A Review on Mycotoxins in Food and Feed: Malaysia Case Study
Fungi are distributed worldwide and can be found in various foods and feedstuffs from almost every part of the world. Mycotoxins are secondary metabolites produced by some fungal species and may impose food safety risks to human health. Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes, deoxynivalenol (DON and T-2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals. Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (70% to 80% during wet seasons). Stored crops under such conditions can easily be contaminated by mycotoxin-producing fungi. The most important mycotoxins in Malaysian foods are AFs, OTA, DON, ZEN, and FMN that can be found in peanuts, cereal grains, cocoa beans, and spices. AFs have been reported to occur in several cereal grains, feeds, nuts, and nut products consumed in Malaysia. Spices, oilseeds, milk, eggs, and herbal medicines have been reported to be contaminated with AFs (lower than the Malaysian acceptable level of 35 ng/g for total AFs). OTA, a possible human carcinogen, was reported in cereal grains, nuts, and spices in Malaysian market. ZEN was detected in Malaysian rice, oat, barley, maize meal, and wheat at different levels. DON contamination, although at low levels, was reported in rice, maize, barley, oat, wheat, and wheat-based products in Malaysia. FMN was reported in feed and some cereal grains consumed in Malaysia. Since some food commodities are more susceptible than others to fungal growth and mycotoxin contamination, more stringent prevention and control methods are required.
Mycotoxins are secondary metabolites produced by some fungal species. Fungi are the eukaryotic and multinucleus organisms that usually show filamentous growth (Hawksworth and others 1996). Fungi are distributed worldwide and can be found in various food and feedstuffs from almost every part of the world (Pfohl-Leszkowicz and others 2002). Fungi and fungal spores are able to colonize and penetrate deep into the matrices of agricultural crops and produce mycotoxins during preharvest and postharvest practices, and processing and storage stages (Bhat and others 2010). More than 300 different mycotoxins have been detected so far (Erber and Binder 2004). Mycotoxins are synthesized under suitable biological, chemical, and physiological conditions. Toxin production is influenced by some ecological and environmental factors such as temperature, type of substrate, moisture content, relative humidity, water activity (aw), occurrence with other fungi, physical damage by insects, use of fungicides, and storage conditions (Zöllner and Mayer-Helm 2006). Several other factors such as poor harvesting practices, improper processing, packaging, drying techniques, and transport activities influence fungal growth and increase the risk of mycotoxin production (Bhat and others 2010).
Climate changes seem to be another important factor affecting mycotoxin contamination of foods and feedstuffs (Paterson and Lima 2010). Depending on the geographical and climate conditions, different fungal species can invade foods and feedstuffs. Aspergillus, Penicillium, and Fusarium species are the most important mycotoxin producers. Penicillium and Aspergillus species can grow at higher temperature and lower aw than Fusarium. Fusarium species grow well at higher aw and lower temperature (Bhat and others 2010). Aspergillus species can be found on nuts, cereals, palm kernels, cocoa, and coffee beans (Kozakiewicz 1996). Depending on the structure and biological origin, mycotoxins can be classified into 4 categories (polycetoacids, terpenes, cyclopeptides, and nitrogenous metabolites) (Bhat and others 2010). Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes (deoxynivalenol (DON) and T-2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals (Bhat and others 2010). AFs, DON, and ergot alkaloids are usually produced at preharvest stages, while FMN and OTA are mainly produced during postharvest activities (Bhat and others 2010). Naturally contaminated crops may contain multiple mycotoxins resulting in more severe effects. Multitoxin occurrence or co-occurrence of mycotoxins results in synergistic effects of mycotoxins, especially in acute toxicities in animals (such as AFs with DON, and T2 toxin, OTA with FMN, and FMN with DON) (Binder and others 2007).
Aspergillus species can produce different types of mycotoxins such as AFs, OTA, cyclopiazonic acid, patulin, citrinin, and ergot alkaloids (Li and others 2003; Flajs and Peraica 2009; Bhat and others 2010). Aspergillus toxins can be classified into 3 groups: carcinogens (AFs), nephrotoxins (OTA), and neurotoxins (territrems) (Ling and others 1979, 1984; Kozakiewicz 1996). Territrems, toxic metabolites produced by Aspergillus terreus, show blue fluorescence under UV light. Territrem A, territrem B, and territrem C can be found in rice and induce tremors and convulsion in mice (Ling and others 1979, 1984). Penicillium species produce AFs, OTA, cyclopiazonic acid, patulin, citrinin, and ergot alkaloids (Goto and others 1996; Li and others 2003; Flajs and Peraica 2009; Bhat and others 2010). FMN and ZEN are produced by some Fusarium species (Minervini and others 2005; Glenn 2007; Cozzini and Dellafiora 2012), while trichothecenes (DON, T-2 toxin, diacetoxyscirpenol, and nivalenol) are produced by several fungal genera such as Fusarium, Trichoderma, Myrothecium, Stachybotrys, Trichotecium, and Phomopsis (Kumar and others 2008). Alternaria toxins are secondary metabolites of Alternaria spp. (Ostry 2008), and Claviceps purpurea is the main producer for ergot alkaloids (Krska and Crews 2008).
Mycotoxins may be inhaled, ingested, or absorbed through the skin. No matter how mycotoxins are entered, they can cause sickness, lower performance, or death in both animals and humans (Bankole and Adebanjo 2004). Mycotoxicosis is the consequence of ingesting mycotoxin-contaminated food or feed by higher animals (Binder and others 2007). Sometimes, mycotoxicosis is caused through indirect ways such as consumption of products from animals (milk or meat) exposed to contaminated feed (Bankole and Adebanjo 2004).
Consumption of mycotoxin-contaminated food or feed results in acute or chronic consequences such as carcinogenic, teratogenic, immunesuppressive, or estrogenic effects (Binder and others 2007). Mycotoxins target the liver, kidney, nervous system, and immune system and common symptoms of mycotoxicosis in human are diarrhea, vomiting, and gastrointestinal problems (Bhat and others 2010).
Mycotoxins in Foods and Their Risk to Human Health
Mycotoxin contamination of foodstuffs is a worldwide problem and a major health threat for humans and animals that cause significant economic losses in both developing and developed countries. Besides, mycotoxin contaminations of agricultural crops pose significant economic losses to both crop producer and handlers who have to give market discounts for the contaminated products. In cases of severely contaminated crops, they have to dispose of the product. Other economic losses related to mycotoxin contamination of foodstuffs are loss of business and product recall (Herrman and others 2002).
Cereals (wheat, rice, maize, and sorghum), oilseeds (sunflower, peanut, cottonseed, and soybean), spices (black pepper, chillies, turmeric, coriander, and ginger), tree nuts (pistachio, almond, coconut, and walnut) are the most important agricultural commodities that can be contaminated with mycotoxins. Milk (animal and human), cheese, and butter can be a source of mycotoxin-contamination if mycotoxin contaminated food or feed is consumed (Bhat and others 2010). Mycotoxin contamination of agricultural crops may occur in 2 ways; the first is when fungi grow as pathogen on a plant and the second one is when fungi grow saprophytically on stored crops (Glenn 2007). It should be mentioned that observation of fungi does not necessarily mean presence of mycotoxins, and not all fungal growth results in mycotoxin production (Binder and others 2007). Fungal growth and toxin production depends on environmental factors (such as warm temperatures and high humidity). Therefore, agricultural products in subtropical and tropical regions are more susceptible to fungal infestation, and consequently, mycotoxin contamination. If subtropical and tropical countries have poorly developed infrastructures (such as processing facilities, transportation, storage, and skilled human resources), more mycotoxin contamination may be observed (Miller and others 1993).
Based on the moisture requirements, mycotoxin producers can be divided into 3 groups: field fungi, storage fungi, and advanced decay fungi. The field fungi mycotoxin producers require a grain moisture content of 22% to 25%, while storage mycotoxin producers usually grow in grain with a moisture content of 13% to 18% (equal to 70% to 90% relative humidity), and advanced decay fungi require over 18% moisture (Agag 2004; Bankole and Adebanjo 2004).
Fardiaz (1995) studied susceptibility of some foodstuffs (cereal grains, rice, soybean, maize, and peanuts) to aflatoxin contamination and found peanuts and maize to be the most susceptible commodities. As peanuts grow in the soil, various fungi contaminate peanut shell, testa, and seed. Any mechanical damage during harvest, drying, and storage increases the chance of fungal contamination and mycotoxin production. Manual harvesting, sorting wet peanuts, and storage under improper conditions favor fungal growth. Besides, aflatoxin producers are more frequently found in warm areas (Fardiaz 1995). Maize is usually contaminated with FMN and ZEN. AFs, OTA, DON, and ergot alkaloids are commonly observed in cereal grains (Puntari and others 2001; Lewis and others 2005; Wu 2006; Fu and others 2008; Hong and Nurim 2010; Reddy and Salleh 2011; Rodrigues and Chin 2012).
In developed countries, severe governmental regulations together with implementation of modern food preservation and handling techniques resulted in fewer mycotoxicosis outbreaks compared to developing countries (Bhat and others 2010). Severity of mycotoxicosis depends on such factors as type and dose of mycotoxin, extent of exposure, health condition, gender, and age of the individual (Bankole and Adebanjo 2004). Depending on these factors, mycotoxin-contaminated foodstuffs can cause chronic and/or acute health problems in humans and animals.
Mycotoxin Contamination of Feedstuffs
Mycotoxin contamination of feedstuffs can impose health hazards to human as mycotoxins appear in animal's tissue and body fluids. Monogastric farm animals (such as poultry and swine) are more susceptible to AFs due to the following 2 reasons: first, cereals are a large part of their diet and, 2nd, they lack the ruminal reservoir of a multitude of microorganisms. Ruminants seem to be less susceptible to mycotoxins rather than other animals as their rumen flora has the ability to convert some mycotoxins into less carcinogen metabolites or biologically inactive compounds (for example, AFB1 is converted to its less carcinogen metabolite AFM1 in a ruminant's body) (Fink-Gremmels 2008). Biotransformation of AFB1 in hens’ liver results in some toxic hydroxylated metabolites that are transferable to eggs (Zaghini and others 2005; Pandey and Chauhan 2007; Aly and Anwer 2009; Herzallah 2009).
Some feedstuffs such as soybean products, corn gluten, peanut cakes, sunflower seeds, palm kernels, and cotton seeds can be contaminated with AFs. Some preserved feed for dairy cows (example, hay, silage, and straw) easily gets contaminated by mycotoxigenic fungi during preharvest or postharvest activities and drying stages (Fink-Gremmels 2008; Bhat and others 2010). Use of mycotoxin-contaminated feed reduces feed intake and less nutrient absorption in the digestive tract of animals and causes decrease in body weight gain, reduction in animal productivity, damage to vital organs, increased incidence of disease due to immunosuppression, interference with reproductive capacity, and death in severe cases (Herrman and others 2002; Fink-Gremmels and Malekinejad 2007; Morgavi and Riley 2007; Bhat and others 2010).
International and National Regulations for Mycotoxins
To date, 100 countries have established regulations to protect consumers from the harmful effects of mycotoxins. Depending on the country, human foods are allowed a total of 4 to 30 ng/g of AFs. The maximum total AF residue limit in human food in the U.S.A. is 20 ng/g, and 4 ng/g is the maximum amount of AFs allowed in food in the EU, which has the strictest standards worldwide (EC 2006; Fellinger 2006; Wu 2006; Zain 2010). According to the European Committee Regulations, the AF maximum permitted level in peanuts, dried fruits, and cereals (for direct human consumption or as an ingredient in foods) is set as 5 ppb for AFB1 and 10 ppb for total AFs (Moss 2002).
Due to the harmful effects of OTA and increasing knowledge of health hazards, many countries have established a limit for OTA in food and feed. At the 37th, 44th, and 56th meetings of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), a provisional tolerable weekly intake (PTWI) of 100 ng/kg BW for OTA was established (JECFA 2001). In a recent proposal from the European Union (EU), which has been effective since October 1, 2006, the maximum tolerated limit for OTA was reduced to below 5 ng/kg BW/d (EC 2006; Fellinger 2006; Wu 2006; Zain 2010). According to the European Commission (Regulation 1881/2006), the maximum contamination level of OTA in processed cereal-based foods and baby foods for infants and young children and dietary foods for special medical purposes intended specifically for infants is 0.5 ng/g, while for unprocessed cereals and coffee, it is 5 ng/g (EC 2006). Based on the FAO's worldwide regulations for mycotoxins in food/feedstuffs, several countries have set the maximum residual limit for ZEN in maize and cereal at less than 100 ng/g (FAO 2004). According to the JECFA, the maximum tolerable daily intake for ZEN is 0.5 μg/kg BW (JECFA 2000), while tolerance levels ZEN in food in Europe are 60 to 200 ng/g (El-Nezami and others 2002). According to European Commission Regulation 2007, the maximum residue limit for ZEN in unprocessed cereals, unprocessed maize, cereal for direct consumption, maize for direct consumption, and processed maize/cereal-based foods is 100, 350, 75, 100, and 20 ng/g, respectively (European Commission 2007) and the maximum level of ZEN in wheat bran (used as ingredient in high fiber breakfast cereals) should not exceed 125 μg/kg (European Commission 2000).
According to the Joint FAO/WHO Expert Committee on Food and Additives, the permitted level of T-2 toxin is 1 μg/kg BW (JECFA 2001). In most food products, the maximum tolerated level for DON is in the range of 500 to 1000 μg/kg (Van Egmond 2002; Bhat and others 2010) but the European Commission Regulation of 2007 has set the maximum permitted level for DON in cereal-based foods for infants and children, as well as cereals intended for direct human use and finished wheat products for human consumption at 200, 750, and 1000 ng/g, respectively (European Commission 2007). Some countries (Russia and France) have set a maximum permitted level for T-2 toxin in malt and unprocessed cereals at 100 ng/g. In Slovakia, the maximum permitted level for T-2 toxin in cereal-based foods for children is set at 0.5 ng/g (FAO 2004). In other European countries, the maximum permitted level for DON in feed is 400 to 5000 ng/g.
According to the European Commission regulation, maximum residual limits of FB1and FB2 in cereals, maize for direct consumption, maize-based breakfast cereals, and maize-based foods/baby foods are 4000, 1000, 800, and 200 ng/g, respectively (European Commission 2007). To avoid apoptosis and sphingolipid metabolism disruption related to the potential carcinogenicity of FMN, daily intake of <1 μg/kg BW/d has been suggested (Petersen and Thorup 2001). The recommended level of FMN in animal feed (for cattle and poultry) is 50 μg/g (Miller and others 1996).
Until 1985, there were no regulations governing mycotoxin contamination of food and feed commodities in Malaysia. However, regulations for aflatoxins in foods set by other countries, such as the United Kingdom and United States, were used as reference. With the introduction and implementation of the Food Regulations 1985, through the Food Act of 1983, the permissible level of aflatoxins in foods was specified. This regulation set the maximum permitted level of 35 ng/g for mycological contaminants (AFs or any other mycotoxins). Since then, this regulation has been enforced and there has been no revision so far. According to the Food Act of 1983, a limit of 15 ng/g of total AFs in nuts and nut products has been established. A lower limit of 5 ng/g of total AFs in spices is permitted by Malaysian regulation (Laws of Malaysia 2006). While there have been few reports of mycotoxin contamination in animal feed in Malaysia (Muniandy 1989; Ali 2000; Lim and others 2008; Khayoon and others 2010; Reddy and Salleh 2011), there are no mycotoxin regulations to date for feed in this country.
Mycotoxin Studies in Malaysia
Mycotoxin contamination of agricultural products has had a strong negative impact on Asian trade, especially with the European Economic Community markets (Fardiaz 1995).
Malaysia's economy is still dependent on agriculture and some important agricultural and industrial products are palm kernel, cocoa, rice, coconut, coffee, pepper, pineapple, tea, and legumes (Abidin and Mat Isa 1995). Studies on mycotoxins in Malaysia started in 1960 when the disease outbreak of pig farms in the state of Melaka occurred due to aflatoxin contaminations in feeds (Lim 1964). Since then, several studies have been carried out on mycotoxins in food and feed commodities in this country. Surveillance and monitoring studies were conducted in various types of imported and locally produced foods. In order to plan for some intervention strategies, a few recent research studies were perfumed to evaluate mycotoxin exposure in Malaysia. This article describes the published studies and reports on different mycotoxins in Malaysia and discusses their occurrences and related health risks.
Factors Affecting Production of Mycotoxins in Foods/Feedstuffs in Malaysia
Due to the high temperature and humidity conditions in tropical and subtropical regions, high presence of mycotoxins can be expected (Bhat and others 2010). Like other tropical countries, Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (50% to 60% and 70% to 80% during the dry and wet seasons, respectively) (Leong and others 2011a). Stored crops under such conditions can easily be contaminated by mycotoxin-producing fungi (Mat Isa and Abidin 1995). As previously mentioned, the rate of peanut consumption in Malaysia is high. Therefore, due to the high mycotoxin contamination of peanuts in tropical countries, consumers are subjected to a great health hazard. The major factors determining infestation of fungi and toxin production are high temperature and humidity stress (Agag 2004). According to Miller (1995), the ecology of aflatoxin-producing fungi in the tropical region is different from other geographical areas. Aspergillus spores are widespread in the soil of farming areas, resulting in high aflatoxin levels of agricultural products in tropical countries. As peanuts grow in the soil, various fungi contaminate peanut shell, testa, and seed. Unshelled peanuts from Malaysia were contaminated with elevated amounts of AFs compared to shelled peanuts or peanut products (Sulaiman and others 2007; Arzandeh and others 2010). Food products sold at open markets in tropical regions are usually displayed under improper conditions. Exposure to fungal spores, from dust and environmental pollution for a long period of time, increases the chance of mycotoxin production in such commodities.
Fusarium, one of the main mycotoxin producing fungi, can invade variety of economically important agricultural crops in Malaysia. Diseases produced by Fusarium species are one of the major threats to the farmers in Malaysia (Latiffah and others 2009). In the 1st comprehensive study on diversity of Fusarium species in Malaysian soil, 8 species were isolated from 30 studied areas (Lim 1971). The most widespread species were F. solani followed by F. oxysporum. In the study conducted by Mohd Izham and others (2005), 5 species of Fusarium were identified in various types of soils in Penang Island. Latiffah and others (2007) identified 4 Fusarium species, namely, F. solani, F. semitectum, F. equiseti, and F. oxysporum from agricultural soils in Peneng. The most prevalent species were F. solani (84%), followed by F. semitectum (7%), F. equiseti (7%), and F. oxysporum (2%). F. solani and F. oxysporum were also isolated from forest soil samples in Teluk Bahang and Bird Valley, Pulau Pinang (Latiffah and others 2009, 2011). Latiffah and others (2010) identified 4 Fuzarium species, namely, F. solani, F. oxysporum, F. semitectum, and F. proliferatum from peat soil of Pondok Tanjung and Sungai Beriah, Perak. According to the studies on occurrence and diversity of Fusarium species in soils and plants from several tropical highland areas in Malaysia, variety of Fusarium species were identified (Manshor and others 2012). Out of the 20 species identified, the most predominant species were F. solani (66.1%) followed by F. graminearum (8.5%), F. oxysporum (7.8%), F. semitectum (5.7%), F. subglutinans (3.5%), and F. proliferatum (3.4%). Seelan and Muid (2010) isolated 8 Aspergillus species from tropical soils in Sarawak, Malaysia. However, their study revealed that low levels of ochratoxin producing Aspergillus were present on the soil of this region compared to the temperate countries.
Mycotoxins have been studied since 1960 when a huge number of turkeys died in England due to consumption of contaminated groundnut meal imported from Brazil (Bankole and Adebanjo 2004; Morgavi and Riley 2007). AFs, secondary metabolites produced by some members of the Aspergillus genus, are highly toxic, carcinogenic, teratogenic, and mutagenic compounds (Bhat and others 2010). AFs are difuranocoumarin derivatives, which are produced by A. flavus and A. parasiticus through a polyketide pathway (Ito and others 2001; Turner and others 2009). Among the 18 different types of AFs identified, B series (AFB1 and AFB2), G series (AFG1 and AFG2), and M series (aflatoxin M1 (AFM1), and aflatoxin M2 (AFM2)) are the most important. M series are the hydroxylated derivatives of B series that can be found in milk, meat, and milk products of dairy cattle and other mammals that have consumed aflatoxin-contaminated food or feed. Milk is the most important product for introducing AFM1 into the human diet (Chu 1991; Chen and others 2005).
AFB2 and AFG2 are dihydroxy derivatives of AFB1 and AFG1, respectively. Generally, AFB2, AFG1, and AFG2 are not reported in the absence of AFB1 (Chun and others 2007).
AFs possess a polycyclic structure, which is derived from a coumarin nucleus attached to a bifuran system. AFB1 and AFB2 are attached to a pentanone, but AFG1 and AFG2 are attached to a 6-membered lactone. The reason that AFB1 and AFG1 are considered more toxic and carcinogenic than AFB2 and AFG2 is that they possess a double bond in the form of vinyl ether at their terminal furan ring, which is the active site and makes their fluorescence more intensive. This active site may undergo a reduction reaction resulting in significant change in their activity (Molyneux and others 2007; Turner and others 2009). According to Herzallah and others (2008), the presence of a lactone ring makes AFs unstable to alkaline hydrolysis. However, AFs are stable at high temperatures with little damage occurring during cooking or pasteurization. In the presence of oxygen, AFs are unstable to UV light, extreme pH values (<3, >10), and oxidizing agents (Herzallah and others 2008). AFB1 is considered the most harmful aflatoxin and induces teratogenic, mutagenic, and hepatotoxic effects in mammals and laboratory animals (Abdel-Wahhab and Ahmed 2006; Nakai and others 2008). According to the Intl. Agency for Research on Cancer, AFB1 has been classified as a class 1 human carcinogen, implicating that its carcinogenic effect in humans has been documented. As possible carcinogens to humans, AFG1, AFB2, and AFG2 are classified in group 2B (IARC 1993a; Chiavaro and others 2001; Villa and Markaki 2009).
Occurrence of AFs
The optimum temperature for growth and aflatoxin production by A. parasiticus and A. flavus is 25 to 35 °C and 28 to 30 °C, respectively (Bhat and others 2010). As that optimum temperature is provided in warm-temperature zones, AFs are a far greater problem in tropical regions than in temperate zones of the world. However, the movement of agricultural commodities around the globe has made almost every part of the world a target, and no region of the world is free of AFs. As previously mentioned, mycotoxins are chemically stable during processing and storage, thus making it critical to avoid the conditions leading to mycotoxin formation during production, harvesting, transport, and storage, which is not usually possible or achieved in practice (Bullerman and Bianchini 2007; Kabak 2009; Fernández-Cruz and others 2010).
AFs are generally found in agricultural products, such as cereals (rice, wheat, maize, barley, and sorghum), spices (black pepper, chili, ginger, coriander, and turmeric), and fat-containing crops including tree nuts (pistachios, almonds, walnuts, and Brazil nuts), peanuts, and oilseeds (cotton, sunflower, sesame, and soybean) (Tabata and others 1993; Nawaz and others 1997; Stroka and others 2000; Ostry and others 2001; Abdulkadar and others 2004; Aycicek and others 2005; Ammida and others 2006; Liu and others 2006; Yentür and others 2006; Zöllner and Mayer-Helm 2006; Cheraghali and others 2007; Chun and others 2007; Molyneux and others 2007; Var and others 2007; Trucksess and others 2008; Adzahan and others 2009; Reddy and others 2009; Fernández-Cruz and others 2010; Salem and Ahmad 2010; Afsah-Hejri and others 2011; Iqbal and others 2011; Firdous and others 2012). By implementation of good storage management techniques, mycotoxin contamination of agricultural crops can be minimized. Proper ventilation, uniform loading, reducing insect infestation, and proper temperature control are the most important factors to minimize mycotoxin contamination at the storage level (Kozakiewicz 1996).
Aflatoxin contamination of milk and milk products is a serious problem. As infants depend on milk as a basic food, it is extremely important to control the level of AFs in milk. Although AFM1 is known as a possible human carcinogen, Codex Alimentarius set the maximum intake limit of AFM1 at 50 ng/kg (Codex Alimentarius Commission 2001). AFM1 has been reported in milk, neonatal cord blood, and urine (Lamplugh and others 1988; Maxwell and others 1989; Galvano and others 1996; Martins and Martins 2000; Mykkänen and others 2005; Gürbay and others 2006; Polychronaki and others 2006; Virdis and others 2008; Nuryono and others 2009; Hussain and others 2010; Mohd Redzwan and others 2012; Sabran and others 2012; Suriyasathaporn and Nakprasert 2012). AFM1 was also reported in pasteurized milk, ultrahigh-treated milk, milk powder, and some milk-based products (Montagna and others 2008; Shundo and others 2009; Ghazani 2009; Fallah 2010). Hens fed with contaminated feed with more than 3300 mg/kg of AFB1 within 28 d have been shown to produce contaminated eggs (Wolzak and others 1985).
Adverse Effects and Toxicity of AFs
AFs can affect a wide variety of animals including fish, swine, poultry, rodents, cattle, and, of course, humans. Although the response to aflatoxin depends on exposure level, health, age, duration of exposure, nutritional status of diet, and environmental factors, no report has been issued on resistant of animal or human to the acute toxic effects of AFs (Etzel 2006; Wagacha and Muthomi 2008). AFs have been linked to kwashiorkor, which is a form of protein energy malnutrition. Reduction in the levels of secretory immunoglobulin A (IgA) has been associated with AFs (Turnerand others 2003). AFs were reported to be responsible for loss of weight among children (in some cases of hepatocellular cancer) (Bhat Ramesh and Vasanthi 2003; Bhat and others 2010). AFs were found in the tissues of patients suffering from Reye's syndrome that is characterized by encephalopathy and visceral deterioration. Patients suffering from Reye's syndrome show kidney and liver enlargement and cerebral edema (Zain 2010).
Tumor in the respiratory tract of animals and humans is linked with respiratory exposure to AFB1-contaminated dust (Agag 2004). As a result of low immunity, humans or animals affected by AFs are more susceptible to bacterial and parasitic infections. Due to contamination of feed with AFB1, an unusual increased incidence of lung cancer has been observed among animalfeed production plant workers in Denmark (Autrup and others 1993).
The liver is considered the main target organ for aflatoxin toxicity and carcinogenicity. AFs exert their mode of action by interfering with the function of the immune system (Higgins and others 1992). Aflatoxin toxicity is the result of interactions with nucleic acids and interfering with protein synthesis. AFs are considered to be cofactors in the higher incidence of liver cancer along with hepatitis-B virus (Williams and others 2004). Increased incidences of human gastrointestinal and hepatic neoplasms in the Philippines, Africa, and China have been associated with AFs (Hussein and Brasel 2001).
AFs are biomonitored through the analysis of aflatoxin metabolites, blood protein adducts, and DNA adducts (Polychronaki and others 2006; Kumar and others 2008). AFs exposure level can be assessed accurately through measurement of aflatoxin-albumin (AFalb) adducts in blood (Turner and others 2003). Mutagenicity and carcinogenicity of AFB1, AFG1, and AFM1 are due to the formation of a reactive epoxide at the 8, 9-position of the terminal furan ring. (Chrevatidis and others 2003; Agag 2004). Other metabolites (AFB2) in which the double bond is saturated are a much less serious threat (Molyneux and others 2007). According to Harris (1991), AFB1 chemically binds to DNA and imposes structural alterations in particular sequences of DNA. Such alterations lead to mutation that is capable of initiating carcinogenic transformations (Harris 1991; Agag 2004). In the liver, AFB1 is metabolically biotransformed to a highly reactive electrophilic compound, and this compound can interact with cellular macromolecules. Some compounds (such as curcumin) can alter the microsomal activation of AFB1 and reduce the AFB1 toxicity by increasing its detoxification (Nayak and Sashidhar 2010).
Four processes are involved in the toxic kinetics of AFs as follows: absorption, distribution, biotransformation, and elimination. After oral ingestion of AFB1, it is efficiently absorbed by body and metabolized prior to excretion through urinary and fecal routes. Unabsorbed AFB1 is excreted in feces, while absorbed AFB1 and its metabolites are excreted in urine. Other metabolites are biliary excreted. The entrance (absorption) of toxins into the body usually occurs through oral intake. The process through which toxins are transferred from their site of absorption to other areas of the body is called distribution and transformation of toxins into new chemicals by body is called biotransformation. In the excretion process, toxins and their metabolites are primarily excreted through feces, urine, or breast milk (in lactating mothers) (Polychronaki and others 2006; Nayak and Sashidhar 2010). According to Coulombe and others (1985), approximately 50% of the oral dose of AFB1 is quickly absorbed in the small intestine and enters the liver. Liver concentrates the main part and kidneys concentrate small portion of absorbed AFB1. Free AFB1 and its water-soluble metabolites can be found in the mesenteric venous blood (Wogan and others 1967). Absorbed AFs are metabolized by members of the cytochrome P450 (CYP) enzyme family (Forrester and others 1990) that convert AFB1 to AFB-8,9-epoxide (carcinogenic form of AFB1). AFB-8,9-epoxide is able to bind covalently to serum albumin (Sabbioni and others 1987) and DNA (Essigmann and others 1977) and form lysine adducts and aflatoxin B1-N7-guanine (AFB-N7 guanine), respectively. CYP enzymes can also oxidize AFB1 to AFM1 and AFQ1 derivatives in addition to aflatoxicol (which is inactive and has urine excretion without further metabolism) (Gorelick 1990). Ruminal flora can partially degrade AFs resulting in formation of aflatoxicol (Fink-Gremmels 2008). Mutagenicity of AFB1 is induced by forming an adduct with the guanine moiety in DNA. This pathway is a consequence of the metabolic activation of AFB1 to the aflatoxin exo-epoxide, which subsequently forms an AFB1-N7-guanine adduct. This AFB1-N7-guanine is the precursor to mutations induced by aflatoxin (Wild and Turner 2002).
Aflatoxicosis in Humans
Like all other toxicological syndromes, aflatoxin contamination can be categorized as acute or chronic. Chronic toxicity is characterized by its low-dose exposure over a long period of time, which results in cancers and other irreversible effects. Acute toxicity of AFs has a rapid onset and an obvious toxic response (Zain 2010). Acute aflatoxicosis or toxic hepatitis precipitates symptoms such as jaundice, diarrhea, depression, low-grade fever, anorexia, liver damage, and decreased essential serum proteins synthesized by the liver. In severe cases, it leads to death. Repetitive incidents of aflatoxicosis have been reported in Kenya, India, and Malaysia (Lye and others 1995; Lewis and others 2005; Shephard and others 2006; Probst and others 2007; Zain 2010). The acute lethal dose for adult humans is 10 to 20 mg and the estimated acute lethal dose for children is approximately 3 mg (Lye and others 1995). For most animal species, the range of LD50 values is 0.5 to 10 mg/kg BW.
Based on the report of the European Union Scientific Committee for Food (SCF) in 1994, low levels of exposure to AFs (as low as 1 ng/kg BW/d or even less) can increase the risk of liver cancer (Leblanc and others 2005). Hepatocellular carcinomas caused by chronic dietary exposure may be compounded by the hepatitis B virus. Regrettably, chronic exposure to AFs results in approximately 250000 deaths in Sub-Saharan Africa and China annually. Death is due to hepatocellular carcinoma, which is attributed to some risk factors such as high daily intake (1.4 L g) of AFs and high incidence of hepatitis B (Wild and others 1992).
Aflatoxin exposure of humans is mainly through direct or indirect consumption of contaminated food. Indirect aflatoxin exposure refers to ingestion of milk and dairy products with AFM1 carried over from contaminated feed. Consumption of eggs and tissues from animals fed with mycotoxin-contaminated feed is another source of indirect exposure (Agag 2004). As mentioned before, respiratory exposure to AFB1-contaminated dust results in tumors in the respiratory tract (Agag 2004). After inhalation of AFB1-contaminated dust, lung cells and nasal mucosal epithelial cells biodegrade AFB1 and subsequently B1-DNA adducts are formed (Daniels and Massey 1992; Tjalve and others 1992). Clinical symptoms of acute aflatoxicosis in human are as follows: vomiting, high fever, highly colored urine, tremors, convulsion, cerebral edema, coma, elevated serum transaminases, hypoglycemia, and fatty degeneration in the liver and kidneys (Agag 2004). Child aflatoxicosis cases increase during the late part of the tropical rainy season in the rural areas. Symptoms of children's aflatoxicosis are convulsions, vomiting, coma, and death. Fatty degeneration of the viscera, cerebral edema, and encephalopathy are then a common cause of death among children (Shank and others 1977; Van Rensburg 1977). In some parts of the world (such as Australia), fatty degeneration of the viscera and encephalopathy in children is referred to as Reye's syndrome with the following symptoms: fever, sore throat, coughing, rhinorrhea, earache, convulsions, vomiting, problems with respiratory rhythm, and loss of muscle tone (Keeler 1983).
According to Kaplan and others (2003), it is estimated that human average intake of AFs is between 10 and 200 ng/kg/d (Kaplan and others 2003). Asia and Africa have had the most reports for human aflatoxicosis. Recurring mycotoxin outbreaks and health risks associated with consumption of contaminated foodstuffs are becoming a worldwide problem. These outbreaks indicated important public health implications for all developing countries and the need for protecting humans and animals by limiting their exposure to mycotoxins.
OTA is the second most important mycotoxin (Bhat and others 2010). In 1965, OTA was discovered as a metabolite of Aspergillus ochraceus (Scott and others 1972). Shortly after, it was recognized as a potent nephrotoxin (Shotwell and others 1980). In historical documents, it has been mentioned that OTA was found in some Egyptian tombs, and it is believed that OTA was responsible for the mysterious deaths of several archaeologists (Pittet 1998). Because OTA has nephrotoxic, carcinogenic, teratogenic, and immunosuppressive effects in many animal species, it has been classified as possibly carcinogenic to humans (group 2B) by the Intl. Agency for Research on Cancer (IARC 1993a). In cases of simultaneous occurrence of the 2 mycotoxins, OTA can increase the mutagenic ability of AFB1 (Wagacha and Muthomi 2008). OTA has been associated with Balkan endemic nephropathy (BEN), which is a kidney disease that occurred in some areas of Balkan countries (Pfohl-Leszkowicz and others 2002). Much evidence has shown that OTA was indeed involved in endemic BEN, which is usually accompanied by upper urinary tract urethral cancer. Animal studies have indicated that OTA is an immune-suppressant, a liver toxin, potent teratogen, and carcinogen (Pfohl-Leszkowicz and Manderville 2007). In Morocco, daily intake of OTA from bread has been estimated to be 126 ng/kg bw/day (Zinedine and others 2007a).
From a chemical point of view, OTA contains a 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-(3R)-methylisocoumarin linked to L-ß-phenylalanine through a carboxyl group (Fernández-Cruz and others 2010). OTA is a white crystalline powder that is stable in food industrial processes, but is unstable when subjected to light. If OTA is hydrolyzed with acid, it will convert to phenylalanine and an optically active lactone acid named ochratoxin α (IARC 1993a).
A. ochraceus is a slow-growing fungus that produces dense and pale yellow colonies with globose-shaped biseriate conidial heads. Several toxic metabolites (such as OTA, penicillic acid, vioxanthin, viomellein, and xanthomegnin) can be produced by A. ochraceus (Kozakiewicz 1996). OTA is produced by some fungi of the genera Aspergillus and Penicillium. Major producers (A. carbonarius, A. ochraceus, and A. westerdijkiae) are classified into the Aspergillus subgenus Circumdati section Circumdati. Aspergillus niger is a less important OTA producer (Benford and others 2001, Bacha and others 2009). A. ochraceus, A. niger, A. carbonarius, and P. verrucosum and some other related Aspergillus spp. such as A. alliaceus, A. sclerotiorum, A. melleus, and A. sulphurew have been reported to produce OTA (Kozakiewicz 1996). Aspergillus spp. are responsible for OTA production in tropical/semi-tropical (Bhat and others 2010). Aspergillus spp. are capable of producing OTA at conditions of high humidity and temperature. Some Penicillium spp. may produce OTA at a very low temperature (5 °C) (Gupta and others 2007). The main OTA producer, A. ochraceus, is a mesophilic xerophile that grows over the temperature range 8 to 37 °C with an optimal temperature of approximately 30 °C. The minimum aw for OTA production is 0.83, and the highest amount of OTA production is at an aw value of 0.98. A. ochraceus is more common in dried foods (especially nuts). Another major OTA producer in the tropics is A. carbonarius. The optimal temperature condition for A. carbonarius is approximately 32 to 35 °C, and the aw for spore germination at 30 °C is 0.83 (Benford and others 2001; Fernández-Cruz and others 2010). Zain (2010) reported that AFB1 is absent or present at low levels when high levels of OTA are present and vice versa. This observation suggests a competition between Aspergillus species and their toxins either at the production level or in their rate of absorption in the gastrointestinal tract.
Occurrence of OTA
OTA is a frequent, natural contaminant responsible for several foodstuffs, such as cereals, coffee beans, cocoa, dried fruits, peanuts, wine, fish, eggs, poultry, milk, and defatted groundnut cake (used as animal feed), rye, brown kidney beans, soya beans, maize, nuts, cornflour, tea, and some herbs (Bonvehi 2004; Rizzo and others 2004; Tafuri and others 2004; Jørgensen 2005; Bugno and others 2006; Batista and others 2009). In tropical countries, OTA is associated with moldy green coffee beans. OTA can even be found in roasted coffee beans and coffee brew (Studer-Rohr and others 1995; Kozakiewicz 1996; Sibanda and others 2002). Contamination of agricultural commodities can occur in the field, at harvest, during processing, in storage, and in shipment. OTA contaminates cereals, cocoa, coffee, spices, and dried fruits during storage, while grapes usually are contaminated in the field (Bhat and others 2010). Some processing techniques can reduce fungal populations on the crops. Human exposure to OTA usually occurs through the consumption of improperly stored food products. OTA can be determined in the tissues and organs of humans and animals (including breast milk, meat, and blood) (Pfohl-Leszkowicz and Manderville 2007; Kumar and others 2008).
OTA in human milk and urine
Reports have demonstrated the transfer of OTA to milk in humans, rabbits, and rats (Breitholtz and others 1991; Zimmerli and Dick 1995; Ueno and others 1998; Galvano and others 2001; Skaug and others 2001). OTA can appear in the blood and internal organs (such as kidneys) of animals fed with contaminated feeds (Jørgensen and Petersen 2002; Bhat and others 2010). There is a relationship between a mother's dietary intake of contaminated food (such as cereals, cheese, cakes, and juices) and OTA contamination of her milk (Bhat and others 2010). OTA has also been observed in human urine (Pascale and Visconti 2001; Fazekas and others 2005; Pena and others 2006; Manique and others 2008).
Little OTA transfer to ruminants (such as cow) milk and milk products have been reported (Skaug 1999; El-Sayed Abd Alla and others 2000) as OTA can be hydrolyzed to a nontoxic compound due to the ability of the rumen microflora to degrade OTA (Ringot and others 2006). Healthy cattle are able to degrade and inactivate up to 12 mg OTA/kg feed by rumen protozoa. Such effective deactivation explains the high tolerance of ruminants to OTA exposure (Hult and others 1976; Fink-Gremmels 2008). Sometimes, small amounts of OTA may be found in milk that can be due to a protein-rich diet that drastically affects the cleavage capacity by rumen microorganisms (Breitholtz-Emanuelsson and others 1993; Muller and others 2001; Fink-Gremmels 2008).
Adverse effects and toxicity of OTA
There is sufficient evidence to show the nephrotoxic, mutagenic, carcinogenic, teratogenic, and immunosuppressive effects of OTA in laboratory animals. OTA is a liver toxin, and tumors of the upper urinary tract have been associated with exposure to OTA (Pfohl-Leszkowicz and Manderville 2007). According to the latest news, OTA induces apoptosis in human lymphocytes and neuronal cells. Induction of apoptosis in neuronal cells contributes to the pathogenesis of neurodegenerative diseases (such as Parkinson's and Alzheimer's disease) (Assaf and others 2004; Zhang and others 2009).
In a human, OTA is distributed through the blood mainly to the kidneys, but in animals, it has been shown to be accumulated in body organs and tissues such as meat, liver, and kidney (Jørgensen 1998; Dragacci and others 2000; Jørgensen and Petersen 2002; Bhat and others 2010). In animal acute toxicity cases of OTA, kidney and liver damage have been reported as the main causes of death in ducklings, chicks, and rats (Scott and others1972). Some evidence has demonstrated that exposure to OTA can cause problems for kidneys (functional and morphological changes) and some harmful effects on the liver and heart. Moreover, OTA can cause morphological abnormalities, gastrointestinal/renal tissue lesions, lymphoid tissue lesions, blood clotting, and reduction in egg production. With regard to laboratory animals, the oral LD50 is 1.0 ng/kg for pigs and 0.2 ng/kg for dogs (Puntari and others 2001).
In most animal species, OTA is usually absorbed in the stomach due to its lipid-soluble, nonionized, and acidic properties (pKa = 7.1). In rats and mice, OTA is rapidly absorbed from the small intestine and stomach. According to Ringot and others (2006), nonruminant species, such as pigs, chickens, rabbits, and rats, may absorb approximately half of the ingested OTA. After ingestion, 4 steps are associated with OTA as follows: absorption, distribution, elimination, and metabolism. Due to its acidic nature, OTA is passively absorbed by the stomach in its monoanion (OTA–) and nonionized forms (Pfohl-Leszkowicz and Manderville 2007). Intestine absorbs OTA, too (Gupta and others 2007). OTA has high binding affinity to plasma proteins that favors its passive absorption (Gupta and others 2007; Pfohl-Leszkowicz and Manderville 2007). Distribution of OTA consists of the following 2 steps: distribution in blood and tissue-specific distribution. Blood distributes OTA to the kidneys and at lower concentrations to the fat, muscle, and liver (Ringot and others 2006). After entering the bloodstream, OTA binds to serum proteins (mostly albumin), thereby, facilitating passive absorption of OTA in its nonionized form. The nonionized form of OTA can strongly bind to some small serum proteins, and these small molecules are able to easily pass through the glomerular membrane in mammals. The nephrotoxic effect of OTA is related to this binding (Pfohl-Leszkowicz and Manderville 2007). According to Gupta and others (2007), concentration of OTA and its metabolites in plasma and tissues depends on several factors as follows: age, gender, toxin dose, duration of administration, health status, route of administration, diet composition, and form of OTA (naturally occurring or crystalline). In pigs, the level of OTA in the blood is approximately 5-fold greater than in the kidney. The OTA tissue distribution in pigs, chickens, rats, and goats after acute treatment is as follows: kidney > liver > muscle > fat. Renal and biliary routes are involved in of OTA. OTA elimination routs vary with the following factors: route of administration and OTA dose. Kidney is the main route of excretion in humans and monkeys (Pfohl-Leszkowicz and Manderville 2007).
There are 2 known metabolic pathways for OTA. According to Ringot and others (2006), the major metabolites include hydroxylated derivatives (4(R)-OHOTA, 4(S)-OHOTA, and OTAalpha (OTa) (lacking the phenylalanine moiety). Almost 10 different OTA derivatives were identified in cell culture and pig kidney microsomes (Ringot and others 2006; Pfohl-Leszkowicz and Manderville 2007). According to Marin-Kuan and others (2008), oxidative stress induced by OTA leads to DNA damage and mutations. Production of oxygen radicals is directly increased by OTA or is an indirect consequence of the inhibition of Nrf2-regulated antioxidant gene expression (Marin-Kuan and others 2008). In addition, OTA triggers a set of complex biological effects, which are related to cell proliferation and tumor development in renal tissue. Depending on the individual cell-specific susceptibility and intracellular OTA concentration, toxicity, apoptosis, or tumor development may occur. OTA's effect on DNA is the result of blocking repair mechanisms and the genotoxicity of OTA is due to its ability to promote DNA adducts formation (Pfohl-Leszkowicz and Manderville 2007). In addition, OTA disrupts blood coagulation and glucose metabolism, resulting in toxic effects in some organs (Gupta and others 2007).
ZEN, (3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2 benzoxacyclotetradecin-1,7(8H)-dione, is a macrocyclic ß-resorcylic acid lactone (Diekman and Green 1992; European Commission 2000; JECFA 2000; Minervini and others 2005; Bhat and others 2010; Cozzini and Dellafiora 2012). ZEN is a nonsteroidal estrogen or mycoestrogen and was previously known as F-2 toxin (Morgavi and Riley 2007; Zinedine and others 2007b). ZEN is biosynthesized through a polyketide pathway by some Fusarium species (Huffman and others 2010). According to the Intl. Agency for Research on Cancer (IARC), ZEN is classified as Group 3 (not carcinogen to humans). Field fungi such as Fusarium graminearum (Gibberella zeae) (formerly named F. roseum) is the main producer of ZEN. F. culmorum, F. verticillioides, F. cerealis, F. Semitectum, F. crookwellense, F. pseudograminearum, and F.equiseti are other ZEN producers (European Commission 2000; Glenn 2007). Some derivatives of ZEN (such as α-zearalenol, α-zearalanol, β-zearalanol, β-zearalenol, and zearalanone) have been found in Fusarium-infected corn stems in the field (Minervini and others 2005).
Occurrence of ZEN
Fusarium species are able to grow in moist and cool conditions and invade crops both in preharvest, postharvest, and under poor storage conditions, but toxin production usually occurs at postharvest activities and storage (European Commission 2000; Zinedine and others 2007b). Crops such as corn, maize, wheat, barley, rice, oats, millet, and sorghum can easily be contaminated by ZEN (European Commission 2000; JECFA 2000; Zinedine and others 2007b). ZEN has been reported in cereal products (for example malt, beer, soybeans, and flour), corn silage, corn by-products, and soya meal (Schollenberger and others 2007; Zinedine and others 2007b). ZEN has also been reported in eggs (Sypecka and others 2004). Very low levels of ZEN and its metabolites (usually below the limit of quantification) might be found in milk (Seeling and others 2005). ZEN is a heat-stable mycotoxin, but under alkaline conditions, a temperature higher than 150 °C can degrade the toxin (European Commission 2000). Due to high consumption rate of cereal-based food products, children are more affected by ZEN-contaminated foods (Bhat and others 2010).
Adverse effects and toxicity of ZEN
ZEN can cause infertility, abortion, reproduction problems (especially in swine), and is associated with cervical cancer (Bhatnagar and others 2002; El-Nezami and others 2002). Ingestion of contaminated feed results in interference with the exocrine and endocrine systems. Like other environmental estrogens, ZEN has the potential to disrupt sex steroid hormone functions (Bennett and Klich 2009; Bhat and others 2010). ZEN and its metabolites bind to estrogen receptors and activate gene transcription. Besides, they interfere with the regular activity of the endocrine glands (Malekinejad and others 2005; Fink-Gremmels and Malekinejad 2007).
In several incidences, ZEN has been associated with some pubertal changes (Kuiper-Goodman and others 1987). Freni-Titulaer and others (1986) observed a significant correlation between the pubertal changes and consumption of ZEN-contaminated soya-based products and meat from animals fed with ZEN-contaminated feed (Freni-Titulaer and others 1986). Due to the estrogenic activity of ZEN, farm animals fed with ZEN-contaminated feed show alterations in the reproductive tract, decrease in fertility, increase in number of fetal resorptions and implementation failure, and reduced litter size (Morgavi and Riley 2007). The alterations in reproductive tract are permanent. Strong hyperestrogenic responses can be observed in animals susceptible to ZEN (such as swine) (Coulombe 1993). Hyperestrogenism in swine has been related to consumption of moldy grain (Bennett and Klich 2009). Among animals, pigs are the most sensitive and poultry are the least affected by ZEN (Bhat and others 2010). In pigs, ZEN poisoning is usually associated with feminizing syndromes or hyperestrogenic activity and causes urinary/genital problems (Danicke and others 2005). According to the Joint FAO/WHO Expert Committee, the safety of ZEN is evaluated on the basis of the dose that has no hormonal effect in pigs (JECFA 2000).
According to Wood (1992), consumption of low-dosed ZEN-contaminated feedstuffs by dairy cows does not pose any health hazards to humans (Wood 1992). The rumen flora can convert ZEN into its hydroxy-metabolite α-zearalenol (almost 90%) and β-zearalenol (Fink-Gremmels 2008). As reported by Takahashi-Ando and others (2002), lactonohydrolases produced by microflora in the large intestines of monogastric mammalian is able to convert ZEN to its nonestrogenic compound. Compared to ZEN, α-zearalenol has higher estrogenic potency, but as its rate of absorption is lower, it affects dairy cattle less than ZEN (Fink-Gremmels 2008). Cytotoxic effects of α and β Zearalenols are due to inhibition of DNA and protein syntheses and inducing oxidative damage (Othmen and others 2008).
In cases that lactating cows are fed with an oral dose of 6000 mg zearalenone (equivalent to 12 mg/kg BW), 6.6 μg/L β-zearalenol, 4 μg/L α-zearalenol, and 6.1 μg/L zearalenone can be detected in the milk (JECFA 2000). ZEN was found in 55.1% and 22.4% of the endometrial tissues from women suffering from endometrial adenocarcinoma and endometrial hyperplasia, respectively (Tomaszewski and others 1998). Zearalanol and ZEN were detected in blood plasma of girls aged 6 mo to 8 y old suffering from premature thelarche in Puerto Rico between 1978 and 1981 (Saenz de Rodriguez and others 1985). Following oral administration, ZEN is rapidly absorbed by body and high level of toxin can be measured in serum. It was reported that oral bioavailability of ZEN can reach to 80% to 85% of the ingested dose. ZEN is widely distributed and slowly eliminated from body tissues (Kuiper-Goodman and others 1987; Fink-Gremmels and Malekinejad 2007). Kuiper-Goodman and others (1987) reported that 67% of total oral dose of ZEN was excreted within 48 h and 45% and 22% of the oral was recovered in urine and faeces, respectively. The rate of renal excretion was higher in rabbits and humans. ZEN can be deposited in body tissues and carry over into milk. As reported by Prelusky and others (1990), ZEN and its major metabolites (α and β Zearalenols) were detected in plasma and milk of lactating cows.
ZEN is reduced to its major metabolites (α and β Zearalenols) by 3-α and 3-β-hydroxysteroid dehydrogenase as catalysts (Olsen and Kiessling 1983; Othmen and others 2008). This biotransformation mostly occurs in liver, but gut microflora and intestinal mucosa were reported to be able to metabolize ZEN (Biehl and others 1993; Kollarczik and others 1994; Danicke and others 2001, 2002). Due to the steroid metabolism in target organs, conversion of ZEN can be observed (Malekinejad and others 2006). ZEN has great affinity to uterine and oviduct estrogen receptors in pig, rat, and chicken (Fitzpatrick and others 1989). In most animal species (except rabbits), ZEN and its metabolites are excreted in the bile in rabbits urine is the main route (Kuiper-Goodmanand others 1987). In the elimination process, the alcoholic metabolites (α and β Zearalenol) are excreted as free compounds and glucuronide conjugates through faeces and urine (Danicke and others 2001; Othmen and others 2008).
Kuiper-Goodman and others (1987), reported low acute oral toxicity of ZEN (with LD50 values 4000 mg/kg b.w.) for rodents. The suggested tolerable daily intake of ZEN is estimated to be 0.05 μg/kg BW/d (Kuiper-Goodman and others 1987).
The causative agents for feed refusal syndrome in the 1930s were probably trichothecenes (Morgavi and Riley 2007). In 1949, the first member of the trichothecenes class was isolated from Trichothecium roseum and named trichothecin (Glenn 2007). The very large family of trichothecenes consists of several chemically related toxins produced by different species of Fusarium, Trichoderma, Myrotecium, Verticimonosporium, Trichotecium, Cephalosporium, Stachybotrys, and Cylindrocarpon (Glenn 2007; Bhat and others 2010). Trichothecenes cover almost 190 different structures all having in common a tetracyclic sesquiterpenoid 12,13-epoxytrichothec-9-ene ring system (Ueno 1983; Desjardins and others 1993; Zöllner and Mayer-Helm 2006). Based on some structural features, trichothecenes are divided into 4 main groups. Group A (T-2 toxin, HT-2 toxin, neosolaniol, and diacetoxyscirpenol) are without a carbonyl group at position C-8 and have an oxygen function at C-8. Group B (DON and its derivatives, nivalenol, 3-acetyl-deoxynivalenol, fusarenon-X, and 15-acetyldeoxynivalenol) have a carbonyl group at C-8. Group C and D have a second epoxy group and a macrocyclic structure, respectively (Ueno 1983; Berthiller and others 2005). Group A trichothecenes are the most toxic (Mirocha and others 2003; Bhat and others 2010). Trichothecenes of concern produced by Fusarium spp. are deoxynivalenol (DON), T-2 toxin, diacetoxyscirpenol, and nivalenol (Glenn 2007). Toxicity of T-2 toxin is 10 times more than DON in mammals (Ueno 1983). DON, the most prevalent toxin in this group, belongs to type B trichothecenes that are more phytotoxic (Mirocha and others 2003; Foroud and Eudes 2009). Trichothecenes are relatively insoluble in water, nonvolatile, and have low molecular weight. All members have a trichothecene ring in their chemical structure and are stable under different environmental conditions (Wannemacher and others 1997).
Trichothecenes are produced by several fungal genus such as Fusarium, Trichoderma, Myrothecium, Stachybotrys, Trichotecium, and Phomopsis (Kumar and others 2008). DON was first reported in Japan and named “Rd-toxin.” The same compound was then isolated from contaminated corn used as feed for pigs with emesis and named “vomitoxin” (Morooka and others 1972; Morgavi and Riley 2007). DON has other names, including amphetamine deoxynivalenol and alpha methyl phenethylamine (Herrman and others 2002; Bhat and others 2010). DON is highly stable and can survive food processing procedures such as milling and powdering. In corn and wheat, DON is mainly produced by F. graminearum (Bhat and others 2010).
Occurrence of trichothecenes
Fusarium graminearum and Fusarium culmorum produce DON that is the most prevalent trichothecene in food/feedstuffs and cause problems also in temperate zones of the world. Fusarium pseudograminearum, F. graminearum, and F. culmorum are responsible for the production of DON grain (Glenn 2007). The main sources for trichothecene contamination are grains such as corn, barley, wheat, and oats (Zöllner and Mayer-Helm 2006). Trichothecenes were detected in cereal products and milk (Sørensen and Elbaek 2005; Spanjer and others 2008). DON and its metabolite were reported in eggs (Sypecka and others 2004). Fusarium-contaminated wheat, millet, and barley were responsible for the syndrome named alimentary toxic aleukia in Siberia, Russia, between 1942 and 1947 (Wannemacher and others 1997). The “red mold disease” of barley and wheat in Japan was attributed to Fusarium spp. Deoxynivalenol and nivalenol were isolated from moldy grains (Wannemacher and others 1997).
Adverse effects and toxicity of trichothecenes
Trichothecenes have been strongly associated with fatal and chronic toxicoses both in humans and animals. Exposure to trichothecenes results in delayed growth in eukaryotes. Humans, mammals, fish, birds, invertebrates, and plants can be affected by trichothecenes (Wannemacher and others 1997). Plant regeneration and mammals reproduction is affected by trichothecenes (Rocha and others 2005). Trichothecenes are phytotoxic to parsnip, wheat, and maize (Desjardins 2006; Pestka 2007). Fusarium spp. cause scab in wheat and root and stalk rots in corn and sorghum. At the flowering stage, plants are more susceptible to fungal infection. Warm and moist conditions in the field favor Fusarium invasion of grain crops and formation of DON or T-2 toxin (Herrman and others 2002). Some of the trichothecene mycotoxicoses include swine feed refusal as observed in the U.S.A., akakabi-byo or red mold disease in Japan, and alimentary toxic aleukia in Central Asia and Russia (Desjardins and Proctor 2007).
Trichothecenes have an amphipathic nature that allows them to cross the cell membrane and interact with endoplasmic reticulum (Yang and others 2000), chloroplast (Bushnell and others 2010), and mitochondria (Pace 1983). They inhibit ribosomal protein synthesis in cells and alter cell membrane structure (McLaughlin and others 1977; Wannemacher and others 1997; Desjardins and Proctor 2007). The primary effect of trichothecenes is inhibition of mitochondrial translation, targeting the peptidyl transferase center (Freid and Warner 1982; Bouaziz and Martel 2009; Bin-Umer and others 2011). According to Shifrin and Anderson (1999), tichothecenes activate a cellular stress response named ribotoxic stress response. Rocha and others (2005) reported that trichothecenes have other effects such as inhibition of cell division, RNA and DNA synthesis, disruption of membrane integrity and structure, as well as mitochondrial function. According to Cundliffe and Davis (1977), T-2 prevents formation of the initial peptide bond, while DON inhibits the elongation step.
Activity of peptidyl transferase and nucleic acid synthesis is inhibited by trichothecenes (McLaughlin and others 1977; Thompson and Wannemacher 1986). Trichothecenes interrupt cell membrane integrity and mitochondrial function induces programmed cell death in plants (Bunner and Morris 1988; Pace and others 1988; Rocha and others 2005) and apoptosis in animal cells (Yoshino and others 1997; Shifrin and Anderson 1999; Islam and Pestka 2003).
Trichothecenes inhibit DNA and protein syntheses, but no mutagenic or carcinogenic effect has been reported. The main target affected by trichothecenes is the digestive system (Ueno 1983). DON and T-2 toxin affest immunity by inhibiting protein synthesis and cell proliferation (Bhat and others 2010). DON decreases antibody and immunoglobulin levels in the body (Richard 2007).
Tricothecenes bind to ribosomes and inhibit protein synthesis (Thompson and Wannemacher 1986; Middlebrook and Leatherman 1989). T-2 toxin interferes with the cell membrane function due to its amphipathic characteristic (Pritchard 1979). T-2 toxin alters cellular immune response, too (Ueno 1984). Extremely high dose of DON (more than 27 mg/kg BW) can cause death but low doses (50 g/kg BW) result in vomiting in animals. Pigs are the most sensitive animals to DON (Pestka 2007).
Trichothecene toxicity in animals usually has the following symptoms: feed refusal, decreased feed conversion, weight loss, vomiting, severe dermatitis, abortion, bloody diarrhea, hemorrhaging, abnormal feathering, lesions at the edges of bird beaks, decreased egg production, and death. (Herrman and others 2002). Nausea, diarrhea, abdominal pain, dizziness, vomiting, and headache are symptoms of trichothecene mycotoxicosis in humans (Bhat and others 2010).
As trichothecenes are lipophilic, they can be easily absorbed through skin (Coulombe 1993), pulmonary mucosa, and gut. Direct dermal application or oral ingestion of trichothecene causes rapid irritation to the skin or intestinal mucosa (McLaughlin and others 1977; Wannemacher and others 1997). As mentioned before, T-2 toxin has high toxicity. Exposure to high concentrations of aerosolized T-2 toxin results in pulmonary edema or lung lesions, while its oral intake causes direct damage to the intestinal mucosa (Creasia and others 1990; Nordby and others 2004). T-2 toxin lethal dose in mice is 5.2 mg/kg. According to Nordby and others (2004), inhalant exposure to grain dust in a grain production environment and mills might be responsible for some hormone-related cancers. Grains (barley, oats, and wheat) have been shown to be contaminated with different levels of trichothecenes (DON and T-2 toxin) (Nordby and others 2004).
Liver is the main organ for metabolism of the trichothecenes and the intestine is capable of doing some metabolic alteration to them. The metabolized toxins can be excreted in urine and feces (Matsumoto and others 1978). Acute oral, dermal, parenteral, or aerosol exposure to trichothecenes results in intestinal and gastric lesions (Wannemacher and others 1997). Trichothecenes have potential to be used as biological weapons. It was claimed by the U.S. government that in Southeast Asia and Afghanistan (during the 1970s) after exploding certain munitions (air-to-surface rocket, aerial bomb), a yellow and oily droplet mist spread in the air and fell on individuals at the explosion site and sickening them. Early symptoms of acute exposure to trichothecenes included nausea, vomiting, skin discomfort, weakness, and dizziness (Wannemacher and others 1997; Haig 1982). Diarrhea (at first watery brown and later grossly bloody) began within an hour, and after 3 h, sore mouth, bleeding gums, coughing and dyspnea, hematemesis, and epistaxis was reported (Haig 1982).
De-epoxy DON is the main metabolite of DON (Yoshizawa and others 1986). De-epoxy DON, mainly produced by rumen and intestinal microflora and to a lesser content by liver, is exerted via animal urine and faeces (Gareis and others 1987; Swanson and others 1988). In contrast to pigs, mice, and rats, human gastrointestinal microflora are not able to transform DON and de-epoxy DON cannot be detected in human urine and faeces (Sundstol and Pettersson 2003).
Although ruminants are less susceptible to DON (Fink-Gremmels 2008), but reduction in milk production, inhibition of reproductive performance and immune function were attributed to DON-contaminated feed (Bhat and others 2010). Compared to pigs, poultry are more resistant to DON but more sensitive to T-2 toxin. T-2 toxin causes reduction in egg production and increases incidence of cracked eggs (Devegowda and others 2005; Morgavi and Riley 2007). Scabby grain toxicosis or trichothecene mycotoxicosis has been observed within hours after ingestion of contaminated food/feedstuffs such as corn, rice, wheat, and their products (Ueno 1970; Wang and others 1993). Chronic poisoning with group A trichothecenes results in immune system malfunction and significant changes in the blood cell count. T-2 toxin is the most important in group A. It can be metabolized by the gut microflora of mammals. Metabolized toxins are excreted via bile (WHO 2002). Immune system problems related to group A trichothecenes include depressed antibody formation, delayed hypersensitivity, changes in leukocyte counts, and depletion of selective blood cell progenitors. Group B trichothecenes cause reduction in dietary consumption in animals especially in pigs (WHO 2002; Bhat and others 2010).
In 1891, consumption of moldy maize (corn) contaminated with FMN caused a disease in equines (Haliburton and Buck 1986). Neurotoxic syndrome equine leukoencephalomalacia (ELEM) the massive liquefaction of the cerebral hemisphere of the brain and is attributed to some neurological manifestations such as lameness, nervousness, ataxia, aimless circling, facial paralysis, abnormal movement, and inability to drink or eat (Coulombe 1993; Marasas 2006). In 1988, a new class of mycotoxins was isolated from cultures of F. moniliforme (today known as F. verticillioides) and named FMN (Gelderblom and others 1988). FMNs are one of the main mycotoxin classes of concern produced by Fusarium species (Glenn 2007). Fusarium species are able to cause seedling diseases, stalk rots, ear rots, root rots, and kernel damage (Munkvold and Desjardins 1997). FMNs are primary amines and 4 series of FMN have been identified A, B, C, and P (Rheeder and others 2002; Cole and others 2003; Gelderblom and others 2007). “A” series members (FA1and FA2) have amides and the “B” series (FB1, FB2, FB3, and FB4) possess a free amine (Gelderblom and others 1992). Fumonisins of series “C” were isolated from wheat cultures of F. oxysporum (Seo and others 1996). Fumonisin B has the same structure as macrofusine (Norred 1993).
Occurrence of FMN
Fusarium spp. are the most important field fungi (especially for maize) (Visconti 2001; Bankole and Adebanjo 2004) and invade a variety of plants. FMNs have been found in several agricultural products including corn, corn products, medicinal plants, herbal tea, dried figs, and bovine milk (Omurtag and Yazicioglu 2004; Sørensen and Elbaek 2005; Gazzotti and others 2009; Karbancioglu-Güler and Heperkan 2009; Seo and others 2009; Moretti and others 2010).
Adverse effects and toxicity of FMN
According to the Intl. Agency for Research on Cancer, FMN are classified as group 2B substances (possibly carcinogenic to humans) (IARC 1993b). There is not enough evidence for human health hazards related to FMN-contaminated food. However, a link between high incidence of human esophageal carcinoma and consumption of FMN-contaminated maize has been reported (Yoshizawa and others 1994; Abnet and others 2001; Marasas and others 2004). FMNs have cancer-promoting activity (Norred 1993; Munkvold and Desjardins 1997). FMNs are responsible for porcine pulmonary edema (Morgavi and Riley 2007). FB1 is a cancer promoter that causes neural tube defects in human babies (Marasas and others 2004; Bhat and others 2010). FB1 has hepatotoxic and nephrotoxic effects on animals. Chronic effects of FMN in animals include impairment in the basic immune function, kidney and liver damage, respiratory difficulties, heart problems, reduction in milk production, weight reduction, and increase in mortality rate (Casteel and others 1994; Norred and others 1998; Diaz and others 2000). FMNs induce apoptosis in rat kidneys and cultured human cells (Seefelder and others 2003). Another sign of FMN toxicosis in dairy cattles included elevated serum enzyme activity of diagnostic liver enzymes that show mild hepatocellular injury (Fink-Gremmels 2008). Cooking fumonisins contaminated crops under alkaline conditions (such as production of tortillas from maize) results in formation of hydrolyzed FB1 that was reported in the feces of nonhuman primates (Shephard and others 1994).
Fumonisins are poorly absorbed through gastrointestinal tract and rapidly cleared from the blood and excreted in bile (Soriano and others 2005). Accumulation of fumonisins in body tissues is low and little residues can be found in liver and kidney (Riley and Voss 2002). Ruminants were reported to have the minimum fumonisin absorption and show tolerance to Fumonisins that are poorly absorbed through of fumonisins (Voss and others 2007). FB1 is able to cross the placenta and reduced reproductive performance in mice (Gelineau-van Waes and others 2005). It was reported that FB1 reduce folate uptake by the embryos and decrease the amount of folate binding protein in the yolk sac membrane (Stevens and Tang 1997).
FB1 has a similar structure to sphingoid bases (such as sphingosine) and acts as an inhibitor for ceramide synthetase, the key enzyme in biosynthesis of sphingolipid. Second type of lipids (sphingolipids) can be found in cell membranes and particularly in brain tissues and nerve cells (Soriano and others 2005). Inhibitory effect of FB1 results in accumulation of free sphingosine and sphinganine and subsequently cell death (Galvano and others 2002; Soriano and others 2005; Voss and others 2007). The inhibition of biosynthesis of glycosphingolipids can be observed a few hours after oral exposure to FB1 (Soriano and others 2005). This inhibition is responsible for the wide variety of health effects (such as high rate of human liver cancer and oesophageal cancer) due to the ingestion of fumonisins contaminated foods (Voss and others 2007).
Ceramide synthase inhibition results in accumulation of sphinganine (Sa) and sphingosine (So) in serum, urine, and tissues. The Sa : So ratio in tissues is auseful biomarkers for exposure to FMN (Voss and others 2007). Another useful biomarker is the Sa concentrations of tissue, urine, or serum (Enongene and others 2002). FA1 is less cytotoxic than FB1, but Van der Westhuizen and others (1998) reported FA1 as a ceramide synthase inhibitor. Haschek and others (1992) showed that FB1 (either orally or IV) resulted in hepatic changes such as necrosis and pulmonary edema in pigs. They mentioned that FB1 alters metabolism of sphingolipid, resulting in release of membranous materials into the blood circulation and consequently hepatocellular damage. Inhibition of ceramide biosynthesis has consequences such as: inhibition of hippocampus neurons cell growth, pulmonary edema, heart failure, liver lesions, and apoptosis (Soriano and others 2005). Besides, FB1 was reported as a possible carcinogen to humans with possible genotoxical effects (as a result of the activation of oxidative pathways) (Mobio and others 2000).
Occurrence of Mycotoxins in Malaysia
Studies on mycotoxins in food/feedstuffs in Malaysia go back to 1965 when samples of peanuts and peanut cooking oils were investigated (Chong and Beng 1965). The most severe report on aflatoxin contamination of food in Malaysia was reported in October 1988. There was an outbreak of acute hepatic encephalopathy resulting in death of 13 Chinese children in the northwestern state of Perak in peninsular Malaysia. The children have consumed Chinese noodle (joh see fun) and the symptoms include fever, vomiting, diarrhea, abdominal pain, and hematemesis. AFs were confirmed in the postmortem samples (Lye and others 1995). Since then, several studies have been carried out on different commodities. Due to the high health risks of AFs, they have been studied more often than other mycotoxins. Samples of imported and locally produced food have been examined. Surveys of aflatoxins in food from Malaysia showed that among all food products, peanuts are highly vulnerable to fungus and aflatoxin contamination, while in spices, oilseeds, and cereals, aflatoxin contamination is due to improper processing methods and poor storage conditions (Mat Isa and Tee 1984; Mat Isa and Abidin 1995; Abdullah and others 1998; Leong and others 2010).
Cereals and grains
Rice is the main product and staple food in Malaysia. Corn, wheat, and barley are not staple food grains in this country and are totally imported from Argentina, China, Indonesia, and Thailand (Warr and others 2008). In a survey on stored paddies, rice and rice flour samples were contaminated with AFs (MARDI 1992). However, AF levels in the positive samples were lower than 4 ng/g. A. flavus was also isolated from some of the AF-negative samples. Contamination of wheat flour from retail markets with AFs has been reported earlier (Abdullah and others 1998). The level of AFs in wheat flour samples was in the range of 11.25 to 436.25 ng/g. Abdullah and others (1998) conducted a survey on fungal colonies in starch-based foods from retail outlets in Malaysia. Aflatoxigenic colonies of Aspergillus were detected in wheat flour (20%), glutinous rice grains (4%), ordinary rice grains (4%), and glutinous rice flour (2%). Ordinary rice samples were contaminated with AFG1 (2.4%) and AFG2 (3.6%). Level of AFs in the positive samples collected from private homes ranged from 3.69 to 77.50 ng/g. About 1.2% of wheat flour samples was contaminated with AFB1 (25.62 ng/g), and 4.8% with AFB2 (11.25 to 252.50 ng/g), 3.6% with AFG1 (25.00 to 289.38 ng/g), and 13.25% with AFG2 (16.25 to 436.25 ng/kg). Higher incidence of AF contamination in wheat flour can be due to the following factors: first, presence of aflatoxin-producing Aspergillus spp. is more often seen in wheat flour than ordinary rice, and second, there are longer storage periods for wheat flour compared to other grain flours. Abdullah and others (1998) concluded that aflatoxin contamination occurred at the consumer level since the percentage of contaminated samples was higher at private homes compared to retail markets.
Some of grains from Kuala Lumpur markets have been screened for AF contamination (Rahmani and others 2010). Rice and wheat samples were contaminated with AF : AFB1 (12%), AFB2 (23%), AFG1 (18%), and AFG2 (18%). The ranges of total AFs in the contaminated cereal samples were 0.01 to 5.9 ng/g. Yazdani and others (2011) collected samples of milled rice from retail markets in 4 provinces of Malaysia and screened them for Aspergillus and Eurotium spp. contamination. Isolates were then tested for their aflatoxin-producing ability. Only A. flavus isolate was able to produce AFB1 and AFB2. In a survey by Hong and Nurim (2010), AFB1 and AFB2 were detected in 45% of corn-based products (0.2 to 101.8 ng/g). Samples were collected from imported and locally produced products at retail shops and local market in Kuala Terengganu. Wheat and barley samples from different markets in the state of Penang were analyzed for the presence of Aspergillus spp. and AFB1. Reddy and Salleh (2010) reported A. flavus and A. niger as the dominant aflatoxin-producing specie in all samples. AFB1 was detected in some of wheat samples at 0.42 to 1.89 ng/g and 1 barley sample at 0.58 ng/g. Barley and wheat samples were imported from Thailand and India, respectively (Reddy and Salleh 2010). Later in 2011, Reddy and his research group identified A. flavus and A. niger as the dominant aflatoxin-producing fungi in rice samples from Penang (Reddy and others 2011). They reported that rice-based products had the highest incidence of A. flavus. About 65.4% of A. flavus isolates produced AFB1 ranging from 1700 to 4400 ng/g, and 31% produced AFB2 ranging from 620 to 1670 ng/g. Their studies revealed that among the various examined food groups, cereal-based foods were the second most susceptible foods to AFB1 contamination. Peanut products were reported to be the most susceptible to AFB1 and 50% to 75% of cereal-based foods were contaminated with AFB1 with a mean level ranging from 1.25 to 3.86 ng/g. High levels of AFB1 contamination were detected in corn-based products (75%), rice (69.2%), wheat (64.2%), and oats (50%) (Reddy and others 2011).
Malaysian commercial cereal samples including rice, wheat, and maize flakes were analyzed for AFs. The results showed that 33.3% of rice samples were contaminated with AFs ranging from 0.01 to 3.96 ng/g (Soleimany and others 2011). Later in 2012, Soleimany and others determined AFs in cereals from Malaysian markets using a more accurate method. About 70% of cereal samples were contaminated with AFs at levels of 0.15 to 4.54, 0.2 to 3.2, 0.26 to 2.59, and 0.12 to 1.94 ng/g for rice, wheat, barley, oat, and maize meal, respectively (Soleimany and others 2012a, b). As AFs were detected at low concentration in rice, therefore, rice and its products can be considered low-risk commodities in Malaysia. There were only a few reports on OTA in cereals from Malaysia. Rahmani and others (2010) found very low levels of OTA ranging from 0.03 to 5.32 ng/g in barley, rice, maize meal, and oat from Malaysian markets (Rahmani and others 2010). In the simultaneous determination of mycotoxins in cereals, Soleimany and others (2011) detected OTA in barley, wheat, maize meal, oat, and rice samples (0.1 to 5.32 ng/g) (Soleimany and others 2011). They also surveyed OTA contamination in commercial samples of rice, wheat, and maize flakes in Malaysia. Low levels of OTA were detected in the samples (0.49 to 5.71 ng/g) (Soleimany and others 2012a). Later, in a broader study, they used UPLC-MS/MS for the detection of mycotoxins in cereals and reported that some samples of rice, wheat, oat, barley, and maize meal were contaminated with OTA (Table 1). However, only 1 maize-meal sample exceeded the proposed regulatory limit of 5 ng/g (Soleimany and others 2012b).
Table 1. Mycotoxin levels (ng/g) in cereals and grains
| ||3.69 to 77.50||–||–||–||–||Abdullah and others (1998)|
| ||0.01 to 3.83||0.05 to 5.32||–||–||2.8 to 73.11||Rahmani and others (2010)|
| ||0.68 to 3.79||–||–||–||–||Reddy and others (2011)|
| ||0.19 to 3.96||0.49 to 5.96||27.85 to 74.67||43.16 to 68.97||2.4 to 6.11||Soleimany and others (2011)|
| ||0.15 to 4.54||0.2 to 4.34||40.1 to 61.5||12.5 to 81.2||1.5 to 51.1||Soleimany and others (2012a)|
| ||0.15 to 4.42||0.2 to 4.34||12.59 to 33.25||6.15e34.92||1.5 to 51.1||Soleimany and others (2012b)|
|Wheat||0.42 to 1.89||–||–||–|| ||Reddy and Salleh (2010)|
| ||0.1 to 5.93||0.1||–||–||ND||Rahmani and others (2010)|
| ||0.55 to 5.07||–||–||–||–||Reddy and others (2011)|
| ||2.90 to 3.98||0.9||80.63||48.85 to 82.73||2.98 to 6.73||Soleimany and others (2011)|
| ||0.2 to 3.2||0.15 to 2.11||42.0 to 75.3||22.8 to 112.5||1.42 to 12.74||Soleimany and others (2012a)|
| ||0.2 to 3.2||0.15 to 2.11||12.15 to 29.35||5.5 to 18.62||1.42 to 12.74||Soleimany and others (2012b)|
|Wheat flour||11.25 to 436.25||–||–||–||–||Abdullah and others (1998)|
|Wheat-based noodle||–||–||–||0.627 to 1.243||–||Moazami and Jinap (2009)|
|Barley||0.58||–||–||–||–||Reddy and Salleh (2010)|
| ||0.1 to 2.86||0.03||–||–||2.38 to 24.43||Rahmaniand others (2010)|
| ||0.26 to 2.59||0.18 to 2.84||45.5 to 97.7||27.9 to 72.5||0.95 to 20.26||Soleimany and others (2012a)|
| ||0.26 to 2.59||0.18 to 2.84||10.75 to 31.21||5.5 to 23.63||0.95 to 20.26||Soleimany and others (2012b)|
|Oat||0.21 to 0.29||0.07||–||–||2.8||Rahmani, and others (2010)|
| ||0.65 to 2.85||–||–||–||–||Reddy and others (2011)|
| ||0.12 to 1.94||0.1 to 0.2||49.5 to 177.3||22.7 to 100.2||ND||Soleimany and others (2012a)|
| ||0.12 to 1.94||0.1 to 0.2||12.12 to 18.85||6.72 to 16.48||ND||Soleimany and others (2012b)|
|Maize meal||0.1 to 0.34||ND||–||–||2.5 to 2.9||Rahmani and others (2010)|
| ||0.15 to 1.8||0.1 to 5.76||48.2 to 209.3||35 to 109||1 to 13.47||Soleimany and others (2012a)|
| ||0.15 to 1.8||0.1 to 5.14||30.15||6.18 to 29.15||1 to 13.47||Soleimany and others (2012b)|
|Corn||0.2 to 101.8||–||–||–||–||Hong and others (2010)|
| ||1.75 to 8.95||–||–||–||–||Reddy and others (2011)|
Rahmani and others (2010) also reported 2.8 to 73.11 and 2.38 to 24.43 ng/g of ZEN in rice and barley samples, respectively. However, they did not detect any ZEN contamination in wheat samples. Soleimany and others (2011; 2012a, b), who screened a broader range of cereal samples, detected ZEN in rice samples from 1.5 to 51.1 ng/g, while the lowest level was found in oat and wheat samples (Table 1). Soleimany and others (2011; 2012a, b) also analyzed cereal and grain samples from Malaysian markets for FMN. In 2011, they examined fumonisins B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in rice, maize, and wheat samples. Wheat samples contained higher levels of FMN (80.63 ng/g). FMN contamination in rice samples ranged from 27.85 to 74.67 ng/g. Later in 2012, they surveyed rice, wheat, barley, oat, and maize meal samples and reported high levels of FB1 and FB2 in maize meal samples (48.2 to 209.3 ng/g) (Soleimany and others 2012a). In another study by Soleimany and others (2012b), low levels of FMN in cereals were reported (10.75 to 33.25 ng/g).
Moazami and Jinap (2009) examined different wheat-based noodle products consumed in Malaysia for trichothecenes. Several types of noodles, composed of yellow alkaline, instant noodle, and white salted noodle, were analyzed. They reported a low occurrence of DON in commercial noodle products ranged from 0.627 to 1.243 ng/g. The incidence of DON was higher in imported noodles as compared to local products. Soleimany and others (2011, 2012a, b) reported DON contamination in cereal samples ranged from 12.5 to 81.2, 22.8 to 112.5, 35 to 109, 5.5 to 72.5, 6.72 to 100.2 in rice, wheat, maize, barley, and oat samples, respectively. More recently, Samsudin and Abdullah (2013) surveyed the occurrence of mycotoxigenic fungi and mycotoxins levels in red rice in Malaysia. Red rice, a fermented product of Monascus spp., was contaminated with mycotoxins due to its traditional preparation method. Monascus spp. as starter fungi were present in all 50 samples followed by Penicillium chrysogenum in 62%, Aspergillus niger in 54%, and Aspergillus flavus in 44% of the samples. Citrinin was detected in all samples ate levels of 0.23 to 20.65 mg/kg, AF in 92% of samples at 0.61 to 77.33 μg/kg, and OTA in all samples at 0.23 to 2.48 μg/kg.
Hsuan and others (2011) studied distribution of Fusarium species on rice, sugarcane, and maize samples obtained from farms in different states in Malaysia. They identified 5 species, namely, F. sacchari, F. fujikuroi, F. proliferatum, F. andiyazi, and F. verticillioides. Izzati and others (2011) studied distribution of Fusarium species in maize grown in different locations throughout Malaysia. They reported 8 Fusarium species in samples from Johor, Selangor, Pahang, Pulau Pinang, and Sabah states. The most frequent species detected were F. proliferatum (29.9% isolates), F. semitectum (22.2% isolates), F. verticillioides (13.7% isolates), and F. subglutinans (12.6% isolates). According to Zainudin and others (2011), several species of Fusarium are associated with corn cultivated throughout Malaysia. They isolated 10 Fusarium species from corn plants cultured in 12 main corn growing locations in Malaysia. The most domenent species were F. proliferatum, F. subglutinans, F. verticillioides, and F. nygamai. The most contaminated samples with Fusarium sopecies were obtained in Semenyih, Selangor. Darnetty and other (2008) also isolated F. proliferatum, F. oxysporum, F. nygamai, F. semitectum, F. solani, and F. verticillioides from corn samples grown in 4 states of Malaysia, namely Pulau Pinang, Perlis, Sabah, and Sarawak.
Nuts and nut products
In Malaysia, peanuts are a common dietary staple consumed in the raw, roasted, or baked form. Peanuts and peanut products have the highest consumption among the nuts produced in Malaysia. Penang adults consume an average of 0.77 grams of total nuts (including peanuts) per day (Leong and others 2010). Raw shelled peanuts can be found in almost all retailed outlets throughout the country and they are widely used as an ingredient in a variety of popular foods and dishes. Peanuts in Malaysia are partially supplied by local production; however, the majority are imported from India, Vietnam, and China. The occurrence of AF in nuts and peanut has been proven (Abdulkadar and others 2004). AF contamination of groundnut and groundnut oil was reported by Chong and Beng (1965). Consumption of such contaminated commodities exposes humans and animals to different levels of AF from nanograms to micrograms per day. Due to consumption of AF-contaminated groundnuts, an outbreak in pig farms in Melaka was reported in 1960 (Lim 1964).
Table 2 shows mycotoxin levels in nuts samples from Malaysian Market. In monitoring studies carried out from 1981 to 1984, Mat Isa and Tee (1984) reported that AFs were found in 59% of raw shelled peanut samples. About 80% of peanut butter samples were reported to be contaminated by AFs. Local peanut butter contained higher levels of AFs than imported peanut butter. Some popular local peanut products, namely, satay sauce and rempeyek, were also contaminated with considerable levels of AFs. Later in 1985, 96 raw shelled peanut samples were analyzed and 88.5% were contaminated with AFs and 53.1% were contaminated with AFs higher than 40 ng/g (MARDI 1987). In another comprehensive study carried out in 1992 to 1995, a total of 403 samples of raw shelled peanut samples from retail markets in all major towns in the states of Selangor, Negeri Sembilan, and Melaka were analyzed for AFs. Only 5.8% of samples from Selangor were suitable for human consumption; the rest contained more than 40 ng/g of AFs (Abidin and Mat Isa 1994; Mat Isa and Abidin 1995). Ali and others (1999) also reported high levels of AFs in peanut products with 65% of samples contaminated with more than 50 ng/g of AFs (maximum 180 ng/g). Their study also showed that levels of AFB1 in peanut and peanut products were higher among all AFs. Samples of locally produced peanut candy and peanut bars were contaminated with AFs ranging from 9 to 180 ng/g (Ali and others 1999). Sulaiman and others (2007) analyzed raw shelled peanuts from the state of Perak for AFs and found quite high contaminations of AFB1 (0.85 to 547.51 ng/g) and AFG1 (1.37 to 375.98 ng/g) in peanut samples. About 50% of samples contained total AFs in the range of 0.85 to 762.05 ng/g in which 45% of them exceeded the maximum permitted levels of 35 ng/g set by Malaysian Food Regulation 1985. In the survey by Hong and others (2010), AFB2 was found in 42% of peanut samples marketed in Kuala Terengganu at concentration levels of 0.2 to 101.8 ng/g (Hong and others 2010). Arzandeh and others (2010) screened raw peanut kernels from Malaysian supermarkets for AFs and found 78.57% of the samples contaminated with AFs, of which 10.71% contained more than 15 ng/g. Total AF concentrations ranged from 2.76 to 97.28 ng/g (Arzandeh and others 2010). In another study, Leong and others (2010) reported very high levels of AFs in coated peanut products and raw shelled peanuts of 514 and 711 ng/g, respectively (Leong and others 2010). Some nuts and nut products from Penang, including walnuts, raw shelled peanuts, roasted shelled peanuts, roasted peanut in shell, coated nut products, peanut cakes (gung tang), pounded peanuts, peanut butters, and peanut bakery and confectionery products, were analyzed and 16.3% of the samples were contaminated with AFs. Total levels of AFs in the samples varied from 16.6 to 711 ng/g. The highest level of contamination was found in raw shelled peanuts and AFB1 was the most frequent found with higher levels compared to the other AFs (Leong and others 2010).
Table 2. Mycotoxin levels (ng/g) in nuts and spices
|Raw shelled peanut||40||–||MARDI (1985)|
| ||0.85 to 762.05||–||Sulaiman and others (2007)|
| ||17.8 to 711||–||Leong and others (2010)|
| ||2.76 to 97.28||–||Arzandeh and others(2010)|
| ||0.2 to 101.8||–||Hong and others (2010)|
| ||16.6 to 711||–||Leong and others (2010)|
| ||5.25 to 15.33||–||Reddy and others (2011)|
| ||0.62 to 977||2.82 to 7.41||Afsah-Hejri and others (2012)|
|Peanut candy and peanut bar||9 to 180||–||Ali and others (2010)|
|Coated nut product||113 to 514||–||Leong and others (2010)|
|Roasted groundnut in shell||29.7 to 179||–||Leong and others (2010)|
|Peanut products||0.33 to 273.63||–||Leong and others (2011a)|
|Honey peanuts||1.47 to 6.25||–||Reddy and other s(2011)|
|Roasted peanuts||3.21 to 8.91||–||“|
|Pistachio||0.66 to 1.09||–||“|
|White pepper seed||0.2 to 4.5||0.18 to 2.4||Jalili and others (2009)|
| || || ||Jalili and others (2010)|
|White pepper powder||0.1 to 4.6||0.21 to 3.4||Jalili and others (2010)|
|Black pepper seed||0.1 to 4.8||0.15 to 13.58||Jalili and others (2010)|
|Black pepper powder||0.7 to 4.9||0.23 to 12.64||Jalili and others (2010)|
|Chili and pepper||0.58 to 4.64||–||Reddy and others (2011)|
|Cumin powders||1.89 to 4.64||–||Reddy and others (2011)|
|Chili||0.2 to 79.71||0.2 to 101.24||Jalili and Jinap (2012a)|
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Using liquid chromatography tandem mass spectrometry, Leong and others (2011b) determined the natural occurrence of AFs in 128 nut samples marketed in Penang. Their result revealed that more than about 50% of the samples were contaminated with AFs ranging from 0.40 to 221.61 ng/g for AFB1 and 0.33 to 273.63 ng/g for total AFs (Leong and others 2011b). More recently, Reddy and others (2011) reported that peanut products were contaminated with considerable amounts of AFB1 ranging from 1.47 to 15.33 ng/g. Raw peanuts showed higher contamination with AFB1 (5.25 to 15.33 ng/g) as compared with roasted peanuts (3.21 to 8.91 ng/g). Their study also found that some pistachio and almond samples were also contaminated with noticeable levels of AFB1 (0.66 to 1.09 ng/g). Afsah-Hejri and others (2013) screened raw peanut samples from supermarkets and retail markets for Aspergillus spp. and AFs contamination. Isolates were then tested for their aflatoxin-producing ability in media. The green Aspergillus spp. from raw peanut samples that were found positive in the screening test and showed a sharp AFB1 peak in their HPLC chromatogram were isolated from peanut samples with high moisture content. According to the report, the high prevalence of Aspergillus spp. and high level of AFB1 contamination in the raw peanut was due to improper storage conditions. As mentioned before, peanut contamination with Aspergillus occurs at preharvest stages when peanuts are in direct contact with soil. Cross-contamination occurs at processing, transportation, and storage. Raw peanut samples from supermarket and retail markets in Kuala Lumpur were analyzed for the presence of Aspergillus spp. and AFB1. Targeting specific genes responsible for aflatoxin production, Afsah-Hejri (2012) reported A. flavus and A. parasiticus as dominant aflatoxin-producing strains in raw peanut samples. AFB1 was detected in all raw peanut samples, ranging from 0.62 to 977 ng/g. Studies on OTA in nuts from Malaysia are very rare. The only study was conducted by Afsah-Hejri and others (2012) who developed an efficient HPLC conditions for OTA determination in peanuts. They examined raw peanut from the Kuala Lumpur market which indicated OTA contamination in samples ranged from 2.82 to 7.41 ng/g (Afsah-Hejri and others 2012; Afsah-Hejri and Jinap 2012). Afsah-Hejri (2012) developed a PCR-based detection method and identified A. ochraceus, A. carbonarius, and A. niger as dominant OTA-producing fungi in raw peanut samples.
In Malaysia, a large variety of spices is used as main ingredients in daily cooking. Malaysia is one of the main producers of spices (especially peppers) in the world and black and white peppers are the major export commodities. However, due to tropical climate conditions, mycotoxin contamination almost always occurs during harvesting, postharvesting, and storage. Therefore, it is necessary to conduct regular monitoring on mycotoxin contaminations in spices. In an early screening study by MARDI in 1984 to 1985, black and white pepper samples from the Pepper Marketing Board (PMB) in the state of Sarawak were analyzed. All pepper samples were positive for AFs. According to the report, aflatoxin contamination of pepper is due to traditional processing and storage methods. However, to reduce the contamination and microbial loads, pepper intended for export is being recleaned and reprocessed (MARDI 1985; Mat Isa and Nazarifah 1986). Later in 1987, MARDI surveyed 19 different types of commonly-used spices including dry and wet spices (MARDI 1987). All samples were contaminated with AFs. According to MARDI (1987), this is probably due to unsuitable storage conditions at retail outlets where the products are kept for long periods. Jalili and others (2009) screened imported and locally produced pepper products from Malaysian markets and found 55.5% of all samples contaminated with AFs (low level of contamination 0.1 to 4.9 ng/g) (Table 2). AFB1 was the highest among other AFs. White peppers were less contaminated compared to black pepper samples collected from the same farm, probably due to the processing effect. In white pepper production there is an extra process of shell removal, which can reduce mycotoxin contamination (Jalili and others 2009). In the study by Reddy and others (2011), 93.3% of analyzed spices were contaminated with AFB1 at 0.58 to 4.64 ng/g. All chili and pepper samples were contaminated with AFB1. Cumin powders contained the highest levels of AFB1 ranging from 1.89 to 4.64 ng/g (Reddy and others 2011). The latest study by Jalili and Jinap (2012a) on AFs in several chilli samples from open markets and supermarkets in Malaysia showed that 65% of all samples taken were contaminated with total AF levels in the range of 0.2 to 79.7 ng/g. AFB1 was the highest as compared to the other examined AFs. Higher levels of AFs were observed in samples collected from open markets (Jalili and Jinap 2012a). Jalili and others (2010; 2012a) detected OTA in 81.25% of chili samples from Malaysian market ranging from 0.2 to 101.2 ng/g (Jalili and others 2010; Jalili and Jinap 2012a). About 95% of samples from the open market and 45% from supermarkets were contaminated with OTA. Higher contamination in samples from open markets can be due to long period and improper storage conditions of spices. Their results also indicated that 16.3% of the samples were contaminated with OTA more than 10 ng/g. In another study, Jalili and others (2012b) examined OTA in commercial peppers which consisted of imported and local black and white pepper in powder and seed form. About 47.5% of samples were contaminated with OTA at levels of 0.15 to 13.58 ng/g, and 33.3% of them exceeded the maximum limit of 5 ng/g (Jalili and Jinap 2012b). However, very low concentrations of OTA were detected in prepacked peppers. Contamination was higher in black pepper compared to white pepper due to different processing methods. In the production of white pepper, peppercorn shells are removed which reduce mycotoxin contaminants.
Vegetables and fruits
In an intensive study on occurrences of Fusarium species in plants from Peninsular Malaysia during the period 1981 to 1986, more than 1000 isolates of Fusarium were obtained from rice, potato, water melon, and chilli (Salleh and Strange 1988). Three species of F. prolijeratum, F. nygamai, and F. longipes were identified on plants. They also reported the association of F. solani and F . oxysporum var. redolens with human diseases. A survey on occurrence of Fusarium species on vegetable fruits from markets in Penang Island reported 6 species namely, F. semitectum, F. oxysporum, F. subglutinans, F. proliferatum, F. solani, and F. equiseti (Nurulhuda and others 2009). The most common species isolated from cucumber (Cucumis sativus), tomato (Lycopersicon esculentum), okra (Hibiscus esculentus), loofah (Luffa acutangula), bitter gourd (Momordica charantia), brinjal (Solanum melongena), and fresh red chilli (Capsicum annuum) were F. semitectum (33%) followed by F. oxysporum (27%) and F. solani (25%). Since all the 6 identified species are able to produce mycotoxins, they suggested that vegetable fruits could pose health hazard. Latiffah and others (2007) isolated different Fusarium species from crops cultivated in Peneng. They isolated F. solani species on lettuce, papaya, starfruit, cabbage, paddy, banana, dragon fruit, longan, and limau kasturi. F. equiseti was reported in lettuce and dragon fruit. F. semitectum was isolated on lettuce and paddy. In a survey on Fusarium species associated with wet market potatoes in Malaysia, 65 Fusarium strains were isolated and identified from samples collected from different regions in Malaysia. All of the 65 isolates belong to F. solani and F. oxysporum species (Chehri and other 2011). Manshor and others (2012) also reported occurrence of F. solani on grape and loofah (petola) fruits grown in highland areas in Malaysia.
The single study on mycotoxin contamination in oilseeds (including sunflower and sesame) from Malaysia showed that 82.1% of samples were contaminated with AFB1. However, the extent of contamination was not too high, varying from 0.54 to 5.33 ng/g (Reddy and others 2011).
In 1986, MARDI surveyed dried cocoa beans from the states of Selangor and Perak. About 31% of the samples collected were contaminated with AFs. Samples with the highest contamination had high moisture contents in the range of 11% to 13.8% (MARDI 1986).
Milk and eggs
In the survey by Abidin and Mat Isa (1992), samples of locally produced fresh milk and eggs from wet markets in the states of Selangor, Negeri Sembilan, and Melaka were examined. Only 1.7% of fresh milk samples from Selangor contained AFM1 at 0.24 ng/g. About 20% of all eggs were contaminated with AFs at 0.16 to 0.41 ng/g.
Traditional herbal medicines called jamau and makjun that commonly consumed in Malaysia were screened for AFs. The incidence of AFB1, AFB2, AFG1, and AFG2 were 70%, 61%, 30%, and 4%, correspondingly. However, the extent of total AF contamination was considerably low at 0.03 to 1.57 ng/g (Ali and others 2005). The authors suggested that low levels of AFs may be attributed to the antifungal effects of herbs that prevent fungal growth, and consequently, mycotoxin production. In another study some of the herbal products, namely pasak buni, maajau ratu, tongkat ali, greennleaf energizer, medicare AM700, and medicare AM800, marketed in Malaysia were examined for mycoflora and AFs. Mold counts were low, but several Aspergillus spp. were isolated from all samples, however, none of them were mycotoxigenic. No AF contamination was observed in herbal products in this particular study (Mohd Fuat and others 2006).
Animal feed are both imported and locally produced in Malaysia. Most of the raw ingredients (including cereal grains, soybean meal, and corn gluten meal) are imported from Thailand, China, India, Argentina, U.S.A., Australia, and Canada. Due to improper storage conditions during production and transportation, there are always reports on feed contamination by mycotoxins. A survey conducted by MARDI from 1981 to 1984 showed that more than 70% of feed samples were contaminated by AFs (Ali 2000). Muniandy (1989) reported AFB1 levels of 5 to 400 ng/g in 10% of analyzed animal feed from Malaysia. Maize is the most commonly used feed ingredient in Asian countries. In the study on mycotoxin contamination in animal feeds from Asia, high levels of AFB1 (106 ng/g) were reported in maize samples from Malaysia (Binder and others 2007). Lee-Jiuan and Li-Mien (2006) surveyed the occurrence of mycotoxins in feedstuffs from Asian countries. They reported corn sample from Malaysia with high mycotoxins contamination including OTA, Afs, ZEN, and FEM. The samples showed OTA levels as high as 143 μg/g. In another survey on imported poultry feed, elevated concentrations of AFB2 were detected in the corn samples, ranging from 0.2 to 101.8 ng/g, which exceed FDA action levels of 20 ng/g (Hong and others 2010). Khayoon and others (2010) studied AF contamination in animal feedstuffs including meals from corn, wheat, soya bean, palm kernel, canola, sunflower, and copra and meals. They reported that 19% of samples were contaminated with AFs at 6.5 to 101.9 ng/g. Sunflower meal and corn germ meal contained the highest levels of total AFs. Feed samples showed higher contamination with AFG1 comparing to other AFs. In a broader survey, samples of corn used for animal feed use were collected from 10 states in Malaysia. A. flavus (87%) was the most prevalent fungus in the samples. AFB1 was detected in 81.2% of samples ranging from 1.0 to 135 ng/g. Levels of AFs in 22.5% of samples were above an international regulatory limits of 20 ng/g assigned for animal feeds (Reddy and Salleh 2011). Binder and others (2007) reported FMN contaminations in maize samples used as feed in Malaysia. They reported the average contamination level of 1335 ng/g of FMN in maize samples. However, Reddy and Salleh (2011) reported very low levels of FMN in com samples used as animal feed.
Health Impact Studies of Mycotoxins in Malaysian Foods
The first warning about aflatoxins in Malaysia was reported after a disease outbreak of 2 pig farms in the state of Melaka in 1960 (Lim 1964). The disease was identified by gross liver damage due to AFs in the feed (Lim and Yeap (1966). The feed had been prepared from imported ingredients including peanut meal and oil cakes. In another incidence, few Chinese children died due to acute hepatic encephalopathy in the state of Perak in 1988. Epidemiologic studies revealed that the incidence was related to the consumption of AF-contaminated Chinese noodle (Lye and others 1995). The pathological observations showed broad coagulative necrosis of the liver with proliferative ductal/ductular metaplasia of the hepatocytes. Bile stasis, central vein sclerosis, giant cell formation, and steatosis were also observed. Renal and hepatic failure was reported as the ultimate cause of death (Chao and others 1991). Toxicological studies showed a high concentration of AFs in tissues of the victims (Cheng 1992).
Studies on dietary exposure to mycotoxins in Malaysia showed that nuts are the main root of mycotoxin exposure due to high consumption rate and high mycotoxin contamination of nut products. Leong and others (2011a) estimated the dietary exposure of AFB1 in nuts and nut products in the Peneng population and reported 0.36 and 8.89 ng/kg BW as the low and high levels of exposure, respectively (Leong and others 2011a). Based on the derived margin of exposure (MoE) values in their study (ranging from 34 to 847), AFB1 was reported to be a public health concern in Malaysia and should be considered as a high priority for risk management actions. Leong and others (2012) studied the relationship between AFB1-lysine adduct levels in serum and dietary intake of AFs from nuts/nut products in the state of Penang (Leong and others 2012). Results showed that 97% of blood samples from healthy adults contained AFB1–lysine adducts higher than 0.4 pg/mg albumin, with a range of 0.20 to 23.16 pg/mg albumin. Individuals in the range of 31 to 50 y old were 3 times more likely to have high AFB1 levels compared to those between 18 and 30 y old. Their study revealed that Penang adults are likely to be exposed to AFB1, but the level of exposure is lower than what causes acute aflatoxicosis or death. They estimated the exposure to aflatoxins as 3 ng AFB1/kg BW/day and a population risk for primary liver cancer of 0.25 cancers/year per 100000 people. As their finding was lower than the incidence rate of 4.9 per 100000 registered for Malaysia (Ministry Health of Malaysia 2006), it was concluded that the Penang population is not at considerable risk for developing primary liver cancer due to AF exposure. Sabran and others (2012) investigated the association between urinary AFM1 level and consumption of milk and dairy products in Malaysian adults (Mohd Redzwan and others 2012; Sabran and others 2012). AFM1 was detected in 61.3% of urine samples ranging from 0 to 0.0747 ng/mL. Individuals with high intake of milk and dairy products (>67.79 g/d) showed considerably high urinary AFM1 level compared to those with low intake. The study found a significant and positive relation between milk/dairy products consumption and urinary AFM1 level. Although the level of AFM1 in urine samples was not too high, long-term exposure to AFM1 may pose negative health effects.
In order to protect Malaysian consumers from the risks of mycotoxin contamination, maximum permissible levels have to be generated for all food products and subjected to control by regulatory authorities. Of the food commodities surveyed, peanuts and their products are the ones most susceptible to growth and consequent mycotoxin contamination. Therefore, methods of control and mitigation of mycotoxins at storage in shops and homes should be developed. Intervention and reduction protocols for contamination in spice commodities are also needed. Rice, as one of the staple foods for Malaysians, was safe regarding mycotoxin contaminations. There is a lack of studies on animal-derived products such as milk and dairy products, meats, and eggs. Most of the studies carried out include surveys and exposure assessments. Hence, studies on interventions, preventions, and detoxifications are needed. Nevertheless, to evaluate the extent of the mycotoxin problem in Malaysia, there is a need for a more extensive and frequent observations of susceptible commodities from farm to table. Consumer awareness programs are also needed to minimize the risk. Like other agriculture communities, mycotoxins contamination in food and feed can have considerable economic implications in Malaysia. Losses from rejected shipments and lower prices for low-quality products can devastate country export markets. Mycotoxin contamination also affects farmers by reduced income from lower selling prices for contaminated commodities. The economic impact of mycotoxin contamination on livestock production includes mortality, reductions in productivity, weight gain, feed efficiency, fertility, and ability to resist disease. The cost of mortality, morbidity, hospitalization, and health care services are also need to be taken into account. However, there is no report on economic analysis or the annual cost of mycotoxins contamination in Malaysia in terms of agricultural products spoilage, losses in livestock productivity, and human health effects, to date. Therefore, the economic losses have to be calculated and the costs of preventing mycotoxins through better production, harvesting, and storage practices must be weighed against the economic losses. High-risk agricultural commodities and high-risk population groups for selected mycotoxins need to be identified. The effect of mycotoxins on national economies and international trade must to be assessed.