Mycotoxins and human health: Significance, prevention and control

Transcription

1 Smart Biomol. Medicine, Edited by Ajay K. Mishra, Ashutosh Tiwari, and Shivani B. Mishra Copyright 2010 VBRI Press, ISBN: Mycotoxins and human health: Significance, prevention and control Njobeh B. Patrick 1*, Dutton F. F Michael 1, Makun A. A Hussaini 2 1Environment and Health Research Group, faculty of Health Scienc University of Johannesburg, South Africa. 2Department of Biochemistry, Federal University of Technology, P.M.B 65, Minna, Niger State, Nigeria.

2 Mycotoxins and human health 133 Abstract Mycotoxins are diverse range of harmful secondary metabolites produced by fungi in various food and feed commodities at different stages in the field, during processing, transportation and storage. Generally, the fungi mainly associated with mycotoxin production belong to the Aspergillus, Penicillium and the Fusarium genera that often contaminate and compromise food safety and quality. Their presence in foods and feeds is inevitable and as such, humans and animals are exposed to them on a daily basis leading to a wide range of health effects. Human exposure to mycotoxins is mainly via ingestion of contaminated foods, but other routes: inhalation and dermal exposures are involved. Even though the problem of mycotoxin is worldwide, the situation in most African (also Asian) countries is more than doubled that of the rest of the world exacerbated by the climatic, socio-economic and political situations in the continent. Because this chapter is devoted to such important mycotoxins as aflatoxins (AF), fumonisins (FB) and ochratoxins (OT) in Africa, an attempt is made to provide an update on the current state of occurrence in different commodities, of some important mycotoxins as well as the degree of human exposure and associated health implications. Furthermore, an effort is made to review some aspects of risk assessment of mycotoxins and control strategies from the African perspective taking into account some of the challenges and needs in mycotoxin analyses. Correspondence/Reprint request: Dr. Njobeh B. Patrick, Environment and Health Research Group, faculty of Health Sciences, University of Johannesburg, South Africa pnjobeh@uj.ac.za Introduction Mycotoxins are secondary metabolites produced by microorganisms that invade and grow on various organic matter including food and plant materials. They represent a range of chemical structures, which therefore, have an equally wide range of physiological properties, ranging from neutral to beneficial and toxinogenic (producing mycotoxins) potentials. If

3 134 Njobeh B. Patrick secondary metabolites are classified based on these properties, then the group produced by the filamentous fungi that are toxigenogenic, are termed mycotoxins, literally meaning fungal arrow poisons. Like all toxigenic substances, the effects of toxicity are mainly related to the degree of human and animal exposure to them, in other words, the amount ingested or absorbed, as first pointed out by Paracelsus in the 16 th Century. Different classes of the most important mycotoxins defined based on their health implications, with further emphasis on their distribution and the different routes of exposure are discussed in this chapter. Substances can be therapeutic at low concentrations or toxic at higher levels. In the case of mycotoxins, most exposures are detrimental but there is a difference between doses that give rise to chronic symptoms and those that may exhibit acute toxicities. The latter can be lethal and may lead to the death of the exposed organism. In the case of mycotoxins, it is a question of definition of the target organism. If we describe a fungal metabolite as beneficial such as an antibiotic, we may thus relate a fungal secondary metabolite as one that kills a pathogenic microorganism or a mycotoxin that kills or harms humans or animals. In the case that is being discussed herein, most mycotoxins can give rise to acute conditions at low concentration doses (see Tables 1 and 2, which gives a list of the more commonly occurring mycotoxins and their LD 50 values). Because all mycotoxins, being highly variable in their chemistries, have different toxicological properties, their effects and symptoms will equally vary significantly. There are several recognized human mycotoxicoses (disease produced by mycotoxins) (Table 2) and other conditions suspected of contributing to the disease (co-factor). These are not to be confused with mycetism, a condition that arises from consuming poisonous mushrooms (toadstools). Though ergotism (also known as St. Anthony s Fire) that caused the death of thousands of people in some parts of Europe in the Middle Ages [1] was reported, the development of the concept on the role of mycotoxins in causing human and animal health problems began with the discovery of aflatoxins (AF) feed poisoning in England in Subsequently, outbreaks of mycotoxicoses such as aflatoxicosis in Kenya [2] and India [3,4] as well as stachybotrycosis (trichothecene toxicosis) in Europe [5] and North America Kuhn et al [6] were reported. Since the discovery of aflatoxins (AF), over 300 of fungal metabolites are known to mankind with quite a few still awaiting discovery. However in practice, this number is much reduced as only a handful occur at significant levels in food and feed commodities, most of which are given in Table 1. Some of these mycotoxins including ochratoxins (OT) [7,8] and fuminisin

4 Mycotoxins and human health 135 (FB) [9] were discovered in S. Africa. This chapter is devoted to the discussion on the degree of human and animal exposure to significant mycotoxins, the associated health implications and the extent of mycotoxins control (also their effects) in Africa, even though an overview of cases of different mycotoxicoses reported elsewhere in the world is also provided. As of the time of writing this chapter, some reports appeared on the news media that over 2.3 million bags of maize were contaminated with lethal doses of AF in Kenya [10,11]. This is the same country where a fatal incidence of mycotoxicosis due to acute AF-maize poisoning in 2004 was largely ever reported worldwide. The main dilemma here is the difficulty of the Kenyan government to recall these products from the market, which is one of the main problems of mycotoxin control in the continent, where the perception on mycotoxins till date is not widely conceived. This is well reflected on the fact that despite the increased incidence of mycotoxin residues in animal products such as milk (including human breast milk) and meat, mouldy food materials are constantly fed to domesticated animals. This issue will be discussed in detail subsequently in this chapter. 1. Fungal colonization and its role in mycotoxin production As always the case, one of the things that has to do with mycotoxin contamination of foods and feeds is prior fungal colonization developing from germinating spores either in the field and or during storage. Fungi are responsible for producing mycotoxins because they adapt very well in our environment by growing and maturing over surfaces and through solid materials. The nature of fungal development or their relative importance as pathogens (or parasites) in causing diseases in man and animals is beyond the scope of this chapter. It is alleged that over 1.5 million species of fungi are known to exist, but only about 70,000 species have been described [12,13] and those considered important due to their toxinogenic capabilities are some members of the genera: Fusarium, Aspergillus and Penicillium, Alternaria, Claviceps and Stachybotrys. In sub-saharan Africa [14,15,16] and to some extent, north of Africa [17], the major genera often encountered in stored cereal (maize, barley and wheat) and legume seeds (peanuts and cow pea) are the Fusarium, Aspergillus and Penicillium. Their levels of infection in these commodities among others, are provoked by insect infestations that cause seed damage and also the use of traditional storage structures available in W. Africa [18,19,20] as history suggests. Because of the importance of these fungi in producing deleterious mycotoxins in food and

5 136 Njobeh B. Patrick feed commodities, there is need to establish a fungal database specific for mycotoxin profiling within each country which at present, is non-existing. This may pave the way for proper control strategies to be put in place. The three genera are singled out and discussed in this chapter. The fungal species within each genera under certain environmental conditions produce specific mycotoxins, singly or in combinations based on their toxigenic potentials under certain conditions that are discussed later. In most if not all circumstances, these group of toxigenic fungi in association with one another, attack the same plant material resulting in a much more serious quality loss. Unlike primary metabolism, secondary metabolism (toxin production) qualitatively depends on fungal species and quantitatively on the fungal strains [21]. The toxigenic fungi are those of particular concern to a mycotoxicologist and therefore described briefly herein. First described in 1729 by Micheli PA, a Florentine priest-mycologist, Aspergillus spp. are among the most abundant and widely distributed organisms [22] in soils, decomposing plants and indoor air environment [23]. There are over 180 species of Aspergillus among which the most important members are A. flavus and A. parasiticus, which are most widely studied because they produce the most notorious mycotoxins, the aflatoxins. They are ubiquitous in the tropics and desert environments. Optimal production of AF by A. flavus, A. parasiticus [24,25] and some strains of A. tamarii and A. nomius [26] is at temperatures between o C [27] and kernel moisture content of about 18% [28]. These fungal species are common infecting cereals and nuts from Africa [14,15,16,17]. Other emerging fungal species in the last decade found capable of synthesizing AFs are A. ochraceoroseus, A. rambellii, Emericella astellata and E. venezuelensis in the north west of South America and West Africa [29,30]. Initially considered a storage problem, AF may be produced in the field [24,31] by A. flavus, especially during drought stress and low soil moisture content. However, the greatest AF production in grains occurs at post harvest under poor conditions [24]. Other important species of the Aspergillus genera are A. ochraceus, A. carbonarius and A. niger as they produce ochratoxins. Production of OT by Penicillium verucosum will be discussed subsequently. Historically, ochratoxins are another group of mycotoxins, which after the discovery of aflatoxins, was discovered in 1965 in South Africa by van der Merwe and co-workers [7,8]. Production of ochratoxins by A. ochraceus seems more likely in the warmer or tropical region [32]. Grains with moisture content above 22% provide optimal condition for OTA production [28]. Ramos et al [33] indicated a w above 0.7 as ideal but will depend on the composition of

6 Mycotoxins and human health 137 the substrate. Klich [22] provides a comprehensive list of toxigenic fungi and the different mycotoxins they produce in different commodities. Like the Aspergillus, Penicillium spp. are filamentous, ubiquitous and opportunistic saprophytes, which probably is why they can grow in almost any environment. It is thus a large genus with over 150 recognized species among which 50 or more are commonly occurring [34]. They are thus frequently isolated from a variety of foods and according to Frisvad and Thrane [35], P. crustosum, P. verrucosum and Moss [36], P. italicum, P. polonicum, P digitatum and P. expansum are the most important foodborne fungal species throughout the world. Some of these Penicillium spp. have been isolated in human foods including cocoa beans and their processed products [15], maize, peanuts, melon seeds and cassava products [16] from Cameroon and previously from Nigerian melon seeds [37]. They contaminate a host of plant species and as such, produce a wide range of mycotoxins such as ochratoxin (OT), patulin (PAT), citrinin (CIT), penicillic acid (PA), etc. Ochratoxins are also produced by P. verrucosum [38] mainly in the colder or temperate regions as it proliferates very well in these regions [32]. At low temperatures around 5 o C, Penicillium spp. may produce OT [39], which is a common phenomenon in the cold or temperate regions. Detailed investigations of the two major OTA producing species on agar media showed that the temperature range for growth of A. ochraceus and P. viridicatum was 8-37 C and 0-31 C respectively, whereas the temperature range for OTA production by A. ochraceus was C with an optimum yield between C and the range for toxin production by P. viridicatum was 4-31 C with maximum OTA production between C [40,41]. The Penicillium spp. are frequently isolated from a variety of foods and as viewed by Frisvad and Thrane [35], P. crustosum, P. nurdicum, P. verrucosum and Moss [36], P. italicum, P. polonicum, P digitatum and P. expansum are the most important food-borne fungal species, particularly in the temperate region. A number of Penicillium spp. are associated with human food supplies inhabiting cereal grains and fruits [42]. Members of this genus are mainly responsible for fruit decay throughout the world and of course produce significant quantities of mycotoxins already mentioned. The genus Fusarium was first described in 1809 and subsequently recognized as one of the most important pathogenic and toxin-producing fungal genera in the world affecting almost all plant species. They are filamentous fungi with fast growing colonies with or without cottony aerial mycelium [23]. The species of Fusarium are ubiquitous soil fungi infecting plants in the field either as primary or secondary invaders [43] common in

7 138 Njobeh B. Patrick tropical and subtropical areas of the world [23]. The world s staple foods mainly maize and other cereals such as wheat and rice are severely affected by different members of this genus. Models predicting the combined interactive effect between a w and temperature on growth of major fumonisin (FB) producers viz a viz F. verticillioides and F. proliferatum in maize have been developed [44]. The findings obtained in these studies found a w and temperature of and 30 o C, respectively, as the best combination in providing maximum growth of these fungi. These findings are in excellent agreement with the previous reports of Soriano and Dragacci [45] who also indicated that Fusarium spp. require higher temperatures of 25 o C for growth which increases with increase in a w, while optimal production of FB by F. verticillioides in maize was found by Marin et al [46] to be at temperature of 30 o C and a w of Several reports found F. verticillioides as the most important toxinproducing Fusarium sp. associated with maize in Africa [47,48] and elsewhere in the world [49]. This is because it has been linked with outbreaks of animal diseases and also associated with human carcinomas. Other members of economic importance are F. graminearium, F. subglutinans, F. culmorum, F. sporotrichioides, F. semitectum, F. oxysporum and F. proliferatum, because of their toxigenic role in producing the various trichothecenes (TH). The Fusarium spp. are usually considered field fungi causing infections, but they may persist after harvest especially when storage conditions (improper drying and high temperatures) are favourable. In cereals and cereal-based products, members of this genera can produce a diverse range of mycotoxins such as FBs, zearalenone (ZEA) and TH such as deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin (T-2). To determine the incidence of mycotoxins in foods, it is necessary (though not always) to primarily identify fungal spp. in that material. By identifying what fungi that contaminate a commodity, it may be possible to predict the likely mycotoxins that are present in that material [50]. Fungal colonization in food materials can be determined by direct observation of visible moulds growing on the material. However, not all fungi present can be determined as some infections take place from within the seed or plant material. Thus fungal identification can effectively be done by cultivation under laboratory conditions. Identification by conventional method is often based on the similarities in morphological characteristics (micro- and macroscopic) between species as well as variability and mutation occurrence in cultures [51]. This detection method however is time consuming, labour and cost intensive, complex and thus require mycological expertise [52]. Due to their sensitivity, rapidity and reliability, polymerized chain reaction (PCR)-based techniques as an alternative have

8 Mycotoxins and human health 139 been introduced [53]. Advances in molecular techniques have therefore made it possible to further elucidate differences between species based on genetic diversity. These methods are considered more accurate and have shed more light and to some extent, contributed to the detection of mycotoxins [54]. Following these techniques, species-specific primers are used depending on the species within the genera to be identified. O Donnell et al [55] have provided a simplified method in identifying Fusarium spp., Samson et al [56] for Penicillium spp. and Manonmani et al [52] for Aspergillus spp. Another taxonomic characteristic to consider when distinguishing fungal spp. is to evaluate the secondary metabolite profile (toxigenic potential) [57]. More recently, the application of microarray technologies [58] and the use of libraries of Expressed Sequence Tags (ESTs) in identifying and monitoring the expression of several genes particularly those involved in secondary metabolism has evolved [59]. However, whichever method to be used must ascertain proper fungal identification to species or possibly to strain level. 2. Mycotoxin screening, distribution in food and feed commodities and exposure Mycotoxin exposure entails the toxin amount an individual is exposed, duration and route of transmission. Humans are exposed to mycotoxins in the following ways: ingestion, dermal, parental and bioaerosol (inhalation) routes and it is supposed that the main source of exposure is mainly by ingestionconsumption of mycotoxin contaminated foods. Dermal route may be related to exposure of the dermis to mycotoxins (mainly via handling contaminated material or those involved in laboratory analyses of mycotoxins), which may react instantly to the skin and cause irritation. Parental exposure is simply mother-to-child transmission of the toxins through the placenta and breast milk. In the case of bioaerosol route, airborne mycotoxins are inhaled and the toxic effects via this route are by all means much more severe than for any other mode of exposure. In order to establish the degree of human exposure to mycotoxins, it is important to provide an overview on mycotoxins in foods (surveillance data), the extent to which these commodities are consumed (food consumption pattern), combined with their distribution pattern in the body. Wagacha and Muthomi [60] provide a comprehensive review on the extent of mycotoxin problem in Africa in relation to their implications on food/feed safety and health as well as the degree with which humans and animals are exposed to them, mainly highlighting significant mycotoxins, while also exploring possible intervention strategies. More to that, Sherif et al [61] also afforded some

9 140 Njobeh B. Patrick useful information on principles for evaluating mycotoxin exposure and the inherent problems associated with such studies. Undesirably, only limited literature data is available in most African countries, a challenge which makes it much more difficult to assess population exposure with some degree of certainty. It is only through assessment of the current situations that well defined policies on mycotoxin regulation and monitoring can be successfully achieved. Distribution of mycotoxins in foods and feeds varies from region to region [62], depending on the prevailing climatic conditions [27,63]. According to CAST [64], FB is common in maize, AF in maize and peanuts in the tropics, OT in barley in the Scandinavian countries, while DON is predominant in wheat in America, Canada and Europe. However, this worldwide distribution pattern seems to differ lately as a result of increased demand on international trade in food commodities. A list (Table 1) is thus provided to summarize the incidence of mycotoxins and the likely fungi producing them in various commodities in Africa in the last decade. In Africa, AFs and FBs are likely of greatest significance and widespread in most dietary staple foods [60]. Therefore, these toxins will be discussed in this chapter, while taking into account, such mycotoxins as DON, T-2, ZEA, CIT and PAT. Before engaging in the discussion on the distribution of mycotoxins, it is important to also review methods employed in mycotoxin screening in food commodities Mycotoxin screening Mycotoxin screening is required to ascertain their distribution in different matrices. Different methods are applied but may be specific to some toxins, matrices and the resources available, but should confirm to international standards as proposed by the European Commission (EC) [65]. Screening procedure involves a series of steps including sampling, sample preparation, actual testing [64,66] and in some cases, may require confirmatory testing. Even though different screening methods are applied, each requires proper sampling to obtain a representative sample from the whole lot. Because of the heterogeneicity in the distribution of mycotoxins in commodities, sampling is assumed to constitute the greatest source of variability associated with mycotoxin analysis than for other aspects of analysis [64,66]. The sample is prepared by grinding into smaller particles (about 2µm particle size), homogenously mixed and sub-sample obtained and stored at low temperature conditions (-20 o C is ideal) for further analysis to prevent microbial growth and further production of mycotoxins. Representative samples are thus subjected to analysis and those for chromatographic separations, require mycotoxin reference standards. An

10 Mycotoxins and human health 141 extensive review of Scudamore [67] discusses the principles and applications of some selected methods of analysis to include thin layer chromatography (TLC), gas chromatography (GC), high performance-tlc (HPTLC), enzyme linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) particularly for AF, FB, OTA, amongst others. Over the years and in recent times, HPLC coupled either with photodiode array (PDA), ultraviolet (UV), fluorescent (FL) or refractive index (RI) as detectors, is highly sensitive and routinely used in analysis of several mycotoxins. In other words, Pohland et al [41] have provided an insightful review of the validated analytical methods followed for OT, while a detailed discussion on some of the methods commonly applied for screening FB [68] and AF [69] is presented. Mycotoxin screening commences with extraction and in most cases, may be proceded by clean-up or purification of the extracted material. Depending on the type of toxin and matrix, some extraction and clean-up methods include solid phase extraction (C18-reversed-phase), strong anion exchange (SAX), column chromatography (CC) and liquid-liquid partitioning using polar solvents the analyte eluted using organic solvents or weak acids. Clean-up procedure is usually essential for chromatographic separation of mycotoxins. A common example is the use of some immunoassay kits and here, the analyte is trapped by the antibody and conjugates it to an enzyme, which further, is eluted and analyte measured using ELIZA reader, capillary electrophoresis system, luminometer, etc. [70] In most if not all situations, several groups of mycotoxins may cocontaminate food materials. Because of the variability in their chemical structures, previously it was not possible to device a common analytical method in screening them as a total entity as the case may be for proteins and carbohydrates. However, recent advancements in multi-analyte approach whereby, several mycotoxins are simultaneous analyzed have made mass spectrometric detection more popular [70]. Liquid chromatographic-mass spectrometry (LC-MS) and HPLC-MS/MS [71,72] have been optimized for the analysis of different mycotoxins in a single run following simple extraction techniques without clean-up procedure and thus, seem to be promising. Detection of mycotoxins following several of these methods have not been widely occommodated in many laboratories in Africa as they are not only sophisticated but sound rather unrealistic mainly due to lack of well equipped laboratories and expertise. A qualitative and semi-quantitative method (TLC) by two-dimensional TLC devised by Patterson and Roberts [73] used in screening mycotoxins in food and feed commodities have received wide criticisms. Some of such condemnations are the non-quantitative nature and that of low recoveries of mycotoxins in most materials. Its main strength may be related to the fact that with an experienced operator, more than 20

11 142 Njobeh B. Patrick mycotoxins with the exclusion of polar toxins such as FB and moniliformin (MN) can be detected at once [74] and high recoveries obtained mainly from low oil-containing matrices such as cereals and some animal feeds. Quantification of AF via this method is widely been used in the United States and can conveniently be applied in Africa. Shephard [68] describes further some other TLC-related methods when dealing with FB following derivatization with OPA. Subsequent analysis (confirmatory testing) of analytes can further be done via HPLC or by other means. Recoveries of mycotoxins in our laboratory were studied in different food commodities including some cereal and legume seeds following the TLC method of Patterson and Roberts [73]. We found that AF can be recovered from spiked maize, rice, beans, soybeans and peanuts at a range of %, whereas the recovery rates of OTA in these materials were between 83-92% (standard deviation range: 7-18%) [75]. To ascertain the performance of analytical systems, a proficiency testing whereby, a reference material of known mycotoxin concentration is analyzed in comparison to similar tests conducted in other laboratories [64]. Recently, some new non-chromatographic methods generally referred to as BioCop that use transcriptomics (genomic fingerprinting), electrochemical and surface plasmon resonance (SPR) sensors for analysis of some TH such as DON, NIV and T-2 in foods have been introduced but still under [76]. One of the simplest, fastest and growing technologies in the last decade is such visual mycotoxin testing as test kits, strips, dipsticks or the lateral flow device [70] are used in mycotoxin analysis for regulatory purposes on-site at all levels of production during processing. They are non-instrumental methods usually used for the rapidly quantifying single or group of related toxins in the same material outside a laboratory environment. VICAM in April 2010, launched VertuTM, a digital lateral flow technology for easy, fast and accurate quantitative mycotoxin screening in food and other agricultural products at various levels of production both on site and in-laboratory screening with no special training or expertise required [77]. This technology does not require special training or expertise. The above-mentioned analytical methods are used for virtually all mycotoxins including AF, OT and FB, except for TLC method devised by Patterson and Roberts [73] which is not used for screening highly polar mycotoxins (FB). As such, the TLC methods reviewed by Shephard [68] are those recommended and used for analysing FB mycotoxins in various matrices.

12 Mycotoxins and human health Mycotoxin distribution in food commodities Aflatoxins Despite having been the first to be discovered, studies on AF still dominate mycotoxin research today likely because, they are the most notorious of all mycotoxins. Although no region of the world can be declared free from AFs exacerbated by international trade, the greatest occurrence of AF in effect is found in the tropics and sub-tropical regions of the world. The common (high-risk) substrates mostly contaminated by AF are peanuts and their byproducts, followed by maize and animal feeds. In West Africa, levels of AF in peanuts and cereals (mainly maize) for human consumption have so far been reported to be less than 1000ppb with a majority (>80%) of these samples having levels that are below tolerable limits (10ppb) (Table 1) levels above this value were obtained in rice (max: 1,642ppb) [78] and sorghum (range: 20-1,164ppb) [79] samples containing visible moulds. Bankole et al [80] found total AF in dry roasted peanuts from Nigeria ranging between ppb with over 44% of the 106 analyzed samples containing AF levels above the tolerable limit of 20ppb recommended by most countries including Nigeria [81]. Less than 40ppb of AF levels (<5% of samples with AF levels above 10ppb) have been reported in stored Cameroon food commodities. In these reports, the highest AFB 1 level in maize was found to be 31ppb [82], while stored cassava chips had AF range of ppb [83] In the same country, similar levels (range: ppb) also featured in cereals (maize, rice), cassava and nuts such as peanuts, melon seeds and soybeans [75]. For other countries in West Africa including Benin [19] and Togo [84], mean AFB 1 levels in maize and peanuts are below 16 ppb. For Ghana, all maize samples from silos and warehouses had AF levels that ranged from 20 to 355ppb [85] and afterwards, these levels were almost doubled (range: 2-662ppb) in the same commodity [86]. The situation in East Africa seems to differ significantly. In Kenya for example, severe cases of aflatoxicosis have been reported in 1982 and According to the 2004 outbreak report, maize was highly associated with the episode as samples had AF levels as high as 46,400ppb [2]. Most peanuts from western Kenya highly consumed across the country are generally safe since only a very small portion of the samples contained very high AF levels [87]. During the same period, Muture et al [88] found maize from the aflatoxicosis affected area (eastern Kenya) with AF of the same magnitude but at higher levels (Max: 58,000ppb). In the north of Africa, El-Sayed et al [89] previously, recovered AFB 1 and AFG 1 at levels as high as 35,000 and 16,000ppb, respectively, from Egyptian maize and maize products. Such levels differ

13 144 Njobeh B. Patrick significantly from those obtained from neighbouring countries such as Tunisia, Morocco and Algeria with far less AF levels (<80ppb) as seen in Table Fumonisins They are the most recently discovered and characterized well known mycotoxins by two independent groups of scientists in South Africa. At least 14 FB analogues are known but those that naturally do occur in foodstuffs of concern are FB 1, FB 2 and FB 3, with FB 1 being the most common and toxic of them. Fumonisin is the most important Fusarium mycotoxin, alongside ZEA and the TH due to their worldwide occurrence in a variety of foods and their toxic effects to both animal and human tissues. They are commonly found especially in all regions where maize is grown, possibly with the exception of colder regions such as those of Canada and North-eastern Europe [90]. Maize and maize-based products are the most common substrates of FB contamination. In Africa, higher levels of FBs mainly FB 1 in specific products such as maize and maize-based products have been well established in many studies and S. Africa, seems to champion these events. A case here was seen in the Transkei region where significantly high FB levels (range: 50-46,900ppb) were recorded [91]. Further, some naturally infected maize samples contained 117,500ppb FB 1 and 22,960ppb FB 2, the highest levels yet recorded in history [92]. These levels were by far higher than those obtained from surveys conducted in other parts of Africa. For instance, data generated by Ngoko et al [82] on stored maize for human consumption showed the highest FB 1 level ever recorded is 26,000ppb in Cameroon, which also did not differ from those obtained by Njobeh et al [75] in the same region. Data from Ghana [86] showed much higher total FB content ranging from 70-52,670ppb in all maize samples. For other cereals such as barley and barley products, FB contents appear to be low relative to maize and maize-based products. According to recent data from S. Africa, Maenetje & Dutton [74] found that the levels of FB recovered from barley and barley products used as raw materials in the S. African breweries were low (max: 5,000ppb) (Table 1). Aside from cereals, it is important to note that FB can also contaminate other food materials including nuts. In the tropical region of West Africa, Sangare-Tigorie et al [93] recovered FB 1 in peanut from Cote d Ivoire at levels as high as 6,000ppb. The work of Njobeh et al [75] found maximum levels of FB 1 1,500ppb in peanut, with much lower FB 1 levels in beans and soybeans from Cameroon.

14 Mycotoxins and human health Ochratoxins They are a group of structurally related compounds and include ochratoxin A (OTA), B (OTB) and C (OTC) among which, OTA is supposedly the most common and toxic. OTA occurs in a wide range of foods worldwide, but at the highest level in animal feeds preceeding cereals but appears to also be contained in nuts, swine products, grape juice, wine, beer and other beverages. Natural occurrence of OTA in various commodities has extensively been reviewed [41]. From the data, there is evidence of extremely higher levels of about 27,000ppb in Danish feed and Canadian grains with up to 70,000ppb from Yugoslavian maize and Australian animal feeds. In Africa, the situations seem to differ notably even though this may be argued based on lack of sufficient data from Africa to support this hypothesis. Comparatively, data reviewed in Table 1 showed to a large extent, lower OTA levels are established in these surveys. However, there have also been concerns on the subject of contamination of coffee with OTA in Africa especially after having analyzed green coffee beans from three continents. In the study, Romani et al [94] found 98.7% samples had OTA and with respect to samples from America or Asia in terms of frequency and level of contamination, African samples were more contaminated especially when two of these samples from Congo had the highest levels of 18 and 48ppb recorded in the survey. In Nigeria, levels of 150ppb OTA were reported in maize Sibanda et al 1997 [95], and a decade later, Dongo et al [96] found lowers levels ranging from ppb in kolanuts and much higher levels (range: ppb) [97] were recovered from cocoa beans. Mouldy rice and sorghum samples from Nigeria were found to contain mean levels of 155ppb [78] and 49ppb [79], respectively. The presence of OTA was also investigated in cocoa products including cocoa beans, cake, butter and chocolate by Bonvehi [98], but a lower range of 0.1-9ppb was found even though >40% of samples had levels exceeding tolerable limit of 5ppb. From neighbouring Cameroon, OTA levels obtained by Mounjouenpou et al [15] from cocoa beans ranged from ppb, while subsequent analysis of Njobeh et al [75] established much lower levels in cereals and legume seeds ( ppb) in the country. Most of the works on OTA in Africa are surveys from north eastern Africa probably because severe OTA problem is very well observed in some countries of the Mediterranean basin. From these studies, some data present high levels (max: 224.5ppb) in breakfast cereals from Morocco [99], Tunisian cereal grains (range: ppb) [100] with several of these samples having levels that exceed largely the maximum tolerable limit (5ppb) in the continent. In beer and wine, the presence of OTA in these products is from grain or grape contaminated with OTA used as raw materials in the brewery industry.

15 146 Njobeh B. Patrick Table 1 shows maximum OTA content of 2,340ppb was found in 13/29 traditional brewed beer [101], but lower levels (max: ppb) were present in industrial [74] beer samples from S. Africa. In the country, Shephard et al [102] formerly recovered OTA from all 24 tested S. African wines at mean levels of 0.16µg/l (ppb) and 0.24µg/l in white and red wines, respectively. Multiple occurrence of AF and other mycotoxins, particularly the possible human carcinogens in the same sample are common and of great concern. Members of the group of AF may co-occur in a particular product [103] with other mycotoxins, although their mixtures may vary in different foodstuffs. McEvoy [104] found AF and OTA as the most important mycotoxins from a human health perspective. Ochratoxin production takes place during pre- and especially post-harvesting, which is predominant in foods and feeds during storage and may co-occur regularly with other mycotoxins especially those originating from the Aspergillus and Penicillium genera. Most of the surveys reported in literature have indicated co-occurrence of OTA with such mycotoxins as CIT [24,105]. Apart from the major mycotoxins discussed above, others that are less problematic than AF, OTA and FB but cannot be ignored because of their sporadic natural occurrence in foods and feeds and include DON, T-2, ZEA, sterigmatocystin (ST), CIT, PAT, cyclopiazonic acid (CPA), penicillic acid (PA) and tenuazonic acid (TA). Though data on the incidence of these minor toxins in Africa are limited, DON has been found in barley in S. Africa [74] and maize from Zambia [130], Cameroon [75,82] and Benin [131]. Also, ZEA was shown to occur in agricultural commodities from S. Africa [74], Cameroon [75,82] and Nigeria [78,79]. Citrinin has also been reported in fermented maize from Ghana [85]. Although these miscellaneous mycotoxins generally exhibit low toxicity in various mammalian species, their simultaneous occurrence with FB, AF or OTA exacerbate the ill-effects of the former. 3. Mycotoxin exposure Exposure is significant and best assessed by estimating excretion of mycotoxins and associated metabolites in bile, urine and faeces as well as their distribution in blood, milk, liver, kidney and semen. Determination of metabolites of mycotoxins in by-products such as milk may serve as biomarkers in understanding their mechanisms of actions in humans as well as estimating the severity of human exposure to such toxins [132,133]. The main issue dilemma is that very limited data on mycotoxins in foods and those from determining mycotoxin excretion or tissue residues are available in African countries, which are not substantial to assess exposure in the continent with

16 Mycotoxins and human health 147 some degree of certainty. This is mainly attributed to the fact that most analytical laboratories are nonexistent, not well-equipped and consist of skeletal benches or shelves and in most cases, are limited in expertise. Limited studies however, have been conducted in only some countries on AFM 1 in milk and milk products (Table 1), AF-adducts in human blood [134,135,136], semen [137] and urine [138] ; OTA in breast milk [139], human blood [140,141] ; FB in faeces [142], sphingolipids (FB biomarkers) in urine or serum [61]. Country Commodity Mycotoxin %Frequency a Range/Mean (ppb) Main producer Reference Algeria Wheat OTA 40 (30) A. ochraceus 17] Benin Yam chips AF 97 (107) 0->15 [106] Maize AFB1 100 % 15.2 [19] FB1 >90% Max 12,000 F. verticiliioides Benin/Togo Maize AFB (502) 0-20 A. flavus [84] Peanut AFB1 4.6 (175) Min 20 A. flavus Botswana Peanut AF 78 (120) [107] Burkina Maize FB1 100 (90) Max 16,000 [108] Faso Cameroon Maize AFB1 1.3 (72) 0-17 A. flavus [82] Maize AFG (72) Max 31 A. flavus Maize FB1 64 (90) ,000 F. verticillioides Cocoa bean OTA 77.8 (36) A. carbonarius [15] Cassava chips AF 25 (72) [83] Variety b AF 51 (82) A. flavus/a. [75] parasiticus Variety b FB1 41 (82) 37-24,225 F. verticiliioides Variety b OTA 2.7 (74) c A. ochraceus Cote Maize AFB1 100 (10) [93] D Ivoire Peanut AFB1 100 (10) Maize FB1 100 (10) 300-1,500 Peanut FB1 100 (10) Maize OTA 100 (10) Peanut OTA 60 (10) Min 0.64 Egypt Maize/maize AFB1 2.5 (57) Max 35,000 [89] products AFG1 Max 16,000 Maize AFB1/AFB2 23 (40) No data [109] Ethiopia Cereals d AFB1 8.8 (352) Trace-26 [110] FB (352) Max 2,117 d OTA 24.3 (352) Max 2,106 Kenya Peanut AF 63.7 (769) e [87] Maize AFB1 54 (480) 0-58,000 [88] Maize products AF 100 (350) 1-46,400 f [2] Gambia Peanut AFs 87 (47) [111] Ghana g Maize FB1 100 (15) 70-33,103 F. verticillioides [86] FB2 67 (15) 60-12,318 FB3 40 (15) 68-7,249 AF 53 (15) Guinea Peanut Maize Rice AFB1 AFB1 61 (46) 22 (9) 0 (66) No data No data AFB1 Libya Milk AFM (49) [113] cheese AFM1 75 (20) Milk AFM (40) 5 [114] Yoghurt AFM1 7.5 (40) 2.2 Morocco Milk AFM (54) [115] Maize flour AF h 80 (20) [116] Wheat flour AF 17.6 (17) Poultry feed AF 66.6 (21) Foods i OTA 40.8 (120) [117] [112]

17 148 Njobeh B. Patrick Rice OTA 26 (100) [118] Breakfast OTA 8.3 (48) [99] cereal Infant cereal OTA 0 (20) - Nigeria Bean AFB1 58 (50) A. flavus [119] Melon seed AF 25 (120) [120] Sorghum j AFB (168) A. flavus/parasiticus [88] OTA 20.5 (112) Rice j AFB (196) 20-1,642 A. flavus/parasiticus [87] OTA 40 (140) 24-1,164 Wheat AFB1 54 (50) Maize k AF 18.4 (103) k A. flavus [121] FB (103) 70-1,780 F. verticillioides FB2 66 (103) F. verticillioides Cocoa l OTA 85 (170) [98] Kola nuts OTA 98 (50) [96] Cocoa beans OTA 90 (59) [97] S. Africa Beer m OTA 44.8 (29) 3-2,340 [101] Wine OTA 100 (24) 0.16 and 0.24 n [102] Beer m FB 100% [122] Barley o AFB1 25 (123) p A. flavus [74] p OTA 13 (123) q A. ochraceus FB1 51 (82) 440-5,000 q F. verticillioides Animal feeds AFB1 74 (23) A. flavus [123] FB1 26(23) 15-5,900 F. verticillioides OTA 0 (23) - Maize r FB (141) 5-10,140 [124] Sudan Animal feed AF 64.3 (56) [125] Cow milk AFM (44) s [126] Vegetable oil AFB (56) [127] Tunisia Foods t AF 50.5 (209) [128] OTA 59.8 (209) Wheat OTA 38 (110) [100] Barley OTA 40 (103) Rice OTA 28 (96) Sorghum OTA 38 (113) Maize FB1 21% Max: 5,000 F. verticillioides [129] Zimbabwe Coffee bean OTA 90.5 (84) [94] a Values in brackets are no of samples analyzed. b Commodities analyzed include maize, rice, peanuts, soybeans, cowpea, cassava flour/flakes and melon seeds. c Maize and peanut samples had OTA levels of 2.1 & 10.8 ppb, respectively. d Samples include barley, sorghum, teff & wheat with FB present in a few samples of sorghum. e AF ranges in Busia and Homabay districts samples are and ppb, respectively, with just 2.1% of samples unsuitable for use as animal feeds (>100 ppb) based on FDA action level. f 55, 35 & 7% of samples had AF levels in maize >20, >100 & 1000 ppb, respectively, with Makueni and Kitui districts with highest reported cases of aflatoxicosis also having the highest AF levels in the study. g Total FB range found was 70-52,670 ppb with only one sample consisting of visible moulds having FB >5,000 ppb; F. graminearum was also found to produce high amounts of FB in the study. h Range of AFB 1 found in maize flour, wheat flour and animal feed samples were respectively, , and ppb. i Includes dried raisins, walnuts, peanuts, dried figs & pistachios. j Samples contained visible moulds and were analyzed by TLC. k Samples analyzed were pre-harvest maize and AFB 1 in19 +ve samples ranged from ppb. l Samples include cocoa bean, cake, powder, butter and chocolate. m Traditional brewed beer. n Mean OTA level in red and white wine were 0.16 and 0.24 ppb, respectively. o Includes barley, maltster malt, and maltster barley and beer; Number of beer samples analyzed (n) = 48. p Levels (ppb) of mycotoxin in samples other than beer. q Range of OTA level in beer (n=48); only barley and malt samples were analyzed for FB 1. r Good home grown maize from Centane and Bizana, respectively in the Eastern Cape province. s 83% of samples had AFM 1 levels above action level of 0.5 ppb. t Includes various cereals, nuts, dried fruits and spices.

18 Mycotoxins and human health 149 For aflatoxin M 1 (AFM 1 ) and M 2 (AFM 2 ), metabolites of AFB 1 and AFB 2, respectively, it is not surprising they are found in milk and milk products worldwide, given the fact that food and feed materials often contain high AF levels. Regarded as secondary exposure, breast milk can be a major source of AF for nursing babies and communities that consume milk and milk products regularly. Nektaria [133] and Polychronaki et al [138,143], reported AFM 1 in 138/388 breast milk samples from nursing mothers in Egypt (range: pg/ml). Such high levels are expected since the levels of AFB 1 and AFG 1 levels (35,000 and 16,000ppb, respectively) are reported in that country. It is estimated that about 1-3% of AFB 1 consumed via feed is converted to AFM 1 in milk [144,145] but the review of Pettersson [146] provides a list of studies that established a range of between % in the estimated carry-over of AF. In animal feeds, AF levels ranging from and ppb were obtained from surveys in S. Africa [123] and Sudan [125], respectively. These levels may likely be reflected in products from animals fed such feeds. The persistent carry-over of OTA into animal products is exacerbated by wide use of OTA-contaminated grains and feeds in the animal production sector. It has long been a practice particular among the rural farmers in West Africa, where most often than not, animals are fed visible mouldy grains due to the poor perception on mycotoxins. This thus results in increased mycotoxin disposition in various tissues. Compared with AF, OT is rapidly absorbed into the body and between 40-66% of the toxin is resorbed into the blood [146]. Data on residual OTA in tissues and other products mainly from pigs and chickens [41,146] fed diets contaminated with known OTA levels are well documented. These data indicate that blood and kidney are the most heavily contaminated tissues having the highest levels of residual OTA. In a study from Tunisia, 28% of human serum samples contained OTA ranging from ppb [147]. But previously in the same country, 84% of blood samples analyzed contained much higher OTA levels (range: ppb) with such levels linked to increased rate of human chronic intestinal nephropathy (CIN) of unknown aetiology [140]. In the case of FB, its estimation in blood is unlikely since it is rapidly excreted via the bile and faeces and thus poorly absorbed by the body as in the case of AF and OT. However, the action of FB to alter sphingolipid metabolism (sphinganine/sphingosine ratio) can be studied in serum or urine [61], but the problem here is the difficulty in recovery in obtaining higher recoveries of these sphingoid bases [142]. FB exposure can thus be appropriately assessed by estimating levels in faeces [142,148,149]. Although there are significantly high levels of FB in maize in the developed [150] as much as in the developing countries [90,151] as for AF, human exposure to these toxins is significantly lower in the developed countries. This is because consumption of maize and maize-

19 150 Njobeh B. Patrick based products in such countries seems to be modest [45] with estimated total daily intake of mycotoxins in European diets found to be low. Unlike in most parts of Africa, Asia and Latin America, where maize is a staple diet, humans are exposed to their toxic effects on a daily and on-going basis [86,152]. Daily maize consumption in a S. African rural population (Transkei region) with a high rate of human oesophageal cancer is estimated to be between g/day [124]. This unusual high intake level contributes greatly to high mycotoxin intake as seen in the study that FB exposure level could be up to µg/kg body weight (bwt) per day. Thiel and co-workers [153] previously, estimated between µg/kg bwt of FB exposure in the same region. Maize daily intake in other African countries is likely to be of similar magnitude as that of Transkei region with estimates in other African states summarized by Shephard et al 2007 [124]. As noted earlier, the presence of multiple mycotoxins in the food chain is common and has evoked worldwide concern. For instance, the Fusarium mycotoxins as well as AF [131] do co-occur in particular feedstuffs [32] at very significant levels. Another compelling issue being that of the interactions of mycotoxins as co-factors with malnutrition (kwashiorkor) and such diseases as hepatitis B & C, HIV/AIDS [135,136] and Burkitt s lymphoma as in most African nations. These interactions may produce much more deleterious effects than those of individual mycotoxins or disease conditions in isolation. Such information on the toxic effects of simultaneous exposures in humans is still very limited. However, in a diverse human diet, exposure will be to multiple toxins, may be at low doses but on an intermittent rate over prolonged periods with the ultimate effects yet to be unravelled [75]. While it is nearly impossible to predict the effects of mycotoxin exposure to multiple dietary exposures, some in vivo and in vitro studies give a clear indication about the synergistic and additive effects they may cause that should be of great concern to the general public [154]. A very recent review of the magnitude of mycotoxin problem in Africa and their implications thereof is provided [60], which can be useful for further details. 4. Significance of human exposure to mycotoxins 4.1. Acute and chronic human mycotoxicoses Acute human mycotoxicoses (Table 2) are quite rare as considerable amounts of the commonly occurring mycotoxins have to be ingested over a short time span. Further, some of these mycotoxins, as listed in Table 2 have higher LD 50 s, i.e., DON or very high ones, e.g., ZEA and FB 1. The list of