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Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, V ... Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, V ... Document Transcript

  • Problems associated with Fusarium mycotoxins in cereals. Angelo Visconti ABSTRACT Fungi of the genus Fusarium are common plant pathogens occurring worldwide, mainly associated with cereal crops. Fusarium species can produce over one hundred secondary metabolites, some of which can unfavourably affect human and animal health. The most important Fusarium mycotoxins, that can frequently occur at biologically significant concentrations in cereals, are fumonisins, zearalenone and trichothecenes (deoxynivalenol, nivalenol and T-2 toxin). These compounds have been implicated as the causative agents in a variety of animal diseases, such as leukoencephalomalacia, pulmonary oedema, infertility, diarrhoea, vomiting, anorexia, leukopenia, immunosuppression, skin and gastrointestinal irritation, hemorraging, etc., and have been associated to some human diseases. The IARC working group on carcinogenic risk to humans has classified the toxins derived from Fusarium moniliforme (including fumonisins) as possibly carcinogenic to humans (Group 2B). Fusarium mycotoxins contamination of cereals can cause economic losses at all levels of food and feed production including crop and animal production, crop distribution and processing. Practical strategies to eliminate these mycotoxins from feed and food are required, although some progress is being made at level of individual compound or group of compounds. Health risks associated with the consumption of cereal products contaminated with Fusarium mycotoxins are worldwide recognized and depend on the extent to which they are consumed in a diversified diet; several countries have recommended maximum tolerated levels for some of these mycotoxins. Further risk assessment and regulatory efforts should be established in order to ensure that Fusarium mycotoxins levels in foods and feeds are kept well below of those levels which can constitute a potential hazard for human and animal health. Key words: Fusarium, mycotoxins, biomarkers, trichothecenes, fumonisins, zearalenone. Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, V.le L. Einaudi 51, I-70125 Bari, Italy
  • INTRODUCTION Fungi of the genus Fusarium are common plant pathogens occurring worldwide, mainly associated with cereal crops. Fusarium species can produce over one hundred secondary metabolites, some of which can unfavourably affect human and animal health. The most important Fusarium mycotoxins, that can frequently occur at biologically significant concentrations in cereals, are fumonisins, zearalenone and trichothecenes (deoxynivalenol, nivalenol and T-2 toxin). These compounds can occur naturally in cereals, either individually or as specific clusters of two or more of them depending on the producing fungal species (or strain); they have been implicated (alone or in combination between themselves and/or with other mycotoxins) as the causative agents in a variety of animal diseases and have been associated to some human diseases. Maize is the crop most susceptible to contamination by all Fusarium mycotoxins (particularly important are fumonisins), while wheat and barley are subjected to contamination of deoxynivalenol, nivalenol and, at lesser extent, zearalenone and T-2 toxin and related trichothecenes. The major fungal species (widely distributed in cereal crops) producing these mycotoxins are F. graminearum, F. culmorum and F. crookwellense, producing zearalenone, deoxynivalenol, nivalenol and related trichothecenes, F. sporotrichioides, producing T-2 toxin and related trichothecens, and F. moniliforme and F. proliferatum, producing fumonisins. Mycotoxin contamination of crops may cause economic losses at all levels of food and feed production including crop and animal production. Contaminated grains result in increased costs for handlers and distributors due to extra drying costs, excess storage capacity, losses in transit, loss in markets. The occurrence of mycotoxins varies from commmodity to commodity, year to year, and region to region. Years of environmental conditions favorable to mycotoxin development will result in higher economic losses. F. graminearum is the primary causal agent of wheat head scab and causes extensive damage to wheat in humid and semihumid wheat growing areas of the world by reducing grain yield and quality. Although the prevention of mycotoxin contamination of grain is the main goal of food and agricultural industries throughout the world, under certain environmental conditions the contamination of various cereal grains with Fusarium fungi and mycotoxins are unavoidable for grain producers. While certain treatments have been found to reduce concentrations of specific mycotoxins, no single method has been developed that is equally effective against the wide variety of mycotoxins that may co-occurs in different cereal grains. The ideal decontamination procedure should be easy to use, inexpensive and should not lead to the formation of compounds that are still toxic or can alterate the nutritional and palatability properties of the grain or grain products. This report brings together some general information about toxicity to man and animals of the most important Fusarium mycotoxins (some details on carcinogenicity, cytotoxicity and immunotoxicity, and acute animal toxicity), the relevant economic losses and detoxification approaches, and regulatory issues worldwide. Some data from recent investigations carried out at the CNR Institute of Toxins and Mycotoxins (Bari, Italy) are also presented with particular emphasis to the use of biomarkers to evaluate human and animal exposure to fumonisins and to in vivo and in vitro assays for fumonisins detoxification by means of non-nutritive adsorbent compounds. TOXICITY OF FUSARIUM MYCOTOXINS Carcinogenicity The International Agency for Research on Cancer (IARC, 1993) has grouped the main Fusarium mycotoxins on the basis of the fungal species producing them as it represents the best way to identify the real situation through which humans become exposed to these naturally occurring toxins. Therefore, the most important mycotoxins, in terms of 2
  • natural occurrence and toxicology, have been grouped in: toxins derived from F. sporotrichioides (T-2 toxin and related trichotecenes), toxins derived from F. graminearum, F. culmorum and F. crookwellense (Deoxynivalenol, Nivalenol, Fusarenone X and Zearalenone), and toxins derived from F. moniliforme (Fumonisins and Fusarin C). Only the group of toxins deriving from F. moniliforme was identified as group 2B, i.e. possible carcinogenic to humans and with sufficient evidence of carcinogenicity towards experimental animals, whereas the data relevant to the other groups of toxins or to individual toxins were not sufficient (or adequate) to make them classifiable as to their carcinogenity to humans (group 3). The following carcinogenicity data were identified by the working group for individual toxins: i) T-2 toxin increased the incidence of pulmonary and hepatic adenomas in mice (additional data from studies with rats were considered inadequate for evaluation); ii) zearalenone caused an increase in hepatocellular adenomas in female mice and in pituitary adenomas in both male and female mice; iii) fusarin C induced papilloma or dysplasia in the forestomach and oesophagus in mice and rats (these data were not considered sufficient to assess the carcinogenicity towards experimental animals); iv) fumonisin B1 induced hepatocellular carcinoma, cholangiofibrosis and cholangiocarcinoma in rats (26 month feeding experiment at level of 50 mg/kg); this toxin was shown to be a strong tumor promoter, but only a weak initiator (similar effects have also been shown for FB 2 and FB3); v) no evidence of carcinogenicity was observed in mice for deoxynivalenol or nivalenol in feeding experiments lasted 26 weeks or two- years. The summary of the IARC evaluation of Fusarium mycotoxins (IARC, 1993) is reported in Table 1. All the above mentioned toxins but fumonisins have shown, at different exent, some genotoxic effect in mammalian cells in vitro, including clastogenic effects, chromosomal aberrations and sister chromatid exchange, or unscheduled DNA synthesis, and T-2 toxin induced DNA damage and chromosomal aberration in rodent also in vivo (IARC 1993). Table 1. Carcinogenicity risk evaluated by IARCa for Fusarium mycotoxins. Toxins Degree of evidence of Overall carcinogenicitya evaluationa In human In animals Toxins derived from: F. graminearum, F. culmorum, F. crookwellense I Group 3 Zearalenone ND L Nivalenol I Fusarenone X I Deoxynivalenol I Toxins derived from: F. sporotrichioides ND Group 3 T-2 toxin L Toxins derived from: F. moniliforme I S Group 2B Fumonisin B1 L Fumonisin B2 I Fusarin C L *From IARC, 1993 a I, insufficient evidence; L, limited evidence; ND, no adequate data; S, sufficient evidence. Group 2B = possibly carcinogenic to humans; Group 3 = not classifiable as to its carcinogenicity to humans. 3
  • Immunotoxic and cytotoxic effects Fusarium mycotoxins may predispose livestock to infectious disease, and this might result in feed refusal and decreased productivity. Increased infections in food-producing animals might also result in increased animal-to-human transmission of pathogens such as Salmonella and Listeria. Ingestion of mycotoxins by humans might contribute to decreased resistance to infectious agents and neoplasms, and these compounds may function as unrecognized etiological factor of immune disfunction diseases (Pestka and Bondy, 1994). The capacity of deoxynivalenol to alter normal immune function is particularly important. There is extensive evidence that deoxynivalenol can be immunosuppressive or immunostimulatory, depending upon the dose and the duration of of exposure. While immunosuppression can be explained by the capacity of inhibiting protein synthesis, immunostimulation can be related to interference with normal regulatory mechanisms. In addition to several trichothecenes, immunomodulatory effects have been shown for fusarochromanone (TDP-1) and its acetyl derivative (TDP-2), metabolites of Fusarium equiseti (Minervini et al. 1992). There are studies showing increased resistance to infections after (oral or intraperitoneal) exposure to both deoxynivalenol or T-2 toxin (Eriksen and Alexander, 1998). Intraperitoneal preinoculation treatment with T-2 toxin can be immunostimulatory and can actually enhance resistance to Listeria, whereas postinoculation T-2 toxin treatment is markedly immunosuppressive. Similarly, enhanced resistance to mastitis pathogens by preinoculation gavage of T-2 toxin or to Staphylococcus hyicus or Mycobacterium avium infections after a single-dose oral pre-treatment with deoxynivalenol in mice has been observed. However, dietary deoxynivalenol exposures to as low as 2 mg/kg for 5 weeks was sufficient to decrease time-to-death intervals in mice challenged with Listeria; these levels of contamination can frequently be found in cereal crops when wheater conditions are favourable to the growth of Fusarium in the field (Eriksen and Alexander, 1998; Pestka and Bondy, 1994). Zearalenone and its analogues are capable of inhibiting mitogen-stimulated lymphocyte proliferation and can induce thymic atrophy and macrophage activation (Forsell and Petska 1985, Visconti et al. 1991). Dietary exposure to relatively high doses (10 mg/kg) of zearalenone for two weeks deacreases resistance to Listeria (Pestka and Bondy, 1994). Fumonisins have been shown to be cytotoxic to dog kidney and rat hepatoma cell lines. Several studies have shown altered immune response in poultry fed diets containing fumonisins at relatively high levels (Pestka and Bondy 1994). Structure-activity relationship of trichothecenes. The 12,13 epoxy ring, and substitutions of the hydroxyl group or hydrogen in some of the positions of the trichothecene skeleton influence the cytotoxicity and lymphocyte blastogenesis or protein synthesis inhibition. The toxicity of trichothecenes varies significantly between different test systems, but the rank order of toxicity between the toxins is similar. The type A toxins, having a functional group other than carbonyl in C-8 position, have a higher cytotoxic effect than type B toxins with a carbonyl group in this position, and trichothecenes are in general more cytotoxic than other Fusarium metabolites. The toxicity of group A toxins decreases in the order of isovaleryl > hydrogen > acetyl > hydroxyl substituents in the C-8 position, i.e. T-2 toxin > diacetoxyscirpenol > 8-acetyl neosolaniol > neosolaniol. Loss of side chains in C-4, C-8 or C-15 by hydrolysis of T-2 toxin decreases the toxicity and losses of more than one side chain in these positions further decreases the toxicity (Visconti et al. 1991). In the type B group, the toxicity is influenced by the substituents in the C-4 position. In particular, the toxicity decreases in the order of acetyl > hydroxyl >hydrogen in the C-4 position in the B-type trichothecenes. (Visconti et al. 1991). The presence of a hydroxyl group in nivalenol increases the toxicity in animals a tenfold as compared to the hydrogen in deoxynivalenol (Eriksen and Alexander, 1998). Fig. 1 shows the structure activity relationship for 11 type A trichothecenes and 5 type B 4
  • trichothecenes produced by Fusarium with respect to cytotoxicity towards cultured human cell lines (K-562 and MIN- GL1) as reported by Visconti et al. (1991). Toxicity towards domestic animals The main toxic effects of the major Fusarium mycotoxins are reported below. Zearalenone may be present in cereal crops in cooler and moist regions worldwide. Zearalenone and related metabolites possess strong estrogenic activity and can result in severe reproductive and infertility problems when they are fed to domestic animals in sufficient amounts. Swine appears to be the most sensitive of the domestic animal species, therefore the most frequently reported with problems caused by zearalenone, which include enlargement or swelling and reddening of the vulva in gilts and sows (vulvovaginitis), swelling of the mammary glands and atrophy of the ovaries, vaginal and rectal prolapses. In young male it can cause swelling of the prepuce, testicular atrophy, enlargement of the mammary glands, while in boars it causes reduced libido and a marginal reduction in sperm quality. Effects in other species are much less pronounced. High concentrations of zearalenone have been associated with infertility and development of atypical secondary sexual characteristics in heifers (Prelusky et al. 1994). Thricothecenes Substituents at CD50 a (b g/ml) C-3 (R1) C-4 (R2) C-15 (R3) C-7 (R4) C-8 (R5) MIN-GLI K-562 Type A T-2 toxin OH OAc OAc H O-Isoval 0.0003b 0.001 HT-2 toxin OH OH OAc H O-Isoval 0.0025 0.02 Acetyl T-2 toxin OAc OAc OAc H O-Isoval 0.025 0.03 T-2 triol OH OH OH H O-Isoval 0.040 0.09 T-2 tetraol OH OH OH H OH 0.200 0.28 8-acetoxyneosolaniol OH OAc OAc H OAc 0.003 0.002 Acuminatin OH OH OAc H OAc 0.0045 0.01 Tetracetoxy T-2 tetraol OAc OAc OAc H OAc 0.022 0.04 Neosolaniol OH OAc OAc H OH 0.05 0.15 4,8-diacetoxy T-2 tetraol OH Oac OH H OAc 0.08 0.10 Diacetoxyscirpenol OH OAc OAc H H 0.001 0.001 Type B Fusarenon-X OH OAc OH OH =O 0.06 0.15 Nivalenol OH OH OH OH =O 0.20 0.30 Deoxynivalenol OH H OH OH =O 0.40 0.30 15-acetyldeoxynivalenol OH H OAc OH =O 2.00 0.40 3-acetyldeoxynivalenol OAc H OH OH =O 7.00 2.00 a 50% cytotoxic dose; b Derived from dose response curves of four data mean values. Fig. 1. Structure-activity relationship for trichothecenes toxicity to cultured human cell lines. 5
  • Deoxynivalenol (also called vomitoxin) is the most important trichothecenes because of its high incidence in cereals, including maize, wheat, barley, oats, etc., but it is not one of the most acutely toxic of this group of mycotoxins (Rotter et al. 1996). At the cellular level the main toxic effect is inhibition of protein synthesis via binding to ribosome. In animals, the main overt effect at low dietary concentrations (> 2 mg/g in feed for pigs) appears to be a reduction in food consumption (anorexia) and weight gain, while higher doses (> 20 mg/g) induce feed refusal, diarrhoea and vomiting. Deoxynivalenol is known to alter brain neurochemicals, and the serotoninergic system appears to play a role in mediation of the feeding behaviour and emetic response. Animals fed low doses of toxins are able to recover from initial weight losses, while higher doses induce more long term changes in feeding behaviour. Swine are more sensitive than other livestock species to the presence of deoxynivalenol in their feed. Extensive degradation of deoxynivalenol to secondary metabolites in the rumen has been reported, and this may explain the higher tolerance to the toxin by ruminants with respect to poultry and pigs (Prelusky et al. 1994, Rotter et al. 1996). Most of the clinical signs caused by the ingestion of deoxynivalenol are also observed with nivalenol, the latter being in general more toxic. T-2 toxin is the most toxic of the Fusarium trichothecenes, though less widely distributed than deoxynivalenol. In pigs clinical signs of T-2 toxicosis includes emesis, posterior paresis, lethargy, and frequent defecation. At natural levels of contamination in the diet T-2 toxin causes reduced feed intake and animal performance. At high concentrations in the diet it produces diarrhoea, emesis and feed refusal. T-2 toxicosis in poultry causes oral lesions, reduced feed consumption and growth rate in young animals and reduced egg production in laying hens. The impact on poultry production become important at dietary concentrations above 2 mg/kg. In ruminants T-2 toxicosis results in a wide range of responses, such as feed refusal, leukopenia, depression, diarrhoea, coagulopathy, enteritis, and posterior ataxia. Reduction of humoral immunity is a common effect for pigs, poultry and ruminants exposed to low concentrations of T-2 toxin in the diet. This increases susceptibility to other diseases and the effects of poor management practices. Most of the clinical signs caused by the ingestion of T-2 toxin are also observed with diacetoxyscirpenol and, at slightly lower extent, with HT-2 toxin (Prelusky et al. 1994). Fumonisins are a group of mycotoxins mainly produced in maize grown under warm and dry conditions by F. moniliforme and F. proliferatum. Fumonisin B1 (FB1), B2 (FB2) and B3 (FB3) are the major compounds present in maize fungal cultures and have been shown to occur naturally at biologically significant levels in maize and a variety of maize-based human foodstuffs and animal feeds in several countries throughout the world (Visconti, 1996, Visconti et al., 1995). FB1 concentrations usually exceed those of FB2 and FB3 of about 3 or more times, although higher concentrations of FB2 or FB3 can also be observed (Visconti et al. 1995, Chulze et al. 1996, Ramirez et al. 1996). Commercially available maize products for human consumption are generally contaminated at levels below 1 µg/g FB1, although individual products in certain countries can reach far higher levels (Doko and Visconti, 1994). Fumonisins (particularly FB1 and in some cases FB2) has been shown to cause fatal toxicoses in animals (Marasas, 1995; Caramelli et al. 1992). The ingestion of fumonisin-contaminated maize has been associated with spontaneous outbreaks of equine leukoencephalomalacia, a neurological syndrome characterized by focal, often extensive, liquefactive necrosis of the white matter of the cerebrum, and with acute pulmonary edema in pigs, a fulminant swelling of the lungs and thoracic cavity (Marasas 1995). Although hepatic injury has been observed in all vertebrate species studied, a number of species-specific effects have been induced experimentally by fumonisins on other target organs, including: - renal injury in rats; - liver cancer in rats; - immunosuppression in chickens; - toxicity to broiler chicks and chicken embryos; - potentiation of atherogenic plaque formation in primates; - oesophageal mucosal hyperplasia in chronically dosed pigs; - nephrotoxicity in rabbits; - leukoencephalomalacia and hemorrhage in the brain of rabbits (Marasas 1995, Visconti et al. 1998). 6
  • 0 3 6 9 12 15 18 21 24 27 Day Fig. 2. Time course of the SA/SO ratio in urine of rats fed for 13 days with diets containing different amounts of fumonisins, followed by 13 days with diets containing low levels of fumonisins (< 1 µg/g, dotted lines). Rats had been previously exposed for 9 days to 0.5 µg/g fumonisins. Biomarkers of fumonisins exposure. The mode of action of fumonisins has been postulated to be through the inhibition of the enzyme ceramide synthase that catalyzes the acylation of sphinganine to form dihydroceramide and thus inhibits the de novo biosynthesis of complex sphingolipids (Merrill et al. 1996). This results in an increase of the concentration of sphinganine (SA) relative to the concentration of sphingosine (SO), one of the end products of the sphingolipid biosynthesis. The increase of SA/SO ratio in biological fluids or tissues can be measured in serum, urine and animal tissues, such as kidney and liver, and provides a useful biomarker to assess human and animal exposure to fumonisin contaminated feed and food (Riley et al. 1994; Solfrizzo et al. 1997a, 1997b). The use of this biomarker is particularly important for the following reasons: i) it is indicative for exposure and toxic effects derived from a biochemical change (disruption of sphingolipid metabolism) generated directly by fumonisins on the exposed individual; ii) the direct measurement of fumonisins or their metabolites within an organism or in biological fluids is not an effective biomarker because these toxins are quickly eliminated after ingestion. The development of effective biomarkers to estimate the fumonisin exposure in support to 7
  • regulatory limits at global level is warranted due to considerable problems related to the sampling of maize and related products to be analyzed and the big variation in maize consumption in different countries (Visconti et al. 1998). The above biomarker has been validated by proving that the increase of the SA/SO ratio in urine of rats exposed to fumonisins is both “time dependent” and “dose dependent” (Solfrizzo et al. 1997b). In particular, experiments with rats exposed through the diet to 0.5 mg/kg fumonisins for 9 days, followed by 13 days at higher fumonisins doses, showed that the minimum effective dose to induce a significant increase of the SA/SO ratio value in rat urine was between 1 µg/ g and 2 µg/g of fumonisins in the diet. The inversion of the SA/SO ratio (from <1 to >1) appeared after 5 days of exposure to the toxin and persisted for at least 4 days (for contaminated diet at levels of 7 µg/g fumonisins or higher) after removal of the exposure (see Fig. 2) (Visconti et al. 1998). On the other hand, no relevant effect could be observed on the SA/SO ratio in urine of rats fed for 7 days (no pretreatment) with 4 mg/kg (see below: detoxification). The duration of the exposure seems, therefore, to play an important role in eliciting the biomarker response. ECONOMIC LOSSES CAUSED BY FUSARIUM MYCOTOXINS Factors that influence the degree of fungal infestation and mycotoxin contamination on cereal production include the prevailing weather conditions (Visconti 1996), the susceptibility of the crop to fungal invasion and mycotoxin contamination (Visconti 1996, Pascale et al. 1997), and growth stage during infestation (Chulze et al. 1996). During seasons of extensive mycotoxin contamination, grain shortages may occur leading to elevate prices and costs for livestock and poultry producers and consumers of grain products. Additional chemical monitoring expenses also have to be added to the cost of the grain. Fusarium infestation may reduce crop yields, germination rates, seedling vigour, and grain quality. Mycotoxin contaminated grain my be downgraded from food to feed grade, and additional cleaning and milling procedures may be required to reduce contamination. Highly contaminated crops may be diverted to non-feed uses such as ethanol production. In some cases it may be more economical to destroy the crop in the field rather than harvest an unmarketable product. Improper drying and storage can lead to increased contamination (Charmley et al. 1995). Restricted market, nonmarketable product, price discounts, increased production costs (pest control, irrigation), increased postharvest costs (on-farm drying, testing and sampling and detoxification), increased transportation costs, inability to obtain loans on stored grain, disposal of useless crops (buried, burning) are common problems faced by crop producers because of mycotoxin contamination (CAST 1989). Representatives of the New York Corn Growers Association estimated, conservatively, that approximately $12 million was lost by corn producers in New York in 1990, due to discounted prices caused by low to moderate contamination with deoxynivalenol and in some cases zearalenone. Nearly 40% of the 1990 crop in western and central New York was not sold because of this contamination. In 1980, when Ontario winter wheat was extensively infested with Fusarium, producers lost domestic sales, shiploads of wheat were embargoed, and the wheat in storage had to be repositioned, all at additional expense (Charmley et al. 1995). Fusarium head blight losses to barley producers in the upper Midwest of the USA over the last five years (1992-1997) have been estimated to as high as $364 million. Deoxynivalenol reduces barley quality, lowering producer prices, and limiting the amount of uninfected barley available for the malting and brewing industries, which are not willing to accept barley with detectable levels of deoxynivalenol because conditions during malting promote Fusarium growth, leading to malted samples containing higher level of the toxin than in the original seeds. The limited amount of uninfected barley in the U.S. has had effects on the malting and brewing industry, which paid and extra $200 million in 1994-1995 to get non-infected grain from Canada (Dahleen 1997). 8
  • Most economic losses due to the consumption of mycotoxin-contaminated diets by farm animals result from reduced animal production and increased disease incidence. Livestock producers are effected by increased costs deriving from higher mortality rates, reproductive failures (abortion), reduced feed efficiency (higher feed costs, lower live weight, infertility syndrome, increased susceptibility to disease), overall quality loss, monitoring and testing. Swine are very sensitive to the presence of Fusarium mycotoxins in their diet. Deoxynivalenol can cause reduced feed intake, and vomiting, reduced body weight gain or body loss. Delay in the time taken for pigs to reach the ideal marketing body weight or marketing pigs below this weight can have serious economic consequences for pig producers. Deoxynivalenol has also been associated with abortions, stillbirths, and weak piglets. Immunosuppression with its associated increases in infection and disease incidence also increases production costs. Zearalenone affects reprodution in swine and can have major economic effects in farrow-to-finish pig enterprises where the number of pigs produced per sow per year determine profit margins. Based on 1991 prices, it was calculated that a 10 or 20% reduction in farrowing rate combined with a 10 or 20 % reduction in growth (as may occur if deoxynivalenol and zearalenone contaminated feed was consumed) would result in a 17 to 44% reduction in profit margins, due to increased feeding and veterinary costs per head, and a decline in the number of pigs marketed (Charmley et al. 1995). Documented outbreaks of contamination of corn in the USA corn belt with Fusarium spp. in several years caused feed refusal and vomiting in swine, and resulted in extensive losses. In nortwest Ohio in 1975, 46% of the grain sample from four counties was contaminated, resulting in additional feed costs of $0.50 per bushel (1 bushel = 0.035 cubic meters). High losses were sustained and many producers faced financial disaster (CAST 1989). Poultry are tolerant to the presence of deoxynivalenol in their diet, and there is little transmission of deoxynivalenol into eggs. On the other hand, T-2 toxin causes adverse effects in laying hens. Moreover, consumption of feed containing a combination of toxins has a greater adverse effect on poultry than when feeds containing a single toxin are fed. Cattle are relatively tolerant to the presence of Fusarium mycotoxins in their diet, but there are a few reports of adverse effects in cattle fed contaminated feed (Charmley et al. 1995). The economic implications of animal feed contaminated with fumonisins are significant, especially if contamination results in death of livestock. A number of reports have appeared on fumonisin contamination of feeds and maize screenings from feed manufacturers, not necessarily implicated in disease outbreaks (Marasas, 1995, Visconti et al. 1995). The presence of T-2 toxin in the fermentation substrate can reduce fermentation efficiency and inhibit yeast efficiency. Although most mycotoxins do not transfer into the alcohol during fermentation processes, their concentration in the spent grains make them unsuitable for animal feed without detoxification (CAST 1989). Mycotoxin exposure in humans increases medical and welfare costs, and reduces income potential of the individual. Consumer problems are related to less nutritious food, increased health risks in years of severe mycotoxin contamination, higher product prices, long term chronic effects from low-level contamination. Social costs are represented by regulatory costs (establishing standards and tolerances, surveillance and assay, enforcement), research and extension, education, lower foreign exchange earnings, increased costs of imports (CAST 1989, Charmley et al. 1995). DETOXIFICATION OF FUSARIUM MYCOTOXINS The Fusarium mycotoxins are relatively stable under most food processing conditions and can be detected in most cereal based foods. Cooking of spaghetti and noodles prepared from wheat reduced the level of deoxynivalenol to 53% 9
  • (Nowicki et al. 1988), while cooking of polenta from corn flour reduced the levels of fumonisins B1 and B2 to 91 and 80%, respectively (Pascale et al., 1995). Several Fusarium toxins, including zearalenone, deoxynivalenol and fumonisins, can be solubilized in beer during brewing, while no mycotoxins have been detected in ethanol produced from contaminated cereals. Specific processes used in food preparation, such as nixtamalization of maize dough (cooking with Ca(OH) 2) reduces consistently the fumonisin levels of contaminated corn, but the product of fumonisin degradation (hydrolyzed fumonisin) appear to retain the original toxicity when fed to rats. Similarly no reduction in toxicity was found when ammoniated fumonisin B1 was fed to rats although the fumonisin B 1 contamination level was reduced by 45% in the treated material (de Nijs 1998, and references therein). Physical removal of the fines or screenings from bulk shipments of corn reduced the fumonisin content by 26-69% (Sydenham et al. 1994). Starch prepared from contaminated corn by wet milling was fumonisin-free. However, the gluten and fiber fractions contained considerable amounts of fumonisin and required further decontamination before they could be used for animal feed (Bennett and Richard, 1996). Gamma irradiation reduced T-2 toxin, zearalenone and deoxynivalenol levels of wheat, corn and soybeans, respectively, with 16, 25 and 33% (Hoosshmand and Klopfebstein 1995), and deoxynivalenol and fumonisins (FB1 and FB2) in corn with 13% and 20%, respectively (O’Neill et al. 1993; Visconti et al. 1996). A wide variety of chemicals, including calcium hydroxide monomethylamine, sodium bisulfite, moist and dry ozone, chlorine gas, hydrogen peroxide, ascorbic acid, hydrochloric acid, sulfur dioxide, formaldehyde, ammonia and ammonium hydroxide, have been found to be effective (at different extents) against several Fusarium mycotoxins, including deoxynivalenol, zearalenone, T-2 toxin, DAS, and fumonisins. In particular sodium bisulfite is one of the most effective against deoxynivalenol in corn, although the treatment is not suitable for direct application to human foods. Often chemical treatments have been used in combination with physical treatments to increase the efficacy of decontamination (Charmley et al. 1995, and references therein). The administration of T-2 toxin specific antibodies neutralized the in vitro inhibitory effects on protein synthesis in human B-lymphoblastoid cultures and protected rats from lethal T-2 toxicosis (Feuerstein et al. 1988). Similarly T-2 toxin monoclonal antibodies inhibited human lymphocytes blastogenesis in a specific and dose-dependent manner, with neutralization occurring in almost equimolar conditions between T-2 and antibody. On this basis a rational for clinical use of T-2 toxin specific monoclonal antibody in prophylaxis and theraphy of T-2 toxemia has been proposed (Minervini et al. 1994). A more appropriate approach to detoxification of mycotoxins involves the use of adsorbent materials with the capacity to tightly bind and immobilize mycotoxins in the gastrointestinal tract of animals, then reducing the bioavailability of the toxin. Dietary addition of zeolite, bentonite, or spent bleaching clay from canola oil refining have been shown to alter the effectof T-2 toxin and zearalenone in rats (CAST 1989). An extensive review on the prevention of toxic effects of mycotoxins by means of non-nutritive adsorbent compounds has been published by Ramos et al. (1996), from which the following informations have been taken out with reference to Fusarium mycotoxins. Cholestyramine has been used to adsorb zearalenone in vitro from gastric and intestinal simulated fluids. This resin, used extensively for in decreasing total and LDL cholesterol, adsorbed almost 100% of the mycotoxin present in the medium when used at concentration over 1%. One gram of cholestyramine was able to adsorb over 1.76 to 2.00 g of zearalenone. Anion exchange resins, such as divinylbenzene-styrene polymers, exhibited benficial effects when added to diet of T-2 intoxicated rats; the growth-depressing effect caused by T-2 toxin was minimized and the reduction in feed consumption was overcome with 5% and 7.5% of resin, respectively. 5% of 10
  • divinylbenzene-styrene polymers (anion-exchange) added to diets of rats supplemented with 10 mg zearalenone per 100 g of body weight (2 weeks) resulted in reduced renal and hepatic residues of zearalenone and its metabolites.The treatment resulted in a major decrease in urinary excretion of conjugated zearalenone and its metabolites. 0.2% polyvinylpyrrolidone, added to the diets of pigs contaminated with deoxynivalenol (5-14 mg/kg of feed), did not apear to alleviate the toxic effect of this toxin when fed to barrows and gilts over a period of 5 weeks. Bentonite has been shown to be effective against T-2 intoxications in rats, but it was ineffective against zearalenone and nivalenol in pigs. The efficacy of aluminosilicates (HSCAS), which are very effective with regard to preventing aflatoxicoses, was limited against zearalenone and practically zero for T-2 toxin, diacetoxyscirpenol and deoxynivalenol. Beneficial effects of activated charcoal have been shown in rats intoxicated with T-2 toxin. Superactivated charcoal (2-3 fold the absorptive capacity of other leading charcoals) had a beneficial effect (70% survival) on rats intubated with T-2 toxin at levels 6 fold the LD50. When administered to rats after 3-5 h from having been given a lethal dose of T-2 toxin, it prevented deaths with the median effective dose being 0.175 g/kg bw. The mechanism of this beneficial effect has been postulated to be the ability, shown in vitro, of the charcoal to bind the mycotoxin, preventing its absorption and especially enterohepatic recirculation (Ramos et al. 1996, and references therein). Alteration of bioavailability of fumonisins by binding agents. Galvano et al. (1997) identified some commercial activated carbons with high affinity for fumonisin B 1 after testing in vitro several activated carbons to establish the relation of adsorption ability to physicochemical parameters, such as surface area, iodine number and methylene blue index. Consequently, we have tested one of the most promising of these materials for its ability to protect rats in vivo from dietary fumonisin toxicity (Solfrizzo et al. 1998a). Rats (four animals Table 2. Measurements of weight and SA/SO ratio of biological samples from rats exposed to fumonisins through Fusarium moniliforme contaminated diet, with 2% or without activated carbon (AC) CONTROL DIET CONTAMINATED DIET REVERSE CONTAMINATED DIET + AC PARAMETER (+2% AC) (4 mg/kg FB1+FB2) (4 mg/kg FB1+FB2 + 2% AC) DIET* Liver weight (g) 7.27 a** 9.45 b 9.58 b 6.94 a SA/SO in liver 0.06 a 0.09 a 0.16 b 0.09 a Kidney weight (g) 0.96 a 1.04 a 1.07 a 0.95 a SA/SO in kidney 0.19 a 1.10 b 0.15 a 0.88 b SA/SO in urine 0.37 a 0.41 a 0.27 a 0.38 a * Rats exposed for one week to fumonisin contaminated diet followed by one week of blank control diet ** Data with same letter in individual row represent means of 4 measurements which have no significant difference, while different letters indicate significant difference (P<0.05) per group) were fed diets containing 0 or 4 µg/g FB1 + FB2 (alone or in combination with 2% activated carbon) ad libitum for one week; adequate amounts of fumonisin producing Fusarium moniliforme maize culture were incorporated 11
  • into blank control diet to obtain contaminated diets. Urine samples were collected from metabolic cages after one week feeding; liver and kidney tissues were collected and weighed after sacrifying the animals. The elevation of SA concentration or SA/SO ratio were used as fumonisins biomarkers. The results of the all experiment are summarized in Table 2. The organ weights and on the biomarker value in urine and organs are reported to indicate the effects of fumonisins administration and the (eventual) reverse effects of the washing out (reverse diet) or of the addition of activated carbon. Rats exposed to contaminated diet showed statistically significant increase of liver weight (P<0.05, compared to control) whereas the kidney weight remained costant. The analysis of SA and SA/SO in kidney, liver and urine of fumonisin treated rats showed statistically significant increase of SA and SA/SO in kidney, and SA in liver. SA mean concentration and SA/SO ratio in liver and kidney of rats exposed to contaminated diet supplemented with activated carbon were lower than the corresponding values of rats fed only with contaminated diet, but the differences were not statistically significant. In contrast, the addition of activated carbon resulted effective (P<0.01) in avoiding the increase of liver weight caused by the diet containing Fusarium moniliforme maize culture (Solfrizzo et al. 1998a). The reversibility of fumonisins to elicite the biomarker was investigated in kidney and liver of four rats exposed consecutively to one week of contaminated diet and one week of blank control diet. Mean values of kidney SA/SO returned to control values after one week feeding blank control diet. In contrast mean value of SA/SO in liver was significantly elevated after one week washing out, indicating that the effect of fumonisins on sphingolipid metabolism in liver last longer than in kidney, although the quantitative effect (increase of SA/SO value) in liver is lesser than in the kidney. The analysis of SA/SO in urine of treated rats did not give any information as mean values were not statistically significant from the control values, although in a previous experiment was demonstrated that 2 µg/g of fumonisins were enough to elicite urinary SA/SO. The disparity of the results between the two experiments could be explained by the longer exposition to fumonisins used in the previous experiment (see above: biomarkers). In particular the rats were exposed for 9 days to 0.5 µg/g of fumonisins followed by 13 days to 2 µg/g of fumonisins whereas in this experiment rats were fed with contaminated diet only for one week. Several adsorbent materials, namely celite, bentonite, activated carbon and cholestyramine, have been tested in our laboratory at various fumonisins concentrations to evaluate the in vitro capacity to adsorb fumonisin B1 (Solfrizzo et al. 1998b). Experiments were performed by incubating the sorbent material (1 mg/ml) with increasing concentrations of fumonisin B1 in water (up to 260 µg/ml). Celite was not effective even at the lowest tested fumonisin concentration (3.2 µg/ml), while bentonite showed a relatively low affinity for fumonisin, with ca. 12% adsorbption from a solution containing 13 µg/ml FB1. Activated carbon and cholestyramine exhibited a good adsorption capacity even at high toxin concentrations (62% and 85%, respectively, at FB1 concentration of ca. 200 µg/ml). The adsorption isotherms of activated charcoal and cholestyramine, shown in Fig. 3, clearly indicated cholestyramine as the best candidate for testing in vivo the adsorbing potential with respect to FB1. The effectiveness of the binding agent in vivo in rats fed with fumonisin contaminated diets was evaluated by using the measurements of the SA/SO ratio as biomarker of the bioavailable fumonisins. The value of the SA/SO ratio decreased significantly (P<0.01), from 2.82 to 1.36, when 2% of cholestyramine was added to a diet containing 20 µg/g of fumonisins (FB1 + FB2) (Solfrizzo et al. 1998b). This result demonstrates the beneficial effect of cholestyramine in reducing the bioavalaibility of fumonisins in vivo in rats at the gastrointestinal level. The effectiveness of this kind of compounds, highly charged quaternary ammonium (strong 12
  • 0 10 20 30 40 50 60 70 80 Unadsorbed FB1 concentration (µg/ml) Fig. 3. Adsorption isotherms of cholestyramine and activated carbon with respect to fumonisin B1. anion exchange) resin, could be due to the sum of two factors, the ion exchange capacity of the adsorbent and its capacity to physically entrap the fumoninins in the polymeric matrix. PUBLIC HEALTH ASPECTS OF FUSARIUM MYCOTOXINS IN FOOD Health risks associated with the consumption of cereal products contaminated with Fusarium mycotoxins are worldwide recognized and depend on the extent to which they are consumed in a diversified diet. Outbreaks of human intoxications associated with wheat contaminated by F.graminearum and relevant mycotoxins have been reported in India, China and Japan, with symptoms including nausea, abdominal pain, throat irritation,
  • Table 3. Worldwide recommended regulatory limits on Fusarium mycotoxins in cereals and cereal products Country Mycotoxin Commodity Limit ng/g Austria Deoxynivalenol Wheat, rye / durum wheat 500 / 750 Zearalenone Wheat, rye, durum wheat 60 Brazil Zearalenone Maize 200 Canada Deoxynivalenol Uncleaned soft wheat (food) 2,000 Diets for: Deoxynivalenol swine, young calves, lactating dairy animals 1,000 cattle, poultry 5,000 HT-2 toxin swine, young calves, lactating dairy animals 25 cattle, poultry 100 All mycotoxins Feedstuffs for reproducing animals 0 France Zearalenone Cereals 200 Hungary T-2, HT-2, DAS, NIV Flour and muesli 300 Zearalenone “ “ 100 Deoxynivalenol “ “ 1,000 “ bran for meal 1,200 “ cereals 2,000 Israel T-2 toxin Grain for feed 100 Netherlands All mycotoxins Cereal(product)s 0 Romania Zearalenone All foods 30 Russia T-2 toxin Cereals (wheat hard and stron type), flour, wheat bran 100 Zearalenone “ “ “ “ “ “ 1,000 Switzerland Fumonisins B1+B2 Maize (products) 1,000 Uruguay Zearalenone Maize, barley 200 U.S.A. Deoxynivalenol Finished wheat products (food) 1,000 “ “ Grains and grain by-products for feed 5,000-10,000* Fumonisins Maize (products) for feed 5,000- 50,000** From: FAO 1997, Eriksen and Alexander 1998 * 5,000 : not exceeding 40% of the diet, destined fir swine (not exceeding 40% of the diet); 10,000: destined for ruminating beef and feed lot cattle older than 4 mos and for chicken (not exceeding 50% of the cattle or chicken total diet). **feed destined to horses (5,000), pigs (10,000), beef cattle and poultry (50,000) diarrhoea, dizziness and headaches (Beardall and Miller 1994). In China several thousands people have been recorded as victims of outbreaks attributed to the consumption of scabby wheat and moldy maize; typically, people became ill from 5 to 30 min after consumption; deoxynivalenol and zearalenone at doses considered toxic were found in samples of the incriminated food. An outbreak of disease affecting about 50,000 people in the Kashmir Valley (India) was attributed to the consumption of bread made from rain-damaged wheat; the wheat associated to mild gastrointestinal tract symptoms was reported to contain several trichothecenes. Pre-puberty was observed in children in Puerto Rico, possibly caused by zearalenone ingestion (Beardall and Miller, 1994). Alimentary toxic aleukia has been widely reported in the former U.S.S.R. since 1913 and has been attributed to the consumption of grain contaminated with Fusarium fungi, which have been shown to produce trichothecenes (T-2 toxin
  • and other type A trichothecenes). The most severe outbreak occurred in the spring of 1944 in the Orenburg district, in which 10% of the population were affected and mortality rates were as high as 60% in some counties. Clinical features of the disease include leukopenia, agranulocytosis, bleading from the nose, throat, and gums, necrotic angina, a hemorragic rash, sepsis, exhaustion of the bone marrow, and fever (Beardall and Miller, 1994). The oesophageal cancer has been somewhat associated with the maize infected by Fusarium moniliforme and/or Fusarium graminearum and relevant mycotoxins (fumonisins or trichothecenes and zearalenone) in some regions of South Africa and China (Beardall and Miller, 1994). Home-grown maize in certain rural areas can be contaminated at >100 µg/g, like Transkei region in South Africa, and Linxian and Cixian counties in China. In some of these areas the consumption of maize contaminated at high levels of fumonisins has been associated with a high incidence of human oesophageal cancer (Marasas 1995). High levels of fumonisins (up to 20 µg/g) have also been found in maize based food (maize, maize flour and polenta) in Italy. Increased human consumption of maize meal in certain regions of Italy has also been associated with increased risk of oesophageal cancer compared to other parts of the country and western Europe, although a direct causal role for fumonisins in the aetiology of such tumors has not been established (Visconti et al. 1998). The hazards of Fusarium mycotoxins to the human and animal health have lead government authorities of several countries to recommend regulatory limits for the presence of some of these mycotoxins in several cereal based foods or feeds (see Table 3). The Nordic Council of Ministers has recently published a monograph for the risk assessment of Fusarium toxins in cereals which reports the temporary tolerable daily intake (tTDI) of some of these mycotoxins for the population of the Scandinavian countries (Eriksen and Alexander 1998). A summary of the report is presented in Table 4. Tolerable daily intakes (TDIs) of fumonisin B1 in the Netherlands, based on a safety factor of 100, have been calculated as following: 500 and 2300 ng/kg bw based on the no observed effect levels (NOELs) estimated in toxicity studies in rats with purified FB1 administered by gavage and amended food, respectively, and 1000 ng/kg bw derived from FB1 related toxic effects observed in horses (de Nijs 1998). The exposure estimate for the Netherlands was 4, 57, and 220 µg FB1/day/person based on consumption of 3, 42, and 162 g of maize and a mean FB 1 content of 1.36 mg/kg in the maize (de Nijs 1998). It was estimated that from the group at risk, people with gluten intolerance such as people with celiac or Duhring’s disease (0.02 % of the entire population), 37% is daily exposed to an intake of > 100 µg FB1 and 97% to levels of > 1 µg FB1 per person, whereas for all people these percentages would be 1% and 49%, respectively (de Nijs 1998). Human exposure estimates or risk assessment have been proposed for fumonisins in other countries including Switzerland, Canada, South Africa, U.S.A.. The results of these risk assessments (depending on safety/uncertainty factors chosen) indicate a range of possibilities from very low risk in Canada (<0.089 µg/kg bw/day over the period 1991-1995) to a very high risk in subsistence farmers on a maize staple diet in parts of rural South Africa. Estimates of human exposure in the Transkei, South Africa, ranged from 14 to 440 mg/kg b.w./day, varying considerably according to the source and extent of maize in the diet as well as the extent of Fusarium kernel rot prevalent in the harvested crop. These illustrate the considerable impact of differing maize consumption patterns by different population groups (Marasas, 1997).
  • Table 4. Critical effect in animal studies, established temporary tolerable daily intake (tTDI) and estimated average Scandinavian intake of some Fusarium toxins.* Toxin Critical effect in animal Proposed tTDI Uncertainty factor Average Scandinavian studies (µg/kg bw) (µg/kg bw) used in the assessment intake (µg/day) T-2 / HT-2 toxins 100-200a 0-0.2 1000 0.14-0.20 DON 50b; 100c 0-1 100 0.4 - 0.7 NIV 0.05 -0.09 ZEN 50d; 16000a 0 -0.1 (matematical 0.02 extrapolation with a risk level of 10-6) FB1 <1000e; 3500a <1 not estimated *From: Eriksen and Alexander, 1998 a proliferative/carcinogenic effect; bvomiting; cgeneral and immunotoxic effect; d hormonal/reproductive effect; e liver/brain damage CONCLUSIONS Trichothecenes, zearalenone and fumonisins are distributed widely in cereal crops, to the extent of ubiquity in certain crops grown in specific regions and seasons. During seasons of extensive mycotoxin contamination, grain shortages may occur leading to elevate prices and costs for livestock and poultry producers and consumers of grain products. Most economic losses due to the consumption of mycotoxin-contaminated diets by farm animals result from reduced animal production and increased disease incidence. Immunosuppression with its associated increases in infection and disease incidence also increases production costs. Animal diseases such as infertility, vaginal or rectal prolapse, anorexia, skin and gastrointestinal irritation, haemorraging, abnornal offsprings, leukoencephalomalacia, pulmonary edema, liver tumours, etc. could be ascribed to the consumption of feed contaminated with these mycotoxins. Many approaches have been used to reduce the toxicity of mycotoxin contaminated feed. However, most methods have been tested on a limited number of specific toxins. Since contaminated cereals may contain a broad range of mycotoxins of differing chemical characteristics, including heat stability, solubility, and adsorbent affinity, a detoxification procedure that works well for individual toxins may not be effective for the diverse mycotoxin combinations that occur naturally. Health risks associated with the consumption of cereal products contaminated with Fusarium mycotoxins are worldwide recognized and depend on the extent to which they are consumed in a diversified diet. To some extent, the presence of small amounts of Fusarium mycotoxins in cereals and related food products is unavoidable; this necessitates risk assessments carried out by regulatory bodies in several countries to help establish regulatory guidelines to protect public health. By assessing the levels in food at which these substances may pose a potential risk to human health, it is possible to devise appropriate risk management strategies. However, several important factors have to be taken into account in making a rational risk management decision, including adequate toxicological data and knowledge of the level of exposure, availability of technically sound analytical procedures (including sampling), socioeconomic factors, food intake patterns and levels of mycotoxins in food commodities which may vary considerably between countries.
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