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been found in samples of pistachio nuts, pecans,

wheat, and green coffee beans as well as reported in

processed foods. Reports claim that its occurrence is

not as frequent as aflatoxins, but this may be due to

the lack of sensitive methods for its detection.

Citrinin

0023 Citrinin was first recognized as a promising antibiotic

but it was later found to cause kidney damage, retard

growth, and eventually cause death in animals. Citri-

nin was isolated in the 1930s and produced by

Penicillium citrinum; however, P. verrucosum is also

known to produce the toxin. It is known as the

yellow-rice toxin discovered in Japan in 1950–60,

when hundreds of tonnes of rice imported from vari-

ous parts of Asia were declared unfit for consump-

tion. It was found in cereal products in parts of

Europe, north America, and Canada and in maize

samples from South-east Asia. A survey in Denmark

detected both citrinin and OTA in barley samples and

it is usually a cocontaminant with OTA, causing

damage to the kidneys.

Penicillic acid

0024 Penicillic acid was first isolated in 1913 from a moldy

corn culture and has never been found in processed

foods as it is an unstable compound. Penicillic acid is

produced by Penicillium genera and is usually found

in apples, corn, and beans at moderate incidence.

Additional fungal species that occur during storage

are able to produce substantial amounts.

Miscellaneous

0025There are many other mycotoxins that have been

known to occur in foods and feeds; however, more

work is required to establish their occurrence pattern

and implications involved. Names of some myco-

toxins are: alternaria toxins, aspergillic acid, kojic

acid, ergotoxins, rubratoxins, the group of tremor-

genic mycotoxins, and many more.

Determination

0026Once mycotoxins are formed, they are difficult to

eliminate and being able to detect them is very im-

portant to ensure consumer protection. Their levels

can vary from mg to pg levels, therefore, identifica-

tion and quantitation at such low levels are challenges

for the chemist. Since their discovery, a large number

of methods have been developed by scientists and

some have been used more extensively than others

as their diversity varies depending on their physical

and chemical properties. The methods used for the

identification and quantitation of mycotoxins must

be sensitive, specific, and as simple as possible. The

presence of mycotoxins in their natural substrate is

extremely unhomogeneous, therefore, it is critical

that a representative sample is selected to avoid

obtaining false results. Since sampling comprises the

highest source of variation in the analytical proced-

ures, a large number of random samples must be

taken from a lot of commodity using the appropriate

device. Although it is not always feasible and

tbl0002 Table 2 The most common methods used for the analysis of various mycotoxins

Mycotoxin Extraction solvents Clean-up Detectionmethod Approval

status

Immunoassays Chemical

Aflatoxins Me, AN, W, DiClMt, Ac IAC ELISA HPLC AOAC

Ochratoxin A Me, AN, W, Cf, phosphoric

acid

IAC ELISA HPLC AOAC

Patulin Ac, EtOAc, Silica gel 60, fluorosil,

celite

P HPLC AOAC

Fumonisins Me, AN, W IAC AOAC

HPLC (derivative)

Trichothecenes Me, AN, W, Cf, DiClMt,

EtOAc

MycoSep ELISA GC/MS (derivative)

Zearalenone EtOAc, Me, AN, Cf, W IAC ELISA HPLC AOAC

Cyclopiazonic acid Me, W, phosphoric acid,

hydrochloric acid

HPLC

Sterigmatocystin Me, KCl, AN P HPLC

Citrinin Cf, phosphoric acid HPLC

Moniliformin AN, W C

bond-elute HPLC

Penicillic acid AN, W, KCl HPLC

AC, acetone, AN, acetonitrile, Cf, chloroform, DiClMt, dichloromethane, EtOAc, ethyl acetate, Me, methanol; IAC, immunoaffinity column; W, water;

P, polyclonal antibody; ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; GC/MS, gas chromatography–

mass spectrometry; AOAC, Association of Official Analytical Chemists; KCl, potassium chloride.

4094 MYCOTOXINS/Occurrence and Determination

economical to obtain a large number of samples, a

balance between sampling variation and costs should

be maintained.

0027 Once appropriate sampling has taken place, the

mycotoxins are isolated from their substrate by ex-

traction. Several aqueous, organic solvents or mix-

tures of them with water and/or acid are used to

assist the partition of the toxin in the solvent. Such

solvents include methanol, acetonitrile, chloroform,

dichloromethane, ethyl acetate and many others,

shown in Table 2. The extract obtained is then

cleaned up by various types of columns, which

remove potential interfering compounds. This is a

crucial step as the level of sensitivity depends on

how clean the sample is. Immunoaffinity columns

(IACs) are the most common clean-up and concen-

tration systems used because they are simple and easy

to use and the fact that the analysis is fully automated

makes them amenable to a large number of samples.

They are coupled with specific antibodies developed

for the target mycotoxin and, when the extract is

passed through the IAC, the toxin is bound and is

then eluted with the appropriate solvents. The eluate

is then ready for analysis either by a chemical or

immuno-based method. Other clean up columns

include MycoSep, fluorosil, C

, and celite.

0028 After clean-up, the sample will be subjected to

analysis to identify and quantitate the mycotoxins

present. The method used for this purpose depends

on the chemical properties of the mycotoxin, as sep-

aration of the contaminants must take place prior to

their quantitation. Thin-layer chromatography (TLC)

was one of the first and most widely used chemical

methods for mycotoxin analysis and relied on the

fluorescent properties of the mycotoxin because the

color produced and its intensity reflect the concen-

tration of the toxin. Although TLC is a powerful tool

in mycotoxin analysis, the data obtained are con-

sidered to be presumptive as they roughly estimate

the quantity of the toxin and as a consequence further

confirmation is required. High-performance liquid

chromatography (HPLC), on the other hand, is con-

sidered a more sophisticated and sensitive technique

which can provide information regarding the identity

and quantity of a compound simultaneously. It is also

possible to increase the fluorescence of a compound

by derivatization, increasing the sensitivity of the

method. Identification of the toxin is made by com-

parison of the retention time with a standard and its

quantity determined by the absorbance value as well

as the area of the elution peak. Therefore, HPLC has

replaced TLC and is now the officially approved

method of testing by the Association of Official and

Analytical Chemists (AOAC). Even though HPLC is

more extensively used, it suffers the disadvantages

that it can only run one sample at a time and the

samples to be tested have to be clean to achieve the

required sensitivity. In addition to other methods, gas

chromatography (GC) is commonly used for myco-

toxins that do not have chromophores and fluores-

cent groups. It is considered one of the most effective

methods for the analysis of trichothecenes and ZEN

but the extract must be derivatized prior to analysis.

Derivatization may differ depending on the detection

method used, for example, acylation agents are used

when flame ionization detection (FID) is used and

trifluoroacetyl (TFA) esters are made when an elec-

tron capture detector (ECD) is used. Confirmation of

the mycotoxin identified by GC is usually done by

mass spectrometry (MS).

0029In order to overcome the problems encountered

with the chemical methods, efforts to develop im-

munoassays have been made over the past 25 years.

Antibody-based bioassays such as enzyme-linked

immunosorbent assay (ELISA) and radioimmuno-

assay (RIA) are constantly under development and

are being used for a number of mycotoxins. When

compared with chemical methods, ELISA is more sen-

sitive, requires smaller volumes of sample, and is more

rapid, as a large number of samples can be processed

on the same ELISA plate. A larger number of anti-

bodies (monoclonal and polyclonal) exist and the

efforts to develop new ones are ongoing. The only

problem with ELISA assays is that the results obtained

are usually semiquantitative and they can roughly

estimate the quantity of the contaminant. Therefore,

it must be used in conjunction with a quantitative

method (such as HPLC) for confirmation. Table 2

gives a summary of the most commonly used reagents

and methods for the analysis of mycotoxins.

See also: Aflatoxins; Mycotoxins: Classifications;

Toxicology

Further Reading

Council for Agricultural Science and Technology (1989)

Mycotoxins: Economic and Health Risks. Task Force

Report 116.

Dhumal SS and Salunkhe DK (1992) Mycotoxins in foods.

In: Tu AT (ed.) Food Poisoning, pp. 291–334. New

York: Marcel Dekker.

Park DL, Ayala CE, Guzman-Perez SE, Lopez-Garcia R and

Trujillo S (2001) Microbial toxins in foods: algal, fungal

and bacterial. In: Helferich W and Winter CK (eds) Food

Toxicology, pp. 94–136. Boca Raton: CRC Press.

Scudamore KA (1998) Mycotoxins. In: Watson DH (ed.)

Natural Toxicants in Food, pp. 147–181. Sheffield:

Academic Press.

Sharma RP and Salunkhe DK (1991) Introduction to

mycotoxins. In: Sharma RP and Salunkhe DK (eds)

MYCOTOXINS/Occurrence and Determination 4095

Mycotoxins and Phytoalexins, pp. 13–80. Boca Raton:

CRC Press.

Smith JE, Lewis CW, Anderson JG and Solomons GL (1994)

Mycotoxins in Human Nutrition and Health. European

Commission, Directorate-General XII, Science Research

and Development.

Trucksess MW and Wood GE (1994) Recent methods of

analysis for aflatoxins in foods and feeds. In: Eaton DL

and Groopman JD (eds) The Toxicology of Aflatoxins:

Human, Veterinary and Agricultural Significance, pp.

409–432. San Diego: Academic Press.

USDA (1999) Grain Fungal Diseases and Mycotoxin Refer-

ence, pp. 1–55. Kansas City: GIPSA.

Toxicology

F S Chu, University of Wisconsin, Madison, WI, USA

Introduction

0001 Developments in the last three decades have revealed

many new mycotoxins that are potentially hazardous

to human and animals. In considering their toxic

effects, it is important to distinguish between

‘mycotoxicosis’ and ‘mycosis.’ Mycotoxicosis is

used to describe the toxic action of mycotoxin(s)

and is frequently mediated through a number of

organs, notably the liver, kidney, and lungs, and the

nervous, endocrine, and immune systems. Part of the

name of the toxin or commodity involved is generally

used to describe mycotoxicosis. For example, ‘afla-

toxicosis’ generally refers to the toxicosis having typ-

ical clinical and pathological symptoms associated

with the ingestion of aflatoxins. ‘Mycosis,’ however,

refers to a generalized invasion of living tissue(s) by

growing fungi.

0002 Owing to their diverse chemical structures, myco-

toxins may exhibit a number of biological effects,

including both acute and chronic toxic effects as well

as carcinogenic, mutagenic/genotoxic, teratogenic,

and immunotoxic effects. Their toxic effects are often

organ-specific. Once mycotoxins are ingested in the

body, the toxins may either directly react with the

target organs to exert toxic effects or be metabolized

to an active intermediate (activated) and then exert

their toxic effects. The toxins and their metabolites

may also be detoxified and excreted. Thus, factors

such as species, age, nutrition, and hormone status,

etc. that affect the metabolism of mycotoxins, can

also manifest mycotoxin toxicity. As will be shown

below, the interaction of mycotoxins, either activated

through metabolism or as intact molecules, with

cellular macromolecules plays a dominant role in

their toxic actions. Modulation of the immunosystem

also plays an important role for the overall toxic

effects, and mycotoxins can be either immunosup-

pressive (most often) or immunostimulatory. Recent

studies on the effect of mycotoxin on apoptosis fur-

ther revealed their mode of action at the cellular level.

Owing to the large body of literature on mycotoxins

that has accumulated over the years, this chapter will

focus only on the toxic effects of selected mycotoxins

(see Table 1) that are the most frequent contaminants

in foods and feed. The mode of their toxic action will

also be highlighted.

Historical Considerations

0003The mycotoxin problem is actually an old problem.

Two classical examples, i.e., ergotism and mushroom

poisons, have been known for centuries. Outbreaks of

other types of mycotoxicoses have also been recorded.

Because intoxications by ergots and poisonous mush-

rooms are a result of ingestion of a fungal body con-

taining toxic metabolites, these two types of poisons

are excluded from the most modern discussion on

mycotoxins. Nevertheless, ergots and poisonous

mushrooms still can be unintentionally introduced

into food chains in the modern days. For a historical

point of view, the toxicology of ergotism and one of

the mycotoxicoses that occurred in World War II are

briefly reviewed here.

Ergotism

0004Ergotism is a human disease that results from con-

sumption of the ergot body in rye or other grains

infected by a parasitic fungus of the genus Claviceps.

Two types of ergotisms have been documented. In the

‘convulsive’ type, the affected persons have general

convulsions and a tingling sensation in the muscles

(such as the feeling of one’s foot going to sleep), and

sometimes, the entire body is racked by spasms. Epi-

demics occurred between 1581 and 1928 in European

and other countries. Although it has been suggested

that the consumption of ergots that cause convulsive

ergotism may play a role in ‘Salem Witchcraft’ inci-

dence, this is still a controversial issue. In the ‘gan-

grenous’ type, the affected parts became swollen and

inflamed with violent, burning pains, hence, the term

‘Fire of St. Anthony.’ The affected area became numb

first, turned black, then shrank, and finally became

mummified and dry. Outbreaks occurred from the

Middle Ages up to the nineteenth century. In some

areas of France, grain contained as much as 25%

ergots. Patients died as a result of consumption of

4096 MYCOTOXINS/Toxicology

100 g of ergot over a few days. In general, 2% ergots

in the grain is sufficient to cause an epidemic. Be-

tween 1770 and 1771, about 8000 people died in

one district alone in France. European and other

countries have a regulatory limit of 0.1–0.2% ergots

in flour. Ergotisms result from the intoxication of

ergoline alkaloids that are produced by the fungus

present in the sclerotia of Claviceps purpurea. The

most active components are amides of d-lysergic acid,

including both the cyclic-type peptide and nonpeptide

amide of ergot alkaloids. These alkaloids cause

smooth-muscle contraction, block neurohormones,

have both vasoconstriction and vasodilation effects,

and also affect the central nervous system. Thus, they

have some therapeutic uses.

Alimentary Toxic Aleukia (ATA)

0005 A well-documented example of mycotoxicosis in

humans is the outbreak of ATA, a disease that

resulted in more than 5000 deaths in humans in the

Orenberg district of the former USSR during World

War II. The cause of this outbreak was later deter-

mined to be trichothecene mycotoxins produced by

fungi growing on grain allowed to stand in the

field during winter. The toxicology of ATA will be

discussed later.

Discovery of Aflatoxin

0006Although a number of antibiotics produced by fungi

were found, in the middle of the 1940s, to be toxic to

animals, renewed interest on toxic fungal metabolites

only begun after the discovery of aflatoxin B1 in the

early 1960s because of its highly potent carcinogenic

effects to test animals, and the name of ‘mycotoxin’

was adopted thereafter.

Toxic Effects of Selected Mycotoxins

Aflatoxins

0007Since various aspects on aflatoxins have been dealt

with elsewhere, the toxicology and mode of action on

this groups of mycotoxins are reviewed only briefly

herein.

0008Toxic effects Aflatoxin B1 (AFB1) is one of the most

potent naturally occurring carcinogens and is the

most toxic member of this group of mycotoxins.

Other AFs are less toxic (B1 > G1 > B2 > G2). Al-

though the main target organ is liver, AFB1 also

affects other organs and tissues, including kidney and

lungs, to a lesser degree. Typical symptoms for afla-

toxicosis in animals include proliferation of the bile

tbl0001 Table 1 Toxicological effects of selected commonly occurring mycotoxins

Major mycotoxins Toxicological effect

Mode of action

Alternaria (AAL) mycotoxins A, Hr, M Inhibition of ceramide synthase

Aflatoxin B1 (AF) and other aflatoxins A, C, H, I, M, T Form DNA adducts and mutate p53 tumor suppressor gene

Citrinin (CT) C(?) Nh, M Inhibition of protein synthesis

Cyclopiazonic acid (CPA) Cv, I, M, Nr, Alteration of Ca

2þ

hemostasis and inhibition of Ca

2þ

-dependent ATPase

Deoxynivalenol (DON) I, Nr Inhibition of protein synthesis

Cyclochlorotine (CC) A, C, H, Binding with DNA

Fumonisins (FM) A, C, Cv, H, Nr, R Disruption of sphingolipid metabolism by inhibiting ceramide synthase

Luteoskyrin (LT) A, C, H, M Binding with DNA

Moniliformin (MN) Cv, Nr Binding with pyruvate dehydrogenase and affecting TCA cycle enzymes

Ochratoxin A (OTA) A, I, Nh, T Inhibition of protein synthesis and several enzymes; enhancement of

lipid peroxidation; impaired cellular Ca

2þ

and cAMP homeostasis

Patulin (PT) C(?), Nr, D, T Inhibition of -SH enzymes

Penicillic acid (PA) C(?), Nr, M Inhibition of -SH enzymes

Rubratoxin B (RB) A, H, T Inhibition of protein synthesis

Sterigmatocystin (ST) H, C, M Same as aflatoxin

T-2 A, ATA, Cv, D, I, T Inhibition of protein synthesis, binding with peptidyl transferase,

membrane proteins and other enzymes

12–13, Epoxy-trichothecenes (TCTC)

other than T-2 and DON

A, D, I, Nr Same as T-2 toxin

Tenuazonic acid Hr Inhibition of protein synthesis

Tremorgenic toxins Nr Binding and inhibition of acetylcholinesterase

Zearalenone (ZE) G, M Binding with estrogen receptor

Letters in parentheses are the abbreviations for the mycotoxin’s name used throughout the text. A, apoptosis, ATA, alimentary toxic leukemia;

C, carcinogenic; C(?), carcinogenic effect is still questionable; Cv, cardiovascular lesion; D, dermatoxin; G, genitotoxin and estrogenic effects;

H, hepatotoxic; Hr, hemorrhagic; I, immunomodulation effects, M, mutagenic, Nh, nephrotoxin; Nr, neurotoxins; R, respiratory; T, teratogenic.

From CAST (1989) Mycotoxins, Economic and Health Risks. Task Force Report No. 116. Ames, IA: Council of Agricultural Sciences and Technology and Chu

(1998) Mycotoxins – Occurrence and toxic effects. In: Encyclopedia of Food and Nutrition – Food Contaminants, 2nd edn., pp. 858–869. New York:

Academic Press.

MYCOTOXINS/Toxicology 4097

duct, centrilobular necrosis and fatty infiltration of

the liver, and hepatomas, in addition to generalized

hepatic lesions. The susceptibility of animals to AFB1

varies considerably with species with a decrease in

sensitivity (LD50: mg kg

1

) in the following order:

rabbits (0.3), ducklings (0.34), mink (0.5–0.6), cats

(0.55), pigs (6–7 kg size, 0.6), trout (0.8), dogs (1.0),

guinea-pigs (1.4–2.0), sheep (2.0), monkeys (2.2),

chickens (6.3), rats (5.5 for male, 17.9 female), mice

(9.0), and hamsters (10.2). In addition to these acute

(hepatotoxic) effects, carcinogenic effects of AFB1 is

of most concern. A 10% incidence of hepatocarci-

noma was observed in male Fisher rats fed a diet

containing only 1 mg AFB1 per kilogram over a period

of 2 years. Rats, rainbow trout, monkey, and ducks

are most susceptible, but mice are relatively resistant

to AFB1. Because of their presence in foods and

strong evidence of their association with human

carcinogenesis, aflatoxins are still a serious threat to

human health, even after more than 40 years of re-

search. Consumption of AFB1-contaminated feed by

dairy cows results in the excretion of AFM1 in milk.

AFM1, a hydroxylated metabolite of AFB1, is about

10 times less toxic than AFB1, but its presence in milk

is of concern for human health.

0009 Immunotoxic effects Animals fed with AFB1 are

more susceptible to infection. While AFB1 primarily

affects cell-mediated immunity (CMI) and phagocytic

cells, it also reduces/suppresses delayed hypersensitiv-

ity (DH). For example, suppression of DH to keyhole

limpet hemocyanin and a decrease in splenic CD4 cell

(T-helper cells) and IL-2 were found in CD-1 and

C57B/16 mice fed with AFB1.

0010 Mode of action AFB1 must first be activated

by mixed-function oxidases, more specifically cyto-

chrome P450 1A2 and 3A4, to a putative short-lived

AFB-8, 9-exo-epoxide before exerting its carcino-

genic effects. The activated intermediate either can

be converted to hydroxylated metabolites, conjugated

to glutathione or glucuronic acid, etc. and then be

excreted, or it can bind to DNA, RNA, and protein

to exert its toxic, carcinogenic, and mutagenic effects.

Adduct formation of this intermediate with DNA

occurs thorough nucleophilic attack primarily at the

N-7 guanine position, which then involves G–Cto

T–AortoA–T nucleotide substitutions and causes

mutations.

0011 Aflatoxin-induced G:C mutations, both G to T and

G to A, have been implicated in the inactivation of

the human p53 tumor suppressor gene, and a high

frequency of mutations has been found at the third

position of codon 249 of p53. The prevalence of

codon 249 mutation, i.e., AGG to AGT (Arg to Ser)

was found to be around 50% in human primary

hepatocellular carcinoma (PHC). In contrast, the

prevalence of such mutations is low in nonPHC

patients. The identification of mutations/inactivation

of p53 at this site has been used as a biomarker for

AFB-induced liver cancers in humans.

0012The role of the covalent interaction between AFB

and specific enzymes/proteins is less clear. Several

aflatoxin metabolites do interact with enzymes and

proteins. Both aflatoxins B2a and G2a, which are

very reactive with proteins through formation of

Schiff’s bases between the dialdehyde groups in

AFB2a and the –NH

groups of proteins, have been

shown to be very effective inhibitors of DNase

in vitro. The activated AFB1 and AFG1 also react

with albumin to form albumin adducts through the

same Schiff-base mechanism, followed by Amadori

rearrangement and subsequent condensation with an-

other aldehyde group. The presence of this adduct in

human serum has been used as an index of human

exposure to AFs.

0013Aflatoxins B1 and G1 also bind with serum albu-

min, nucleic acid, and proteins such as lysine-rich

histone and chromatin noncovalently. Aflatoxicol, a

major AFB metabolite, interacts with estrogenic re-

ceptors. Although the role of such interaction was not

clearly defined, NMR analysis of the binding of AFB1

and AFG1 with double-stranded d(ATGCAT)

d(GCATGC)

and B-DNA and gel-shift analysis of

the binding of both mycotoxins with plasmid

pBR322 revealed that intercalation of aflatoxins be-

tween the base pair was involved. Intercalation of

stable epoxide-AFB1 with these nucleotides has also

been demonstrated. These data suggest that intercal-

ation may be significant for subsequent covalent at-

tachment of the carcinogen at the N7 position of

guanine in DNA. The activity of several enzymes,

such as deoxyribonuclease and RNA polymerase, is

inhibited by AFB1, but these inhibitory effects are

secondary. Recent data also showed that oxidative

DNA damage is involved in the genotoxicity of AFB.

0014Apoptosis (APT) Induction of apoptosis by AFB has

been observed in several cell systems. In the HL-60

human promyelocytic leukemia cells, the dose of

AFB1 needed for induction of APT by AFB1 was

considerably higher (1–20 mgml

1

) than those needed

by trichothecene mycotoxins (0.001–0.2 mgml

1

Such an effect was attributed to their inhibitory effect

on RNA synthesis as a result of its binding with DNA

and the inhibitory effect of DNA-dependent RNA

polymerase.

0015Aflatoxin and human carcinogenesis Whereas AFB1

has been found to be a potent carcinogen in many

4098 MYCOTOXINS/Toxicology

animal species, the role of AF in carcinogenesis in

humans is complicated by hepatitis B virus (HBV)

infections in humans. Epidemiological studies have

shown a strong positive correlation between AF levels

in the diet and PHC incidence in some parts of the

world (China, Kenya, Mozambique, Philippines,

Swaziland, Thailand and Transkei of South Africa).

Aflatoxin–DNA and AF–albumin adducts, two of the

major biomarkers for human exposure to AFB, as

well as several AF metabolites, mainly AFM1, have

been detected in serum, milk, and urine of humans in

these regions. However, the prevalence of HBV infec-

tion is also correlated to liver cancer incidence in

these regions and HBV in humans. Data on the

enhancement of mutation of the p53 gene suggest a

synergistic effect of these two risk factors for PHC in

humans. (See Aflatoxins; Cancer: Carcinogens in the

Food Chain.)

Ochratoxins

0016 Toxic effects Ochratoxin A (OA), the most toxic

member (LD50 about 20–25 mg kg

1

) and also most

commonly found toxin in this group, is a potent

nephrotoxin causing kidney damage, including de-

generation of the proximal tubule, in many animal

species. Liver necrosis and enteritis were also ob-

served. Other than acute toxic effects, OA also acts

as an immunosuppressor and teratogen. Although

OA has never been shown to be mutagenic, a weak

genotoxic effect has been demonstrated in several

systems. Ochratoxin A is only a weak nephrocarcino-

gen because a high level of toxin and an extended

period of exposure are necessary to induce the

tumors. A dose-related induction of renal tubular

cell tumors was found in Fisher rats (F344/N) with a

significant increase in renal tubular cell tumors at

levels of 70 and 210 mg of OA per kg of body weight

per day; but cancer incidence was not significantly

different from the control at lower levels (21 mgof

OA per kg of body weight per day). Recent studies

showed that OA also causes liver cancer in rats.

0017 Modulation of immune system Animals receiving

OA show general immunotoxic signs, including lym-

phocytopenia and depletion of lymphoid cells, espe-

cially in the thymus, bursa of Fabricius and Peyer’s

patches. Increased counts of total leukocytes and

neutrophils in the blood with reduced lymphocyte

levels were observed in the weaner pigs receiving

OA. A striking increase in the counts of eosinophils

and of apoptotic phagocytes was also found. De-

pressed humoral immune responses, including lower

serum IgM and IgG levels and suppressed antibody

response to SRBC and other antigens in mice, have

been observed in animals fed or injected with OA.

DH was also suppressed. The immunosuppressive

effects of OA were prevented by phenylalanine both

in vitro and in vivo. OA also stimulates cytokine

production with increase in IL-2 nd IL-5 in the

EL4.IL-2 cell system.

0018Mode of action The mode of the toxic effect of OA

is not as clearly defined as those of AFB. OA inhibits

several enzymes, including phenylalanine hydroxyl-

ase, phenylalanine-tRNA synthetase and renal phos-

phoenolpyruvate carboxykinase, which may play a

role in its toxic effects. The synthesis of the latter

two enzymes was found to be inhibited by OA. In

addition, it is also a strong inhibitor for carboxypep-

tidase A. Enhancement of lipid peroxidation is con-

sidered as one of the manifestations of cellular

damage in OA toxicity. The induction of free radicals

in a bacterial model system by OA was found to be

regulated by Ca

2þ

ions. Superoxide dismutase and

catalase have been shown to have some protective

effect for OA-induced nephrotoxicity in rats. OA-

induced lipid peroxidation in the Vero cell was also

found to be decreased in the presence of these two

enzymes, but the effects of aspartame and piroxcam

were less pronounced. In the renal epithelial cells, OA

interferes with hormonal Ca

2þ

signaling, leading to

altered cell proliferation. Thus, OA may impair cellu-

lar Ca

2þ

cyclic AMP (cAMP) homeostasis.

0019Ochratoxin A binds strongly with serum albumin;

thus, the toxin has a long half-life in animal (35 days

with a single dose of OA) and human bodies. Such an

interaction has not only played a significant role in

the toxic effect of this mycotoxin because of its

heterogenous body distribution, but also been used

as a biomarker for human exposure of OA. Although

OA has been found to bind to several proteins and

enzymes noncovalently, administration of labeled OA

to rats showed no covalent binding of OA to DNA of

kidney and liver. Using the P-32 postlabeling tech-

nique, however, OA–DNA adducts were found in

kidney, liver, and several other organs of mice and

rats with OA. Such adducts may be due to the prod-

ucts derived from OA-mediated cytotoxicity. OA

induces unscheduled DNA synthesis in cell cultures

in vitro.

0020Apoptosis OA induces APT in lymphocytes and

several other cell systems. The apoptotic effect was

attributed to its inhibition of protein synthesis. Spe-

cific cell types with distinct members of the mitogen-

activated protein kinase family were involved in the

APT at OA concentrations at which no acute toxic

effect can be observed. Induction of apoptosis via the

c-jun amino-terminal-kinase pathway can explain

some of the OA-induced changes in renal function

MYCOTOXINS/Toxicology 4099

as well as part of its teratogenic action. Exposure of

human proximal tubule-derived cells and renal epi-

thelial cell lines to low OA concentrations can lead to

direct or indirect caspase 3 activation and, subse-

quently, apoptosis. (See Immunology of Food.)

0021 Ochratoxin A and human health Ochratoxin A has

long been considered to be associated with the neph-

ropathy of people residing in certain areas of Tunisia,

Scandinavia, and the Balkan regions, where exposure

may be ‘endemic.’ The pathological lesions of neph-

ropathy in humans are similar to those observed in

endemic porcine nephropathy, which is due to the

consumption of feeds contaminated with OA. Since

a high proportion of the patients suffering from

endemic nephropathy in the Balkan region develop

tumors of the renal pelvis and ureter, the possible

involvement of nephropathy in tumor development

was suggested. Ochratoxin A has been found in

human serum in Tunisia and several European coun-

tries, including Bulgaria, Poland, Yugoslavia, and

Germany. In certain areas of the Balkans and Tunisia,

OA levels in the foods and in human serum in

endemic regions are higher than in the nonendemic

areas. OA also has been found in human milk and

kidneys in some endemic regions.

0022 Human exposure to OA could occur through con-

sumption of OA-containing cereals or animal foods.

The mean daily intake of OA by humans in Euro-

pean Union member countries, as calculated from

data on OA levels in foods and in human bloods,

was estimated to be 1.8 ng of OA kg

1

(range: 0.7–

4.7) and 0.9 ng kg

1

(range: 0.2–2.4), respectively.

The main dietary sources (55%) were from cereals

and cereal products (0.2–1.6 mgkg

1

). Thus, the EU

Committee for Food has recommended that the OA

daily intake in humans should be below 5 ng kg

1

Coffee (mean level 0.8 mgkg

1

), beer, pig meat,

blood products, and pulses also contribute to the

OA intake in humans. These findings reemphasize

the possible involvement of OA in human carcino-

genesis and the health hazard of exposure to OA in

humans. Among 77 countries that have regulations

for different mycotoxins, eight have specific regula-

tions for OA, with limits ranging from 1 to 20 mgkg

1

in different foods.

Fumonisin (Fm)

0023 Toxicologic effects Although FmB1 was originally

found to be a potent cancer promoter, it is now rec-

ognized as a carcinogen. It is primarily a hepatotoxin

and carcinogen. Feeding the toxin to rats has resulted

in cirrhosis and hepatic nodules, adenofibrosis,

hepatocellular carcinoma-ductular carcinoma, and

cholangiocarcioma. In addition to liver, lesions were

also observed in kidney, including renal carcinoma.

Tubular nephrosis was found both in rats and in

horses of field cases associated with equine leukoen-

cephalomalacia (ELEM). The effective dose of FmB1

for cancer initiation in rat liver depended on both the

level and duration of exposure. All of the three major

Fms, i.e., FmB1, FmB2, and FmB3, show cancer initi-

ation and promoting activities in rats. In cell-culture

systems, FmB1 has been found to be a mitogen, cyto-

toxic to the cells, and can alter the expression of genes

associated with the cell cycle. It induces in vivo geno-

toxicity, but has no mutagenic effect in the Salmonella

system.

0024FmB1 was identified as an etiological agent respon-

sible for ELEM in horses and other Equidae (donkeys

and ponies), and for porcine pulmonary edema (PPE)

in pigs. ELEM was originally found to be a seasonal

disease occurring in late fall and early spring. The

disease primarily causes neurotoxic effects in animals,

including uncoordinated movements and apparent

blindness. Liver damage has also been reported.

Death sometimes occurs with no nervous symptoms.

Clinically, the disease is characterized by edema in the

cerebrum of the brain. The levels of FmB1 and FmB2

in feeds associated with confirmed cases of ELEM

range from 1.3 to 27 p.p.m. In pigs, PPE occurs only

at high FmB levels (175 p.p.m.), whereas liver

damage occurs at much lower concentrations with

a no observable adverse effect level (NOAEL) of

< 12 p.p.m. Cardiovascular function is altered by

FmB1. The FmB1-induced PPE is caused by left-

sided heart failure, and not by altered endothelial

permeability. In the cattle, renal injury, hepatic

lesions, and alteration of sphingolipid in various

organs were observed. The level causing disease in

poultry is also high. Fms are also toxic to some plants:

jimsonweed, black nightshade, duckweeds and toma-

toes with the asc/asc genotype being most susceptible.

0025Mode of action The primary effect of FmB1 is

due to its disruption of sphingolipid metabolism

by inhibiting the enzyme sphinganine (sphingosine)

N-acyltransferase (ceramide synthase). Because com-

plex sphingolipids and ceramides are heavily involved

in cellular regulation, including cell differentiation,

mitogenesis, and apoptosis, such effects play an im-

portant role in the early phase of most of the diseases

discussed above. The target organs may be very

sensitive to the sphingolipid dysregulation. Because

sphingolipids are natural inhibitors of Ca

2þ

activated-protein kinase C (PKC), it has been sug-

gested that FmB may affect PKC-regulated functions.

A direct interaction of diacylglycerol binding site

of PKC to phorbol esters, which are well-known

tumor promotors, by FmB1 has been observed. The

4100 MYCOTOXINS/Toxicology

alteration of sphingolipid metabolism results in a

dramatic increase in the free sphingolipid bases, i.e.,

sphinganine (Sa) and sphingosine (So), in tissues and

a decrease in complex sphingolipid levels. Significant

increases in the So/Sa ratio were found in serum of

pigs fed a diet containing as little as 5 p.p.m. of total

FmB. Thus, Sa/So ratios in serum and urine are now

being used in testing as an early biomarker of FmB

exposure in animals and humans. FmB1 disrupts o-6

fatty acid metabolic pathway and/or prostaglandin

synthesis; such an effect is likely to be an important

event in the mitoinhibitory effect of FmB1 on growth-

factor responses. FmB1 was also found to inhibit the

cytochrome P-450 enzyme selectively with most sig-

nificant effect on CYP2C11. Such inhibition was

considered to be due to a suppressed activity of

protein kinase activity resulting from the inhibition

of sphingolipid biosynthesis.

0026 Apoptosis Whereas the role of FmB1 on immuno-

system is not entirely clear, the effect of FmB1 on

apoptosis in many cell lines has been extensively

studied. Immune cells are one type of target cell. For

example, an increase in nitric oxide and prosta-

glandin E-2 production was found when a murine

macrophage cell line was grown in the presence of

FmB1. The kidney, liver, and tomato cells, and other

animal or plant cells treated with FmB or TA (one of

the major AAL toxins), showed typical apoptotic

characters with the degree of injury being related to

the dose and time of exposure. The fundamental

elements of apoptosis, as characterized in animals,

are conserved in plants. Inhibition of the ceramide

synthase by FmB and related mycotoxins are con-

sidered to play a key role in the apoptosis of the

cells of the target organs/tissues. FmB1 diminished

the cytotoxic effects of the lipids on esophageal

tumor cells. The marked apoptosis induced by

PGA2 and the PG2- and AA-induced p53 levels

were lowered. It repressed specific isoforms of protein

kinase C and cyclin-dependent kinase activity and

induced expression of CDK inhibitors in monkey

kidney cells (CV-1). Such effects lead to cell-cycle

arrest and apoptosis. Eight genes induced by FmB1

have been identified. The ability of FmB1 to alter gene

expression and signal transduction pathways are con-

sidered to be necessary for its carcinogenic and toxic

effects. Since FmB1 is not genotoxic in bacterial

mutagenesis screens and does not affect unscheduled

DNA synthesis, the apoptotic necrosis, atrophy, and

consequent regeneration may play an essential role in

its carcinogenic effects. FmB1 may be the first

example of an apparently nongenotoxic (nonDNA-

reactive) agent producing tumors through such a

mechanism.

0027Impact on human and animal health While Fms are

commonly detected in corn-based foods and feeds,

the impact of low levels of Fms in human foods is

not known. Current data suggest that they may have

more impact on the health of farm animals than on

humans. Thus, the Mycotoxin Committee of the

American Association of Veterinary Laboratory

Diagnostics recommends that the FmB1 levels be

limited to 5, 10, 50, and 50 p.p.m. for feeds to be

used for horses, swine, beef cattle, and poultry, re-

spectively. Several reports indicated that significantly

higher levels of Fms, sometimes together with AFs,

trichothecene mycotoxins, and carcinogenic nitros-

amines, were present in corn samples collected from

areas with high rates of human esophageal cancer.

Thus, FmB1 in combination with several other etio-

logical agents may play an important role in carcino-

genesis in humans. Currently, only Switzerland

regulates the Fm level (1.0 p.p.m.) in foods.

Selected Important Trichothecene (TCTC)

Mycotoxins

0028General consideration of toxic effects The struc-

tural diversity of TCTC mycotoxins results in differ-

ent toxic effects in animals and humans. Unlike

AFB1, OA, and Fm, where the primary effects and

clinical manifestations are well defined, TCTC myco-

toxicoses are difficult to distinguish because they

affect many organs, including the gastrointestinal

tract, hematopoietic, nervous, immune, hepatobili-

ary, and cardiovascular systems. Ingestion of foods

and feeds that contain TCTC mycotoxins cause many

types of mycotoxicoses in humans and animals, in-

cluding moldy corn toxicosis, scabby wheat toxicosis

(red-mold, akakbi-byo disease, or scabby barley

poisoning), feed refusal and emetic syndrome

(swine), fusaritoxicoses, hemorrhagic syndrome, and

alimentary toxic aleukia (ATA). More than 100

TCTC have been identified in the laboratory; only a

few selected toxins that have been found in foods are

described herein.

0029T-2 toxin and other type A TCTCs Although T-2

toxin occurs naturally in cereal grains, contamination

with T-2 toxin is less frequent than with deoxyniva-

lenol (DON). However, T-2 toxin (LD50 in mice:

2–4mgkg

1

) is much more toxic to animals, perhaps

also to humans, than DON (LD50 in mice: 50–70 mg

1

). Almost all the major TCTCs, including T-2

toxin, are cytotoxic and cause hemorrhage, edema,

and necrosis of skin tissues. Inflammatory reactions

near the nose and mouth of animals are similar to

some lesions found in humans suffering from ATA

disease. The severity of lesions is also related to chem-

ical structure. Macrocyclic toxins such as verrucarin

MYCOTOXINS/Toxicology 4101

A are most active, followed by group A toxins (T-2

toxin), and least with type B toxins, such as nivalenol.

Neurologic dysfunctions, including emesis, tachy-

cardia, diarrhea, refusal of feed/anorexia, and

depression, were also observed. T-2 toxin and

some TCTCs also induce major GI lesions, including

perioral dermatitis, stomatitis, esophagitis, gastritis,

radiomimetic lesions, and sometimes hemorrhage in

the intestines. However, the major lesion of T-2 toxin

is its devastating effect on the hematopoietic

system in many mammals, including humans. Typic-

ally, there is a marked initial increase in the number

of circulating white blood cells, especially lympho-

cytes, followed by a rapid decrease to 10–75% of

normal values. Platelet counts are also reduced.

There is also extensive cellular damage in the bone

marrow, intestines, spleen, and lymph nodes, and in

severe cases, complete atrophy of bone marrow and

marked alteration of plasma coagulation factors. T-2

toxin and related TCTCs are the most potent im-

munosuppressants of the known mycotoxins and

cause significant lesions in lymph nodes, spleens,

thymus, and the bursa of Fabricus. The heart

and pancreas are other target organs for T-2 toxin

intoxication. Although urinary and hepatobiliary

lesions have been observed for T-2 toxin and diace-

toxyscirpenol (DAS), these effects are considered to

be secondary.

0030 Deoxynivalenol (DON) Deoxynivalenol, a major

type B TCTC, causes feed refusal and emesis in

swine; the name ‘vomitoxin’ is also used. Although

DON is considerably less toxic than most other TCTC

mycotoxins, the level of contamination of DON

in corn and wheat is generally high (usually above

1 p.p.m., sometimes greater than 20 p.p.m.). Con-

tamination of DON in the cereal grains is also

world-wide. Toxicologically, DON induces anorexia

and emesis in both humans and animals. Swine are

most sensitive to feed contaminated with DON. Be-

cause of the frequent occurrence of high levels of

DON in wheat and corn, its stability, and reported

food poisoning outbreaks in humans, contamination

of cereals with DON is a major concern of both the

government and food and feed industries. Contamin-

ation of DON in wheat and corn may be associated

with other toxic effects because other Fusarium

toxins, including zearalenone and other TCTCs may

be present also. Other type B TCTCs such as

nivalenol and acetylated-DON are more toxic than

DON to test animals.

0031 Modulation of immune systems Because TCTC

mycotoxins such as T-2 toxin exert a major toxic

effect on the bone marrow, lymph nodes, thymus,

and spleen in mammals, modulation of the immune

system by this group of mycotoxins has been studied

most extensively.

0032Effects on humoral immune response Trichothe-

cenes have been shown to have a marked effect on

humoral immunity. Administration of T-2 toxin and

DAS to animals results in a reduction of their resist-

ance to infection and a decrease in antibody forma-

tion. T-2 toxin may selectively affect subpopulations

of T-suppressor cells or their precursors, and this

suppression of antibody synthesis may be due to im-

pairment of either antibody-forming or T-helper cell

activities. Immunosuppressive effects have also been

observed for other TCTCs, including the macrocyclic

types. Mice fed a diet containing more than 10 p.p.m.

of DON experienced atrophy of the thymus and other

structural changes, decreased antibody formation,

as well as suppressed B- and T-cell proliferation.

However, DON is one TCTC that has both immune

stimulation and suppression effects in experimental

animals. Serum and saliva immunoglobulin (Ig) A was

significantly increased in mice fed with high levels (25

p.p.m.) of DON, whereas serum IgM and IgG de-

creased. The increased IgA production is related to

IgA-mediated nephropathy in mice; thus, it was pos-

tulated that DON might be one of the etiologic agents

in IgA nephropathy, which is the most common glo-

merulonephritis in humans world-wide.

0033Cell-mediated immunity Lymphocyte proliferation

is inhibited in vitro by T-2 toxin and DAS. Although

the response of both T- and B-cells to mitogens was

inhibited, the T-cell response was more sensitive.

There is a stimulating effect due to a low concentra-

tion of T-2 toxin. Structure–activity studies showed

that T-2, HT-2, and 3

-OH T-2 toxins were most

effective in inhibiting proliferation of mitogen-

stimulated human lymphocytes, whereas 3

-OH HT-

2, T-2 triol, and T-2 tetraol toxins were 50–100 times

less effective. The macrocyclic TCTCs, roridin, and

verrucarin A, were 75–100 times more potent than

the T-2 toxin. The differences in the immunotoxic

effects of various TCTCs were attributed to the

differences in both the uptake and metabolism of

toxins by the cells. Interleukin-1 and 2 (IL-1, 2)

production in spleen cell cultures was stimulated

by trace amounts of TCTC. Hyperinduction of

cytokines in T-helper cells by DON (most) as well

as several other mycotoxins (cyclopiazonic acid,

OA, and alpha-zearalenol) has been found, but

patulin and T-2 toxin were inhibitory. Induction of

expression of mRNA of ILs-2, 4, 5, 6 in a T-cell model

EL4.IL-2 by DON was found at levels required

for partial or maximal protein synthesis inhibition.

4102 MYCOTOXINS/Toxicology

A single oral gavage with DON is sufficient to

induce IL-1 beta, IL-2, IL-6, TNF a mRNA levels in

Peyer’s patches and spleen. The effect in IL-6-

deficient mice was refractory to DON-induced dysre-

gulation of IgA production and development of IgA

nephropathy. TCTC-induced cytokine superinduc-

tion could lead to the terminal differentiation of

immunoglobulin-secreting cells via T-cell-mediated

polyclonal differentiation of B-cells or its precursors.

The Peyer’s patch might be particularly sensitive to

such dysregulation.

0034 Rather than suppression, an apparent increase in

DH by T-2 toxin was found when the toxin was

administered within a few days after the mice were

sensitized with sheep red blood cells. The toxin treat-

ment caused a marked decline in the cell population

of the thymus. Thus, T-2 toxin may interfere with

the generation of suppressor cells. Pretreatment of

animals with either T-2 toxin or DAS was also

found to prolong skin-graft survival time.

0035 Mode of action

TCTCs are potent protein synthesis inhibitors Al-

though a number of mycotoxins, including AFB1,

OA, citrinin, PR-toxin, and tenuazonic acid (TzA),

inhibit protein synthesis, most of these effects are

secondary. For example, AFB1 inhibits protein syn-

thesis at the transcriptional level. Inhibition of pro-

tein synthesis, however, is one of the early events in

the manifestation of toxic effects of TCTC myco-

toxins, which act at different steps in the translation

process. The potency of inhibition varies considerably

with the chemical structure of the side-chain. In

general, T-2, HT-2, NIV, FS-x, DAS, verrucarin A,

and roridin A affect at the initiation step, whereas

verrucarol, trichotecin, and crotocin affect elonga-

tion. Binding of TCTC with ribosomes, specifically

peptidyl transferase, is a key step leading to the inhib-

ition of protein synthesis. Inhibition of the elongation

step by DAS and fusarenon X has also been demon-

strated. Some of the less well-known TCTCs such as

trichodermol and trichodermone affect the termin-

ation step.

0036 Binding with membrane proteins/enzymes TCTC

may have a direct effect on cells through interaction

with other cellular components such as cell mem-

branes in addition to their effects on protein synthe-

sis. Both T-2 toxin and fusarenon X interact with

a number of ‘–SH enzymes,’ thus inhibiting their

activity.

0037 Apoptosis Several TCTCs, including roridin A, T-2

toxin, nivalenol, and DON, induce APT in HL-60

human promyeloytic leukemia cells at significantly

low levels with a minimum effective dose ranging

from 0.001 to 0.2 mgml

1

. Depending on the lympho-

cyte set, tissue source, and glucocorticoid induction,

DON can either inhibit or enhance apoptosis in

murine T, B, and IgA cells. Whereas suppression of

protein synthesis was considered the mechanism

for induction of APT by TCTC, a later study

showed that the T-2-induced apoptosis was media-

ted by the elevation of Ca

2þ

ion levels. Epidermal

cells developed apoptosis after T-2 toxin was

applied to the dorsal skin of hypotrichotic WBN/

ILA-Ht rats. Such an effect was found to be associ-

ated with the induction of c-fos and perhaps also

c-jun mRNAs.

0038Impact of TCTC on human and animal health Be-

cause of their diverse toxic effects and also because of

frequent contamination by toxins or toxigenic fungi

in foods and feeds, TCTCs are also potentially

hazardous to human and animal health. However,

among the many types of TCTC mycotoxicoses

mentioned earlier, only ATA and scabby wheat tox-

icosis have been demonstrated in human populations.

The former, ATA, was attributed to the human

consumption of overwintered cereal grains colonized

by Fusarium sporotrichioides and F. poae; this

caused the deaths of hundreds of people in the former

USSR between 1942 and 1947. Later studies indi-

cated that T-2 toxin and related TCTCs were

the primary cause. The signs and symptoms of ATA

disease, which include skin inflammation, vomiting,

damage to hematopoietic tissues, leukocytosis, and

leukopenia, are common in humans and animals,

including cats, cattle, guinea-pigs, poultry, monkeys,

and swine.

0039Deoxynivalenol has been found to be primarily

responsible for outbreaks of scabby wheat toxicosis

in humans that are quite common in several coun-

tries, but these toxicoses rarely cause death. For

example, between 1961 and 1985, 35 outbreaks in-

volving 7818 cases were found to be caused by con-

sumption of foods made from either scabby wheat or

moldy corn in China. The symptoms, which occurred

within 15 min to 1 h after consumption of foods made

from moldy corn meal, included: nausea (90%),

emesis (61%), headache and drowsiness (78%), and

5–6% had abdominal pain, diarrhea, and a mild

fever. People generally recovered 2–4 days after con-

sumption of the foods. Analysis of the leftover moldy

corn revealed that the samples had 0.34–93.8 p.p.m.

of DON; no T-2 toxin or nivalenol (NIV) were found.

Similar cases have been reported in people consuming

scabby wheat flour.

0040The widespread natural occurrence of DON in

wheat in Canada and the USA in the late 1970s and

MYCOTOXINS/Toxicology 4103