Van Egmond, 1989). In addition to aflatoxins B1 and B2, A. flavus also produces many other mycotoxins such as cyclopiazonic acid, kojic acid, beta-nitropropionic acid, aspertoxin, aflatrem and aspergillic acid (Goto et al., 1996). The disease caused by ingestion of aflatoxins in contaminated food or feed is called aflatoxicosis. Acute aflatoxicosis occurs when aflatoxins are consumed at moderate to high levels. Depending on the level and duration of exposure, aflatoxins possess both hepatotoxic and carcinogenic properties. Symptoms in humans include vomiting, abdominal pain, alteration in digestion, limb and pulmonary edema, convulsions, rapid progressive jaundice, swollen liver, high fever, coma, and death. The predominant damage is to the liver (Scholl & Groopman, 1995); (Fung & Clark, 2004; Lewis et al., 2005), but acute damage to the kidneys and heart have been found (Richard & Payne, 2003). In liver aflatoxins irreversibly bind to protein and DNA to form adducts such as aflatoxin B1-lysine in albumin and a guanyl-N7 adduct in DNA (Skipper & Tannenbaum, 1990). Disruption of the proteins and DNA bases in hepatocytes causes the toxicity (Tandon et al., 1978; Azziz-Baumgartner et al., 2005). Major outbreaks of acute aflatoxicosis from contaminated food in humans were reported in developing countries (Centers for Disease Control and Prevention, 2004; Lewis et al., 2005). For example, in western India in 1974, 108 persons died among 397 people affected with aflatoxin poisoning in more than 150 villages (Krishnamachari et al., 1975). A more recent incident of aflatoxin poisoning occurred in Kenya in July 2004 leading to the death of 125 people among 317 reported with illness due to consumption of aflatoxin contaminated maize (corn) (Centers for Disease Control and Prevention, 2004; Lewis et al., 2005). Acute toxicosis is not the only concern. World health authorities warn that low doses and long term dietary exposure to aflatoxins is also a major risk as chronic exposure can lead to hepatocellular carcinoma (Bressac et al., 1991; Hsu et al., 1991; Wogan, 1992; Fung & Clark, 2004). Among the four major types of aflatoxins, aflatoxin B1 is the most toxic and the most potent carcinogen in humans and animals including nonhuman primates, birds, fish, and rodents. Chronic exposure can result in suppressed immune response, malnutrition, proliferation of the bile duct, centrilobular necrosis and fatty infiltration of the liver, hepatic lesions, and even hepatomas. In animal models, aflatoxin B1 is modified into a more toxic and carcinogenic by-product during detoxification by a cytochrome P450 monooxygenase in liver (Ngindu et al., 1982; Hsieh, 1989; Eaton & Gallagher, 1994; Lewis et al., 2005). The epoxide form of aflatoxin binds to guanine residues in DNA, forms guanyl-N7 adducts, and induces mutations. One mutation, a G to T transversion (Baertschi et al., 1989; Bressac et al., 1991) at the third base of codon 249, a mutation hot spot of the p53 tumor suppressor gene, is generally believed to be the mechanism for initiating hepatocarcinoma formation (Busby & Wogan, 1981; Hsu et al., 1991; Ozturk, 1991; Coursaget et al., 1993). The p53 gene encodes a transcription factor involved in cell cycle regulation. It is commonly mutated in human liver cancers (Groopman et al., 1994). Aflatoxin B1 is also a potential immunosuppressive agent (Raisuddin et al., 1993). Chronic low level exposure of growing vertebrates to aflatoxins may enhance their susceptibility to infection and tumorigenesis (Raisuddin et al., 1993). AFB1 also affects other organs and tissues, such as the lungs and the entire respiratory system (Kelly et al., 1997). Human hepatocarcinomas are also associated with hepatitis B virus (HBV) and C virus (HCV) infections (Peers et al., 1987; Hsieh, 1989; Wild et al., 1992). Together with aflatoxins these viruses significantly increased the risk of hepatoma in hepatitis patients (Chen et al., 1996a; Chen et al., 1996b; McGlynn et al., 2003; Arsura & Cavin, 2005). In developing countries, many children are exposed to aflatoxin before birth (Turner et al., 2007), while nursing (Polychronaki et al., 2007) and after weaning (Gong et al., 2004). An association of hepatocellular carcinoma and dietary exposure with aflatoxins has been established from patients living in high-risk areas of China, Kenya, Mozambique, Phillippines, Swaziland, Thailand, Transkei of South Africa (Lancaster et al., 1961; Zuckerman et al., 1967; Wong et al., 1977; Hsieh et al., 1985; Zhu et al., 1987; Huang & Hsieh, 1988; Wilson, 1989; Wogan, 1992; Eaton & Gallagher, 1994; Lewis et al., 2005). Aspergillus flavus can grow in immunocompromised warm blooded mammals and can cause invasive and non-invasive aspergillosis in humans and animals (Denning et al., 1991; Denning, 1998; Mori et al., 1998; Denning et al., 2003). A. flavus is the second leading cause of aspergillosis slightly behind A. fumigatus. The incidence of aspergillosis caused by Aspergilli is rising due to the increase of immunocompromised patients in the population due to HIV infection (Denning, 1998; Nierman et al., 2005; Ronning et al., 2005). A. flavus is a weak and opportunistic plant pathogen, affecting many agricultural crops such as maize (corn), cotton, groundnuts (peanuts), as well as tree nuts such as Brazil nuts, pecans, pistachio nuts, and walnuts. Preharvest contamination of these crops with aflatoxins is common. A. flavus also causes the spoilage of post harvest grains during storage. Because A. flavus lacks host specificity (St Leger et al., 2000) and can attack seeds of both monocots and dicots, and seeds produced both above ground (corn) as well as below the ground (peanuts). Under weather conditions favorable for its growth, A. flavus can cause ear rot on maize, resulting in significant economic losses to farmers (Robens, 2001; Richard & Payne, 2003; Robens & Cardwell, 2005).