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

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

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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 carcinogenicitya

Overall evaluationa

In human In animals

Toxins derived from: F. graminearum,F. culmorum, F. crookwellense I Group 3

Zearalenone ND LNivalenol I

Fusarenone X IDeoxynivalenol I

Toxins derived from: F. sporotrichioides ND Group 3

T-2 toxin L

Toxins derived from:F. moniliforme I S Group 2B

Fumonisin B1 LFumonisin B2 I

Fusarin C L*From IARC, 1993a 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.

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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

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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 (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 toxinHT-2 toxinAcetyl T-2 toxinT-2 triolT-2 tetraol

8-acetoxyneosolaniolAcuminatinTetracetoxy T-2 tetraolNeosolaniol4,8-diacetoxy T-2 tetraol

Diacetoxyscirpenol

OHOHOAcOHOH

OHOHOAcOHOH

OH

OAcOHOAcOHOH

OAcOHOAcOAcOac

OAc

OAcOAcOAcOHOH

OAcOAcOAcOAcOH

OAc

HHHHH

HHHHH

H

O-IsovalO-IsovalO-IsovalO-IsovalOH

OAcOAcOAcOHOAc

H

0.0003b

0.00250.0250.0400.200

0.0030.00450.0220.050.08

0.001

0.0010.020.030.090.28

0.0020.010.040.150.10

0.001

Type B

Fusarenon-XNivalenolDeoxynivalenol15-acetyldeoxynivalenol3-acetyldeoxynivalenol

OHOHOHOHOAc

OAcOHHHH

OHOHOHOAcOH

OHOHOHOHOH

=O=O=O=O=O

0.060.200.402.007.00

0.150.300.300.402.00

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.

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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 g/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

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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).

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

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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

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

g/g and 2 g/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

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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).

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

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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%

(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 B1 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

10

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

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 B1 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)

PARAMETERCONTROL DIET

(+2% AC)CONTAMINATED DIET

(4 mg/kg FB1+FB2)REVERSE

DIET*CONTAMINATED DIET + AC

(4 mg/kg FB1+FB2 + 2% AC)

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)

11

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

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

Unadsorbed FB1 concentration (µg/ml)

0 10 20 30 40 50 60 70 80

Ad

sorb

ed F

B1

amou

nt

(µg/

mg)

0

25

50

75

100

125

150

175

200

CholestyramineActivated carbon

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

Zearalenone

Wheat, rye / durum wheat

Wheat, rye, durum wheat

500 / 750

60

Brazil Zearalenone Maize 200

Canada Deoxynivalenol

Deoxynivalenol

HT-2 toxin

All mycotoxins

Uncleaned soft wheat (food)

Diets for:

swine, young calves, lactating dairy animals

cattle, poultry

swine, young calves, lactating dairy animals

cattle, poultry

Feedstuffs for reproducing animals

2,000

1,000

5,000

25

100

0

France Zearalenone Cereals 200

Hungary T-2, HT-2, DAS,

NIV

Zearalenone

Deoxynivalenol

“

“

Flour and muesli

“ “

“ “

bran for meal

cereals

300

100

1,000

1,200

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

Zearalenone

Cereals (wheat hard and stron type), flour,

wheat bran

“ “ “ “ “ “

100

1,000

Switzerland Fumonisins

B1+B2 Maize (products) 1,000

Uruguay Zearalenone Maize, barley 200

U.S.A. Deoxynivalenol

“ “

Fumonisins

Finished wheat products (food)

Grains and grain by-products for feed

Maize (products) for feed

1,000

5,000-10,000*

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 g/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 FB1 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

studies (µg/kg bw)

Proposed tTDI

(µg/kg bw)

Uncertainty factor

used in the assessment

Average Scandinavian

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 extrapolation witha risk level of 10-6)

0.02

FB1 <1000e; 3500a <1 not estimated

*From: Eriksen and Alexander, 1998a proliferative/carcinogenic effect; bvomiting; cgeneral and immunotoxic effect; dhormonal/reproductive effect; eliver/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.

REFERENCES

Beardall J.M., and Miller J.D. 1994. Diseases in humans with mycotoxins as possible causes. In: Miller J.D. and Trenholm H.L. (Eds.), Mycotoxins in grain. Compounds other than aflatoxins. Eagan Press, Pt. Paul (MN) USA, pp.487-539.

Bennett G.A., and Richard J.L. 1996. Influence of processing on Fusarium mycotoxins in contaminated grains. Food Technology, 50, 235-238.

Caramelli M., Dondo A., Cantini Cortellazzi G., Visconti A., Minervini F., Doko M.B., Guarda F. 1993. Leucoencefalomalacia nell’equino da fumonisine: prima segnalazione in Italia. Ippologia, 4, 49-56. *CAST (Council for Agricultural Science and Technology). 1989. Mycotoxins - Economic and health risks. Task Force report no. 116. CAST, Ames, Iowa, USA. 92 p.

Charmley L.L., Trenholm H.L., Prelusky D.B., and Rosenberg A. 1995. Economic losses and decontamination. Natural Toxins, 3, 199-203.

Chulze S.N., Ramirez M.L., Farnochi M.C., Pascale M., Visconti A., March G. 1996a. Fusarium and fumonisin ocurrence in Argentinian maize at different ear maturity stages. Journal of Agricultural and Food Chemistry, 44, 2797-2801.

Dahleen L.S. Barley Fusarium head blight research. 1997. ARS Fusarium Workshop, Richard Russell Research Center, Athens, Georgia, USA, September 16-17, pp. 19-20.

de Nijs M. 1998. Public health aspects of Fusarium mycotoxins in food in The Netherlands - A risk assessment. PhD thesis, Landbouwuniversiteit Wageningen, The Netherlands, 140 p.

Doko M.B., and Visconti A. 1994. Occurrence of fumonisins B1 and B2 in corn and corn-based human foodstuffs in Italy. Food Additives and Contaminants, 11, 433-439.

Eriksen G.S., and Alexander J., Eds. 1998. Fusarium toxins in cereals - a risk assessment. Copenhagen, Nordic Council of Ministers., 146 p.

FAO. 1997. Worldwide regulation for mycotoxins 1995. A compendium. FAO Food and Nutrition paper n. 64. FAO, Rome, Italy. 46 p.

Feuerstein G., Powell J.A., Knower A.T., and Hunter K.W. 1988. Monoclonal antibodies to T-2 toxin. In vitro neutralization of protein synthesis inhibition and protection of rats against lethal toxemia. Journal of Clinical Investigation, 76, 2134-2138.

Forsell J.H., and Pestka J.J. 1985. Relation of 8-ketotrichothecene and zearalenone analogue struture to inhibition of mitogen-induced human lymphocyte blastogenesis. Applied and Environmental Microbiology. 50, 1304-1309.

Galvano F., Pietri A., Bertuzzi A., Bognanno T., Chies M., De Angelis L., and Galvano M. 1997. Activated carbons: in vitro affinity for fumonisin B1 and relation of adsorption ability to physicochemical parameters. Journal of Food Protection, 60, 985-991.

Hoosshmand H., and Klopfebstein C.F. 1995. Effects of gamma irradiation on mycotoxins disappearance and amino acid contents of corn, wheat and soybeans with different moisture contents. Plant Foods Human Nutrition, 47, 227-238.

IARC. 1993. Monographs on the evaluation of carcinogenic risks to humans. Vol. 56. Some naturally occurring substamce: food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC, Lyon, France, 599 p.

Marasas W.F.O. 1995. Fumonisins: their implications for human and animal health. Natural Toxins, 3:193-198.

Marasas W.F.O. 1997. Risk assessment of fumonisins produced by Fusarium moniliforme in corn. Cereal Research Communications, 25, 399-406.

Merrill A.H. jr., Wang E., Vales T.R., Smith E.R., Schroeder J.J., Menaldino D.S., Alexander C., Crane H.M., Xia J., Liotta D.C., Meredith F.I., and Riley R.T. 1996. Fumonisin toxicity and sphingolipid biosynthesis. Advances in Experimental Medicine and Biology, 392, 297-306.

Minervini F., Gyongyosi-Horvath A., Lucivero G., Visconti A., Barna-Vetro I., and Solti L. 1994. In vitro neutralization of T-2 toxin toxicity by a monoclonal antibody. Natural Toxins, 2, 111-114.

Minervini F., Lucivero G., Visconti A., and Bottalico C. 1992. Immunomodulatory effects of fusarochromanones TDP-1 and TDP-2. Natural Toxins, 1, 15-18.

Nowicki T.W., Gaba D.G, Dexter J.E., Matsuo R.R., and Clear R.M. 1988. Retention of the Fusarium mycotoxin deoxynivalenol in wheat during processing and cooking of spaghetti and noodles. Journal of Cereal Science, 8, 189-202.

O’Neill K., Damoglou A., and Patterson M.F. 1993. The stability of deoxynivalenol and 3-acetyldeoxynivalenol to gamma irradiation. Food Additives and Contaminants, 10, 209-215.

Pascale M., Doko M.B., and Visconti A. 1995. Determinazione di fumonisine nella polenta mediante cromatografia HPLC. In: Dugo G, and Cotroneo A. (Eds) Atti 2° Congresso Nazionale di Chimica degli Alimenti. La Grafica Editoriale, Messina, Italy, pp. 1067-1071.

Pascale M., Visconti A., Pronczuk M., Wisiniewska H., and Chelkowski J. 1997. Accumulation of fumonisins in maize hybrids inoculated under field conditions with Fusarium moniliforme Sheldon. Journal of the Science of Food and Agriculture, 74, 1-6.

Pestka J. J., and Bondy G.S. 1994. Immunotoxic effects of mycotoxins. In: Miller J.D. and Trenholm H.L. (Eds.), Mycotoxins in grain. Compounds other than aflatoxins. Eagan Press, Pt. Paul (MN) USA, pp. 339-358.

Prelusky D.B., Rotter B.A., and Rotter R.G. 1994. Toxicology of mycotoxins. In: Miller J.D. and Trenholm H.L. (Eds.), Mycotoxins in grain. Compounds other than aflatoxins. Eagan Press, Pt. Paul (MN) USA, pp. 359-404.

Ramirez M.L., Pascale M., Chulze S.N., Reynoso M.M., March G., and Visconti A. 1996b. Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids grown in Argentina. Mycopathologia, 135, 29-34.

Ramos A.J., Fink-Gremmels J., and Hernandez E. 1996. Prevention of toxic effects of mycotoxins by means of non-nutritive adsorbent compounds. Journal of Food Protection, 59, 631-641.

Riley R.T., Wang E., and Merrill A.H. jr. 1994. Liquid determination of sphinganine and sphingosine: use of the sphinganine-to-sphingosine ratio as a biomarker for the consumption of fumonisins. Journal of Association of Analytical Chemists International, 77, 533-540.

Rotter B., Prelusky D.B., and Petska J.J. 1996, Toxicology of deoxynivalenol (vomitoxin). Journal of Toxicology and Environmental Health, 48, 1-34.

Solfrizzo M., Avantaggiato G., and Visconti A. 1997a. Rapid method to determine sphinganine/sphingosine ratio in human and animal urine as a biomarker for fumonisin exposure. Journal of Cromatography B, 692, 87-93.

Solfrizzo M., Avantaggiato G., and Visconti A. 1997b. In vivo validation of the sphinganine/sphingosine ratio as a biomarker to display fumonisin ingestion. Cereal Research Communications, 25, 437-442.

Solfrizzo M., Avantaggiato G., Carratù M.R., Galvano F., Pietri A., and Visconti A. 1998a. The use of biomarkers to assess the in vivo effect of activated carbon on fumonisins fed through diets contaminated with Fusarium moniliforme. Revue de Medicine Veterinaire, 149, 667.

Solfrizzo M., Torres A., Chulze S., and Visconti A. 1998b. Cholestyramine as a binding agent for detoxification of fumonisins: in vitro studies and determination of its effectiveness in rats. (manuscript in preparation)

Sydenham E.W., Stockenstrom S., Thiel P.G., Shephard G.S., Koch K.R., and Marasas W.F.O. 1994. Fumonisin-contaminated maize: physical treatment for the partial decontamination of bulk shipments. Food Additives and Contaminants, 11, 25-32.

Visconti A. 1996. Fumonisins in maize genotypes grown in various geographical areas. Advances in Experimental Medicine and Biology, 392, 193-204.

Visconti A., Minervini F., Lucivero G, and Gambatesa V. 1991. Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay. Mycopathologia 113, 181-186.

Visconti A., Boenke A., Doko M.B., Solfrizzo M., and Pascale M. 1995. Occurrence of fumonisins in Europe and the BCR - Measurements and Testing Programme. Natural Toxins, 3, 269-274.

Visconti A. Solfrizzo M:, Doko M.B., Boenke A., and Pascale M. 1996. Stability of fumonisins at different storage periods and temperatures in gamma irradiated maize. Food Additives and Contaminants, 13, 929-938.

Visconti A., Avantaggiato G. and Solfrizzo M. 1998. Biomarker to display the ingestion of fumonisins through contaminated diet. - Analysis of sphinganine/sphingosine ratio in biological samples. In: Marengo G. and Pastoni F. (Eds.), Proceedings of Sixth International Symposium on Microbiology of Food and Cosmetics in Europe. pp. 260-280.