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Comment; Fungal organisms like all types of life forms have the capacity to evolve, mutate, change to meet the demands placed upon them in order to survive.  This paper reviews numerous new toxins that have been produced and are being changed, altered and produced that we need to be aware of as they are modifications of known toxins with undocumented (at the moment) effects on other organisms—such as us!

■ INTRODUCTION
Filamentous fungi show a remarkable potential to produce
secondary metabolites. Although the exact number of fungal
metabolites is unknown, a significant fraction of the ∼170,000
currently known natural products1 are of fungal origin, and it
can be assumed that many more compounds are yet to be
discovered. Fungal metabolites include important pharmaceut-
icals such as penicillin or statins, potent poisons such as
aflatoxins or trichothecenes, and metabolites that are both toxic
and pharmaceutically useful, such as the ergot alkaloids.2 As
fungi can grow on basically any organic material, also food and
feed are regularly contaminated with fungal metabolites.
Poisonous fungal metabolitescommonly referred to as
mycotoxinsare of major importance to food and feed safety.
One of the best definitions of mycotoxins states that they “are
natural products produced by fungi that evoke a toxic response
when introduced in low concentration to higher vertebrates and
other animals by a natural route”.3 In addition to animal
toxicity, mycotoxins may exert phytotoxic or antimicrobial
effects. A variety of fungi are known to produce mycotoxins,
including Aspergillus, Fusarium, and Penicillium species.4 The
most important classes of mycotoxins5 include the highly
carcinogenic aflatoxins (e.g., aflatoxin B1, AfB1),6 trichothe-
cenes (e.g., deoxynivalenol, DON),7 fumonisins (e.g., fumoni-
sin B1, FB1),8 ochratoxin A (OTA),9 and zearalenone (ZEN).10
These toxins and several others are regulated in many countries
of the world11 after thorough risk assessment, taking into
account toxicity, occurrence, and consumption data as well as
economic and political considerations.
Sound analytical methods are key to ensuring food safety,
and dozens of new methods are developed and published each
year.12,13 Of those, liquid chromatography−mass spectrometry
(LC-MS) based methods are becoming more and more

popular, as they allow the sensitive simultaneous determination
of multiple fungal metabolites in many matrices. These
methods also sometimes reveal “surprising” findings, such as
known mycotoxins in untypical matrices (e.g., fumonisin B2 in
grapes14) or in unusual geographical regions (e.g., aflatoxins in
Europe15), for which global warming might partly be
responsible.16 LC-MS is of tremendous help in the discovery
of new mycotoxins (e.g., NX-toxins17), as well as masked18 or
other modified19 forms of mycotoxins. Moreover, “emerging
mycotoxins”, which are in the scope of this review, can occur in
high frequency and sometimes also in high concentrations in
cereals and in other food- and feedstuffs.
The term “emerging mycotoxins”, although often used
nowadays for certain fungal metabolites, is not clearly defined.
One of the first papers to use this term was published in 2008
and deals with the Fusarium metabolites fusaproliferin (FP),
beauvericin (BEA), enniatins (ENNs), and moniliformin
(MON),20 and the term has mostly been used for these
compounds ever since. However, in a more recent paper,
emerging mycotoxins were defined as “mycotoxins, which are
neither routinely determined, nor legislatively regulated;
however, the evidence of their incidence is rapidly increasing”.21
According to this definition, many more fungal metabolites
with known (or at least suspected) toxicity would fall in the
category of emerging mycotoxins.

Special Issue: Public Health Perspectives of Mycotoxins in Food
Received: July 30, 2016
Revised: August 30, 2016
Accepted: September 6, 2016
Published: September 7, 2016

Review
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J. Agric. Food Chem. 2017, 65, 7052−7070

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The aim of this review is to critically assess the toxicity and
occurrence of different fungal metabolites that are currently not
regulated in food or feed. We deliberately chose the most
“interesting” fungal compounds based on the frequency of
occurrence and concentrations in food and feed, as well as
current scientific interest. The following sections will deal with
the Fusarium metabolites ENNs, BEA, MON, FP, fusaric acid
(FA), culmorin (CUL), and butenolide (BUT), the Aspergillus
metabolites sterigmatocystin (STE) and emodin (EMO), the
Penicillium metabolite mycophenolic acid (MPA), and the
Alternaria metabolites alternariol (AOH), alternariol mono-
methyl ether (AME), and tenuazonic acid (TeA).
■ FORMATION, TOXICITY, AND OCCURRENCE OF
EMERGING MYCOTOXINS
Enniatins. ENNs are cyclic hexadepsipeptides that are
produced by several Fusarium species, such as F. avenaceum,
F. oxysporum, F. poae, or F. tricinctum, which grow mainly on
cereals. In total, 29 species of ENNs are known.22 ENNs A, A1,
B (Figure 1), and B1 are most frequently detected in food and

feed. The toxicity of ENNs is based on their ionophoric
properties. They facilitate the transport of mono- or divalent
cations such as K+ or Ca2+ across membranes, thereby
disrupting normal physiological concentrations of these
ions.23 Low micromolar concentrations of ENNs were shown
to be cytotoxic to different cell lines24−28 and to reduce the
motility of boar spermatozoa.29 Cell death was shown to be
mediated by the induction of apoptosis28,30,31 via the
mitochondrial pathway24,32 or by the induction of necrosis
linked to lysosomal damage.33,34 There are conflicting data on
whether35 or not31 ENNs trigger the production of reactive
oxygen species (ROS). Several studies found no genotoxic
effect of ENNs.30,31,34 Interestingly, ENN B1 and T-2 toxin
showed an antagonistic toxic effect on cultured intestinal
epithelial cells and intestinal explants, which indicates that ENN
B1 down-modulates the gastrointestinal toxicity of T-2 toxin.36
ENNs were shown to interact with proteins. In rat liver
microsomes ENNs inhibited the activity of acyl-CoA:ch-
olesterol acyltransferase.37 ENN B was shown to bind
calmodulin and to inhibit 3′,5′-cyclo-nucleotide phosphodies-
terase.38 ENNs furthermore interact with multidrug resistance
ATP-binding cassette (ABC) transporters. The overexpression
of P-glycoprotein (ABCB1) and breast cancer resistance
protein (ABCG2) counteracted the cytotoxic effect of ENNs
on human cancer cell lines.39 Ivanova and co-workers40 found
that two multidrug resistance ABC transporters facilitate the
transport of ENN B1 across a monolayer of the human
intestinal cell line CaCo-2 in the apical to basolateral direction.
Furthermore, exposure to ENNs counteracted the protective
effect of ABC transporters against cytotoxic compounds in

yeast41,42 and human cancer cell lines,39 and it may
consequently alter the bioavailability of pharmaceuticals in vivo.
Several studies investigated the in vivo toxicity of ENNs in
rodents. Most of these studies show low toxicity. A single dose
of 50 mg ENNs/kg body weight (BW) did not induce signs of
toxicity in rats.43 Oral administration of ENN A to mice at a
dosage of 1 mg/kg BW/day for 6 days or subcutaneous (sc)
administration of ENN A to mice at a dosage of 0.5 mg/kg
BW/day for 6 days did not elicit a toxic effect.44 A daily
intraperitoneal (ip) ENN B dose of 5 mg/kg BW administered
for 9 days did not affect BW, food intake, or behavior in mice.45
In rats fed an ENN A contaminated diet (465 mg/kg feed,
corresponding to 20.9 mg/kg BW/day) for 28 days, no effects
on body and organ weight, histology of duodenum tissue,
biochemical blood parameters,46 or any visible signs of illness47
were found. Although altered peripheral blood lymphocyte
levels were detected, their consequences are yet unclear. In the
sole study that showed toxic effect of ENNs, ip administration
of 10−40 mg/kg BW at an interval of 8 h was lethal for
immune-deficient mice within 2−5 days, whereas lower levels
caused only weight loss.48
Two studies investigated the distribution of ENNs in
different tissues of rats, and the highest levels were detected
in jejunum, liver, and fat tissue.45,46 These findings led the
authors to suggest that the jejunum is a site of ENN
absorption46 and that ENNs primarily accumulate in fat-rich
tissues owing to their lipophilic properties.45 Dietary ENNs
were also shown to accumulate in the meat, skin, and liver of
broilers,49 and traces of the toxin were detected in a high
fraction of Finish egg samples.50 ENNs were shown to cross the
blood−brain barrier in mice.51
Bioavailability and toxicokinetics of ENN B were investigated
in pigs.52 The toxin had a high (∼91%) oral bioavailability and
was rapidly absorbed, distributed, and eliminated. Hence, the
authors suggested that rapid metabolization rather than low
bioavailability may explain the low in vivo toxicity of ENNs.
Several metabolites of ENNs have been reported. Mono-
oxygenated, dioxygenated, and N-demethylated metabolites
were detected in liver microsomes of rats, dogs, humans, and
chickens.53−55 The same metabolites were detected in liver,
plasma, and egg samples of chickens54 and in liver and colon
samples of mice.45
A mixture of enniatins termed “fusafungine” is used as a
nasal/oromucosal spray with antibiotic and anti-inflammatory
properties.56,57 However, in April 2016, the European
Medicines Agency of the European Union recommended to
revoke the authorization of fusafungine sprays “due to serious
allergic reactions and limited evidence of benefit”.58
ENNs were detected in 37, 68, and 76% of food (n = 4251),
feed (n = 3640), and unprocessed grain (n = 2647) samples
collected in Europe between 2000 and 2013.59 Maximum
reported concentrations in grains were 950, 2000, 18,300, and
5720 μg/kg for ENN A, ENN A1, ENN B, and ENN B1,
respectively. Maximum reported concentrations in cereal-based
food were, 42, 125, 832, and 980 μg/kg for ENN A, ENN A1,
ENN B, and ENN B1, respectively. ENNs were detected in
96% of samples (n = 83) of feed and feed raw materials with
median and maximum concentrations of 30 and 5441 μg/kg,
respectively.60 In a survey of Chinese medicinal herbs, ENN A,
ENN A1, ENN B, and ENN B1 were detected in 8, 7, 12, and
7% of all analyzed samples (n = 60) with maximum
concentrations of 355, 253, 291, and 40 μg/kg, respectively.61

Figure 1. Chemical structure of enniatin B.

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In summary, ENNs are toxic in vitro, but most in vivo data
indicate no or only low toxicity. ENNs are frequently detected
in food, feed, and grains, but feeding studies conducted with
rodents indicated a high tolerance to dietary ENN levels 1
order of magnitude higher than the reported maximum levels.
In their scientific opinion from 2014, the European Food Safety
Authority (EFSA) concluded that acute exposure to ENNs is
not a concern to human health, whereas no conclusion could be
drawn with respect to chronic exposure due to the lack of
relevant in vivo toxicity data.59 Interestingly, effects of ENNs on
the bioavailability of pharmaceuticals have been suggested by in
vitro data and should be further clarified before any valid
conclusions on that aspect can be drawn.
Beauvericin. BEA is a cyclic hexadepsipeptide (Figure 2),
produced by several Fusarium species, such as F. proliferatum,

F. subglutinans, F. verticillioides, or F. oxysporum. BEA is an
ionophore; it transports mono- or divalent cations, for example,
K+ or Ca2+, across membranes and thereby disrupts normal
physiological concentrations of these ions.62,63 Accordingly, low
micromolar concentrations of BEA were shown to be cytotoxic
to different cell lines in vitro.25,26,31,64 Cell death was shown to
be mediated by apoptosis31,65−67 via the mitochondrial
pathway32,68 or by necrosis.69 There are conflicting reports
on whether35,70 or not31 oxidative stress is involved in BEA
toxicity. BEA showed no mutagenicity in the Ames test71 and
intercalated into DNA only when applied at high concen-
trations (>100 μM).31 However, it showed a mutagenic
potential in the alkaline Comet assay.72 Low micromolar BEA
concentrations affected the development of immune cells in
vitro. BEA disturbed the maturation process of dentritic cells
and decreased the endocytosis ability of macrophages when
applied during their differentiation process.73 The toxin was
furthermore shown to act as an enzyme inhibitor in liver
microsomes. It inhibited cytochrome P450 enzymes74 and acyl-
CoA:cholesterol acyltransferase, the enzyme that causes the
accumulation of cholesteryl ester in atherogenesis.37 BEA was
shown to interact with P-glycoprotein (ABCB1) and breast
cancer resistance protein (ABCG2) in vitro.39 The data suggest
that BEA is both a substrate and an inhibitor of the investigated
transporters. It was concluded that the presence of homologues
of these transporters in the gastrointestinal tract may decrease
the bioavailability of BEA. Furthermore, the inhibitory effect of
BEA on these transporters may affect the bioavailability of
pharmaceuticals.
In vivo, toxicity and pharmacological behavior of BEA were
studied in mice and poultry. A daily ip administration of 5 mg/
kg BW for 9 days did not affect BW, food intake, or behavior in

mice.45 The LD50 values in mice were ≥100 and ≥10 mg/kg for
oral and ip administration, respectively.75 When the distribution
of BEA in different tissues was investigated, the highest
concentrations were detected in the liver and in fat tissue,
suggesting an accumulation in fat-rich tissue due to the
compound’s lipophilic properties.45 BEA was shown to cross
the blood−brain barrier in mice.51 The effect of chronic dietary
exposure to BEA was investigated in poultry. In turkey and
broiler feeding trials, diets contaminated with ≤2.5 and ≤12
mg/kg BEA, respectively, and cocontaminated with other
Fusarium mycotoxins, had no effect on performance parame-
ters, biochemical blood parameters, or meat quality.76−79
However, increased heart weight was detected in one broiler
trial.76 Dietary exposure of broilers and laying hens to 10 and 9
mg/kg BEA, respectively, did not affect growth, feed uptake, or
egg production.49 Surveys of poultry products in Finland found
trace amounts of BEA in a high fraction of analyzed egg
samples and in a low fraction of analyzed meat and liver
samples.50,80 The authors suggested that BEA accumulates in
egg yolk owing to its lipophilicity.
BEA was detected in 20, 21, and 54% of food (n = 732), feed
(n = 861), and unprocessed grain (n = 554) samples collected
in Europe between 2000 and 2013.59 Maximum reported
concentrations for BEA in grains and in cereal-based food were
6400 and 844 μg/kg, respectively. BEA was detected in 98% of
samples (n = 83) of feed and feed raw materials with median
and maximum concentrations of 6.7 and 2330 μg/kg,
respectively.60 In a survey of Chinese medicinal herbs, BEA
was detected in 20% of all analyzed samples (n = 60) with a
maximum concentration of 125 μg/kg.61
In summary, BEA isalthough toxic in vitronot toxic to
rodents and poultry in vivo. Dietary BEA concentrations
applied in poultry feeding trials were comparable to maximum
concentrations reported in feed and feed raw materials. In vivo
toxicity data for other species are missing. BEA presumably
accumulates in fat-rich tissue including eggs of laying hens.
However, detected levels should be negligible concerning an
acute risk to human consumers. In their scientific opinion from
2014, EFSA concluded that acute exposure to BEA is not a
concern to human health, whereas no conclusion could be
drawn with respect to chronic exposure due to the lack of
relevant in vivo toxicity data.59 Effects of BEA on components
of the immune system and on the bioavailability of
pharmaceuticals were suggested by in vitro studies and should
be investigated in vivo.
Moniliformin. MON (1-hydroxycyclobut-1-ene-3,4-dione;
semisquaric acid; Figure 3) is a small, water-soluble molecule

originally isolated from a Fusarium strain, initially called F.
moniliforme, in the 1970s.81 MON is among the most acidic
organic acids and therefore occurs in nature typically as sodium
or potassium salt. It is produced by a large variety of Fusarium
spp. (e.g., F. proliferatum,82 F. subglutinans,83 F. avenaceum,82
F. tricinctum,84 F. fujikuroi,85 F. nygamai,86 F. pseudonygamai,87
F. temperatum,88 or F. thapsinum87) and only recently has been
shown to be a metabolite of Penicillium melanoconidium.89

Figure 2. Chemical structure of beauvericin.

Figure 3. Chemical structure of moniliformin.

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The primary mode of action of MON seems to be the
inhibition of thiamin pyrophosphatase dependent enzymes,
which compromises the tricarboxylic acid cycle. Soon after the
discovery of impaired oxidation of pyruvate and α-ketoglutarate
by MON,90 inhibition of pyruvate dehydrogenase was
demonstrated in vitro.91,92 Later, several enzymes sharing
thiamin as common cofactor (pyruvate dehydrogenase, α-
ketoglutarate dehydrogenase, pyruvate decarboxylase, and
acetohydroxy acid synthase) have been shown to be inhibited
by MON.93 The authors concluded that MON meets the
criteria for an active site-directed, irreversible affinity label for
enzymes utilizing thiamin and that the inhibition of pyruvate
dehydrogenase is cofactor-directed and likely caused by the
structural similarity of MON and pyruvate.
MON was shown to by phytotoxic to wheat and corn, where
it caused necrosis, affected growth regulation, and rendered
leaves distorted.81 Its toxicity on animal cells was tested
extensively in vitro, and its effects are highly dependent on the
used cell lines. For instance, MON showed no inhibitory effects
on the proliferation of human white blood cell progenitors or
human platelet progenitors, but was cytotoxic to human red
blood cell progenitors with a half-maximal inhibitory
concentration (IC50) value of 4.1 μM.94 After treatment of
human hepatocellular carcinoma cells for 2 or 3 days with
MON, IC50 values of 39.5 μg/mL (403 μM) and 26.8 μg/mL
(273 μM) were recorded.95 With regard to chicken primary cell
cultures, MON was not toxic to chondrocytes and macro-
phages, but was toxic to splenocytes, cardiac (IC50 = 95 μM),
and skeletal myocytes (IC50 = 42 μM).96 Literature reports on
genotoxicity are slightly ambiguous. MON did not show any
activity in gene mutation assays with Salmonella typhimurium or
Escherichia coli with or without metabolic activation.97
However, the same authors elucidated that MON caused
pronounced dose-dependent chromosomal aberration in
primary rat hepatocytes. Compared to the control, a 9-fold
increase was seen after 3 h of exposure to 1 μg/mL (10 μM).
Previously, studies with 5−500 μM MON on the same cell type
showed no genotoxic effects as measured with the unscheduled
DNA synthesis test.98 The most recent study that investigated
genotoxic effects of MON used human peripheral blood
lymphocytes.99 Chromosomal aberrations, sister-chromatid
exchanges, and micronucleus frequencies were significantly
increased in a dose-dependent manner compared with the
negative control after treatment of lymphocytes with MON
concentrations between 2.5 and 25 μM.
MON shows even more severe effects in vivo. Incomplete
uptake of the polar toxin through the cell membranes in cell
culture studies may be a possible explanation for this
discrepancy. LD50 values of MON have been reported as 4.0
mg/kg BW in 1-day-old cockerels (oral),81 as 2.2−2.8 mg/kg
BW in 9-month-old female mink (ip),100 as 1.4 mg/kg BW in 7-
week-old female broiler chickens intravenous (iv),101 as 20.9
and 29.1 mg/kg BW in female and male mice (ip),102 as 3.7
mg/kg BW in 7-day-old ducklings (oral),103 and as 41.6 and
50.0 mg/kg BW in female and male rats (oral).103 Besides
damage to the heart muscle, including myocardial lesions and
increased relative heart weights,100,103−105 also muscular
weakness,103,104 respiratory distress,103,104 decreased feed intake
and BW gains,101,105 and impaired immune function104,106 were
noted frequently. In summary, these studies highlight that
poultry are the most affected species and that the heart is the
main target organ in all investigated animals. Potential
synergistic effects of the cardiotoxic MON with the co-

occurring BEA and ENNs107 or FB1105,106 were disproved for
the investigated conditions.
Occurrence of MON in cereals seems to be rather frequent
in various regions of the world, albeit at rather low levels on
average. For instance, 40% of 151 Finnish cereal samples
collected in 2005 were positive for MON, with a mean level of
190 μg/kg and a maximum level of 850 μg/kg.108 The toxin
was also found in maize samples from all agroecological zones
of Lesotho from the 2009/2010 and 2010/2011 seasons with
levels reaching 1.2 mg/kg.109 Furthermore, 93% of naturally
contaminated maize samples from northwestern Italy (n = 108)
collected in 2008, 2009, 2010, and 2011 showed detectable
levels of MON with maximum concentrations of 2.6, 0.5, 0.9,
and 0.4 mg/kg for the respective years.110 MON was detected
in 26 of 50 samples of poultry feed mixtures of Slovak origin in
concentrations up to 1.2 mg/kg.111
Two excellent reviews compiled general information on
MON112 or data on the negative health effects caused by
MON.113 The latter paper stated that “MON per se does not
pose a clear threat to human health at current levels”, if an
internal no observed adverse effect level (NOAEL) of 10 mg/
kg BW/day was applied. However, a more recently published
study indicated a severe impact of MON on the immune
system of Sprague−Dawley rats and a lowest observed adverse
effect level (LOAEL) value of 3 mg/kg BW.114 Also, it was
assumed a few years ago that MON “is not likely to be a
genotoxic carcinogen”,113 which at least has to be questioned
given the latest data,99 and further studies should be conducted
to answer this question. The European Commission Direc-
torate-General for Health and Consumers requested a scientific
opinion from EFSA about the risks for public health related to
MON in feed and food,115 which is due soon. Disregarding
potential genotoxicity, current average contamination levels of
cereals imply that the compound is likely not a problem for
human health. Still, guidance levels for MON in poultry feed
might be warranted to better protect animals from occasional
high concentrations and to provide scientific support to poultry
farmers.
Fusaproliferin. FP is a bicyclic sesterterpene (Figure 4). It
is produced by Fusarium species such as F. proliferatum,

F. subglutinans, and F. verticillioides. FP was moderately
cytotoxic to lepidopteran SF-9 cells and to human B
lymphocyte IARC/LCL 171 cells. In the case of SF-9 cells,
the CC50 values (concentration resulting in 50% cell viability)
were 100 and 70 μM after 24 and 48 h of exposure,
respectively.116 Fornelli and co-workers found a CC50 value
of >100 μM after 48 h of exposure.25 For IARC/LCL 171 cells
the CC50 values were 60−65 and 55 μM after exposure times of
24 and 48 h, respectively.116 Moreover, exposure to 30 μM FP
inhibited the proliferation of IARC/LCL 171 cells.116 When the
transport of FP across a monolayer of Caco-2 cells was
investigated, 80−83% of FP that had been applied to the apical
side was detected on the basolateral side after 4 h of incubation,
which suggests a high bioavailability in vivo.117

Figure 4. Chemical structure of fusaproliferin.

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In vivo toxicity data are limited to brine shrimp (Artemia
salina) larvae and chicken embryos. FP was toxic in the brine
shrimp larvae bioassay with an LD50 of 53.4 μM or 23.7 μg/
mL.116 FP exerted teratogenic and pathogenic effects on
chicken embryos when applied at concentrations of 1 or 5
mM.118 The toxin was shown to noncovalently interact with
oligonucleotides in vitro.119 Although interactions with DNA
may account for the teratogenic effect of FP, they have not yet
been confirmed in living organisms or cells.
FP was detected in 17% of samples (n = 83) of feed and feed
raw materials with median and maximum concentrations of 2.6
and 14.8 mg/kg, respectively.60 A review of FP occurrence data
published between 1997 and 2011 shows its occasional
occurrence in grains and grain-based foodstuffs120 at concen-
trations up to 500 mg/kg.121
In summary, high levels of FP occasionally occur in grains
and grain-based food and feed. However, its toxicity and mode
of action have not been comprehensively investigated so far.
Toxic effects on chicken embryos and brine shrimp larvae
suggest a potential hazard to humans and animals that should
be clarified by further experiments.
Fusaric Acid. FA (5-butylpiconilic acid; Figure 5), is a
phytotoxin produced by a great variety of Fusarium species,

such as F. oxysporum, F. moniliforme, F. proliferatum, F. sub-
glutinans, F. verticillioides, F. crookwellense, F. napiforme, and
F. fujikuroi.122,123 This off-white to yellowish crystalline
compound, a precursor of the beta blocker bupicomide,124 is
understudied for its mode of action and its effects in livestock
and humans. FA has been shown to exhibit cytotoxic and
growth inhibitory effects in different normal and cancer cell
lines. Cell proliferation was suppressed by 500 μM FA in
human WI-38 fibroblasts and colorectal adenocarcinoma cell
lines.125 Furthermore, FA was cytotoxic to dog kidney (IC50 =
10 μg/mL), McCoy mouse (IC50 = 25 μg/mL), and Chinese
hamster ovary fibroblast cells (IC50 = 10 μg/mL), as well as to
rat hepatoma cells (IC50 = 50 μg/mL).126 FA was shown to
induce oxidative and mitochondrial stress, as well as to cause
apoptosis and necrosis in the human hepatocellular carcinoma
cell line HepG2.127 May and co-workers found that FA inhibits
the growth of the rumen microorganisms Ruminococcus albus
and Methanobrevibacter ruminantium at concentrations >84
μM.128 Another in vitro study demonstrated that FA inactivates
several clinical and soil isolates of Acanthamoeba, with an IC50
value of 0.31 μM.129 As a phytotoxin, FA is known to play a
critical role in accelerating the development of Fusarium wilt in
numerous plants, such as banana and tomato plants.130−132
The LD50 values of FA in mice were 100 and 80 mg/kg BW
for iv and ip administration, respectively.133 The average oral
bioavailability of FA (25 mg/kg) in Sprague−Dawley rats was
58%.134 Several studies indicated a neurochemical effect of FA.
Researchers demonstrated a hypotensive effect of FA in
humans, rabbits, rats, cats, and dogs that was based on the
inhibition of dopamine-β-hydroxylase,133,135,136 the enzyme
that converts dopamine to norepinephrine. FA was furthermore
shown to alter brain and pineal neurotransmitters in rats.137

Oral administration of FA (200 mg/kg BW) to swine caused
neurochemical changes and vomiting behavior.138 Co-admin-
istration of different doses of FA and DON to swine decreased
weight gain and feed consumption, which led the authors to
suggest that FA might act synergistically with trichothecenes.139
Chickens that received FA-contaminated diet (up to 150 mg
FA/kg diet), on the other hand, did not show any abnormalities
in behavior, feed intake, weight gain, and appearance of visceral
organs.140 FA exerted a teratogenic effect in zebrafish. The
compound caused malformations at the notochord due to
copper chelating.141 A synergistic cytotoxic effect was detected
for in ovo administration of FA and FB1.142 Interestingly, oral
administration of 1 mg/mL FA inhibits tumor growth of head
and neck cancer in an in vivo murine model.143
Porter and co-workers showed that FA is a naturally
occurring contaminant in several types of cereal grain and
mixed livestock and poultry feeds and is synthesized together
with other Fusarium mycotoxins.137 Smith and Sousadias
reported that in 48 investigated swine feed samples, 85%
were positive, with the highest concentration of 136 mg/kg
FA.144
Because FA is produced by the most common fungal genus,
Fusarium, due to its neurochemical effects and due to possible
synergistic effects with predominant mycotoxins (DON and
FB1), this substance might pose a problem to humans and farm
animals. FA is not yet regulated in any country or monitored
regularly. For an appropriate risk assessment, more studies
covering in vivo and in vitro trials as well as occurrence are duly
needed.
Culmorin. CUL is a sesquiterpene diol (Figure 6) produced
by several Fusarium species, such as F. culmorum, F. graminea-

rum, F. crookwellense, and F. venenatum (summarized by
Pedersen and Miller145) and by the marine ascomycete
Leptosphaeria oraemaris.146 In addition, the formation of various
related compounds, namely, hydroxyculmorins (5-, 12-, 15-
hydroxyculmorin), culmorone, and hydroxyculmorones, has
been reported for different Fusarium strains.147,148
Information on the toxicological relevance of CUL and its
hydroxylated forms is limited. CUL was shown to be
phytotoxic149,150 and to possess antifungal activity.146 Pedersen
and Miller demonstrated that this compound results negative in
the Ames test.145 In baby hamster kidney cells BHK-21, only
the highest tested CUL concentration (20 μg/mL; 84 μM)
exhibited mild cytotoxicity.147 The LD50 for CUL in the chick
embryotoxicity screening test (CHEST) was between 68.0 μg/
kg (incubation period of 22 days) and 78.2 μg/egg (incubation
period of 7 days).151 Thus, CUL was approximately 10 times
less toxic than DON and approximately 25−30 times less toxic
than STE in the same test. In an attempt to extrapolate data
from the CHEST, the mouse LD50 (ip) for CUL was estimated
to range between 250 and 1000 mg/kg BW, indicating low
mammalian toxicity.145
So far, in vivo studies on the toxicity of CUL are restricted to
caterpillars and swine. Administration of a CUL-contaminated

Figure 5. Chemical structure of fusaric acid.

Figure 6. Chemical structure of culmorin.

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diet (25 mg/kg) for 7 days did not affect the mortality or
weight of corn earworm (Heliothis zea) and fall armyworm
(Spodoptera frugiperda) larvae.152 Similarly, CUL exposure (2
mg/kg diet, 21 days) had no negative impact on the
performance of growing piglets.153 Interestingly, CUL even
seemed to slightly enhance the growth of H. zea larvae and to
increase the weight gain of pigs. In addition, a potential
interaction of CUL and DON was investigated in both studies.
The combination of CUL (10 mg/kg diet) with DON (25 mg/
kg diet) resulted in a significantly increased mortality and
decreased weight of H. zea larvae when compared to DON
alone. The authors assumed a synergistic effect, which is
probably related to an impaired DON detoxification in insects
in the presence of CUL.152 While an interaction of these two
mycotoxins could not be confirmed in pigs,153 CUL was
recently proposed to increase DON toxicity in wheat seeds.150
The latter is of certain interest, because the levels of CUL as
well as hydroxyculmorins strongly correlate with those of DON
in cereal grains.154,155 In a recent study, CUL was detected in
95−100% of Norwegian barley, oat, and wheat samples (n =
76). Highest contamination levels were found in oats (n = 28),
where median and maximum CUL concentrations reached 2.0
and 31.5 mg/kg, respectively.155 Streit and co-workers found
63, 63, 13, and 7% of feed and feed raw materials (n = 83)
positive for CUL, 15-hydroxyculmorin, 5-hydroxyculmorin, and
15-hydroxyculmorone.60 In contrast, hydroxyculmorins were
more frequently found than CUL in Italian malting barley
samples156 and groundnuts from Cameroon.157 Besides factors
such as region, season, and sensitivity of the analytical method,
variations in the hydroxyculmorin profile between individual
Fusarium species148 may account for these differences.
On the basis of available literature data, CUL seems to have
limited acute toxicity in mammals. However, due to its frequent
occurrence in cereal grains, the mode of action of this
mycotoxin should be elucidated and its potential interaction
with DON (and other trichothecenes) should be clarified.
Butenolide. BUT (4-acetamido-4-hydroxy-2-butenoic acid
γ-lactone; Figure 7) is produced by several Fusarium species,

such as F. tricinctum, F. equiseti, F. graminearum, or
F. sporotrichioides. BUT was cytotoxic to different established
mammalian cell lines126,158 and to primary cultures of neonatal
rat and murine cardiomyocytes.159,160 Minimal effective
concentrations ranged from 1 to 25 μg/mL. In the case of
human chondrocytes, exposure to BUT concentrations ≥4 μg/
mL had a cytotoxic effect, whereas lower concentrations (1−2
μg/mL) caused an increase in cell viability.161 ROS production
was shown to be a mechanism of BUT cytotoxicity in HepG2
cells.158 Furthermore, ROS production due to BUT exposure
caused lipid peroxidation159,161 and DNA damage160 in
mammalian cell cultures. BUT was shown to induce
mitochondrial dysfunction in rat cardiomyocytes, which may
trigger ROS production.162
The effect of BUT exposure was investigated in rodents.
Intragastric administration of 10 or 20 mg BUT/kg BW to rats
for ≥7 weeks caused a loss of BW163 and injuries to the
myocardium159 and liver.163 Markers of oxidative stress and

oxidative damage were increased in myocardium, liver, and
serum.159,163 Exposure of mice to 0.5 mg BUT/mL drinking
water for 3 weeks (corresponds to consumption of ∼2.95 mg
BUT/day) had no effect on BW.164 BUT showed adverse
effects on the growth and development of rat embryos in vitro
when applied at a concentration of ≥1.25 mg/L, whereas no
such effects were observed at 0.625 mg/L.165 In chicken
embryos, oxidative damage was detected in the myocardium,166
liver, and kidney167 after in ovo injection of a single dose of
BUT (10−100 μg). The toxicity of BUT was investigated in a
small number of cows. Application of ≥1.5 mg BUT to the skin
of heifers induced local inflammatory encrusted lesions.168
Intramuscular administration of 1.1 g BUT/day for 3 months,
oral administration of 4.5 g BUT/day for 2 months,168 or
intramuscular administration of 3.8 mg BUT/kg BW/day for 90
days169 caused weight loss and necrosis of the tail tip. Oral
administration of 39 and 68 mg/kg BW was lethal to steers
within 3 and 2 days, respectively.170 Oral administration of 31
mg/kg BW/day for 46 days caused weight loss and esophageal
and gastric ulcers.170 BUT was detected in 52% of samples (n =
83) of feed and feed raw materials with median and maximum
concentrations of 23 and 1490 μg/kg, respectively.60
In summary, the toxicity of BUT was shown in vivo and the
compound’s potential to trigger oxidative stress and oxidative
damage has been reported. However, very high doses of BUT
were administered in previous studies, and it is unclear whether
BUT levels that can be reached by dietary exposure would also
cause adverse effects. Additional experiments with lower
concentrations of BUT should be conducted in vivo to study
the long-term toxicity. Also, more occurrence data are needed.
Sterigmatocystin. STE (Figure 8) is a toxic precursor of
aflatoxins and structurally closely related to AfB1. Its toxic effect

is mediated by its furofuran ring structure, which forms DNA
adducts after metabolic activation to an epoxide.171 STE is
produced by several fungal species within the genera Aspergillus,
Bipolaris, Emericella, Chaetomium, Botryotrichum, and Humicola
and the species Penicillium luteum. The main producers are
Aspergillus fungi, such as Aspergillus flavus, A. parasiticus,
A. nidulans, and especially A. versicolor.172 The International
Agency of Research in Cancer classified STE as a group 2B
carcinogen (possibly carcinogenic to humans).173,174
Several studies could demonstrate that STE exerts genotoxic
and cytotoxic effects on different cell lines, for example, on
immortalized ovarian hamster cells (CHO-K1 cells) for which
an IC50 value of 12.5 ± 2.0 μM was detected after 72 h of
incubation.175 The IC50 value of STE was determined to be 7.3
μM by measuring the protein synthesis in liver hepatocellular
(HepG2) cells.176 In addition, Gao and co-workers investigated
the STE-induced DNA damage in HepG2 cells. They showed a
significant dose-dependent increase of DNA strand breaks and
an increased intracellular ROS level if cells were exposed to 3
and 6 μM STE.177 Also, in the human lung adenocarcinoma cell

Figure 7. Chemical structure of butenolide.

Figure 8. Chemical structure of sterigmatocystin.

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line A549 (IC50 = 3.7 μM)178 and in transformed rat fibroblasts
(AWRF)179 the cytotoxic effect of STE was confirmed.
Furthermore, Sun and co-workers reported the induction of
apoptosis in human peripheral blood lymphocytes incubated
with 2 μg/mL STE.180 STE induced DNA damage in primary
cultured human esophageal epithelial cells and immortalized
human esophageal epithelial cells (Het-1A) and caused G1 and
G2 phase arrest, respectively.181
Only a few recently conducted in vivo studies are available in
livestock, fish, and other animals. Whereas the effects of STE
are similar to those of AfB1, its acute toxicity is much lower.
The LD50 values in rat are 120−166 and 60−85 mg/kg BW for
oral and ip administration, respectively.182 In monkeys, the
LD50 upon ip administration was determined to be 32 mg/kg
BW.183 Studies in ruminants found a negative effect of a STE-
contaminated diet in cattle,184 but not in sheep.185 Dietary
exposure of dairy cows to ≤12 mg STE/kg feed caused bloody
diarrhea, reduced milk production, and was lethal in some
cases.184 In one pig trial with STE (30 μg/kg feed), negative
effects, such as decreased feed intake, incidental diarrhea, and
necrotic alterations of the liver tissue, were observed.186 An
LD50 of 5−7 μg was reported for 5-day-old chicken embryos,
whereas 10 μg killed 90−100% of the embryos.187 Newly
hatched male chicks suffered from liver cirrhosis and altered
biochemical serum parameters after 7 weeks of dietary STE
exposure.188 Also, Sreemannarayana and co-workers described
the hepatotoxic and nephrotoxic impact of STE in
poultry.189−192 In carp and catfish, the mycotoxin caused a
decrease in body growth and crude protein content.193 In Nile
tilapia fish, STE showed toxic and clastogenic effects indicated
by a significantly decreased BW and an increased incidence of
micronucleated red blood cells as well as chromosomal
aberrations in the kidney.194 STE down-regulated TNF-α,
interleukin (IL) 6, and IL-12 mRNA expression in isolated
peripheral blood mononuclear cells and peritoneal macrophage
cells of BALB/c mice, as well as TNF-α and IL-6 protein levels
in serum, suggesting an immunosuppressive effect.195 Already a
single sc dose of STE (5 μg/g of BW) induced tumors in lung
and liver in newborn mice. Males seemed to show an even
higher susceptibility to those effects than females.196
Chromosome aberrations were found in rat bone marrow
cells, when a dose of 31.2 mg STE/kg BW was injected ip.197
Scarce data about the natural occurrence of STE was
available until 2015. An external scientific report, assigned by
EFSA, presents data on 1259 samples of cereal grains, cereal
products, beer, and nuts collected over a period of 14 months
and analyzed for STE by a validated LC-MS/MS method.198
STE was detected in 10% of all samples, and the highest
frequency of contamination was seen in rice (21%) and oat
grains (22%) intended for human consumption. In beer,
peanut, and hazelnut samples, no STE concentration was
found.198 A review from 2010 reported the occurrence of STE
in green coffee beans, spices, nuts, beer, and the outer layer of
hard cheese colonized by A. versicolor.199
The EFSA Panel on Contaminants in the Food Chain
(CONTAM) published a scientific opinion in 2013 about the
presence of STE in food and feed without characterizing its
possible risk due to a lack of occurrence data and the need for
more sensitive analytical methods.172 With new data available
now, the Joint FAO/WHO Expert Committee on Food
Additives (JECFA) is currently working on a safety assessment
of STE proposed by the Codex Committee on Contaminants in
Foods.200 Due to the high STE concentrations used in vitro, a

clear relevance of the observed results is arguable. Thus, follow-
up experiments with low-dose STE treatments should be
conducted in vivo to study the long-term toxicity.
Emodin. EMO, an orange-red crystalline compound, is a
1,3,8-trihydroxy-6-methylanthraquinone (Figure 9) and is

produced by many Aspergillus species, but has also been
found to be produced by the genera Penicillium and
Talaromyces. Furthermore, it occurs as an active ingredient in
many plants,201,202 mainly in Fabaceae (Cassia spp.),
Polygonaceae (Rheum, Rumex, and Polygonum spp.), and
Rhamnaceae (Rhamnus and Ventilago spp.).203 In plants, the
anthraquinones (also aloe-emodin, chrysophanol, physcion or
rhein) are present as aglycones or as glycosides, such as
emodin-8-glucoside.203
In traditional Chinese medicine, EMO, mainly extracted
from the rhizome of Rheum palmatum L., is claimed to be a
therapeutic alternative for the prophylaxis and treatment of
various diseases and physical complaints. Several studies could
demonstrate that EMO inhibits the growth and proliferation of
cancer cells in vivo and in vitro.204,205 EMO induced apoptosis
in the human lung squamous carcinoma cell line CH27206 and
in murine leukemia WEHI-3 cells.207 It might also act as a
JAK2/STAT3 inhibitor, and it down-regulates the expression of
anti-apoptotic Mcl-1, thereby inducing cell death in multiple
myeloma cell lines.208 Antiviral activities were shown in
different studies. EMO reduced the entry and replication of
Coxsakievirus in HEp-2 cells209 and inhibited hepatitis B virus
and Herpes simplex virus type 1 in vitro.210,211 EMO exerted
antibacterial activity against Bacillus subtilis (minimal inhibitory
concentration (MIC) = 28.9 μM), Staphylococcus aureus (MIC
= 14.4 μM)212 and methicillin-resistant or sensitive S. aureus
strains (MIC = 64 μg/mL; 237 μM).213 EMO was suggested to
be purgative, as it was shown to induce contraction of rat
isolated ileum tissue by releasing endogenous acetylcholine.214
EMO was also shown to possess antidiabetic activity by
activating the PPARγ215 or the AMPK216 signaling pathway.
Furthermore, EMO is considered to have anti-osteopor-
otic217,218 and antiallergic effects.219 A recently published
review describes several positive effects in detail.220
In contrast to EMO’s manifold beneficial effects, its negative
effects have not been thoroughly studied. In human
mononuclear cells, EMO (3−30 μM) caused an immunosup-
pressive reaction by decreasing IL-1 and IL-2 production and
IL-2 receptor expression.221 It induced apoptosis in human T
cells222 and a human proximal tubular cell line (HK-2) via the
mitochondrial pathway.223 Furthermore, it inhibited the
stimulation of human peripheral blood mononuclear cells and
decreased the plasma IL-2 level.224 Published studies about the
genotoxicity of EMO are controversial. Mueller and co-workers
showed a genotoxic effect using the micronucleus test and
mutation assay in mouse lymphoma L5178Y cells.225,226 Morita
and co-workers observed an induction of 6-thioguanine-
resistant mutation in the mouse mammary carcinoma cell line

Figure 9. Chemical structure of emodin.

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FM3A by 1−10 μg/mL EMO.227 Other researchers, however,
could not demonstrate a mutagenic effect of EMO.228,229
Furthermore, Westendorf and co-workers discovered only a
slight response in the V79-HGPRT mutagenicity assay.230
In vivo, therapeutic effects and toxicity were investigated
mainly in mice and rats. Two studies with collagen-induced
arthritis mice showed that EMO exerted an anti-inflammatory
effect by inhibition of the NF-κB-pathway231 and reduction of
TNF-α and IL-6 in plasma, PGE2 production, and COX-2
protein expression.232 Following successful in vitro experi-
ments, Liu and co-workers confirmed the inhibition of the
Coxsakie virus also in mice.209 Another mouse trial showed the
positive effect of EMO on diesel exhaust particle induced lung
inflammation.233 EMO also reduced aldose reductase in the
lens of mice, which might play an important role in the
development of diabetes cataract.234 Negative effects of EMO
(0.25−2 μg/mL; 0.92−7.4 μM) were demonstrated for the
survival rates and hatching success of zebra fish embryos.235
Furthermore, EMO induced nephrotoxicity in Sprague−
Dawley rats.236
Long-term dietary exposure to EMO (≤2.5 g/kg) for a
period of 2 years showed no carcinogenic effect in rats or
mice.237 Similarly, neither prenatal mortality nor morphologic
or genotoxic changes were observed in EMO-fed mice and rats,
and NOAELs of 2500 and 850 mg/kg were defined for mice
and rats, respectively.238
EMO is probably the most ubiquitous anthraquinone found
worldwide, especially in higher plant families but also in fungi,
trees, shrubs, lianas, and herbs.203 However, EMO receives little
attention as a fungal metabolite, and occurrence data of fungal
EMO in food and feed are scarce. One study reports the
detection of EMO in 89% of the analyzed 83 feed samples, at
median and maximum concentrations of 9.8 and 1570 μg/kg.60
Because of the high NOAEL in rodents238 and its use in
alternative human medicine for decades, the risk to animal or
human health from EMO is very likely marginal.
Mycophenolic Acid. MPA ((4E)-6-(4-hydroxy-6-methoxy-
7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoic acid; Figure
10) was isolated already 1893 by an Italian physician,

Bartolomeo Gosio, from Penicillium brevicompactum (reviewed
by Bentley239). Besides its production by this fungus, MPA can
also be produced by P. roqueforti and P. carneum or by
Byssochlamys niveaa known producer of patulin.4 MPA holds
the distinction of being the first ever purified antibiotic,239 but
it is widely used nowadays both as an immunosuppressive drug
for prophylaxis and treatment of organ rejection in trans-
plantations and as an antirheumatic drug.240 The main mode of
action of MPA is the specific inhibition of the enzyme inosine
monophosphate dehydrogenase, which is highly expressed in
proliferating cells such as T- and B-lymphocytes. The inhibition
of lymphocyte proliferation leads to immunosuppression.241
Although MPA is sometimes referred to as a mycotoxin, its
acute toxicity in animals is low, with LD50 values of around 450
mg/kg BW in rats and around 1900 mg/kg BW in mice after

oral application.242 The same authors assessed subacute toxicity
in rats and detected no adverse effects at 10 mg/kg BW. At
higher doses growth retardation and anemia were diagnosed in
rats. Dogs died after 12 days when given daily doses of 80 mg/
kg BW and at lower doses showed anorexia, diarrhea, and
enteritis.242 MPA was not mutagenic, did not produce
chromosome aberrations, and was nontoxic when given to
nonhuman primates in higher doses than required for human
therapy.243 Used as an antirheumatic drug in human medicine,
daily doses of 1−3 g were generally tolerated, with gastro-
intestinal intolerance being the major observed side effect.243 In
the following years, a multitude of effects on different cell types,
e.g. lymphocytes, monocytes, neutrophils as well as on
dendritic, mesangial, mast, vascular smooth muscle and
endothelial cells were discovered.244 The authors concluded
in their literature review that while rheumatic patients still
largely benefited from MPA treatment, it holds the risk of
immune suppression, and therapy has been associated with
several, sometimes life-threatening, infections by viruses.
MPA has been shown to occur in various food and feedstuffs
in relatively high concentrations compared to other fungal
metabolites. Dry-cured ham and liver pa
̂

té were shown to be
good substrates for P. brevicompactum, and after inoculation,
levels of 11−14 mg/kg of MPA were found on the food
surfaces.245 All investigated Roquefort cheese samples were
found positive for MPA with levels up to 1.2 mg/kg, whereas
German blue-white soft cheese contained even 11 mg/kg.246 In
lower concentrations, MPA was also found to occur in red
wine247 and ginger.248 In silage, MPA was found in 32% of 233
investigated sampled at levels up to 35 mg/kg249 or even up to
48 mg/kg.250
Given the low toxicity of MPA, the only concern is its role in
unwanted immunosuppression.251 The extensive use of MPA in
medicine has indicated that these concerns are minimal.239
Levels in food do not even come close to therapeutic doses in
humans, so it is safe to assume that MPA should also not be of
any dietary concern. Although in feed the concentrations can be
higher, it was verified that 300 mg of MPA per sheep and day
from contaminated silage does not show any immunodepres-
sive effects.252 In conclusion, MPA should not be termed a
“mycotoxin” as it is neither toxic nor does it show any other
adverse effects on humans or animals at the concentration
levels found in food and feed.
Alternariol and Alternariol Monomethyl Ether. AOH
and its monomethyl ether AME are dibenzopyrone derivatives
(Figure 11), produced by a large variety of Alternaria spp.,
which mainly grow on vegetables, fruits, and cereals.253 In vitro,
AOH and AME were shown to be cytotoxic to HeLa cells, but
less toxic to Bacillus mycoides.254 AOH was furthermore shown
to be cytotoxic to human colon carcinoma cell lines, and
cytotoxicity was shown to be mediated by the activation of the
mitochondrial pathway of apoptosis.255 In Caco-2 cells, AOH

Figure 10. Chemical structure of mycophenolic acid.

Figure 11. Chemical structure of alternariol (R = H) and alternariol
monomethyl ether (R = CH3).

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induced toxicity via cell cycle disruption, induction of
apoptosis/necrosis, and changes in mitochondrial membrane
potential.256 AOH and AME induced DNA strand breaks and
chromosomal aberration in different cell lines.253,257−259 The
DNA damaging effect of these compounds was traced back to
their binding to the minor groove of DNA and their action as
topoisomerase poisons.258,260 Furthermore, in mammalian cell
lines, AOH induced oxidative stress that resulted in oxidative
DNA damage in some studies,261,262 but not in others.263,264 In
line with these observations of DNA-damaging effects, AOH
was shown to be mutagenic in mammalian cell lines.265,266
Mutagenicity assays in bacteria, on the other hand, produced
contradictory results. Both compounds were suggested to be
strongly mutagenic according to the B. subtilis rec-assay267 and
the E. coli ND160 reverse mutation assay.268−270 In the Ames
Salmonella test, the compounds were not or only weakly
mutagenic to strains TA98 and TA100,271−273 and AOH was
mutagenic to strain TA102.274 Effects of both AOH and AME
on reproductive organs and the immune system have been
suggested on the basis of in vitro data. An estrogenic potential
of AOH was detected in a human endometrial adenocarcinoma
cell line; however, this potential was 10,000-fold lower
compared to the endogenous hormone 17β-estradiol.257
Interestingly, AOH showed synergistic estrogenic effects in
combination with ZEN or its phase I metabolite α-
zearalenol.275 AOH and AME specifically inhibited progester-
one secretion in cultured porcine granulosa cells, which
suggests that contaminated food may affect the reproductive
performance of pigs and other mammalian species.276 AOH
induced autophagy and senescence in murine macrophages277
and modified the morphology and cytokine secretion of murine
and human macrophages.278
With regard to in vivo toxicity, no mortality or teratogenic
effects of AOH or AME on chicken embryos were detected at
doses up to 1 mg per egg.279 Chicks exposed to dietary AME at
levels up to 100 mg/kg feed for 4 weeks showed no mortality
or significant loss in performance.279 No evidence of toxicity
was observed in rats when AME and AOH were fed for 21 days
at levels up to 24 and 39 mg/kg feed, respectively.280
Furthermore, no toxic effects were observed when 3.75 mg of
AME was given to rats by oral gavage daily for 30 days.281 In a
study performed by Schuchardt and co-workers, AOH was
shown to be nontoxic to mice after single or repeated oral
application of doses as high as 2000 mg/kg BW.282 An in vivo
oral toxicokinetic study using 200 or 1000 mg/kg AOH
revealed low systemic absorption and rapid metabolization, and
thus it was concluded that target organ toxicity would likely be
restricted to the gastrointestinal tract.282 Furthermore, neither
the micronucleus assay applied to bone marrow nor the Comet
assay applied to liver tissue indicated systemic genotoxicity.
Whether AOH exhibits local genotoxicity in the gastrointestinal
tract remains to be determined.
The metabolism of AOH and AME was investigated in
different animal species and cell lines, and different classes of
metabolites have been observed. AOH and AME were shown
to be hydroxylated at their aromatic carbon atoms by
cytochrome P450, leading to the formation of catechols,282−284
and they were shown to be conjugated with glucuronic acid or
sulfate at their phenolic hydroxyl groups by UDP-glucuronosyl-
transferases and sulfotransferases, respectively.285,286 AME was
furthermore shown to be demethylated to AOH in vitro.284
Two studies investigated the fetotoxicity of AOH and AME.
AME was maternally toxic and fetotoxic when administered ip

to Syrian golden hamsters at a single dose of 200 mg/kg BW,
whereas no such effects were observed at lower doses.281 In
mice, sc administration of AOH (100 mg/kg BW) resulted in
an increased percentage of dead and resorbed fetuses/litter and
runts/litter, whereas lower doses of AOH or AME did not elicit
a fetotoxic effect.254 Interestingly, a synergistic fetotoxic effect
of AOH and AME was observed when both substances were
administered at a dose of 25 mg/kg. AOH and AME were
suggested as etiological agents of human esophageal
cancer,253,287 but a causal relationship has not been
demonstrated so far. Interestingly, precancerous changes were
detected in the esophageal mucosa of mice fed 50 or 100 mg
AME/kg BW/day for 10 months.288
EFSA concluded in 2011 that the estimated chronic dietary
exposure of humans to AOH and AME exceeds the threshold
of toxicological concern for potentially genotoxic substances,
indicating a need to create additional toxicity data.289 According
to a survey of published occurrence data, AOH was detected in
31% and AME was detected in 6% of samples (n ∼ 300) of
European feed and agricultural commodities at maximum
concentrations of 1840 and 184 μg/kg for AOH and AME,
respectively.289 Also, in tomato-based foodstuffs collected in
Belgium, AOH and AME exceeded the threshold of
toxicological concern assigned by EFSA for potentially
genotoxic substances.290
In conclusion, AOH and AME showed genotoxic effects in
vitro. However, administration of high doses that couldon
the basis of currently available data on the occurrence in food
and feedhardly be achieved in vivo did not elicit toxic or
genotoxic effects in rodents. Nevertheless, given the genotoxic
potential, additional toxicity data should be generated. Effects
of AOH and AME on reproductive organs and the immune
system were suggested by in vitro data and should be
investigated in vivo.
Tenuazonic Acid. TeA is a tetramic acid derivative (Figure
12) produced by Alternaria species that grow on vegetables,

fruits, and cereals253 and by plant pathogenic fungi such as
Phoma sorghina and Magnaporthe oryzae.291,292 The toxic effect
of TeA is presumably based on its interference with protein
biosynthesis.293 The compound was nonmutagenic in the Ames
Salmonella test with or without metabolic activation or
nitrosylation.272−274
TeA was toxic to mice and rats, and LD50 values were
determined.294,295 Observed symptoms included diarrhea,
muscle tremor, and convulsion. For mice, LD50 values upon
oral administration were 81 mg/kg BW for females and 186 or
225 mg/kg BW for males. Upon iv administration the LD50
value was 115 mg/kg BW for females. Upon ip, sc, or iv
administration, LD50 values were 125−162 mg/kg BW for
males. For rats, LD50 values were 168 mg/kg BW for females
and 180 mg/kg BW for males upon oral administration and 157
mg/kg BW for females and 146 mg/kg BW for males upon iv
administration. After dietary exposure of mice to 25 mg TeA/
kg BW/day for 10 months precancerous changes were detected

Figure 12. Chemical structure of tenuazonic acid.

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in the esophageal mucosa.288 TeA was furthermore toxic to
beagle dogs (4 oral doses of 2.5 mg/kg BW per day) and
monkeys (daily oral dose of 89.6 mg/kg BW).294 Symptoms
included diarrhea, vomiting, and hemorrhages and were lethal
in most cases. In the case of monkeys, no adverse effects were
detected for daily doses up to 48.8 mg/kg BW. TeA was toxic
to chickens. The compound was toxic in the chicken embryo
assay with an LD50 of 548 μg/egg, but it showed no teratogenic
effect.279 For day-old chicks, the LD50 upon oral administration
was 37.5 mg/kg BW.296 Dietary exposure of broilers to 10 mg/
kg feed for 3 weeks or daily oral administration of ≥1.25 mg/kg
BW to broilers and layers caused macro- and microscopic
lesions in different organs and led to decreased weight gain and
feed efficiency.296 In the same study, no adverse effects were
detected for doses of 0.63 mg/kg BW. The toxicokinetics of
TeA were investigated in pigs and broilers.297 Oral or iv
administration of single doses of 0.05 mg TeA/kg BW had no
adverse effects. The orally administered doses were completely
absorbed in both species and rapidly eliminated in pigs but not
in broilers.
According to a survey of published occurrence data
conducted by EFSA, TeA was detected in 15% of samples (n
∼ 300) of European feed and agricultural commodities at
concentrations between 500 and 4310 μg/kg.289 In a survey of
feed and feed raw materials, TeA was detected in 65% of
samples (n = 83) at median and maximum concentrations of 68
and 1980 μg/kg, respectively.60 Interestingly, high levels of TeA
(up to 1200 μg/kg) were found in Sorghum-based infant
food.298
In summary, administration of TeA was toxic to rodents and
chickens. In their scientific opinion from 2011, EFSA
concluded, on the basis of exposure estimates and due to a
lack of data indicating genotoxicity, that TeA is unlikely to be a
human health concern.289 However, more recent occurrence
data suggested that health risks cannot be excluded for infants
consuming larger quantities of Sorghum-based food and that
more data on the subject should be generated. EFSA
furthermore concluded that on the basis of in vivo toxicity
data296 and estimated exposure, adverse health effects cannot
fully be ruled out for chickens.289 The recent finding that TeA
shows a high bioavailability and slow elimination in chickens
reinforces the notion that the toxin may be a cause for concern
for this species and that more data on the subject should be
collected.
■ CONCLUSIONS
Fungal spoilage of agricultural goods is accompanied by the
formation of mycotoxins, which pose a significant health risk to
humans and livestock. As climatic conditions favor the growth
of different fungi (and hence the formation of different toxins)
in different parts of the world, some populations are more
affected than others, but mycotoxins are a global challenge.
Without any doubt, aflatoxins, due to their high acute and
chronic toxicity, have the most severe impact on human and
animal life. In addition, several other mycotoxins including
trichothecenes, fumonisins, OTA, or ZEN are of high
toxicological significance and have been extensively studied,
and regulations are in force in many countries with the aim of
ensuring tolerable levels in food and feed. Efficient and reliable
analytical methods have been developed and are used to control
foodstuffs for their compliance with regulations. During the
past decade, advances inparticularly LC-MS basedmethods
used for food and feed analysis have been enormous with

regard to their detection limits, speed, and capability to
determine multiple compounds.299 Nowadays, certain modern
analytical methods are able to detect hundreds of different
compounds, for instance, fungal metabolites,300 in a variety of
food and feed samples. Many of these metabolites are irrelevant
in terms of food and feed safety, but for the first time in history
occurrence data can be collected on a large scale before the
toxicity of the detected fungal metabolites is (and can be)
assessed. Sticking to a basic principle in toxicology, sola dosis
facit venenum (the dose makes the poison), both toxicity and
occurrence data are indispensable for risk assessment. Instead
of prioritizing research on natural toxins based on the most
bioactive compounds, we might have reached a point where
assigning priorities based on actual occurrence data in
foodstuffs is at least conceivable.
Given the enormous number of bioactive fungal metabolites,
every attempt to provide exhaustive coverage of their potential
health impact is doomed to fail. As a limited number of
currently nonregulated compounds of fungal origin had to be
selected for this paper, it is plausible that equally or even more
important ones were missed. Still, our aim was to critically
scrutinize the impact of the emerging mycotoxins covered here
and to propose a ranking list for the research community to
prioritize research efforts. The bottom of this list is clearly
reserved for EMO and MPA. Both compounds have already
been used in human medicine for decades with negligible side
effects even at doses that cannot be reached due to ingestion of
contaminated food. CUL would probably rank above these
compounds on our imaginary list. Although levels of
occurrence can be high, the toxicity of this compound in
mammals seems to be marginal. Yet, the mode of action of this
substance has not been elucidated and, in addition, its frequent
co-occurrence with DON may be a cause for concern.
Therefore, it should be clarified whether synergism with
trichothecenes, as hinted in a few studies, actually exists.
Arguably next on the list are ENNs and BEA. Whereas these
compounds are clearly toxic in vitro, most in vivo data indicate
limited toxicity. The EFSA risk assessment for these
compounds stated no immediate concern for human health,
but also highlighted the need for long-term studies to assess
potential chronic toxic effects in vivo.59 Although BUT is toxic
in vivo and causes oxidative stress to cells, it is currently
unknown if the occurring levels are sufficient to cause adverse
effects and toxicity studies with lower doses should be
conducted in animals. A similar picture can be drawn for
STE, as long-term in vivo studies at lower doses are currently
lacking that would be needed to assess chronic toxicity.
Whereas the Alternaria metabolites AOH and AME showed
genotoxic effects in vitro, genotoxicity could so far not be
confirmed in vivo, and further studies on this subject would be
warranted. Other effects caused by these compounds, for
example, on the reproductive or immune system, should also be
clarified using in vivo trials. In the case of FP and FA there is
clearly a need for more studies. Whereas FP levels in cereals can
be high and toxic effects were seen on larvae and chicken
embryos, further animal studies are currently missing. Even less
is known about FA other than that it shows neurochemical
effects and that it is possibly synergistic to co-occurring
mycotoxins such as DON or fumonisins. MON would probably
end up on the upper end of the ranking. Although its acute
toxicity and occurrence levels are unproblematic for humans,
the situation is different for poultry. Furthermore, the
discrepancies between the outcomes of studies investigating

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7061

genotoxic effects of MON should be clarified as quickly as
possible. The situation is similar for TeA. Although toxic to
animals, EFSA concluded that this compound is likely of no
human health concern mainly due to its low average
occurrence.289 Considering recent studies, risk cannot be
excluded, though, both for chickens and for infants consuming
Sorghum-based food.
With regard to Alternaria metabolites, interestingly, AOH,
AME, and TeA made only a minor contribution to the
genotoxicity of certain A. alternata extracts, whereas a high
genotoxic activity was attributed to unidentified compounds.263
Also, altertoxins were shown to be mutagenic in vitro,273,301,302
and altertoxin I was detected in 42% of analyzed feed samples
(n = 83) in a recent survey.60 Altertoxins are just one example
that several fungal metabolites, not explicitly discussed in this
paper, show high toxic potential. Because of this, together with
the recently available information on frequent occurrence, more
data on the exposure of humans and animals as well as in vivo
studies clarifying possible toxic effects are warranted.
■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.6b03413.
ChemDrawings (ZIP)

■ AUTHOR INFORMATION
Corresponding Author
*(F.B.) E-mail: franz.berthiller@boku.ac.at. Phone: +43 1
47654 97313.
Funding
We thank the Austrian Federal Ministry of Science, Research
and Economy, the Austrian National Foundation for Research,
Technology and Development and BIOMIN Holding GmbH
for funding the Christian Doppler Laboratory for Mycotoxin
Metabolism. The financial support of the Austrian Research
Promotion Agency (FFG, Grant 84821) is greatly acknowl-
edged.
Notes
The authors declare no competing financial interest.
■ ABBREVIATIONS USED
ABC ATP-binding cassette
AfB1 aflatoxin B1
AME alternariol monomethyl ether
AOH alternariol
BEA beauvericin
BUT butenolide
BW body weight
CC50 concentration resulting in 50% cell viability
CHEST chick embryotoxicity screening test
CONTAM Panel on Contaminants in the Food Chain
CUL culmorin
DON deoxynivalenol
EFSA European Food Safety Authority
EMO emodin
ENNs enniatins
FA fusaric acid
FB1 fumonisin B1
FP fusaproliferin
ip intraperitoneal
iv intravenous

IC50 half-maximal inhibitory concentration
IL interleukin
JECFA Joint FAO/WHO Expert Committee on Food
Additives
LC-MS liquid chromatography−mass spectrometry
LD50 dose that is lethal to 50% of test subjects
LOAEL lowest observed adverse effect level
MIC minimal inhibitory concentration
MON moniliformin
MPA mycophenolic acid
NOAEL no observed adverse effect level
OTA ochratoxin A
ROS reactive oxygen species
sc subcutaneous
STE sterigmatocystin
TeA tenuazonic acid
ZEN zearalenone

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Dr. Raymond Oenbrink
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