Mycotoxin mechanisms of action and health impact: ‘ in vitro ’ or ‘ in vivo ’ tests, that is the question

Abstract

The aim of this paper is to present examples of in vitro and in vivo tests for mycotoxin mechanisms of action and evaluation of health effects, with a focus on the gut environment and toxicity testing. In vivo investigations may provide information on the net effects of mycotoxins in whole animals, whereas in vitro models represent effective tools to perform simplified experiments under uniform and well-controlled conditions and a suitable alternative to in vivo animal testing providing insights not achievable with animal studies. The main limits of in vitro models are the lack of interactions with other cells and extracellular factors, lack of hormonal or immunological influences, and lack or different levels of in vitro expression of genes involved in the overall response to mycotoxins. The translation of in vitro data into meaningful in vivo effects remains an unsolved problem. The main issues to be considered are the mycotoxin concentration range in accordance with levels encountered in realistic situations, the identification of reliable biomarkers of mycotoxin toxicity, the measurement of the chronic toxicity, the evaluation of single- or multi-toxin challenge. The gastrointestinal wall is the first barrier preventing the entry of undesirable substances. The intestinal epithelium can be exposed to high concentrations of mycotoxins upon ingestion of contaminated food and the amount of mycotoxin consumed via food does not always reflect the amount available to exert toxic actions in a target organ. In vitro digestion models in combination with intestinal epithelial cells are powerful tools to screen and predict the in vivo bioavailability and digestibility of mycotoxins in contaminated food and correctly estimate health effects. In conclusion, in vitro and in vivo tests are complementary approaches for providing a more accurate picture of the health impact of mycotoxins and improved understanding and evaluation of relevant dietary exposure and risk scenarios.

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Editor-in-chief: Hans P. van Egmond, RIKILT Wageningen UR, Business unit Contaminants & Toxins, the Netherlands
Section editors
•-omics Deepak Bhatnagar, USDA, USA
•feed, toxicology Johanna Fink-Gremmels, Utrecht University, the Netherlands
•toxicolog y Isabelle P. Oswald, INRA, France
•pre-harvest Alain Pittet, Nestlé Research Center, Switzerland
•post-harvest Naresh Magan, Cranfield University, United Kingdom
Paola Battilani, Università Cattolica del Sacro Cuore, Italy
•analysis Sarah de Saeger, Ghent University, Belgium
•food, human health, analysis Gordon S. Shephard, University of Stellenbosch, South Africa
•economy, regulatory issues Felicia Wu, Michigan State University, USA
•industrial challenges and solutions Michele Suman, Barilla, Italy
Editors
Paula A lvito, National Institute of Health, Portugal; Diána Bánáti, ILSI Europe, Belgium; Lei Bao, ACSIQ, China
P.R.; Franz Berthiller, BOKU, Austria; Catherine Bessy, FAO, Italy; Wayne L. Bryden, University of Queensland,
Australia; Pedro A. Burdaspal, Centro Nacional de Alimentación, Spain; Jeffrey W. Cary, USDA, USA; Sofia N. Chulze,
Universidad Nacional de Rio Cuarto, Argentina; Mari Eskola , EFSA; Piotr Goliński, Poznań University of Life Sciences,
Poland; Tetsuhisa Goto, Shinshu University, Japan (retired); Clare Hazel, RHM Technology, United Kingdom; Rudolf
Krska, University of Natural Resources and Life Sciences, Vienna, Austria; Antonio F. Logrieco, Institute of Sciences
of Food Production, Italy; Rebeca López-García, Logre International, Mexico; Chris Maragos, USDA, USA; Monica
Olsen, National Food Administration, Sweden; Roland Poms, MoniQA Association, Austria; James J. Pestka, Michigan
State University, USA; Michael Rychlik, Technical University München, Germany; Helen Schurz Rogers, CDC/
NCEH/DEEHS, USA; Hamide Z. Şenyuva, FoodLife International Ltd., Turkey; Joseph R. Shebuski, Cargill Corporate,
USA; Trevor K. Smith, University of Guelph, Canada; Martien Spanjer, VWA, the Netherlands; Jörg Stroka, European
Commission, IRMM; János Varga, University of Szeged, Hungary; Frans Verstraete, European Commission, DG Health
and Consumer Protection; Cees Waalwijk, Plant Research International, the Netherlands; omas B. Whitaker, USDA,
USA; Christopher P. Wild, IARC, WHO
Founding editor: Daniel Barug, Bastiaanse Communication, the Netherlands
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World Mycotoxin Journal, 2015; 8 (5): 573-589 WageningenAcademic
Publishers
ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2014.1864 573
1. Introduction
Mycotoxins pose significant threats to food and feed safety,
with economic and international trade implications as
well (Wu, 2007, 2015; Zain, 2011). Since the discovery
of aflatoxins in 1960 and subsequent recognition that
mycotoxins are of substantial concern for the health of
both humans and animals, mycotoxins have received
considerable attention as biotoxins in the food supply chain.
Mycotoxins enter the body via ingestion of contaminated
food and feed and elicit some complicated and overlapping
acute and chronic effects in sensitive species (Bryden,
2012; D’Mello et al., 1999; Fink-Gremmels, 1999; Wild
and Gong, 2010; Zain, 2011). To be used for a science-based
risk assessment, a precise evaluation of the impacts of
mycotoxin contamination on health must consider the effect
of mycotoxins on the gastrointestinal (GI) environment.
The GI tract represents the first barrier preventing the
entry of undesirable substances. The intestinal epithelium
can be exposed to high concentrations of mycotoxins
upon ingestion of contaminated food and feed, and direct
intestinal damage can be exerted by mycotoxins (Bouhet
and Oswald, 2005). In ruminants, the rumen microflora
can degrade and inactivate mycotoxins, and as a result,
ruminants are the least susceptible animal species. However,
rumen detoxification capacity may be saturable and can
Mycotoxin mechanisms of action and health impact: ‘in vitro’ or ‘in vivo’ tests, that is
the question
F. Cheli*, C. Giromini and A. Baldi
Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Via Trentacoste 2, 20134, Milano,
Italy; federica.cheli@unimi.it
Received: 11 December 2014 / Accepted: 2 June 2015
© 2015 Wageningen Academic Publishers
REVIEW ARTICLE
Abstract
The aim of this paper is to present examples of in vitro and in vivo tests for mycotoxin mechanisms of action and
evaluation of health effects, with a focus on the gut environment and toxicity testing. In vivo investigations may
provide information on the net effects of mycotoxins in whole animals, whereas in vitro models represent effective
tools to perform simplified experiments under uniform and well-controlled conditions and a suitable alternative to
in vivo animal testing providing insights not achievable with animal studies. The main limits of in vitro models are
the lack of interactions with other cells and extracellular factors, lack of hormonal or immunological influences, and
lack or different levels of in vitro expression of genes involved in the overall response to mycotoxins. The translation
of in vitro data into meaningful in vivo effects remains an unsolved problem. The main issues to be considered are
the mycotoxin concentration range in accordance with levels encountered in realistic situations, the identification
of reliable biomarkers of mycotoxin toxicity, the measurement of the chronic toxicity, the evaluation of single- or
multi-toxin challenge. The gastrointestinal wall is the first barrier preventing the entry of undesirable substances.
The intestinal epithelium can be exposed to high concentrations of mycotoxins upon ingestion of contaminated
food and the amount of mycotoxin consumed via food does not always reflect the amount available to exert toxic
actions in a target organ. In vitro digestion models in combination with intestinal epithelial cells are powerful tools
to screen and predict the in vivo bioavailability and digestibility of mycotoxins in contaminated food and correctly
estimate health effects. In conclusion, in vitro and in vivo tests are complementary approaches for providing a more
accurate picture of the health impact of mycotoxins and improved understanding and evaluation of relevant dietary
exposure and risk scenarios.
Keywords: in vivo models, in vitro models, mycotoxicity, gastrointestinal tract

F. Cheli et al.
574 World Mycotoxin Journal 8 (5)
vary with changes in the diet, the mycotoxin burden, the
duration of exposure, and the health and production status
of the animal herd (Fink-Gremmels, 2008). Therefore, the
amount of mycotoxins consumed via food does not always
reflect the amount available to exert toxic action in a target
organ of the body as only a part of the ingested compound
will be bioavailable.
Despite efforts to control fungal contamination, extensive
mycotoxin contamination has been reported to occur in
both developing and developed countries. It has been
estimated that up to 25% of the world’s crops grown for
feed and food may be contaminated with mycotoxins (Fink-
Gremmels, 1999; Hussein and Brasel, 2001). Recent surveys
were carried out to evaluate the incidence of mycotoxins
in food and feed samples. On a global level, up to 81% of
food and feed samples were found to be positive for at least
one mycotoxin and 38% were found to be co-contaminated
(Binder et al., 2007; Marin et al., 2013; Rodrigues and
Naehrer, 2012; Streit et al., 2012, 2013). Furthermore,
according to the possible carry-over of each toxin, feed
contamination can also represent a hazard for the safety of
food of animal origin and contribute to mycotoxin intake
in humans (Fink-Gremmels, 2008).
In addition to health risks, the economic impact of
mycotoxin occurrence in the food supply chain is also an
important consideration. Many factors must be taken into
account when attempting to determine economic losses
in animal production, such as reduced crop production,
disposal of contaminated food and feeds, world trade,
reduced livestock production, increased mortality,
analytical and regulatory costs, and investment in research
(Bryden, 2012; Wu, 2015). The economic costs and impact
on the international trade associated with mycotoxin
contamination are difficult to assess in a consistent and
uniform manner and impossible to determine accurately.
Quantitative estimates of economic losses associated with
mycotoxin contamination in different commodities may
range from hundreds of millions to billions of US$ annually
(Bryden, 2012; Wu, 2007, 2015). In response to health
concerns, mycotoxin regulations have been established in
over 100 countries (FAO, 2004; Van Egmond et al., 2007).
However, no universal standardisation of regulatory limits
for mycotoxins exists, and maximum acceptable limits
vary greatly from country to country. The European Union
(EU) harmonised regulations for the maximum levels of
mycotoxins in food and feed among its member nations
(Cheli et al., 2013, 2014a).
It is difficult to gain a complete understanding of and to
adequately model mycotoxin health effects due to the
complexity of the interactions between the numerous
factors affecting the magnitude of mycotoxin toxicity. In
vivo investigations may provide information on the net
effects of mycotoxins in whole animals, whereas cell-specific
answers may result from in vitro investigations.
The aim of this paper is to present examples of in vitro and
in vivo tests for mycotoxin research, with a focus on the gut
environment and toxicity testing. Advantages, drawbacks
and technical problems regarding specific applications of
each model test, the link between in vitro and in vivo tests,
the relevance of in vitro tests compared to in vivo tests, and
the predictive efficacy of in vitro tests will be discussed.
2. Mycotoxins: animal and human health
implications
Scientists first isolated specific toxins from their fungal
source and demonstrated the causative link with a disease
in 1961, when Turkey X disease was linked to the presence
of aflatoxins in feed (Dickens and Jones, 1963; Wannop,
1961). Since that time, research, ideas and methodologies in
the mycotoxin field have changed frequently, and data from
50 years ago have been revised, confirmed or improved.
The role of mycotoxin co-contamination (aflatoxin and
cyclopiazonic acid mycotoxin) was only fully explained
for Turkey X disease after the original reports were re-
analysed (Bryden, 2012). How many mycotoxins exist is
not known, but toxic metabolites of fungi may potentially
number in the thousands (CAST, 2003). The number of
mycotoxins of concern for human and animal diseases is
considerably less, however, and includes metabolites of
Aspergillus spp., Fusarium spp., and Penicillium spp., which,
with the exception of the Fusarium plant pathogens, may
include commensals in addition to pathogens (Murphy et
al., 2006). The major classes of mycotoxins are aflatoxins,
trichothecenes, fumonisins, zearalenone (ZEA), ochratoxin
A (OTA), and ergot alkaloids. The impact of mycotoxins
on human health was evaluated in 1993 by the WHO-
International Agency for Research on Cancer (WHO-IARC,
1993). Naturally occurring aflatoxins were classified as
carcinogenic to humans (Group 1), whereas OTA, aflatoxin
M
1
, and fumonisins were classified as possible carcinogens
(Group 2B). Trichothecenes and ZEA were not classified
as human carcinogens (Group 3). However, new fungal
metabolites are still emerging, and their potential and
synergistic contributions to diseases in humans and animals
have yet to be assessed.
Mycotoxin contamination is unavoidable and unpredictable.
Despite efforts to control fungal contamination, mycotoxins
are ubiquitous in nature and occur regularly worldwide in
food and feed. The health hazards of mycotoxins have been
reviewed extensively in recent years (Bryden, 2012; Zain,
2011; D’Mello and MacDonald, 1997; Marin et al., 2013;
Wild and Gong, 2010). In health risk assessment, ingestion
of food is thought to be the primary route of exposure to
mycotoxins, although contact and inhalation exposure
have also been reported (Bush et al., 2006). Mycotoxin

Mycotoxicity in vivo and in vitro
World Mycotoxin Journal 8 (5) 575
effects on humans and animals can be categorised as
acute or chronic. Acute toxicity generally has a rapid toxic
response, whereas chronic toxicity is characterised by low-
dose exposure over a long time period. Bryden (2012), in
a review of implications for animal productivity and feed
security, stressed that the major problem associated with
mycotoxin contamination of the animal feed supply chain
is not acute disease episodes but rather low levels of toxin
ingestion that cause an array of metabolic disturbances.
Moreover, animals may vary considerably in their response
to toxin exposure, and the pattern of target organs may
differ by species. For example, in the case of fumonisin B1
(FB1) toxicity, the lungs, liver and pancreas are affected in
pigs, whereas in rats and mice, the liver and kidneys are the
primary target organs (Stockmann-Juvala and Savolainen,
2008). Ruminants are among the least susceptible animal
species as rumen microflora can degrade and inactivate
mycotoxins. However, exposure to complex mixtures of
toxins over a long time period can result in an impairment
of rumen microbiota, reduced detoxifying capacity and liver
function, and immunosuppressive effects (Fink-Gremmels,
2008; Santos and Fink-Gremmels, 2014).
The scientific literature on mycotoxins is rich in reports
investigating cellular mechanisms, cellular toxicity, and
associated pathology (Bony et al., 2006; Bouhet et al., 2004;
Cui et al., 2010). Data with regard to their effects on the
GI tract are, however, comparatively limited (Grenier and
Applegate, 2013; Maresca, 2013; Pinton et al., 2012; Smith
et al., 2012). The GI tract is the initial site for interaction
of ingested mycotoxins, and it exerts a crucial role in
mycotoxin absorption and bioavailability in animals and
humans. A complete evaluation of health risks associated
with mycotoxin exposure requires the knowledge and
elucidation of mycotoxin bioavailability. Studies in animals
and humans have shown that the food bioavailability of
compounds can differ significantly depending on the food
source, food processing or method of food preparation
(Versantvoort et al., 2005). Modulation of intestinal
functions following mycotoxin ingestion is another factor
to be considered. Moreover, the toxicokinetic pathways,
including adsorption, distribution, elimination, metabolic
biotransformations, and the toxicodynamic pathways,
must also be considered. Thus, the amount of ingested
mycotoxins does not necessarily reflect the amount that is
available to the body (Maresca, 2013; Pestka, 2010; Ringot
et al., 2006; Upadhaya et al., 2010).
The difficulty in obtaining a thorough understanding of the
impact of mycotoxins on health is a result of the complexity
of the interactions involved in the intoxications and the
relationships with host factors, such as species sensitivity,
level and time of exposure, individual sensitivity, age, health,
nutritional status, bioaccessibility, mechanisms/modes of
action, metabolism, and defence mechanisms (Hussein
and Brasel, 2001).
3. Mechanisms of mycotoxicity
The potential number of biochemical targets that may be
available to the diverse array of mycotoxins poses a challenge
to understanding their mode of action. The mechanisms of
mycotoxicity were recently reviewed (Bryden, 2012; CAST,
2003; Omar, 2013; Pestka, 2010; Speijers and Speijers, 2004;
Stockmann-Juvala and Savolainen, 2008; Voss et al., 2007).
The major classes of mycotoxins, the most commonly
contaminated food products, and the main mechanism of
mycotoxicity in vivo and in vitro on sensitive species are
reported in Table 1.
4. Mycotoxins: in vitro and in vivo tests
Considering the complex health impacts of mycotoxin
contamination in the food supply chain, a large amount
of research has been conducted using in vivo and in vitro
tests. The in vivo investigations provide information on
mycotoxin net effects in whole animals, whereas cell-
specific answers result from in vitro investigations. Within
in vitro tests, cell-based tests provide well-defined and
reproducible experimental conditions for the evaluation
of mycotoxin toxicity, mechanisms of action and biological
functions, and represent a suitable alternative to in vivo
animal testing. (Cheli et al., 2014b).
However, in vitro tests are not free of problems. Cell-based
models are in vitro systems, which may not reflect the
in vivo conditions of cells and tissues in the organism,
where signalling and interaction between organs play
important roles. The in vitro culture environment
lacks several systemic components involved in in vivo
homeostatic regulation. Moreover, in vitro studies do
not take into consideration the effects of bioavailability,
pharmacokinetics, metabolism, distribution and interaction
with binding and transport proteins, or other biological
processes that occur in the intact organism, all of which
may influence the biological effects of mycotoxins. The
extrapolation of in vitro data to the in vivo situation
remains problematic. The main issues to be considered
are the mycotoxin concentration range in accordance with
levels plausibly encountered in realistic situations, the
identification of reliable biomarkers of mycotoxins, the
toxicity in vivo and in vitro, the measurement of the chronic
mycotoxin toxicity (that is, the actual situation in the field),
and the evaluation of single- or multi-toxin challenge.
Acute mycotoxin toxicity: in vitro and in vivo tests
The in vivo acute toxicity (LD
50
[lethal dose 50%])
evaluation has been criticised because it uses too many
animals and sacrifices a large proportion of the animals
tested (Stallard and Whitehead, 2004). There are many
cell models available for in vitro toxicity testing. Gutleb
et al. (2002) and Cheli et al. (2014b) have noted that the

F. Cheli et al.
576 World Mycotoxin Journal 8 (5)
collection of reliable in vitro toxicity data requires that
several factors be considered, including cell specificity and
sensitivity, the best culture conditions for optimal growth,
the endpoints to measure, and the most appropriate cell
culture system. With this in mind, the issue may be less
how to collect in vitro toxicity data than how to translate
in vitro toxicity data into meaningful in vivo effects. Overall
results indicate that to be useful and predictive of in vivo
effects, in vitro toxicity screening models must be well
characterised and should have the capacity to test a large
Table 1. In vivo versus in vitro mycotoxicity of selected mycotoxins.
Mycotoxins Contaminated
products
Animal
affected
In vivo toxicity In vitro toxicity References
Aflatoxins maize, peanuts,
cottonseed,
tree nuts, dairy
products
swine, dog,
cat, cattle,
sheep, young
birds, humans
hepatotoxicity (DNA strand breakage,
oxidative damage, inhibition of protein
synthesis)
teratogenicity, carcinogenic effect
(chromosomal aberration, gene
expression alteration, inhibition of
DNA methylation)
immunomodulation (reduced
lymphocyte proliferation, reduced
antibody production)
DNA adduct; oxidative
damage; gene
mutation (in bacterial
cell in vitro)
McLean and Dutton, 1995
Hamid et al., 2013
Deoxynivalenol wheat, barley,
maize, oats
swine, dairy
cattle, poultry,
horses,
humans
emetic effects
dose-dependent immune
dysregulation (IgA dysregulation, IL-6
and COX-2 up-regulation, leukocyte
apoptosis)
carcinogenesis (inhibition of
protein synthesis, gene expression
regulation)
cell apoptosis;
inhibition of protein
synthesis; gene
up-regulation (TNF-α,
IL-8, IL-6)
Pestka, 2010
Pestka and Smolinski, 2005
Pestka et al., 2004
Tiemann and Dänicke, 2007
Sergent et al., 2006
Ochratoxins cereal grains,
coffee, grapes
swine, poultry,
humans
nephrotoxicity, hepatotoxicity,
teratogenicity, immunotoxicity, genetic
mutations (radical formation, lipid
peroxidation, apoptosis)
inhibition of protein
synthesis; membrane
lipid peroxidation; DNA
damage
Ringot et al., 2006
Sorrenti et al., 2013
Heussner et al., 2006
Kuroda et al., 2015
Fumonisins maize, cereal
grain, silage
horse, swine,
humans
nephrotoxicity and hepatotoxicity
(apoptosis, necrosis, modulation of
urine enzyme levels, hepatocellular
hyperplasia)
carcinogenicity (oxidative damage,
sphingolipid metabolism alteration)
inhibition of eukaryotic protein
synthesis
inhibition of de
novo sphingolipid
biosynthesis; lipid
peroxidation; apoptosis
(caspase-3 activation
or DNA fragmentation);
inhibition of eukaryotic
protein synthesis
Stockmann-Juvala and
Savolainen, 2008
Karuna and Sashidhar, 2008
Zearalenone maize, hay swine, dairy
cattle
abortion
infertility
vulvovaginitis (impairment embryo
development, inhibition of uterine cell
proliferation)
non-estrogenic toxicities: hepatic and
kidney toxicity
DNA-adduct formation
in in vitro HEp2 cells,
intestinal and kidney
cells
Abid-Essefi et al., 2004
Tiemann and Dänicke, 2007
Minervini et al., 2001
Other
trichothecenes
(T-2 and
HT-2 toxin,
diacetoxyscirpenol,
nivalenol)
wheat, barley,
maize, oats
swine, dairy
cattle, poultry,
horses,
humans
thymic disfunction (modulation of
macrophages, B cells and T cells
proliferation)
apoptosis; DNA
fragmentation
Pestka et al., 2004
Weidner et al., 2012
Islam et al., 1998

Mycotoxicity in vivo and in vitro
World Mycotoxin Journal 8 (5) 577
number of molecules in a short period of time and the data
should provide information on both potential mechanisms
of toxicity and potential subcellular targets. It is unlikely
that any one in vitro model would suffice as a final decision
point for toxicity evaluation, but rather a tiered series of
models that provide important information should be used.
To successfully develop cell-based models that are suitable
for a specific research objective, the first step is to choose
the most adequate cells. With regard to mycotoxin research,
careful consideration should be given to the choice of
the species and the specific target organ of interest. It is
well known that specific toxins may affect many target
organs, tissues and systems, notably the liver, kidney,
nervous system, oral and gastric mucosa, reproductive
tract, and endocrine and immune systems (Hussein and
Brasel, 2001; Murphy et al., 2006). Once the suitable cells
have been chosen, their properties and the physiological
and developmental state of the humans or animals from
which the cells are obtained represent other critical factors
affecting the effectiveness of the model. The availability of
a large number of human and animal cell lines with diverse
genotypes and origin of tissues provides a broad base for
models to draw from for the study of various biological
processes. Great differences in sensitivity to mycotoxins
between different cell lines have been reported (Cheli et al.,
2014b; Gutleb et al., 2002). Examples of different cell lines
used for testing deoxynivalenol (DON) and OTA toxicity
are shown in Table 2, with results suggesting that there
are large differences in sensitivity to these toxins between
cell lines, with IC50 (inhibitory concentrations 50%) values
ranging from 0.4 to 125 and from 1 to 800 µM for DON and
OTA, respectively. Cumulatively, the results indicate that
the response to mycotoxins is quite complex, depending
Table 2. Examples of different cell lines used for the development of cell-based models in mycotoxin toxicity test for ochratoxin
A and deoxynivalenol.
Cells Origin/cell type Species IC50, (µm)1References
Ochratoxin A
VT79 lung/fibroblast Chinese hamster 19
133
Behm et al., 2012
Palma et al., 2007
GES-1 foetal gastric mucosa/epithelial human 67.72 Cui et al., 2010
J774A.1 blood/ macrophage mouse 22.4 Ferrante et al., 2008
HK-2 renal/epithelial human 800 Arbillaga et al., 2007
SK-N-MC brain/epithelial human 66 Baldi et al., 2004
MDCK kidney/epithelial canine 36 Baldi et al., 2004
AML-12 liver/hepatocyte mouse >99 Baldi et al., 2004
LLC-PK1 kidney/epithelial pig 66 Baldi et al., 2004
BME-UV1 mammary gland/epithelial bovine 1 Baldi et al., 2004
Deoxynivalenol
HCT116 colon/epithelial human 125 Bensassi et al., 2012
IEC-6 small intestine/epithelial rat 50.82 Bianco et al., 2012
IPEC-1 small intestine/epithelial pig 2.2 Diesing et al., 2011a
IPEC-J2 small intestine/epithelial pig 7.2 Diesing et al., 2011a
PBMC peripheral blood mononuclear cells human 0.4 Taranu et al., 2010
PBMC peripheral blood mononuclear cells pig 0.54 Taranu et al., 2010
HT 29 colon/epithelial human 10 Bensassi et al., 2009
U937 lymphoma human 0.95 Nielsen et al., 2009
Caco-2 colon/epithelial human 10
3.4
1.39
Bony et al., 2006
Cetin and Bullerman, 2005
Alassane-Kpembi et al., 2013
HepG2 liver carcinoma/epithelial-like human 0.67
28.2
1.89
Calvert et al., 2005
Cetin and Bullerman, 2005
Nielsen et al., 2009
CHO-K1 ovary/epithelial-like Chinese hamster 0.91 Cetin and Bullerman, 2005
C5-O skin/keratinocyte Balb/c mice 1.82 Cetin and Bullerman, 2005
VT9 lung/fibroblast Chinese hamster 1.55
0.7
Cetin and Bullerman, 2005
Behm et al., 2012
1 IC50 = mycotoxin concentration inducing 50% loss of cell viability.

F. Cheli et al.
578 World Mycotoxin Journal 8 (5)
not only on the particular cell line and mycotoxin but also
on exposure dose and time. Therefore, the application of
more than one cell line of different origins to establish a cell-
based model for the purposes of acute toxicity screening
is recommended. When choosing established cell lines,
it is important to keep in mind that these models may
rely on immortalised or transformed cells in an artificial
environment and, during passage, may de-differentiate and
lose some functional properties observed in vivo. Therefore,
cell lines involved in single- or co-culture models must be
sufficiently characterised prior to their use.
The predictive power of in vitro tests for mycotoxin toxicity
may be improved with the inclusion of several endpoints
associated with toxicity. Cell growth or cell viability involves
the presence of metabolic activity and membrane integrity.
The tetrazolium and water soluble tetrazolium salts (MTT/
MTS/WSTs) reduction test is the most widely used for
these factors and is a reliable method that may be amenable
to the use of high-throughput screening because of its
relatively low cost and high sensitivity. Other assays, such
as resazurine reduction (Alamar Blue), neutral red uptake
assay, and cell lactate dehydrogenase release into the culture
medium, have been used to assess cell viability. These
methods are the most frequently used acute-marker assays
for cell viability and cytotoxicity, providing a quantitative
estimation of cell metabolic activity and membrane
integrity. Acute markers should be balanced with chronic
indicators of toxicity, such as glutathione depletion and
lipid peroxidation. According to the cytotoxic endpoint
used, the measured IC50 values for the same mycotoxin
may differ (Table 3). These results may be related to the
differential sensitivities of the cell line, the physiological/
differentiation state of the cells, and the sensitivity of
the biomarkers considered. Therefore, the evaluation of
multiple parameters provides a more accurate toxicity
assessment, providing insights into the mechanisms of
action and the cellular target of mycotoxins as well as the
pathways involved in cellular damage, stress, and death.
Whether in vitro cytotoxicity data are predictive of in
vivo acute toxicity remains a crucial question. In vivo
toxicity LD50 (mg/kg feed) has been calculated from in
vitro measured IC
50
(µM) values for mycotoxins, using
cell lines of different origin in terms of species and tissue/
organs using the Spielmann equation (Braicu et al., 2010;
Creppy et al., 2004; Spielmann et al., 1999). Calculated LD
50
values are not always in line with experimentally determined
LD
50
values in animal tests, which confirms both that some
species are more sensitive than others and that the target
organs are not necessarily those predicted. Species-specific
differences may be overcome using specific animal cell-
culture models. Interestingly, several cell lines of different
origin have been shown to exhibit a higher sensitivity to
OTA than cells of renal origin (Baldi et al., 2004; Creppy
et al., 2004). This is surprising for a mycotoxin classified
as a nephrotoxic compound. Such results confirm that the
extrapolation of in vitro data to the in vivo situation remains
a crucial problem.
In light of the importance of mycotoxin combinations,
toxicological interactions between mycotoxins have been
evaluated using both in vivo and in vitro assays (Alassane-
Kpembi et al., 2013; Bianco et al., 2012; Huff et al., 1988;
Table 3. Cell viability assays: relative cytotoxicity of mycotoxins in different cell lines.
Cells/mycotoxins1IC50 (µM)2References
MTT/MTS NR AB DNA3
Caco-2/ DON 1.39 1.19 Alassane-Kpembi et al., 2013
RAW 264.7/ ENN-B 4.7 2.6 Gammelsrud et al., 2012
HepG2 / DON >25 >25 >25 Sahu et al., 2010
WRL68/ DON 8.5 5 2
MH1C1/ DON 1 2 5
BNL CL2/ DON 7.5 >25 5
Caco-2 dividing cells /DON 10 3.7 Bony et al., 2006
Caco-2 differentiated cells/ DON >10 >10
Caco-2/ DON 25 21.5 1.7 Kouadio et al., 2005
Caco-2/ ZEA 25 15 10
Caco-2/ FB121 >150 20
1 DON = deoxynivalenol; ENN-B = enniatin B; FB1 = fumonisin B1; ZEA = zearalenone.
2
IC
50
= mycotoxin concentration inducing 50% loss of cell viability; AB = Alamar Blue assay; NR = Neutral Red assays; MTT/MTS = tetrazolium and
water-soluble tetrazolium salt assay.
3 Double stranded DNA content.

Mycotoxicity in vivo and in vitro
World Mycotoxin Journal 8 (5) 579
Kolf-Clauw et al., 2013; McKean et al., 2006; Ruiz et al.,
2011a,b; Tajima et al., 2002; Tavares et al., 2013). Only a
small number of in vivo studies on mycotoxin combinations
are available, given that in vivo tests require a large sample
size for evaluation. Therefore, despite the various limits,
in vitro tests may represent powerful tools to establish a
relative potency scale for mycotoxins and to assess their
combined effects in terms of additive, antagonistic or
synergistic toxicity. In Table 4, the combined effects of
combinations of mycotoxins of major concern to human
and animal health are reported. Different methodological
approaches can be used for dose-response assessment
of mycotoxin mixtures, such as the arithmetic model of
additivity, factorial designs and the theoretical biology-
based models of additivity (Oswald and Alassane-Kpembi,
in press). Isobologram analysis is still the most popular and
reliable tool for analysing experimental data obtained from
cellular systems and allows a quantitative assessment of
interaction between mycotoxins tested in a mixture (Chou,
2006; Chou and Talalay, 1984).
The results indicate that the effects of mycotoxin in
combination may differ (by being synergistic, additive
or antagonistic) depending on the dosage used. The
simultaneous presence of several mycotoxins in food
products and diets, even at low doses, may be more toxic
than predicted from single mycotoxins. To date, only a few
studies have examined the interaction between different
Table 4. Combined effects of mycotoxin mixtures: in vitro tests.
Cell line Mycotoxins1Effect2References
IPEC-1 DON+NIV Syn Alassane-Kpembi et al., in press
DON+3-ADON Syn/Ant3
DON+15-ADON Syn
3-ADON+15-ADON Syn
NIV+FX Ad
DON+FX Ant
Caco-2 AFM1+OTA Ant Tavares et al., 2013
CHO-K1 BEA+DON Ant Ruiz et al., 2011a
BEA+T-2 Syn
DON+T-2 Syn
BEA+DON+T-2 Syn
Vero BEA+DON Ant Ruiz et al., 2011b
BEA+T-2 Ant
DON+T-2 Ant
BEA+DON+T-2 Ant
HUVEC AFB1+AFB2Syn Braicu et al., 2010
AFG1+AFG2Ad
HFL/A2780 AFB1+AFB2/AFG1+AFG2Ad
HepG2 AFB1+FB1Weak Ant McKean et al., 2006
BEAS-2B AFB1+FB1Syn
LLC-PK1 OTA+CIT/OTB+OTA/
PAT+OTA/CIT+OTB/PAT+OTB
Syn Heussner et al., 2006
Caco-2 DON+ZEA Ad Kouadio et al., 2005
C6 glioma
Caco-2
Vero
OTA+FB1Syn Creppy et al., 2004
OTA+FB1Syn
OTA+FB1Syn
L929 T-2+NIV Syn Tajima et al., 2002
DON+NIV Syn
ZEA+NIV Syn
T-2+DON+NIV+ZEA+FB1Less than Ad/Ad3
1 AFM1 = aflatoxin M1; AFB1, AFB2 = aflatoxin B1, B2; AFG1, AFG2 = aflatoxin G1,G2; BEA = beauvericin; CIT = citrinin; DON = deoxynivalenol; 15-ADON
= 15-acetyldeoxynivalenol; 3-ADON = 3-acetyldeoxynivalenol; FB
1
= fumonisin B
1
; FX = fusarenon X; NIV = nivalenol; OTA = ochratoxin A; T-2 = T-2
toxin; ZEA = zearalenone.
2 Ad = additive; Ant = antagonistic; Syn = synergistic.
3 Different effects were observed for different doses tested.

F. Cheli et al.
580 World Mycotoxin Journal 8 (5)
mycotoxins, and existing studies have focused primarily on
the acute effects of high levels of mycotoxin combinations
on livestock. Only limited information exists regarding
the interaction of mycotoxins at the lower levels that are
commonly found in naturally contaminated diets (Grenier
and Oswald, 2011). Considering the frequent co-occurrence
of mycotoxins in diets and the concentrations of toxins
to which consumers are normally exposed, knowledge of
mycotoxin interactions is of high relevance. The use of in
vitro tests may therefore represent a new risk assessment
strategy by providing scientific data to regulatory bodies
to implement regulatory standards for a great variety of
mycotoxins occurring both individually and in combination
(Alassane-Kpembi et al., 2013).
Biomarkers of mycotoxin toxicity: in vitro and in vivo
tests
When ingested, mycotoxins may cause mycotoxicosis,
which may be acute or chronic. Chronic conditions have
a much greater impact on human and animal health.
Chronic diseases include reduced or refused intake, and
neurological, oestrogenic, hepatotoxic and immunotoxic
effects (D’Mello et al., 1999; Fink-Gremmels, 2008;
Scudamore and Livesey, 1998; Wilkinson, 1999). Mycotoxins
produce chronic diseases at doses that are usually lower
than those responsible for acute effects. In vitro testing may
contribute to a greater understanding of the mechanisms
of mycotoxin-induced toxicity. To be useful and predictive
of in vivo effects, two critical points must be considered:
the mycotoxin dosage and the choice of biomarkers linking
health impact and exposure.
The mycotoxin doses used in in vivo and in vitro studies
must reflect in-field conditions. Based on mycotoxin
surveys and legislative limits, realistic doses can be
calculated and used in in vivo tests (Binder et al., 2007;
Schatzmayr and Streit, 2013). The conversion of in vivo
doses to in vitro concentrations requires knowledge of the
bioavailability, pharmacokinetics and pharmacodynamics
of each mycotoxin. Sergent et al. (2005, 2006), in a Caco-2
model cell, evaluated OTA and DON transport across the
intestinal epithelium at realistic concentrations. The levels
plausibly encountered in the GI tract after consumption
of food contaminated by these mycotoxins were calculated
assuming ingestion in one meal, taking into account dilution
by the GI fluid, and assuming total bioaccessibility of the
mycotoxins. Insights into how mycotoxins in the diet affect
the GI environment and enter the organism are specifically
discussed elsewhere in the paper.
The choice of biomarkers that best link health impacts and
exposure is critical if data obtained in vitro is to reflect
in vivo situations. Lü et al. (2003) carried out a DNA
microarray analysis to assess OTA-specific expression
profiles in vivo (rats) and in vitro (primary rat proximal
tubular cells). The authors of that study identified 215
model-independent, differentially regulated genes. These
represent 85% of all transcriptional changes, indicating a
high correspondence of in vitro and in vivo data. Sahu et al.
(2008) observed a significant DON-induced necrotic cell
death effect in rat liver clone-9 cells in culture and a DON-
induced liver necrosis in a corresponding in vivo study with
rats, concluding that the hepatotoxicity of DON in clone-9
cells in vitro was in close agreement with that in rats in
vivo as the biomarker of liver damage was the same in both
studies. Rached et al. (2008) evaluated putative biomarkers
of nephrotoxicity of OTA in vivo and in vitro, identifying
six sensitive endpoints for acute kidney injury in rats in
vivo but detected no significant increase in the expression
of the in vivo marker genes, and proteins were evident in
vitro using kidney NRK-52E cells after exposure to OTA.
The authors concluded that these six endpoints may not
be suitable for sensitive detection of nephrotoxic effects
in vitro. The inconsistent results reported in the literature
confirm that it is critical to identify the biomarkers that best
link health effects with exposure. Moreover, it is necessary
to carefully consider the limits of a chosen in vitro model,
such as lack of interactions with other cells, extracellular
factors, lack of hormonal or immunological influences, and
lack or different levels of expression of genes involved in
the overall response to mycotoxins in vitro to validate the
model and support a correct interpretation of the results
for the in vivo situation.
Within the wide spectrum of toxicological effects of
mycotoxins, the ability of some mycotoxins to alter
normal immune function is particularly interesting.
The immunological response involves many different
cell types. Depressed T- or B-lymphocyte activity and
effects on cytokine production have been reported in
mycotoxin-induced immunosuppression in vivo (Oswald
et al., 2005). From the most recent literature, cell-based
models based on either primary cells, such as phagocytes
and lymphocytes isolated from blood, or cell lines have
been used for immunotoxicity screening (Berek et al., 2001;
Ferrante et al., 2008; Gammelsrud et al., 2012; Nogueira
da Costa et al., 2011; Pei and Gunsch, 2013; Pestka and
Zhou, 2006; Richetti et al., 2005; Schmeits et al., 2013).
In vitro tests confirm the immunotoxicological effects of
mycotoxins observed in vivo. However, when cell lines
are used, careful consideration should be given to the
species of origin of the cells, as interspecies differences
in mycotoxin-induced immunomodulation and sensitivity
have been reported (Meky et al., 2001). Attention must be
given on the effects of mycotoxins on the local intestinal
immune response. Because of their location, intestinal
epithelial cells could be used as a powerful biomarker
for the intestinal immune system, following ingestion
of mycotoxin contaminated food and feed. Bouhet et al.
(2006) reported that FB
1
alters the intestinal immune
response by decreasing IL-8 expression both in pig intestine

Mycotoxicity in vivo and in vitro
World Mycotoxin Journal 8 (5) 581
and in the porcine intestinal epithelial cell line IPEC-1.
The impact on the immune system has been evidenced at
doses below those that cause cellular toxicity.
5. Modelling the gastro-intestinal tract
In vitro bioaccessibility and absorption of mycotoxins
Mycotoxins enter the body via consumption of
contaminated food and feed. To enable a more precise
evaluation of the impact of food and feed mycotoxin
contamination on health and to facilitate science-based
risk assessments, it is necessary to provide insights into
how mycotoxins found in food affect the GI environment
and enter the organism to begin their journey from the
gut to the internal organs. The GI wall is the first barrier
preventing the entry of undesirable substances, and the
intestinal epithelium can be exposed to high concentrations
of mycotoxins upon ingestion of contaminated food.
The amount of mycotoxins consumed via food does not
always reflect the amount available to exert toxic action
in a target organ of the body as only part of the ingested
compounds will be bioavailable. The food/feed matrix,
type of mycotoxin contamination, presence of masked
mycotoxins, food/feed treatments, and additives influence
the level of internal exposure to mycotoxins. Bioaccessibility
and digestibility data are needed to evaluate the amount
of toxin that becomes available for absorption through
the intestinal epithelium and to subsequently estimate the
health effects correctly. For this purpose, in vitro digestion
models have been widely used and validated for nutritional
studies in both humans and animals (Cheli et al., 2012;
Hur et al., 2011). The use of in vitro digestion models to
assess mycotoxin bioaccessibility/absorption, and thereby
avoiding the use of more complex cell-culture techniques
or the use of animals in expensive in vivo experiments, has
been reviewed by González-Arias et al. (2013) (Table 5).
Briefly, the static models simulate the pig or human GI tract
and consist of a two- or three-step procedure simulating
digestive processes in the mouth, stomach and small
intestine, following the addition of simulated physiological
juices and incubation at 37°C for a time relevant to the
specific compartment (Minekus et al., 2014). The TIM
models, extensively validated based on data obtained from
animal nutritional research, are multi-compartmental,
dynamic computer-controlled models (Minekus et al.,
1995, 1999). TIM-1 simulates the digestive processes of the
stomach and small intestine of monogastric animals, and
TIM-2 simulates the colon and includes a rich microbial
gut-derived flora (Minekus, 2005).
Application of in vitro models to evaluate mycotoxin
bioaccessibility/absorption from naturally or artificially
spiked contaminated food dates from the beginning of
this century (Avantaggio et al., 2003, 2007; De Angelis et
al., 2014; Kabak and Ozbey, 2012a,b; Kabak et al., 2009;
Versantvoort et al., 2005; Raiola et al., 2012) (Table 6).
Results indicate that the bioaccessibility of aflatoxins is high,
up to 90%, and ranges between 46% and 90%, whereas lower
values are observed for DON and ZEA. The bioaccessibility
of OTA has proven to be extremely variable; values ranging
between less than 30% and up to 84% have been recorded.
The high degree of variability of results exhibited by
different mycotoxins, and even for the same mycotoxin in
various matrices, highlights the need for further studies on
bioaccessibility of these fungal metabolites, wherein the
number and types of food examined should be increased.
These in vitro models are fast, simple and reasonably low
cost, and in vitro bioaccesibility/absorption data fall within
the range of mycotoxin absorption reported from in vivo
studies (Grenier and Applegate, 2013).
Table 5. Main properties and characteristics of in vitro digestion models used for mycotoxin bioaccessibility/absorption determination
(modified from González-Arias et al., 2013).
TIM-1 Döll RIVM Gil-Izquierdo
Type of model Dynamic Static Static Static
Simulated physiology Pig Pig Human Human
Simulation
Mouth No Yes Yes Yes
Stomach Yes Ye s Yes Ye s
Small intestine Yes Yes Ye s Yes
Large intestine No Yes No No
First time assayed with mycotoxins 2003 2004 2004 2012
Mycotoxins assayed1ZEA, AFB1, DON, OTA,
NIV, FB
DON, ZEA AFB1, OTA ENN, BEA, PAT, DON
1 AFB1 = aflatoxin B1; BEA = beauvericin; DON = deoxynivalenol; ENN = enniatin; FB = fumonisins; NIV = nivalenol; OTA = ochratoxin A; PAT = patulin;
ZEA = zearalenone.

F. Cheli et al.
582 World Mycotoxin Journal 8 (5)
In this context, in vitro models to evaluate mycotoxin
bioaccessibility/absorption represent effective tools
to perform simplified experiments under uniform and
well-controlled conditions and may provide insights not
achievable with animal studies. However, these methods
do have their disadvantages; for one, they do not take into
account important physiological factors such as the lack
of the intestinal mucosa, enterohepatic cycling, or the
immune system (González-Arias et al., 2013). The human
Caco-2 cancer cell line, cultivated in a bicameral system,
is one of the most frequently used cell models for research
on absorption, metabolism and bioavailability of drugs
and xenobiotics (Artursson et al., 2001). Differentiated
monolayers of Caco-2 (absorptive-type) cells alone and in
co-culture with HT29-MTX (secretive-type) cells have been
used to study the mechanisms of mycotoxin absorption
and transport (Burkhardt et al., 2009; Caloni et al., 2002;
Meca et al., 2012; Sergent et al., 2005, 2006; Videmann
et al., 2008; Wan et al., 2014). In these experiments,
mycotoxin absorption was tested using purified standards.
Combined use of in vitro digestion models with intestinal
epithelial cells may offer further insight into the effects
of the matrix on the oral bioavailability/absorption of
naturally occurring mycotoxins in food/feed, leading to
more accurate assessments of health risks (Versantvoort et
al., 2005). This model has been used to evaluate digestibility
and absorption of deoxinivalenol-3-β-glucoside (De Nijs et
al., 2012). There is also increasing interest in the occurrence
of masked mycotoxins in food as concerns about the
potential effects of masked mycotoxins on human health
are growing, given that they consistently occur along with
their parent compounds in food and feed (Berthiller et al.,
2013; Dall’Erta et al., 2013). In vitro methods may represent
a powerful approach for evaluating masked mycotoxin
bioavailability, chemical modifications occurring within
the GI tract, and their toxicity.
A recent approach to preventing mycotoxicosis in livestock
involves the addition of adsorbents in the livestock diet that
bind mycotoxins in the GI tract, thereby reducing their
bioavailability (Binder, 2007; Kabak et al., 2006). Dynamic
GI models have been used and shown to be a reliable tool
to study the effectiveness of adsorbent materials in reducing
the bioaccessibility of mycotoxins and thus may represent
viable alternatives to the more difficult and time-consuming
studies involving domestic livestock (Avantaggiato et al.,
2005, 2007). Moreover, GI models associated with in vitro
models using intestinal cell lines for testing the efficacy of
mycotoxin binders have been developed (Cavret et al., 2010;
Devreese et al., 2013). In addition to mycotoxin absorption
efficiency evaluation, these complementary in vitro tests
can be used to evaluate the effects on cellular viability and
identify possible toxic reaction products (formed between
binders and toxins) as well as significant toxic effects of
the binders themselves. One obvious advantage of in vitro
models is their ability to rapidly screen for effects of many
different substances, thus facilitating pre-selection of
products. However, in vivo experiments remain mandatory
for assessing the efficacy of mycotoxin binders under field
conditions (EFSA, 2010).
In conclusion, all results together indicate that the in
vitro digestion models in combination with intestinal
epithelial cells transport may be powerful tools to screen
and predict the in vivo bioavailability and digestibility of
food contaminating mycotoxins and to support the risk
assessment evaluation.
Table 6. Food/feed mycotoxin bioaccessibility/absorption: in vitro results.
Mycotoxins Food (contamination)1Bioaccessibility/absorption (%) References
Aflatoxin B1wheat (S) 88 Kabak and Ozbey, 2012b
maize (S) 90 Kabak and Ozbey, 2012b
ground maize (S) 95 Simla et al., 2009
peanut slurry (N) 80 Versantvoort et al., 2005
feed (S) 46 Zeijdner et al., 2004
Aflatoxin M1milk 80-86 Kabak and Ozbey, 2012a
Deoxynivalenol pasta (N) 30-66 Raiola et al., 2012
grain (N) 25 Avantaggio et al., 2007
Ochratoxin A buckwheat (N) 22-84 Versantvoort et al., 2005; Kabak et al., 2009
Zearalenone feed (N) 32 Zeijdner et al., 2004
grain (N) 25 Avantaggio et al., 2007
1 (S) = spiked; (N) = naturally contaminated.

Mycotoxicity in vivo and in vitro
World Mycotoxin Journal 8 (5) 583
Impacts of mycotoxins on the intestinal functions: in
vitro vs in vivo tests
The maintenance of a healthy GI tract improves the
welfare and health of humans and animals by ensuring that
nutrients are absorbed at an optimum rate and by providing
protection against pathogens through the organism’s
own immune system and gut microbial community. The
absorption of mycotoxins and their fate within the GI
tract suggests that the epithelium is repeatedly exposed
to these toxins at higher concentrations than are other
tissues. It has been reported that epithelial integrity, which
is critical for the maintenance of a physical but selective
barrier between external and internal environments, may
be altered by several mycotoxins (Bouhet and Oswald,
2005; Diesing et al., 2011a,b; McLaughlin et al., 2009;
Mahfoud et al., 2002; Maresca et al., 2002; Pinton et al.,
2012; Sergent et al., 2006; Van de Walle et al., 2010). Over
the past decade, intestinal research has received significant
interest. Grenier and Applegate (2013), in a review on the
modulation of intestinal functions following mycotoxin
ingestion, reported the results of a meta-analysis of nearly
100 published in vitro, ex vivo and in vivo experiments.
The review focused on mycotoxins of concern in terms of
occurrence and toxicity (aflatoxins, OTA, and Fusarium
toxins) and focused on seven intestinal processes (nutrient
digestibility, enzyme activity, nutrient uptake, digestive
microflora interaction, barrier integrity, mucosal immunity,
and pathogen clearance). The experiments included in
the meta-analysis provide important and interesting
information regarding the ‘test’ approach to evaluating the
effects of mycotoxins on intestinal processes. The barrier
integrity of the epithelium is the process that has been
most commonly examined via in vitro tests. The results
indicate that mycotoxins compromise several key functions
of the GI tract, causing decreased surface area available for
nutrient absorption, modulation of nutrient transporters,
or loss of barrier function. In addition, some mycotoxins
facilitate the persistence of intestinal pathogens and trigger
intestinal inflammation.
Epithelial integrity is critical in maintaining a physical
but selective barrier between external and internal
environments. This barrier function is maintained by well-
organised intercellular structures, including tight junctions,
adherence junctions and desmosomes surrounding the
apical region of epithelial cells (Gumbiner, 1993). Intestinal
cells become a very useful tool once they are differentiated
into a polarised monolayer. Three-dimensional models of
the intestinal barrier have been developed using several
intestinal cell lines, such as Caco-2, INT-407, IPEC-
1, and IPEC-J2 cells, and their applications in food and
feed evaluation of nutritional and other properties of
functional food and feed have been reviewed (Cencič and
Langerholc, 2010; Cheli and Baldi, 2011; Cheli et al., 2012;
Purup and Nielsen, 2012). The trans-epithelial electrical
resistance (TEER) of cell monolayers is considered to be a
good indicator of epithelial integrity and of the degree of
organisation of the tight junctions over the cell monolayer.
Since 2000, a number of studies have focused on the effects
of several mycotoxins (DON, OTA, FB
1
and patulin) on
in vitro intestinal barrier integrity (Bouhet et al., 2004;
Bouhet and Oswald, 2005; Diesing et al., 2011a,b; Mahfoud
et al., 2002; Maresca et al., 2001, 2002; McLaughlin et al.,
2004, 2009; Pinton et al., 2009, 2010, 2012; Sergent et al.,
2006; Van de Walle et al., 2010). The results of this body
of research indicate that mycotoxins, particularly DON,
affect epithelial integrity and significantly reduce the TEER.
The mechanisms involved in the disturbances of the TEER
caused by mycotoxins have been examined. The influence of
mycotoxins on the expression of tight junction proteins has
been reported (Diesing et al., 2011; McLaughlin et al., 2004;
Pinton et al., 2012). In the case of FB
1
, glycosphingolipids,
the biosynthesis of which is inhibited by the toxin, may also
play a role in regulating the electrical properties of epithelial
cells (Leung et al., 2003). Modelling the epithelial barrier
by growing cells on inserts represents a valuable in vitro
approach to evaluating mycotoxin mechanisms of action
and their health effects. The question is, however, are in
vitro data correlated with the results from in vivo animal
studies? Toxicity and the mechanism of toxicity of Fusarium
toxins have been assessed using in vitro (intestinal epithelial
cell line), ex vivo (intestinal explants), and in vivo (animals
exposed to mycotoxin-contaminated diets) models (Kolf-
Clauw et al., 2009, 2013; Pinton et al., 2012). Specifically,
these models have been used to compare the intestinal
toxicity of several mycotoxins to investigate their synergistic
effects and to compare the mechanisms of action. A good
correlation between data from in vitro and in vivo tests
was found as the most suitable and sensitive biomarkers
and endpoints to measure were in agreement between the
different tests. These results confirm the relevance of in
vitro tests for the analysis of the effects and interactions of
mycotoxins at the GI level and as risk assessors of dietary
mycotoxin exposure.
6. Conclusions
Mycotoxins have adverse effects on human and animal
health, with differences in species sensitivity to mycotoxins.
Mycotoxins enter the body via ingestion of contaminated
food and feed and elicit complicated and overlapping acute
and chronic effects in sensitive species. The magnitude of
mycotoxin toxicity is influenced by several factors, including
species sensitivity, level and time of exposure, individual
sensitivity, age, health, nutritional status, bioaccessibility,
mechanisms/modes of action, metabolism, and defence
mechanisms. An understanding of the mode of action
in simple in vitro systems can provide a rational basis for
predicting the health effects of single- and multi-mycotoxin
contamination. Despite the limits of in vitro models, such
as the lack of interactions with other cells, extracellular

F. Cheli et al.
584 World Mycotoxin Journal 8 (5)
factors, lack of hormonal or immunological influences,
and lack or different level of expression in vitro of genes
involved in the overall response to mycotoxins, in vitro
tests represent simple and economically efficient tools
for providing insight into the mechanisms of action of
mycotoxins to ascertain potential health effects prior to
the initiation of animal or human clinical studies. However,
extrapolating in vitro data to the in vivo situation remains
problematic. Bridging this gap will be a major challenge for
mycotoxin research in the future. In conclusion, in vitro and
in vivo tests are complementary methods of gaining insight
into how mycotoxins can affect human and animal health
as well as for understanding relevant dietary exposure and
risk scenarios.
Important areas of future research include the characte-
risation of the effects of food and feed contaminated by
several mycotoxins. Combinations of mycotoxins occur
frequently in naturally contaminated diets, and therefore,
additive, synergistic or antagonistic effects may result.
Novel risk assessment strategies should take into account
the potential for toxicological interactions among multiple
mycotoxins. As such, government regulatory standards that
address the health risks associated with exposure to many
different mycotoxins, individually and in combination,
are needed.
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