Ochratoxin A
Michael Trabold
Thomas Wagner
Jakob Weyel
Table of contents
Mycotoxins in general
3
Basic information about ochratoxin A
3
Toxicity
6
Structure of ochratoxin A
7
3D structure of ochratoxin A
8
Biosynthesis of ochratoxin A
9
List of literature
11
List of figures
11
2
Mycotoxins in general
Mycotoxins are secondary metabolites produced by ascomycetes. They can be toxic in very low
amounts and many of them are suspect of being carcinogen. There are about 200 different toxins
known. Some of them are just produced by specific moulds but many of them produce the same
toxin or several. Production of most secondary metabolites, mycotoxins as well, depends on cell
differentiation status. Especially for microorganisms the production of secondary metabolites is
associated with the end of logarithmic growth where the growing rate is decreasing. This leads
for fungi to a relation between the time of sporulation and the production of mycotoxins. For
everyday life a simple rule results: A mould changes its colour from white to another one when it
sporulates. At this time the mould gets really toxic.[1,2,9]
It is not really clear why fungi produce mycotoxins and how they developed them through their
evolution, especially because mycotoxins are no homogenous group of chemicals. [9]
Basic information about ochratoxin A
Ochratoxins are mycotoxins produced by several penicillium and aspergillus strains. They were
initially described by Scott (1965) who primarily found them in Aspergillus ochraceus.[2,3,9]
Ochratoxin A is the most common Ochratoxin. It is a natural contaminant of mouldy food and
animal feed. Examples for contaminated food are grain, pork, coffee, wine grapes and sometimes
even beer. Due to that it is traceable in tissue and organs of animal and human blood and breast
milk.[1-3,9]
Regarding to GHS-Identification Ochratoxin A is very poisonous and listed as CMR (carcinogen,
mutagen and toxic to reproduction).[8,9]
Tabelle 1 Identificational properties of ochratoxin A.[10]
Name
Ochratoxin A
IUPAC Name
N-{[(3R)-5-chloro-8-hydroxy-3-methyl-1oxo-3,4-dihydro-1H-isochromen-7-yl]
carbonyl}-L-phenylalanine
CAS Number
303-47-9
M [g/mol]
403,81
Formula
C20H18ClNO6
Tm
169°C
3
Ochratoxin A-producing Fungi
Ochratoxin A is mostly produced by Aspergillus and Penicillium species. Initially it was described
in Aspergillus ochraceus, which gave the name. Maybe the most familiar fungus that produces
Ochratoxin A is Aspergillus niger, the black mould.[2,8,9]
Figure 1: Conidiophores of Aspergillus sp.
Figure 2: Conidiophores of
Penicillium sp.
4
Sources of Ochratoxin A in dependence of climate conditions
Studies show that the source fungi of ochratoxin A are very climate specific. In moderate
climates like in Germany and Scandinavia ochratoxin A is mostly produced by Penicillium
species. In warmer areas like Spain and Italy the main source is Aspergillus carbonarius. This
leads to the conclusion that Penicillium and Aspergillus do have different temperature optima for
the synthesis of ochratoxin A.[2,8,9]
Laboratory studies have shown that Aspergillus ochraceus grows between 8 and 37°C with an
optimum at 24°C while ochratoxin A is just produced within a range of 12 to 37°C with a
optimum at 31°C. But the production of Ochratoxin A in Aspergillius seems to be extremely
dependent on many factors like pH, strain, the medium and other culture conditions. In case of
ochratoxin A the growth state has not such a big influence like it has with other mycotoxins. [2,8,9]
On the other hand ochratoxin A production in Penicillium seems to be very stable even with
changing environmental conditions.[2,8,9]
5
Toxicity
Aspergillus ochraceus is a mold species in the genus Aspergillus. Aspergillus is known to produce
the mycotoxin ochratoxin A but it is also built by Penicillium. Food is often contaminated by
these mycotoxins. It also produces the dihydroisocoumarin mellein (structure p.7). [1,2,8,9]
Ochratoxin A is a ubiquitous mycotxin produced by fungi of improperly stored food products.
Ochratoxin A is nephrotoxic and associated to urinary tract tumours. It also has genotoxic,
immunossuppresive, teratogenic and carcinogenic effects. The international Agency for Research
in Cancer has classified ochratoxin A as a carcinogen group 2. Group 2 means that the substance
is suspected of being carcinogenic for humans. Furthermore ochratoxin A is genotoxic following
the oxidative metabolism. This activity is divided into direct (covalent DNA adduction via
guinone formation) and indirect (oxidative DNA damage) mechanisms of action. An important
fact is that there are some synergistic effects between different mycotoxins so they can be toxic
or mutagenic below the tolerable daily intake level. Ochratoxin A boosts the mutagenic effect of
aflatoxin for example. Another important example for those synergistic effects is the combination
of Ochratoxin A and citrinin. ochratoxin A is assumed to cause Balkan Endemic Nephropathy
(BEN), a chronic human kidney disease when it is digested in combination with mycotoxin
citrinin. This is a special problem because it is known that some Penicillium verrucosum strains
are able to produce both substances. In humans and animals, ochratoxin A can be metabolized in
the kidney, liver and intestines. Hydrolysis, hydroxylation, lactone-opening and conjugation are
the major metabolic pathways. The exact molecular mechanism of the toxic effects of ochratoxin
A so far remains unknown. Remarkable is the fact that the toxicity of ochratoxin A is specific to
species, cell and even gender.[1,2,8,9]
Reaction of animals
Especially pigs and poultry are very sensitive regarding ochratoxin A (critical concentration 20200 μg/kg fodder). Typical symptoms are: less fodder intake, depression on performance and
kidney disease. The lethal dose (LD50) of ochratoxin A depends on the species and is between 2
and 20 mg/kg bodyweight.[8,9]
Effect on Humans
Studies from the European scientific committee on food show that the intake of ochratoxin A in
different European countries lies between 15 and 60 ng/kg bodyweight per day. In regard to the
LOAEL (lowest observed adverse effect level) of pigs calculated with a safety factor the European
food safety authority has set a tolerable amount of a weekly intake of 120 ng/kg bodyweight. [8,9]
6
Structure of ochratoxin A
Ochratoxin A (IUPAC: N-{[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-isochromen7-yl]carbonyl}-L-phenylalanine) consists of two main components, L-phenylalanine and
ochratoxin α which are linked by a peptide bond.[1,2]
Figure 3: Ochratoxin A and its main components.
Ochratoxin α is the in pos. 5 chlorinated form of ochratoxin β which is the in pos. 7 carboxylated
variant of the polyketide mellein.[1,2]
Figure 4: Isocumarin, mellein, ochratoxin β and ochratoxin α in comparison.
7
3D structure of ochratoxin A
Figure 5: Ball and stick model of ochratoxin A
Figure 6: Stick model for ochratoxin A
8
Biosynthesis of ochratoxin A
The biosynthetic pathway of ochratoxin A is not completely cleared up. It may take a different
step sequence in the construction of the isocumarin derivate ochratoxin α and its linking to
phenylalanine but the basic reactions remain the same. The biosynthesis discussed here was
published by Huff and Hamilton in 1979.
Ochratoxin A consists of two peptide-linked subunits, phenylalanine and an isocumarin derivate
called ochratoxinα. Their biosynthetic construction until the final step of peptide linking will be
regarded separately.[1-3]
a)
Biosynthesis and activation of Phenylalanine:
Phosphoenolpyruvate and erythrose-4-phosphate react via the shikimate pathway to form
phenylalanine which is activated for connection to other substances as for example
ochratoxin α by esterification of its carboxyl group.[1,2,4]
b)
Biosynthesis and activation of ochratoxinα:
The polyketide synthase links 4 molecules of malonyl-CoA and one molecule
acetyl-CoA according to the ketide-synthesis-pathway creating a pentaketide. The
pentaketide is cyclized, aromatized and reduced to form mellein. Mellein is
methylated in pos. 7 by a methyltransferase and S-Adenosyl methionine. An oxidase
creates the carboxylic acid of the newly added methyl group creating ochratoxin β.
Ochratoxin α is produced by bringing a chlorine in pos. 5 due to chloroperoxidase.
Linking phosphate to the carboxyl group activates ochratoxin α.[1,2,5]
Those two parts are linked by a peptide bond and deesterificated via the ochratoxin Asynthase. This forms the target molecule ochratoxin A. Important by-products are the
unchlorinated ochratoxin B or the still esterificated form ochratoxin C.[1,2,6,7]
9
Figure 7: Schematic biosynthesis of ochratoxin A as published by Huff and Hamilton.
10
List of literature
[1]
Martin Reinsch, 07.06.2006, Dissertation:Entwicklung von Analyseverfahren zur
Bestimmung von Ochratoxin A in Lebensmitteln, Humboldt-Universität zu Berlin,
pp. 15-21
[2]
Ellen Mühlencoert, 30.10.2003,Dissertation: Ochratoxin A Production by Aspergillus
ochraceus, Technische Universität München, pp. 1-8
[3]
Huff, W.E., Hamilton, P.B., 1979. Mycotoxins- their biosynthesis in fungi: ochratoxinsmetabolites of combined pathways. Journal of Food Protection 42, 10, pp. 815-820
[4]
J. M. Berg, J. L. Tymoczko, L. Stryer: Biochemie. 6. Auflage. Spektrum Akademischer
Verlag, Elsevier GmbH, München 2007; S. 773–775
[5]
https://de.scribd.com/doc/43403135/Polyketide, 23.12.2016
[6]
Ellen Mühlencoert, 30.10.2003, Dissertation: Ochratoxin A ProductionbyAspergillus
ochraceus, Technische Universität München, p. 7
[7]
http://www.vis.bayern.de/ernaehrung/lebensmittelsicherheit/unerwuenschte_stoffe/ochr
atoxine.htm, 23.12.2016
[8]
Georg Stettner, 21.12.2000, Dissertation: Nachweis und Verhalten von Deoxynivalenol
und Ochratoxin A während der Bierbereitung
[9]
http://www.vis.bayern.de/ernaehrung/lebensmittelsicherheit/unerwuenschte_stoffe/
ochratoxine.htm, 24.12.2016
[10]
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.docountry=DE&
language=de &productNumber=O1877&brand=SIGMA&PageToGoToURL=http%3A
%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fo1877%3Flang
%3Dde, 24.12.2016
List of figures
Fig.1: Provided by Doemens e.V.
Fig.2: Provided by Doemens e.V.
Fig.3: http://content.answcdn.com/main/content/img/oxford/oxfordBiochemistry/
0198529171.ochratoxin.1.jpg, 23.12.2016
Fig.4: http://content.answcdn.com/main/content/img/oxford/oxfordBiochemistry/
0198529171.ochratoxin.1.jpg,23.12.2016
Fig.7: Ellen Mühlencoert, 30.10.2003, Dissertation: Ochratoxin A ProductionbyAspergillus
ochraceus, Technische Universität München, p. 8
11