Aflatoxins: Biosynthesis, Occurrence, Toxicity, and Remedies

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Critical Reviews in Food Science and Nutrition
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Aflatoxins: Biosynthesis, Occurrence, Toxicity, and Remedies
Muhammad Abrar , Faqir Muhammad Anjum , Masood Sadiq Butt , Imran Pasha , Muhammad Atif Randhawa , Farhan Saeed & Khalid Waqas
To cite this article: Muhammad Abrar , Faqir Muhammad Anjum , Masood Sadiq Butt , Imran Pasha , Muhammad Atif Randhawa , Farhan Saeed & Khalid Waqas (2013) Aflatoxins: Biosynthesis, Occurrence, Toxicity, and Remedies, Critical Reviews in Food Science and Nutrition, 53:8, 862-874, DOI: 10.1080/10408398.2011.563154
To link to this article: http://dx.doi.org/10.1080/10408398.2011.563154
Accepted author version posted online: 01 Aug 2012.
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Aflatoxins: Biosynthesis, Occurrence, Toxicity, and Remedies
MUHAMMAD ABRAR, FAQIR MUHAMMAD ANJUM, MASOOD SADIQ BUTT, IMRAN PASHA, MUHAMMAD ATIF RANDHAWA, FARHAN SAEED, and KHALID WAQAS National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan
Food contagion with aflatoxins is the modern concern and has received a great awareness during the last few decades. The intermittent incidence of these toxins in agricultural commodities has negative role on the economy of the affected regions where harvest and postharvest techniques for the prevention of mold growth, are seldom practiced. Aflatoxins are difuranocoumarin derivatives produced by a polyketide pathway by the fungus Aspergillus flavus and Aspergillus parasiticus via polyketide pathway which are highly hepatotoxic, hepatocarcinogenic, teratogenic, and mutagenic in nature and con- taminate a wide variety of important agricultural commodities before, during, and after harvest in various environmental conditions. The production of aflatoxins in innate substrates depends upon the various factors, that is, type of substrate, fungal species, moisture contents of the substrate, minerals, humidity, temperature, and physical damage of the kernels. These toxins cause several ailments such as cancer, hepatitis, mutation abnormalities, and reproduction disorders. Minimization and inactivation of aflatoxins contaminants through proper crop management at farm level and with physical, chemical, and biological techniques are the limelight of the article.
Keywords Aflatoxins, toxicity, mutation, control, radiations, chemical
INTRODUCTION
Aflatoxins are assemblage of about 20 related fungal metabo- lites and occur in a broad variety of imperative foods, includ- ing spices, cereals, figs, nuts, and dried fruit. Even though, the highest contents are produced in food crops grown and stored in the tropical areas of the world, the international trading of these significant foodstuffs ensures that aflatoxins are not only a problem for producing countries but are also of concern for importing countries (Prandini et al., 2008). These components constitute toxigenically and chemically heterogeneous clusters that are grouped together only as the member can cause disease and death in human beings and other vertebrates. Of course, numerous aflatoxins exhibit overlapping toxicities to plants, in- vertebrates, and microbes. Aflatoxins are highly toxic secondary metabolites produced by the species of Aspergillusi, especially Aspergillusi flavus and Aspergillusi parasiticus (Turner et al., 2009). These fungi can grow on a wide variety of foods and feeds under favorable temperature and humidity. These have a
Address correspondence to Farhan Saeed, Ph.D. Scholar, National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan. E-mail: [email protected]
polycyclic structure derived from a coumarin nucleus attached to a bifuran system. B type aflatoxins are attached to a pen- tanone; while G types are attached to a 6-membered lactone. Aflatoxins are highly soluble in somewhat polar solvents, for instance; methanol, chloroform, and dimethylsulfoxide while inadequately soluble in water. Aflatoxin B 1 is the most toxic aflatoxin for mammals which induces mutagenic, teratogenic, and hepatotoxic effects (Nakai et al., 2008). Contamination of aflatoxins can take place at any point along the food chain from the field, harvest, handling, shipment, and storage (Giray et al., 2007; Commission Regulation EC No.1881/2006, Commission Regulation EU No 105/2010). Aflatoxins contaminate a wide variety of vital agricultural commodities in whole world, for example, spices, wheat, corn, rice, dried fruits, and nuts. The compounds are highly toxic, mutagenic, teratogenic, and car- cinogenic, and implicated as causative agents in human hepatic and extra hepatic carcinogenesis (Castells et al., 2008, Santos et al., 2010, Rubert et al., 2010).
The contrivance toward aflatoxins came into being in 1960 owing to severe epidemic of the turkey “X” disease in the United Kingdom which resulted in the mortality of more than 100,000 turkeys and other farm animals (Blount, 1961; Lund et al., 2000). The cause of the disease was attributed to a feed containing
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Figure 1 Aflatoxin gene cluster. (Yabe and Nakajima, 2004).
Brazilian peanuts which were heavily infested with A. flavus. Subsequent analysis of the feed using thin-layer chromatogra- phy revealed that a series of fluorescent compounds, later termed aflatoxins, were responsible for the outbreak (Davis and Diener, 1979). The main responsible toxin metabolites were identified as aflatoxin B1 (AFB1), B2, G1, and G2, with AFB1 being the most abundant and toxic metabolite in this group.
The International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) classifies activities that evaluated for carcinogenicity in humans into 4 groups (IARC, 2006). The USA Government’s Annual Report on Carcinogens has also made a similar classification for aflatoxins (National Toxicology Program, 2005). The categories of carcinogens that are distinguished in Group 1 [The agent (mixture) is carcino- genic to humans], Group 2A [The agent (mixture) is probably carcinogenic to humans] and Group 2B, [The agent (mixture) is possibly carcinogenic to humans], Group 3 [The agent (mixture) is not classifiable as to its carcinogenicity to humans], and Group 4 [The agent (mixture) is probably not carcinogenic to humans].
BIOSYNTHESIS
Intricate processes involving multienzymatic reactions are used for the biosynthesis of aflatoxins. Aflatoxin biosynthetic pathway starts with the detection of the toxins structures. Con- versely, at a molecular level, the main biochemical steps and the subsequent genetic constituents of aflatoxins biosynthesis have been illuminated only in the last decade (Bhatnagar et al., 2002; Yu, 2003). In general, the aflatoxin biosynthesis genes of A. parasiticus and A. flavus are significantly homologous and the arrangement of the genes within the cluster being the same (Yu et al., 1995). Minto and Townsend (1997) determined that aflatoxins are synthesized in two steps from malonyl CoA, former with the formation of hexanoyl CoA, and latterly with the formation of a decaketide anthraquinone. For transfer of acetyl coenzyme A (acetyl CoA) to its ultimate products, that is, AFB1, AFB2, AFG1, and AFG2, at least 18 enzyme steps are required (Yabe and Nakajima, 2004; Roze et al., 2007). Numerous genes encoding the enzymes and the transcription factors have been cloned and characterized. They are located within a huge gene cluster of about 70 kb in the genomes of A. parasiticus and A. flavus. The direction of each transcription is unique in each gene (Woloshuk and Prieto, 1998; Brown et al., 2001; Schmidt-Heydt and Geisen, 2007) and the structures of the gene clusters in A. flavus and A. parasiticus are mostly the same. The aflatoxin gene cluster is given in Fig. 1 and aflatoxin biosynthesis has been presented in Fig. 2 (Yabe and Nakajima,
2004). Cleveland et al. (1987), Yabe et al., (1999), and Yabe and Nakajima (2004) provide the currently accepted scheme for aflatoxin biosynthesis which is: hexanoyl CoA precursor —>
norsolorinic acid, NOR —> averantin, AVN —> hydroxyav- erantin, HAVN —> averufin, AVF —> hydroxyversicolorone, HVN—> versiconal hemiacetal acetate, VHA —> versiconal, VAL —> versicolorin B, VERB —> versicolorin A, VERA —> demethyl-sterigmatocystin, DMST —> sterigmatocystin, ST —> O-methylsterigmatocystin, OMST—> AFB1 and afla- toxin G1, AFG1.
Secondary metabolites are low-molecular-weight natural products with restricted taxonomic distribution, often synthe- sized after active growth of the fungus has ceased, the ß lactam antibiotic penicillin, synthesized by an nonribosomal peptide synthetase, as well as polyketides aflatoxin and sterigmato- cystin synthesized through a polyketide pathway, are among the best studied fungal secondary metabolites (Lori et al., 2003). The discovery that genes involved in secondary metabolites production are clustered, as are the genes that code for the pro- duction of the majority of other secondary metabolites that have been studied, has important implications for gene regulation and evolution. Putative biosynthetic pathway genes for secondary metabolism can easily be detected by in silico analysis of ge- nomic data (Keller et al., 2005). The recent characterization of Louisiana Art Educator’s Association (LaeA), which is a global regulator of secondary metabolism, provides a tool for detecting gene clusters and might reveal novel chromatin-based mecha- nisms of transcriptional control of these clusters. The LaeA gene has been found in all Aspergilli examined to date. However, it remains to be seen if LaeA also functions in other fungi (Keller et al., 2006).
OCCURENCE
Many agricultural commodities are liable to infestation by aflatoxigenic molds and thereby contaminated with aflatoxins. Major crops, that is, maize, rice, and cottonseed have also been found to be infested by aflatoxigenic molds in different coun- tries. The prevalence of aflatoxins in foods and feeds is relatively higher in tropical and subtropical regions where the warm and humid weather provides optimal conditions for the growth of the molds (European Commission, 2006; Rahimia et al., 2010; Luttfullah and Hussain, 2010). The growth of A. flavus and pro- duction of aflatoxin in natural substrates are influenced by the type of substrate, fungal species, moisture content of the sub- strate, mineralskpokrtmj, humidity, temperature, and physical damage of the kernels (Molina and Giannuzzi, 2002; Ribeiro
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et al., 2006; Schmidt-Heydt and Geisen, 2007). The minimum temperature ranges for A. parasiticus growth is 6–8C, the max- imum is 44–46C, and the optimum is 25–35C. A. flavus can produce aflatoxin at a wide range of temperature between 12C and 34C and inhibit the aflatoxin production at 36C while the maximum production is at a temperature of 28–30C (Brackett, 1989). Many factors affect the growth of Aspergillus fungi and the level of aflatoxin contamination in food. Contamination oc- curs at any stage of food production from preharvest to storage (Wilson and Payne, 1997; Giorni et al., 2008). Factors that af- fect aflatoxin contamination include the climate of the region, the genotype of the crop planted, soil type, minimum and max- imum daily temperatures, and daily net evaporation (Bankole and Mabekoje, 2004; Brown et al., 2001; Fandohan et al., 2005;
Ono et al., 1999). Aflatoxin contamination is also promoted by stress or damage to the crop due to drought prior to harvest, insect activity, poor timing of harvest, heavy rains at harvest and postharvest, and inadequate drying of the crop before stor- age (Hawkins and Windham, 2005; Hell et al., 2000; Ono and Sasaki 2002; Turner et al., 2005). Humidity, temperature, and aeration during drying and storage remain the most important factors.
LEGISLATIONS
The perilous nature of aflatoxin to humans and animals has demanded the need for establishment of control measures and
Figure 2 Aflatoxin biosynthesis, (Yabe and Nakajima, 2004).
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Figure 2 Continued.
866 M. ABRAR ET AL.
tolerance levels by International Authorities. Different countries have diverse regulations for aflatoxin contamination. The indus- trialized countries generally set lower tolerance levels than the developing countries where most of the vulnerable commodities are produced. For instance, the acceptance level for aflatoxin in foods is 5 μg kg−1 in Sweden (Akerstrand and Bller, 1989), 10 μg kg−1 in Japan (Aibara and Maeda, 1989) whereas it is 30 μg kg−1 in Brazil (Sabino et al., 1989). Though, such lack of concord may give rise to complexities in the trade of some products. The first Legislative Act was assumed in 1965 by the Food and Drug Administration of the United States which pro- posed a tolerance level of 30 μg kg−1 of afla-toxins Bl + Gl
+ B2 + G2. On the other hand, with increasing understand- ing of aflatoxins as potent toxic substances, the proposed level was lowered to 20 μg kg−1 in 1969. The European Economic Community (EEC) in 1973 ascertained legislation on maximum permitted levels of AFBl in different types of feedstuffs (EEC, 1974) and the legislation has been often amended since then. However, the main framework of the legislation remains more or less the same. The maximum allowable levels of AFBl in animal feeds as established by the EEC are given in Table 1. The legislation has been in action since November 30, 1991. Contamination of agricultural commodities with aflatoxin and hence establishment of regulations has been a major concern to the Joint FAO/WHO Expert Committee. The committee recom- mended that the presence of aflatoxin in food should be limited to “irreducible levels.” An irreducible level is defined as: “the concentration of a substance that cannot be eliminated from a food without involving the discarding of that food altogether or severely compromising the ultimate availability of major food supplies” (FAO/WHO, 1987). The current regulations for afla- toxin established by the EEC (Otsuki et al., 2001) and Joint FAO/WHO Committee (FAO/WHO, 1990, 1992) are given in Tables 2 and 3. The Dutch Authorities undertook a worldwide enquiry (1986–1987) from 66 countries which were requested to report on their regulations and tolerance limits for mycotoxins in food and feeds. Of the 66 countries, 50 had enacted or proposed legislation for aflatoxins in foods, 35 for aflatoxins in feedstuffs, and 14 for aflatoxin Ml in milk and dairy products. An extensive overview of aflatoxin regulations in these countries is provided by van-Egmond (1989). In most of the countries surveyed, the rationales for establishment of tolerance limits and regulations for aflatoxins were based on vague unsupported statements of carcinogenic risk for humans due to exposure to aflatoxins. Now aflatoxin is regulated in more than 75 countries (Van-Egmond and Jonker, 2004).
In developing countries like Pakistan, problem of aflatoxins prevails especially after harvesting and in storage conditions. Crops are usually sun dried in open fields and then stored in poor conditions. There are a lot of chances of aflatoxin production in agricultural commodities. Pakistan is the sixth largest exporter of chilies. Export of red dried chilies from Pakistan has declined after detection of aflatoxin by European Union food authorities. The export of red chilies had grown from Rupees 770 million Pakistani Rupees in 1999–2000 which touched 1.127 billion
Table 1 Maximum permitted level of aflatoxin Bl in different animal feeds established by the EEC
Feed Level (μg kg−1)
Straight feeds Peanut, copra, palm kernels, cotton seed, babassu, maize
and products derived 20
Compound feeds Complete feeds Feeds for cattle, sheep, and goat (except dairy cattle,
calves, and lambs) 50
Feeds for pigs and poultry (except young animals) 20 Other complete feeds 10 Complementary feeds Feeds for cattle, sheep, and goats (with exception of dairy
animals) 50
Feeds for pigs and poultry (with exception of young animals)
30
Table 2 The current European Union legislative limits
Aflatoxins μg kg−1
Total aflatoxins Product type B1 (B1+B2+G1+G2) M1
Groundnuts, nuts, dried fruit, and processed products thereof for direct human consumption or as a food ingredient
2 4
Groundnuts to be subjected to sorting or other physical treatment, before human consumption or as a food ingredient
8 15
Nuts and dried fruit to be subjected to sorting or other physical treatment, before human consumption or as a food ingredient
5 10
Cereals and processed products thereof for direct human consumption or as a food ingredient
2 4
Chilies, chili powder, cayenne pepper, paprika, white and black pepper, nutmeg, ginger, and turmeric
5 10
Milk (raw milk, milk for the manufacture of milk-based products and heat-treated milk)
0.05
NOTE: (FAO/WHO, 1990:1992; Otsuki et al., 2001).
Table 3 Aflatoxin legislations set by the Joint FAO/WHO Expert Committee
Tolerance level Aflatoxin (μg kg−1) Food/feed
Bl 5 Feed for dairy cattle Ml 0.05 Milk Bl +Gl+B2+G2 15 Raw peanut for human consumption
NOTE: (FAO/WHO, 1990:1992; Otsuki et al., 2001).
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AFLATOXINS AND THEIR TOXICITIES 867
Pakistani Rupees mark in 2003–2004 but declined to Rupees 846 million in 2004–2005 and further declined in 2005–2006 by a considerable margin. Pakistan exported 10,707 tons of red chilies in 2001–2002 and reached the peak at 15,543 tons during 2003–2004 and then aflatoxin contamination in red dried chilies was detected by the EU food Authorities. After the detection of aflatoxin, the volume of red chilies export plunged to 11,656 tons in 2004–2005 (Anonymous, 2006). Russell and Paterson (2007) examined samples of red chili pods and red chili powder from different areas of Pakistan and found contaminated with aflatoxins. Few samples contained higher level of aflatoxins than the EEC regulations.
METABOLISM
Hepatic microsomal cytochrome P450s (P450) is responsi- ble for metabolism of aflatoxins to the electrophilic, reactive epoxide which binds to DNA and other critical cellular macro- molecules (Coulombe, 1993; Gallagher et al., 1996; Guengerich et al., 1996). Aflatoxins are “procarcinogen” and enzymatic bioactivation is a prerequisite for carcinogenic action. Even though, the mechanism related to the toxicity of aflatoxin is not fully implicited; however, several researches suggested that toxicity may arise through the generation of intracellular reac- tive oxygen species, that is, superoxide anion, hydroxyl radical, and hydrogen peroxide (H2O2) during the metabolic processing of AFB1 by cytochrome P450 in the liver. These species may attack soluble cell compounds as well as membranes, eventu- ally leading to the impairment of cell functioning and cytolysis (Towner et al., 2003; Sohn et al., 2003; Berg et al., 2004). After absorption from the small intestine, AFB1 readily binds to plasma albumin, which serves as the major transporter of AFB1 in blood (Tessari et al., 2010). In liver, AFB1 is oxidized by microsomal mixed-function oxidase (cytochrome P450) to several water-soluble metabolites (Towner et al., 2000). Prin- ciple metabolism of AFB1 leading to reactive metabolites and biomarkers has shown in Fig. 3. It is generally believed that the formation of AFB1 8, 9-epoxide, an active metabolite and its subsequent covalent binding to DNA, RNA, and proteins play a critical role in both acute and chronic toxicity (Eaton et al., 1997; Josse et al., 2008). Animals, which fail to produce the epoxide, are relatively resistant to both, but those animals which produce the epoxide, but do not effectively metabolize it further, may be at the highest risk to the carcinogenic activity of AFB1, but are relatively resistant to the acute toxicity. Those animals that not only produce the epoxide but also effectively remove it with a hydrolase enzyme, thus producing a very re- active hydroxyl-acetal are most sensitive to the acute toxicity (Moss, 1996; Aninat et al., 2006). In most animal models exam- ined, glutathione S-transferases are probably the most critical AFB1-detoxicity enzymes. The extreme sensitivity of animal species such as turkeys, to AFB1 may be due to a combination of efficient activation by cytochrome P450 and deficient detox- ification as a result of the absence of AFB1-specific glutathione
S-transferase activity in the liver (Klein et al., 2000). Isozyme is articulated as null phenotype in approximately 50% of the individuals and Caucasian population, so expressing it, may be at an increased risk of developing chemically induced cancers (Ryberg et al., 1997).
The epoxide of AFB1 is known to react with guanine residues in DNA and can cause subsequent depurination, while the hy- droxyacetal derivate reacts with proteins through such residues as lysine (Klein et al., 2000; Zhang, 2010). The parent molecule may thus be seen as a very effective delivery system, having the right properties for absorption from the gut and transmission to the liver and other organs of the body. It is, however, the manner in which it is subsequently metabolized in vivo which deter- mines the precise nature of the animal’s response (Moss, 1996). An alternative biotransformation of AFB1 to aflatoxicol is medi- ated by a reductase enzyme and the product can be…