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4 Mycotoxins in Cereal Grains, Nuts and
Other Plant Products
J.P.F. D’Mello*
Formerly of The Scottish Agricultural College, West Mains Road, Edinburgh
EH9 3JG, UK
Introduction
Mycotoxins are a diverse and ubiquitous group of fungal compounds specifically associated with the precipitation of deleteri- ous effects in humans and animals. Viewed globally, food safety is regularly compro- mised by the presence of mycotoxins occur- ring in cereal grains, nuts, fruit and green coffee beans. If feeds are contaminated with mycotoxins, associated residues and meta- bolites may appear in animal products. The mycotoxins of major concern in human health emanate from the secondary meta- bolism of Claviceps , Aspergillus , Penicillium , Fusarium and Alternaria genera. Mycotoxins may be categorized and, indeed, named on the basis of their fungal origin. Mycotoxins may also be classified on the basis of their biosynthetic origin from key primary inter- mediates. Thus, the polyketide mycotoxins are derived from acetyl coenzyme A, while the terpene mycotoxins are synthesized from mevalonic acid. Amino acids are incorpo- rated in the formation of a third group of mycotoxins comprising cyclic polypeptides and their derivatives. It is salutary to note, however, that mycotoxin production may be strain specific. Thus both toxigenic and
atoxigenic strains exist within the Aspergillus flavus species. It is conventional to subdivide toxigenic fungi into ‘field’ (or plant patho- genic) and ‘storage’ (or saprophytic/spoil- age) organisms. Claviceps , Fusarium and Alternaria are classical representatives of field fungi, while Aspergillus and Penicillium exemplify storage organisms. This distinction is academic since the inoculum for postharvest spoilage of grain and fruit, for example, frequently originates from field sources such as soil or plant debris. Further- more, mycotoxins from storage fungi fre- quently are detected on grain, nuts and fruit prior to harvest. Mycotoxigenic species may be distinguished further on the basis of geographical prevalence, reflecting specific environmental requirements for growth and secondary metabolism. Thus, A. flavus , A. parasiticus and A. ochraceus readily proliferate under warm, humid conditions, whereas Penicillium expansum and P. verrucosum are essentially temperate fungi. Consequently, the Aspergillus mycotoxins predominate in plant products emanating from the tropics and other warm regions, while the Penicillium mycotoxins occur widely in temperate foods, particularly cereal grains and infected fruit. Fusarium fungi are more ubiquitous, but even
©CAB International 2003. Food Safety: Contaminants and Toxins (ed. J.P.F. D’Mello) 65
- E-mail: f.dmello@ed.sac.ac.uk
this genus contains toxigenic species which are associated almost exclusively with cereals from warm countries. The diverse ill effects caused by these compounds are incorporated within the generic term ‘mycotoxicosis’, including dis- tinct conditions and syndromes which may add to or occur concurrently with existing disorders such as kwashiorkor and gastro- enteritis. In this chapter, the mycotoxins likely to prejudice human health are reviewed in terms of origin and chemical nature, distribu- tion in foods, toxicology and risk manage- ment. Particular emphasis is placed on recent evidence indicating continuing human expo- sure to these fungal toxins.
Origin and Nature of Compounds
The foodborne mycotoxins most frequently implicated in human disorders are presented in Table 4.1, which also indicates the fungal origin of these compounds. The pathways of biosynthesis are summarized in Table 4.2. In historical terms, the ergot alkaloids, synthe- sized by Claviceps purpurea , have occupied a central position by virtue of their assumed role in widespread gangrenous and convul- sive manifestations in Europe during the Middle Ages. Current concerns relate to the aflatoxins, ochratoxins, fumonisins and patulin. However, the trichothecenes and zearalenone have emerged recently as global
66 J.P.F. D’Mello
Mycotoxins Fungal species Foods
Ergot alkaloids Aflatoxins Cyclopiazonic acid Ochratoxin A
Citrinin Patulin Citreoviridin T-2 toxin (type A trichothecene) Diacetoxyscirpenol (type A trichothecene) Deoxynivalenol (type B trichothecene) Zearalenone
Fumonisins; moniliformin; fusaric acid Tenuazonic acid; alternariol; alternariol methyl ether; altenuene
Claviceps purpurea Aspergillus flavus;A. parasiticus A. flavus A. ochraceus;Penicillium viridicatum;P. cyclopium P. citrinum;P. expansum P. expansum P. citreo-viride Fusarium sporotrichioides;F. poae
F. sporotrichioides;F. poae
F. culmorum;F. graminearum
F. culmorum;F. graminearum; F. sporotrichioides F. moniliforme
Alternaria alternata
Cereal grains Nuts; maize kernels; dried fruits Nuts Cereal grains and products; pig products; raw coffee Cereal grains Apple products Rice Cereal grains
Cereal grains
Cereal grains
Cereal grains
Maize kernels
Fruit; vegetables; cereal grains
Table 4.1. Principal foodborne mycotoxins of confirmed or potential relevance in human health.
Primary metabolite Pathway Mycotoxins
Acetyl coenzyme A
Mevalonic acid
Amino acids
Polyketide
Isoprenoid
Peptide synthesis
Patulin, citrinin, ochratoxins, zearalenone, moniliformin, aflatoxins, fumonisins Trichothecenes: deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, diacetoxyscirpenol Ergot alkaloids
Table 4.2. Biosynthesis of the major foodborne mycotoxins.
foods and, of the two, OTA is more ubiquitous, often occurring with another pentaketide mycotoxin, citrinin, in cereals and associated products. Citrinin is synthesized by a number of Penicillium species.
Patulin and citreoviridin
Several Penicillium species are also capable of synthesizing patulin (Fig. 4.4), a low molecu- lar weight hemiacetal lactone with antibiotic properties. Penicillum expansum is of particu- lar relevance since it is commonly associated with storage rot of apples and a wide variety of other fruits. The occurrence of patulin in apple juice has been attributed to the use of mouldy fruit. Other species of Penicillium contaminating rice from Italy, Spain, Thailand, Burma and other countries are now recognized as producers of an open-chain nonaketide derivative known as citreoviridin.
Fusarium mycotoxins
The natural occurrence of mycotoxins from Fusarium species is generally associated with temperate countries, since many of these fungi require somewhat lower temperatures for growth and mycotoxin production than the aflatoxigenic Aspergillus species. How- ever, extensive data exist to indicate the global scale of contamination of cereal grains
with a number of Fusarium mycotoxins. Indeed, F. moniliforme and its mycotoxins are associated primarily with foods from tropical and subtropical regions. Fusarium species are important pathogens of cereal plants, causing diseases such as fusarium head blight (FHB). The very same species may also synthesize a wide range of mycotoxins, of which the most important from the point of view of human health are the trichothecenes, zearalenone, moniliformin and the fumonisins (D’Mello et al ., 1997). Following episodes of FHB, residues of these mycotoxins may contami- nate harvested grain. The co-occurrence of Fusarium mycotoxins in cereal grains has now emerged as an intractable issue with regard to risk assessment and establishment of regulatory or advisory directives.
Trichothecenes
The trichothecenes comprise four basic groups, with types A and B representing the most important mycotoxins. Type A trichothecenes include T-2 toxin, HT-2 toxin, neosolaniol and diacetoxyscirpenol (DAS), while type B trichothecenes include deoxynivalenol (DON, also known as vomitoxin) and its 3-acetyl and 15-acetyl derivatives (3-ADON and 15-ADON, respec- tively), nivalenol (NIV) and fusarenon-X. All trichothecenes possess a basic tetracyclic sesquiterpene structure with a 6-membered oxygen-containing ring and an epoxide group. These features are illustrated in the structure for DON (Fig. 4.5). The synthesis of the two types of trichothecenes appears to
68 J.P.F. D’Mello
Fig. 4.4. Patulin (Moss, 1996; reproduced with permission from Mycological Research ).
Fig. 4.5. Deoxynivalenol (Moss, 1996; reproduced with permission from Mycological Research ).
be characteristic for a particular Fusarium species. Thus, for example, production of type A trichothecenes predominates in F. sporotrichioides and possibly also F. poae , whereas synthesis of type B trichothecenes occurs principally in F. culmorum and F. graminearum.
Zearalenone
A common feature of many Fusarium species is their ability to synthesize zearalenone (ZEN), and its co-occurrence with certain trichothecenes raises important issues regarding additivity and/or synergism in the aetiology of mycotoxicoses in humans. ZEN (also known as F-2 toxin) is a phenolic resorcyclic lactone (Fig. 4.6), which also occurs as a hydroxy derivative in the form of α-zearalenol. The presence of appropriate
reductases in animal tissues implies that α-zearalenol may be the active form of ZEN in animals.
Fumonisins and moniliformin
With respect to the co-occurrence of myco- toxins, the secondary metabolism of F. moniliforme is of particular significance since it is capable of producing at least three myco- toxins: the fumonisins, moniliformin and fusarin C. The fumonisins are relatively recent additions to the list of mycotoxins, but their significance as major contaminants of maize has already been established and linked with the incidence of cancer in humans. Several structurally related forms of fumonisins (FBs) have been characterized, with FB 1 , FB 2 and FB 3 occurring regularly in maize from different geographical sources. FB 1 (Fig. 4.7) is 2-amino-12,16-dimethyl-3, 5,10,14,15-pentahydroxyicosane with a pro- pane-1,2,3-tricarboxylate substituent at C- and C-15, whereas FB 2 and FB 3 are, respec- tively, the C-10 and C-5 deoxy analogues of FB 1. In addition, FB 1 is structurally similar to sphinganine and sphingosine, intermediates in the biosynthesis and degradation of sphingolipids. Moniliformin occurs as the Na or Ksalt of 1-hydroxycyclobut-1-ene-3,
Mycotoxins in Cereal Grains, Nuts and Other Plant Products 69
Fig. 4.6. Zearalenone (Moss, 1996; reproduced with permission from Mycological Research ).
Fig. 4.7. Fumonisin B 1 (Moss, 1996; reproduced with permission from Mycological Research ).
may contain total aflatoxin concentrations of up to 149 μg kg−^1. In The Netherlands, AFB 1 levels as high as 165 μg kg−^1 have been reported for pistachio nuts, with much lower concentrations in shells (up to 8 μg kg−^1 ). In whole dried figs, UKdata (Ministry of Agri - culture, Fisheries and Food, 1993) showed that between December 1988 and April 1992, the percentage contaminated with aflatoxins (total) at levels above 4 μg kg−^1 fell from 26 to 16%. However, samples containing up to 427 μg kg−^1 were found. The incidence of aflatoxins in fig paste samples above the 4 μg kg−^1 level also fell during this period from 50
to 14%. The maximum concentration of total aflatoxins found in fig paste also declined from 165 to 15 μg kg−^1. These findings attracted comment by COT, who were clearly concerned by the high levels of contamination of pistachio nuts, dried figs and fig pastes but were satisfied that consignments exceeding the 10 μg kg−^1 limit were refused entry by the UKport health authorities. The results of a recent survey of Egyptian foods indicated high incidence and unacceptable levels of AFB 1 in spices, herbs and medicinal plants. As will be seen later, contamination of spices is the subject of scrutiny by EC authorities, but
Mycotoxins in Cereal Grains, Nuts and Other Plant Products 71
Food Aflatoxin
Incidence of contamination (%)
Mean/range (μg kg−^1 ) Country
Maize
Maize-based gruels Peanuts
Pistachio nuts
Peanut butter: ‘smooth’ ‘crunchy’
Dried figs Fig paste Date fruits
Spices
Total Total B 1 B (^2) B (^1) B (^2) Total B (^1) B (^2) G (^1) G (^2) Total B (^1) B (^2) G (^1) G (^2) Total Total B (^1) B (^1)
Total Total B (^1) B (^2) G (^1) G (^2) Total Total Total B (^1) G (^1) B (^1)
19
81 56
25
52
28–
11 28
71 64 24
40
17 0– 0– 0– 0– 0– 0.002–19. 0.8– 1.6– 1.6– 1.6– 3– 0.8–10. 0.2–1. 0.1–21. 0.4–4. 4.1– up to 149 up to 165 0.8–
4.1– 4.1– 3.2– 1.6– 3.2– 1.6– 1.6– 4– 4.1– 113 133 25
Zambia Costa Rica
Indonesiaa
Nigeria Botswana
Japan
UK California, USA The Netherlands Japan
UK
Botswana
UK UK United Arab Emirates
Egypt
aSee also Table 4.6.
Table 4.3. Aflatoxin contamination of foods.
there may be a case for surveillance of other imported foods not currently controlled by legislation.
Ochratoxin A
Ochratoxin A is ubiquitous in foods (Table 4.4; see also D’Mello and Macdonald, 1998), occurring principally in cereal grains (Vrabcheva et al ., 2000), dried vine fruit (MacDonald et al ., 1999) and green coffee beans (Blanc et al ., 1998). The relatively high values in Bulgarian cereals were associated with grain samples taken from villages with a high incidence of Balkan endemic nephro- pathy. A recent study in France indicated consistent contamination of cereals and oilseeds with OTA, the values ranging from 0.6 to 12.8 μg kg−^1 in positive samples. In the UK, OTA analyses of dried vine fruit imported from Greece and other countries (Table 4.4) indicated that 88% were contami- nated with levels in the range 0.2–53.6 μg kg−^1. The OTA data for green coffee beans shown in Table 4.4 are at the lower end of a range of other published values, which included a maximum of 360 μg kg−^1 (Blanc et al ., 1998). Use of contaminated grain in brewing and as animal feed regularly results in transfer of residues into beer and offal. Some OTA contamination of porcine organs has been reported in a survey conducted in the UK (Ministry of Agriculture, Fisheries and Food, 1993). Of 104 samples of kidney, 12% were contaminated with OTA at 1–5 μg kg−^1 , while
3% had concentrations of up to 10 μg kg−^1. Of the black pudding samples analysed, 13% were contaminated with OTA in the range 1–5 μg kg−^1. Citrinin often occurs with OTA in cereal grains. In naturally contaminated samples of Bulgarian wheat, citrinin levels up to 420 μg kg−^1 were detected (Table 4.4).
Patulin
The occurrence of patulin in fruit juice has been a cause of concern in the UK and else- where in Europe (D’Mello and Macdonald, 1998). In recent years, there has been a marked increase in the production of cloudy apple juices prepared by pressing the fruit and stabilizing with vitamin C prior to pasteurization of the juice. The reduction in processing steps, as compared with the pro- cedure for production of clear juices, means that patulin losses during fining and filtration are restricted, with higher residual levels of the mycotoxin in the cloudy juices. A comparison of the patulin concentrations in the two types of juices has been published recently for samples from the UK and Spain. Although the UK data were derived from a relatively small number of samples, it was apparent that the incidence of patulin contamination was higher in cloudy juices, with a median value of 28 μg kg−^1 , compared with 0–10 μg kg−^1 for clear juices. Four cloudy samples had patulin concentrations in excess of 50 μg kg−^1 , compared with only one of the clear juice samples. In two cloudy samples,
72 J.P.F. D’Mello
Source Food Ochratoxin A (range/mean) Citrinin
Bulgaria
UK
Greece
Several countries Thailand
Wheat Oats Bran Wheat Barley Oats Currants Sultanas Raisins Green coffee beans
< 0.5– 0.9– < 0.5–3.
< 0.2– < 0.2– < 0.2– 4.1–22.
< 5– < 5– < 5–
Table 4.4. Ochratoxin A and citrinin contamination of foods (μg kg−^1 ).
1997). The highest values for ZEN in Table 4. (11 and 15 mg kg−^1 ) relate to two barley samples from the Fukuoka region of Japan (Yoshizawa, 1997).
Fumonisins
The widespread contamination of maize with fumonisins is unmistakable and likely to remain an issue of overriding concern. Recent surveillance has confirmed the extensive distribution of fumonisins, particularly in maize produced in the tropics (Table 4.6, adapted from D’Mello and Macdonald, 1998). In most instances, the predominant
fumonisin is FB 1. Outstanding features include high FB 1 concentrations in samples from Thailand (Yamashita et al ., 1995), China (Wang et al ., 1995a) South Africa (cited by Shephard et al ., 2000) and Kenya (Kedera et al ., 1999). Highest levels of FB 2 were reported in Argentinian (Chulze et al ., 1996) and South African samples. In the Philip- pines, Thailand and Indonesia, FB 1 and FB (^2) occurred in over 50% of maize samples, while incidence rates of 82–100% were recorded for samples from Italy, Portugal, Zambia and Benin. In Honduras, Julian et al. (1995) detected FB 1 in all 24 samples of maize tested. In Costa Rica, significant regional differences were observed in contamination of maize with FB 1 , while in Mexico concern has been
74 J.P.F. D’Mello
Country FB 1 FB 2 FB 3 Total
Benin Botswana Mozambique South Africa (Transkei) Malawi Zambia Zimbabwe Tanzania Kenya Honduras Mexico Argentina Costa Rica Italy Portugal USA Vietnam China China
Taiwan The Philippines Thailand Indonesia Nepal
n.d.a–2, ,35– 240– < 50–46, n.d.– 20–1,420, 55–1,910, n.d.– 110–12,000, 68–6,555, 1,000–1,800b,,, 85–8,791, 1,700–4, 10–2,330, 90–3,370, n.d.– 268–1,516, 160–25,970, < 500–8,800c^ , < 500–5,700d^ , < 500–7,200 e^ , ,0–1, 57–1,820, 63–18,800, 226–1,780,
n.d.– n.d.– 75– < 50–16, n.d.– n.d.– n.d.– n.d.–
n.d.–11,300,
n.d.– n.d.–1,080,
155– 160–6,770,
0– 58–1, 50–1, 231–
n.d.– 25–
n.d.
n.d.– n.d.
,n.d.–3,
101– , 110–4,
35– 340–
n.d.–
,55–2, n.d.–
, 85–16,
,10–2, ,90–4,
,524–2, ,430–36,
110–8,
aNot detectable. bMasa and tortillas. c (^) Maize (corn) meal. dUnfermented batter. eFermented batter.
Table 4.6. Worldwide contamination of maize kernels and products with fumonisins B 1 , B 2 and B 3 , (μg kg−^1 ). Data for maize products are identified by footnotes.
expressed at the higher levels in masa and tortillas compared with similar products imported from the USA. The data for maize meal and batter (Groves et al ., 1999) prepared in the Shandong Province of China may also be viewed with disquiet. As with ZEN, a disturbing feature is the co-occurrence of fumonisins with other mycotoxins.
Co-occurrence
The co-occurrence of several mycotoxins in the same sample of cereal grains has pro- voked worldwide concern. Of considerable significance are consistent reports of co-occurrence of Fusarium mycotoxins (D’Mello and Macdonald, 1998). In the Lublin region of south-eastern Poland, type A trichothecene contamination of barley grain was linked with the natural incidence of FHB, in which the predominating organism was F. sporotrichioides (Perkowski et al ., 1997). Of 24 barley grain samples, 12 were positive for T-2 toxin with a range of 0.02–2.4 mg kg−^1. In five of these samples, co-contamination with HT-2 toxin occurred with a range of 0.01–0.37 mg kg−^1. The findings of another study in Poland indicated that infection with F. graminearum can result in contamination of cobs simultaneously with DON and 15-ADON. Concentrations of DON and 15-ADON in Fusarium -damaged kernels ranged from 4 to 320 mg kg−^1 (Table 4.5) and from 3 to 86 mg kg−^1 , respectively, but the axial stems of the cobs were more heavily contaminated at 9–927 mg kg−^1 and 6–606 mg kg−^1 , respectively. A study of Japanese barley samples confirmed the co-occurrence of DON with NIV (Table 4.5; Yoshizawa, 1997). In addition, an appreciable number of the barley samples were found with 3-ADON at levels of up to 19 mg kg−^1. In highly contaminated grains, a positive correlation occurred between levels of DON and its acetyl deriva- tives. DON levels were always higher than those of 3-ADON and 15-ADON, with ratios ranging from 3 to 155. Regional differences were also observed in that DON was the major contaminant in grain from northern districts of Japan, whereas in central districts
NIV was the predominant trichothecene. These differences were correlated with chemotype variants of Fusarium species. Furthermore, Yoshizawa (1997) and Lauren et al. (1996) revealed the occurrence of relatively high levels of ZEN with DON and NIV in cereal samples from Japan and New Zealand, respectively (Table 4.5). Of the 29 cereal samples tested in The Nether- lands, 90 and 79% were positive for DON and NIV, respectively, with 76% containing both mycotoxins together, while ZEN occurred as a third contaminant, albeit at low levels (Table 4.5). In China, FB 1 and AFB 1 co-occurred in 85% of maize samples (Wang et al ., 1995a), while, in the Philippines, Thailand and Indonesia, FB 1 and FB 2 co-occurred with aflatoxins in 48% of maize samples. These fumonisins also co-occurred with NIV and ZEN (Yamashita et al ., 1995). Multiple contamination of maize with fumonisins, DON, NIV and AFB 1 was also observed in north Vietnam (Wang et al ., 1995b). Of additional concern is the co-occurrence of FB 1 , fusaproliferin and beauvericin in Italian samples of maize.
Uptake and Disposition
The principal route of exposure to myco- toxins in humans is through consumption of contaminated diets. Uptake of foodborne mycotoxins is implied from the appearance of these compounds and associated deriva- tives (e.g. adducts) in body fluids. A wide array of factors may affect absorption. For example, FB 1 absorption is greater in fasted than in fed rats, with potentially profound implications for undernourished humans. Uptake of mycotoxins may also be affected by the onset of other conditions such as gas- trointestinal disorders. Mycotoxin form can influence both uptake and disposal. Thus, studies with animal models indicate that hydrolysed FB 1 is absorbed more readily than FB 1 itself, and urinary excretion is also greater. Mycotoxin metabolism is an important feature preceding events such as carcino-
Mycotoxins in Cereal Grains, Nuts and Other Plant Products 75
Human Disorders
Mycotoxins have long been implicated in specific human conditions (Table 4.8). How- ever, conclusive evidence for such an associa- tion has yet to emerge for several of these disorders.
Ergotism
One of the ancient European episodes of mycotoxicoses in humans relates to ergotism (St Anthony’s Fire) caused by the bioactive alkaloids produced in the sclerotia of C. purpurea. The alkaloids cause constriction of peripheral blood capillaries leading to oxygen starvation and gangrene of the limbs (Flannigan, 1991). Occasional cases of ergotism still occur to the present day.
Aflatoxicosis
Aflatoxins can induce acute effects in humans, and field cases continue to occur despite worldwide awareness of the toxico- logical and health implications. Thus, in 1974, an outbreak of liver disease occurred in India following the consumption of mouldy grain containing aflatoxins (see D’Mello and
Macdonald, 1998). Of 997 subjects, 97 were reported to have died during this episode. Principal pathological features in the liver included destruction of centrilobular zones, thickening of central veins and cirrhosis. Chronic aflatoxin exposure may modulate immune function, thereby increasing sus- ceptibility to infection. Indeed, it has been suggested that aflatoxins are the major cause of kwashiorkor in children.
Ochratoxicosis
Balkan endemic nephropathy is a chronic disease occurring in rural populations of Bulgaria, Romania and the former state of Yugoslavia (see D’Mello and Macdonald, 1998). In affected subjects, the kidneys are markedly reduced in size and, histologically, the disease is characterized by tubular degen- eration, interstitial fibrosis and glomerular defects. Tubular function is also impaired. The similarities to porcine nephropathy are striking and have led to the conclu- sion that OTA is also the causative agent in Balkan endemic nephropathy. However, the co-occurrence of OTA with citrinin in cereals from Bulgarian villages with a history of Balkan endemic nephropathy suggests an interaction between the two mycotoxins in the aetiology of this condition (Vrabcheva
Mycotoxins in Cereal Grains, Nuts and Other Plant Products 77
Mycotoxin Disease Food/source Countries
Ergot alkaloids Aflatoxins
Cyclopiazonic acid Ochratoxins
Citreoviridin T-2 toxin Fumonisins
Moniliformin
Ergotism (St Anthony’s Fire) Liver cancer; kwashiorkor, cirrhosis; acute hepatitis; Reye’s syndrome ‘Kodua poisoning’ Balkan (and possible Tunisian) endemic nephropathy ‘Shoshin-kakke’ Alimentary toxic aleukia Oesophageal cancer Primary liver cancer ‘Keshan disease’
Rye Peanuts; maize
Millet Cereal grains
Rice Cereal grains Maize Maize Maize
Europe East and West Africa; India; Taiwan; Thailand; the Philippines India Bulgaria; Romania; former Yugoslavia; Tunisia Japan Former USSR South Africa China China
Table 4.8. Mycotoxins implicated in human disease.
et al ., 2000). It will be noted that the two mycotoxins have similar properties in animal models (Table 4.7). A possible endemic ochratoxin-related nephropathy has also been suggested to occur in Tunisia. Affected subjects were classified into those with chronic interstitial nephropathy, chronic glo- merular nephropathy and chronic vascular nephropathy.
Cancer
Current concern over mycotoxins centres on their carcinogenic potential in humans. In tropical countries, particularly East and West Africa, India, Thailand, the Philippines and China, aflatoxin exposure is a continuing health issue among the indigenous popula- tions. There is now good epidemiological evidence linking aflatoxin exposure with the incidence of liver cancer (Smith, 1997). In one study, it was possible to demonstrate that men were more sensitive than women to the carcinogenic effects of aflatoxins but that in both cases there was a linear effect of dose on the development of liver cancer. The epidemiological data should be inter- preted with caution, as other factors such as malnutrition and disease may have contrib- uted to the incidence of liver cancer. Further- more, interactions may occur. Thus, Wang et al. (1996) indicated that aflatoxin exposure may enhance the carcinogenic potential of hepatitis B virus. The generally accepted order of carcinogenicity is AFB 1 > AFG 1 > AFB 2 > AFG 2. In toxicological classification, AFB 1 has been designated as a group 1 carcin- ogen (i.e. sufficient evidence in humans for carcinogenicity), whereas AFM 1 falls in the group 2B category (i.e. probable human carcinogen). Epidemiological evidence has also been presented to link human oesopha- geal cancer in South Africa with dietary exposure to the fumonisins. In addition, it has been suggested that, in China, fumonisins may promote primary liver cancer initiated by AFB 1 and/or hepatitis B virus (Ueno et al ., 1997).
Continuing Human Exposure to
Foodborne Mycotoxins
Despite enhanced awareness and the adop- tion of legal or advisory guidelines, human exposure to foodborne mycotoxins continues on a global scale, even in developed countries (see D’Mello and Macdonald, 1998). Recent evidence is summarized in Table 4.9 for aflatoxins and in Table 4.10 for OTA. The tables are not designed to be exhaustive but rather illustrative of widespread exposure to these mycotoxins. The evidence of exposure generally is based on mycotoxin residues in body fluids, mother’s milk and tissue specimens. In addi- tion, the association between mycotoxin expo- sure and cancer relies on presumptive intake of contaminated foods, rather than direct determinations of metabolites or DNA adducts. However, efforts are now focusing on measurements of the major adducts in tissues and fluids.
Aflatoxins
The widespread contamination of maize and peanuts with aflatoxin is reflected in the analyses of faeces, urine, blood and breast milk samples of people in different parts of Africa (Table 4.9). In addition to the four forms of aflatoxin, metabolites such as aflatoxicol, AFM 1 and AFM 2 may appear in body fluids and tissues. One study showed widespread fetal exposure to aflatoxins in East and West Africa, as demonstrated by analysis of cord and maternal blood samples (Maxwell, 1998). Aflatoxins were also detected in breast milk samples of mothers. Thus, there is widespread pre- and post-natal exposure of infants to aflatoxins, which may predispose children to infection. Indeed, a hypothesis has been advanced implicating aflatoxin exposure with the pathogenesis of kwashiorkor in African children. It will be noted that despite stringent EU regulations, detectable levels of AFB 1 –albumin adducts have been recorded for individuals in the UK (Table 4.9; Turner et al ., 1998). Detection of adducts is clearly a highly sensitive means of
78 J.P.F. D’Mello
OTA-related nephropathy is thought to occur with similarities to the Balkan syn- drome. Three subsets were identified in affected subjects: those with chronic inter- stitial nephropathy, chronic glomerular nephropathy and chronic vascular nephro- pathy. Patients with chronic interstitial nephropathy had the highest blood OTA lev- els in comparison with the other subgroups or with the general population. However, even the latter group had overall blood OTA levels which were in excess of those seen in Sweden. In Sierra Leone, monitoring of breast milk samples showed that only 9% were mycotoxin-free, with 35% containing OTA. It is clear that infants and mothers in Sierra
Leone are exposed to OTA at levels greater than the current allowances of TDI (Table 4.10). The urinary excretion of OTB by infants in Sierra Leone was quantitatively similar to that of OTA (Jonsyn, 1999). Other individuals at risk may be patients with renal disorders. Although OTA and citrinin are established nephrotoxins, any association with conditions such as the Balkan and Tunisian endemic nephropathies still remains tentative. Thus, the higher incidence and concentrations of OTA in blood of patients requiring haemodialysis and in those with urothelial cancer await elucidation to distinguish between cause and effect (Table 4.10; Jimenez et al ., 1998; Wafa et al ., 1998).
80 J.P.F. D’Mello
Region/country Basis of evidence Observations
Sweden Norway
UK Croatia Italy
Hungary
Spain
Egypt
Tunisia
Sierra Leone
Sierra Leone
Canada
Japan
Blood analysis Breast milk analysis
Blood and urine analysis Blood analysis Breast milk analysis
Blood and colostrum analysis
Blood analysis
Blood and urine analysis
Blood analysis
Blood and urine analysis
Breast milk analysis
Blood analysis
Blood analysis
OTA levels below 0.3 μg l−^1 OTA detected in 33% of samples at levels of 10–130 ng l−^1 ; 12% of samples contained > 40 ng l−^1 All blood and 92% of urine samples contained OTA OTA levels 2–50 ng ml−^1 Significant exposure of babies to OTA at levels exceeding tolerable daily intakes estimated from animal models 52% of random blood samples with 0.2–12.9 ng ml−^1 ; 41% of colostrum samples with 0.2–7.3 ng ml−^1 53% of healthy donors and 78% of patients undergoing haemodialysis positive for OTA; mean concentrations 0.7 and 2 ng ml−^1 , respectively OTA levels of 0–10 and 0–8 ng ml−^1 , respectively, in blood and urine of patients with nephrotic syndrome; OTA levels of 0–3.4 and 0–0.3 ng ml−^1 , respectively, in urine of potential kidney donors and healthy volunteers Chronic forms of interstitial, glomerular and vascular nephropathy; OTA levels of 25–59 μg l−^1 in patients with interstitial nephropathy, 6–18 μg l−^1 in other groups and 0.7–7.8 μg l−^1 in the general population OTA detected in 25% of cord blood samples at levels of 0.2–3.5 ng ml−^1 ; 24% of urine samples contained OTA; 20% of urine samples contained OTB Confirmed exposure of infants to combinations of OTA and various aflatoxins OTA levels 0.6–1.4 ng ml−^1 depending on geographical location OTA levels 0.004–0.28 ng ml−^1
Table 4.10. Continuing human exposure to ochratoxins.
Combinations
The pre-natal and neonatal exposure of chil- dren in Sierra Leone to combinations of aflatoxins and OTA is noteworthy (Jonsyn, 1998). Of 64 cord blood samples analysed, 94% contained either OTA, aflatoxins or both (Tables 4.9 and 4.10). It is suggested that pre-natal exposure to such combinations may have resulted in low birth weights and premature mortality of infants. Continued exposure post-natally is likely in view of con- tamination of breast milk and cereal grains with combinations of OTA and aflatoxins. Of particular concern, however, is the apparent absence of direct determinations of human exposure to combinations of aflatoxins and fumonisins in countries where peanuts and maize constitute the staple foods (Tables 4. and 4.6).
Tolerable daily intakes
The foregoing account demonstrates that mycotoxin intake is inevitable even in coun- tries with stringent regulatory and process controls. In instances where there are ade- quate toxicological data, TDI have been esti- mated for humans (Table 4.11). As previously stated, the actual intakes in many countries may exceed the TDI allowances. In the case of AFB 1 , the TDI estimates have been based on studies conducted in certain tropical coun- tries where infection with hepatitis B virus is an additional carcinogenic factor. In coun- tries where this virus is not a major risk, the TDI for AFB 1 may be set considerably higher. It will be noted that, for the fumonisin carcin- ogens, TDI limits have yet to be established.
Regulatory Control
The ubiquitous distribution, acute effects and carcinogenic potential of mycotoxins have resulted in the imposition or adoption of reg- ulations for maximum permitted levels of these contaminants in primary foods and associated products. Regulations also apply to feedingstuffs in order to reduce transmis- sion of mycotoxins to edible animal products. Van Egmond and Dekker (1995) indicated that 90 countries had regulations relating to maximum permissible levels of mycotoxins in various commodities. However, 13 coun- tries were known to have no regulations and, for some 50 countries, mostly in Africa, no data were available. It is unlikely that the sit- uation has changed significantly since 1995. Virtually all developed countries have statu- tory regulations for the aflatoxins and advi- sory directives for a limited number of the other mycotoxins. Of particular concern, however, is the lack of statutory or advisory regulations for control of fumonisins in foods.
Rationale
With the aflatoxins, the underlying rationale is based on the need to reduce contamination to ‘irreducible levels’, defined as the concen- tration which cannot be eliminated from a food without involving the complete rejection of the food, thereby severely limit- ing the ultimate availability of major food supplies. However, in the evolution of statu- tory regulations for aflatoxins, the guiding principle has remained unaltered, which is to reduce contamination to the lowest level that is ‘technologically achievable’, taking into account advances in analytical methodolo- gies. In the preparation of proposals, comments received through groups such as the World Trade Organization are taken into account. The resulting regulations, therefore, represent a compromise between avoidance of international trade disputes with producer countries and maintenance of consumer protection.
Mycotoxins in Cereal Grains, Nuts and Other Plant Products 81
Mycotoxin TDI
Aflatoxin B (^1) Ochratoxin A Deoxynivalenol
Zearalenone Fumonisins
0.11–0.19 ng 1.5–5 ng 1.5 μg (infants) 3.0 μg (adults) 100 ng Inadequate data
Table 4.11. Tolerable daily intakes (TDIs) of major mycotoxins (kg−^1 body weight).
Mycotoxins in Cereal Grains, Nuts and Other Plant Products 83
Aflatoxins: maximum levels
Country Foods B 1 B 1 +B 2 +G 1 +G 2 M (^1)
European Union
South Africa Taiwan Thailand Japan USA
Groundnuts, nuts and dried fruit, and processed products thereof intended for direct human consumption or as an ingredient in foodstuffs Groundnuts to be subjected to sorting or other physical treatment before human consumption or use as an ingredient in foodstuffs Nuts and dried fruit to be subjected to sorting or other physical treatment before human consumption or use as an ingredient in foodstuffs Cereals and processed products thereof intended for direct human consumption or as an ingredient in foodstuffs Spices a Milk All foods Cereals All foods All foods All foods Milk
2 8 5 2 5 5
10
4
15
10
4
10
10 50 20
20
aProposals under consideration.
Table 4.12. Examples of worldwide regulations for control of aflatoxins in human foods (μg kg−^1 ).
Country Aflatoxins Feedingstuffs
Maximum levels Status
Indonesia
European Union
Taiwan
USA
B 1 +B 2 +G 1 +G (^2)
B (^1)
B 1 +B 2 +G 1 +G (^2)
B 1 +B 2 +G 1 +G (^2)
Copra Groundnut Sunflower seed meal Straight feedingstuffs except: groundnut, copra, palm kernel, cottonseed, babassu, maize and products derived from the processing thereof Complete feedingstuffs for cattle, sheep and goats (with the exception of complete feedingstuffs for calves, lambs and kids) Complete feedingstuffs for pigs and poultry (except those for young animals) Other complete feedingstuffs Feed, oilseed meals for feed under 4% of mixed feed Cottonseed meal Maize and groundnut products intended for breeding beef cattle/pigs or mature poultry Maize and groundnut products intended for finishing beef cattle
1000 200 90 50 20
50
20
10 1000
300 100
300
Proposal
Statutory
Statutory
Statutory
Table 4.13. Examples of worldwide regulations for aflatoxins in animal feedingstuffs (μg kg−^1 ).
Advisory directives
In several countries, advisory directives exist which are not enforceable by law. However, the limits suggested have been used to reduce human exposure to mycotoxins. In the USA and Canada, the advisory level for DON is 1000 μg kg−^1 in finished wheat products such as flour and bran. For apple products including juice, cider and puree, the advisory level for patulin in the UKis set at 50 ppb.
Methodologies
Specific methodologies are prescribed for the aflatoxins, OTA and DON. Of particular relevance are the protocols for the legally controlled aflatoxins.
Sampling
Due to the heterogeneous distribution of mycotoxins in foods, adequate sampling is a primary consideration. In the official control of aflatoxins in the EU, samples are taken according to prescribed methods. Three types of samples are identified. An incremental sample is the quantity of food taken from a single position in a lot or sublot. An aggre- gate sample represents the combined total of all the incremental samples taken from the
particular lot. Laboratory samples are derived from the mixed aggregate sample. The number and size of incremental samples are laid down in the provisions, and specific protocols are prescribed for nuts and dried fruit, milk and derived products. Treatment of laboratory samples is also described in detail.
Analytical
Specific methods for the determination of aflatoxins are not prescribed at the EC level, and laboratories may select any method pro- vided that it is consistent with a number of criteria based on recoveries and precision parameters such as repeatability and repro- ducibility. However, adequate laboratory standards must be demonstrated through participation in proficiency testing and inter- nal quality control schemes. In practice three principal methods are employed in aflatoxin analysis worldwide. Thin-layer chromato- graphy (TLC) remains the method of choice in many countries and its efficacy has been enhanced by new technology including the use of immunoaffinity columns in clean-up and the application of densitometry for quantification. Other methods include liquid chromatography and enzyme-linked immu- nosorbent assay. For DON, the official methods used for regulatory purposes in the USA and Canada are TLC and gas chromatography.
84 J.P.F. D’Mello
Suggested/action limits
Food/beverages Categories OTA DON
Cereals
Coffee
Dried vine fruit Spices Beer Wine
To be subjected to sorting or other physical treatment prior to human consumption or use as an ingredient in foodstuffs Cereals and processed products thereof intended for direct human consumption or use as an ingredient in foodstuffs Green beans Roasted beans and coffee products Currants, raisins and sultanas
5
3
8 4 10 10
0.2–1.
750 a
500
aFlour used as raw material in food products; monitoring level for raw cereals.
Table 4.14. Permitted levels (μg kg−^1 ) for ochratoxin A (OTA) in foods and beverages and action levels for deoxynivalenol (DON) in cereals: measures under discussion within the European Commission.
