Mycotoxins in maize silageDetection of toxins and toxicological aspects
PhD defense by Rie Romme RasmussenRie Romme RasmussenSeptember 20th 2010
Outline
• Introduction
• Aims of the study
• Experiments and Results:p
Cytotoxicity of fungal metabolites
Detection of myctoxinsDetection of myctoxins
Occurrence in maize silage
• Conclusions
• Perspectivesp
National Food Institute, Technical University of Denmark
Maize silage a feed product
Natural fermentation of maize plants by lactic acid bacteria
l H 3 4low pH = 3-4
low O2 < 2%
high CO2 20-90%
Stored for up to 14 months
National Food Institute, Technical University of Denmark
Maize silage a feed product
T Dairy cow
6,000,000
Tonnes DK productionDairy cow
~ 40 kg silage pr. day
50 75% of the daily diet5,000,000
50-75% of the daily diet
Unexplained health problems
4,000,000 Decrease in milk yield
Illness, death
Mycotoxins from fungi2,000,000
3,000,000
? N
NO
S
OS
CH3
OH
CH3O
O
O
OH
1,000,000
O
OOH
OH OH
CH3 HS
OHCH3
01992 1996 2000 2004 2008
National Food Institute, Technical University of Denmark
Year
Maize silage a feed product
T Dairy cow
6,000,000
Tonnes DK productionDairy cow
~ 40 kg silage pr. day
50 75% of the daily diet5,000,000
50-75% of the daily diet
Unexplained health problems
4,000,000 Decrease in milk yield
Illness, death
Mycotoxins from fungi2,000,000
3,000,000
? N
NO
S
OS
CH3
OH
CH3O
O
O
OH
1,000,000
O
OOH
OH OH
CH3 HS
OHCH3
01992 1996 2000 2004 2008
National Food Institute, Technical University of Denmark
Year
Mycotoxins
• Toxic secondary metabolites from filamentous fungi
Cancer hormonal imbalance liver and kidney damage or • Cancer, hormonal imbalance, liver and kidney damage or reduced immune defence
• Production
– type of fungi (species)Fungi
g
– growth conditions
MycotoxinsGrowth
conditions
National Food Institute, Technical University of Denmark
Maize silage pre-harvest fungigi
est
fung
re-h
arv
Fusarium infection
Pr
LJL
Fusarium infection
o:
Fangea
t
Søre
nse
n J
Photo
Photo
:
National Food Institute, Technical University of Denmark
Maize silage pre-harvest fungigi
est
fung
re-h
arv
Pr
Fusarium
Alternaria
Epicoccum
Fusarium
DKEpicoccum
Phoma
Aspergillus flavusAspergillus flavus
Aspergillus parasiticus
National Food Institute, Technical University of Denmark
Maize silage post-harvest fungi
ngi
vest
fun
ost
-harv
Penicillium infection
Po
Penicillium infectional
krup H
Photo
: Ka
National Food Institute, Technical University of Denmark
Maize silage post-harvest fungi
ngi
Maize silage
vest
fun
ost
-harv
Po
Penicillium roqueforti
Penicillium paneumPenicillium paneum
Zygomycetes (Mucor and Rhizopus)
Aspergillus fumigatusAspergillus fumigatus
Byssochlamys nivea
Monascus ruber
National Food Institute, Technical University of Denmark
Maize silage post-harvest fungi
• Penicillium roqueforti / Penicillium paneum– Very common fungal contaminant– Optimal pH: 4-5– Growth at 0.3% O2, 25% CO2
G th t 5oC– Growth at 5oC
• Secondary metabolites• Secondary metabolites– PR-toxin (acute toxic)– Roquefortine C (neurotoxic)q ( )– Mycophenolic acid (immunosuppressive)– Patulin (genotoxic, immunosuppressive)– Marcfortines (no adverse effects described)– Andrastines– and more
National Food Institute, Technical University of Denmark
– and more…
Joint project (2005-2010)”Mycotoxin carry-over from maize silage via cattle into dairy products”
Ph
y y g y p
oto
: Sto
rm IM
L+ LDPho
Preharvest fungi and their mycotoxins in maize, Sørensen, JL (2009)
Post-harvest fungal spoilage of maize silage, Storm IMLD (2009)
oto
: Shutterstoo
ck.com
National Food Institute, Technical University of Denmark
Joint project (2005-2010)”Mycotoxin carry-over from maize silage via cattle into dairy products”
Ph
y y g y p
oto
: Sto
rm IM
L+ LDPho
Preharvest fungi and their mycotoxins in maize, Sørensen, JL (2009)
Post-harvest fungal spoilage of maize silage, Storm IMLD (2009)
oto
: ShutterstoMycotoxin hazard for cattle
Aim
ock.co
m
yfeeding on maize silage
evaluated by chemical and cyto-toxicity test methods
National Food Institute, Technical University of Denmark
Experiments mycotoxins and other Experiments – mycotoxins and other fungal metabolites
Cytotoxicity Detection Occurrence
Comparison of Method 27 fungal
• Mycotoxinsdevelopment
&metabolites
&• Fungal agar• Maize silage
validation performance
• Silage extraction• LC-MS/MS
• Hot-spots• Fresh & ensiled
maize silage
National Food Institute, Technical University of Denmark
Cytotoxicity assay
• Cell viability assay => model system for acute toxicity
• Limit: Absorption, distribution, metabolism, excretion
• Dairy cows
– Rumen fermentation by bacteria, protozoa and fungi
National Food Institute, Technical University of DenmarkDrawings: The Department of Animal Science at Texas A&M University
Cytotoxicity assay
Caco-2 = human colon cancer cell line
Alamar Blue/resazurin assay/ y
11 1210987654321
A 10,000 cells/wellB
C
D
E
growth medium5% CO2, 37ºC
F
E
G
H
48 h toxin exposure
Viable cell
Reduction by it h d i ll
National Food Institute, Technical University of DenmarkResazurin
mitochondria cell enzyme Resorufin, flurescent
Cytotoxicity of mycotoxin standards
Concentration response graph
100100Viability (%)
48 h exposureTwo distinct test days
Concentration-response graph
80
100
80
100Viability (%)
6060
404050%
IC50= 0.6 g/mL
202050%
IC00 001 0 01 0 1 1 1000 001 0 01 0 1 1 10
IC50
Patulin (g/mL)National Food Institute, Technical University of Denmark
0.001 0.01 0.1 1 100.001 0.01 0.1 1 10 Patulin (g/mL)
Cytotoxicity of mycotoxin standards
Viability (%)
80
100
80
100
6060 IC50> 50 g/mL IC50= 48 g/mL
401 10 100
401 10 100
Andrastine A (g/mL) Roquefortine C (g/mL)
Viability (%)
10080
100 IC50> 100 g/mLIC50= 0.6 g/mL
60
80
40
60
50%
50 g
40
60
0.01 0.1 1 10 1000
20
0.001 0.01 0.1 1 10
IC50
National Food Institute, Technical University of Denmark
. Mycophenolic acid (g/mL)Patulin (g/mL)
Cytotoxicity of mycotoxin standards
Caco-2 cells, IC50
Toxin 48 hmean
(µg/ml) SDToxin, 48 h (µg/ml) SDT-2 toxin 0.0037 +/- 0.0008Gliotoxin 0.035 +/- 0.003Deoxynivalenol 0.29 +/- 0.17Patulin 0.62 +/- 0.07R f ti C 48 +/ 2Roquefortine C 48 +/- 2Zearalenone 58 +/- 6Citrinin 83 +/- 32Citrinin 83 / 32N6-formyl-roquefortin-C >46Andrastin A >50Mycophenolic acid >1001-hydroyeremophil-7(11),9(10)-dien-8-one >280
National Food Institute, Technical University of Denmark
IC50 = 50% inhibit concentrationSD = sample standard deviation of 2 independent experiments
Cytotoxicity of fungal agar extracts
• Crude extracts (8 fungal species)
Alternaria tenuissima, Fusarium avenaceum, F. graminearum,Aspergillus fumigatus, Byssochlamys nivea, Monascus ruber,Penicillium paneum, P. roqueforti
• 13-14 days old cultures, 25º C
YES Yeast extract sucrose agarYES Yeast extract sucrose agarPDA Potato Dextrose agarCYA Czapek yeast extract agarSA Maize silage agarSA Maize silage agar
• 15 cm2 plugs with fungal growth extracted with
C Cl O (3 2 ) % ( / ) f i idEtAc:CH3Cl2:MeOH (3:2:1), 1% (v/v) formic acid
• Re-dissolved in 2 ml MeOH
National Food Institute, Technical University of Denmark
Cytotoxicity of Penicillium paneum
Viability %
Mean of 2 tests120
Viability %
Mean of 2 tests100
YES80
YESCYAPDA
40
60 SA
20
40
fungal extract f IBT b
0
National Food Institute, Technical University of Denmark
of IBT number28545 28544 28542 28543PatulinPatulin >2.9 µ>2.9 µg/mLg/mL
Cytotoxicity of Penicillium paneumMajor metabolites: YES, CYA, PDA and SA agar
marcfortine Apatulin
j , , g
citreoiscoumarin
patulin
> IC50
andrastin A
roquefortine C
citreoiscoumarin
< IC50q
< IC50
CytotoxicNon-toxic extracts
National Food Institute, Technical University of Denmark
CytotoxicityFungal agar and silage extracts
Fungi Sec. metabolitesdetected in fungal agar extracts
ll li f i C d iPenicillium paneum patulin, roquefortine C, andrastin A,citreoisocoumarin, macfortine A, …
Penicillium roqueforti (mycophenolic acid), roquefortine C, andrastine A, Interactions or Other; PR-toxin, roquefortine A, …
Aspergillus fumigatus gliotoxin, + Other; verrucologen, fumitremorgin A & B, fumigaclavine C, ...
Byssochlamys nivea patulin, mycophenolic acid, byssochlamic acid, ...
Monascus ruber Citrinin, Other; monacolin K, ankaflavin, …
Fusarium graminearum Zearalenone, deoxynivalenol, Interactions or Other; Aurofusarin, fusarin C, rubrofusarin, …
Red = present in cytotoxic concentrationsGray = present in non-toxic concentrationsBlack = present
National Food Institute, Technical University of Denmark
Black = present
Cytotoxicity of Penicillium roquefortiMajor cytotoxic metabolite in fungal YES extracts ?
#300 P roq raw TTB dilu ted
1 2e+1
R IR O _0929s24 3: D iode A rray R ange: 1 .256e+19.23 1-Hydroxyeremophil-7(11),9(10)-dien-8-one
j y g
1.0e+1
1.05e+1
1.1e+1
1.15e+1
1.2e+1
700 n
m
Fractions
• by RT time
PR-toxin& mycophenolic acid
7.5
8.0
8.5
9.0
9.58.39
8.70
D 2
00-7
• by RT time• targeting peaks
PR toxinPR-toxin analogues (?)N6-formyl-roquefortin-C
AU
5 .5
6 .0
6.5
7.0 5.17
11.34nse
, D
AD PR-toxin
O
OO
OCH
PR toxin analogues (?)
Andrastin A
roquefortin C
3.0
3.5
4.0
4.5
5.0
0.707.61
7.177.92
17.39
12.5622 30
Res
pon O
O
CH3
CH3
CH3CH3
H
5 0e 1
1.0
1.5
2.0
2.5
4.42
3.441.45
2.30
7.17
6.425.57
6.08 10.87
13.26
16.59
22.30
19.37
21.05
Roquefortine C
National Food Institute, Technical University of Denmark
. T im e2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
0.0
5.0e-1
minRoquefortine C
Cytotoxicity of maize silage
Inoculated silage
Viability (%)Byssochlamys niveaPhoto: Birgitte ML Raun
100Healthy silage
Viability (%)QuEChERS extract of maize silage
Evaporated and re-dissolved in MeOH
60
80 Secondary metabolites+ Byssochlamic acid
M h li id
40
60B. nivea
+ Mycophenolic acid+ Other undescribed metabolites- Patulin
20 No significant cytotoxicity
01 10 100 1000
Penicillium roqueforti (+ PR-toxin)Penicillium paneum (- patulin)Aspergillus fumigatus (- gliotoxin)M b ( it i i )
National Food Institute, Technical University of Denmark
maize silage (mg/mL) Monascus ruber (+ citrinin)
Summery of cytotoxicy
• Caco-2 resazurin assay ≈ sensitivty as other in vitro assays
• Applications:pp
fungal agar extracts, screening
fraction of fungal agar extracts fraction of fungal agar extracts
PR-toxic an important toxic metabolite from P. roqueforti
silage extracts silage extracts
• Cytotoxic pre- and post-harvest speciesAlternaria tenuissima Fusarium avenaceum F graminearumAlternaria tenuissima, Fusarium avenaceum, F. graminearum,Aspergillus fumigatus, Byssochlamys nivea, Monascus ruber,Penicillium paneum, P. roqueforti
• Interactions or other metabolites than tested here often contributed to the cytotoxicity
National Food Institute, Technical University of Denmark
Experiments mycotoxins and other Experiments – mycotoxins and other fungal metabolites
Cytotoxicity Detection Occurrence
Comparison of Method 27 fungal
• Mycotoxinsdevelopment
&metabolites
&• Fungal agar• Maize silage
validation performance
• Silage extraction• LC-MS/MS
• Hot-spots• Fresh & ensiled
maize silage
National Food Institute, Technical University of Denmark
Detection techniques
High performance liquid chromatograph
(separation of analytes)
Mass spectrometer
(detector)(detector)
ent.
com
Photo
: ag
ile
National Food Institute, Technical University of Denmark
P
LC-MS/MS
Electrospray
(ionization)
Quadrupole 1
(mass filter)
Quadrupole 2
(Collision cell)
Quadrupole 3
(mass filter)
Quadrupole 2
(Collision cell)
Quadrupole 3
(mass filter)
Quadrupole 1
(mass filter)(ionization) (mass filter) (Collision cell) (mass filter)(Collision cell) (mass filter)(mass filter)
ConeCapillary, high voltage DetectorDetector
Sample, HPLC eluentp ,
National Food Institute, Technical University of Denmark
LC-MS
Electrospray
(ionization)
Quadrupole 1
(mass filter)
Quadrupole 2
(Collision cell)
Quadrupole 3
(mass filter)(ionization) (mass filter) (Collision cell) (mass filter)
ConeCapillary, high voltage Detector
Sample, HPLC eluentp ,
Mass filter
Photo: waters.com
National Food Institute, Technical University of Denmark
Detection - Multi-mycotoxin analysis
• 32 fungal metabolites• Size: 150-700 Da O
OO
OH CH3O
OOH
• Size: 150 700 Da O
OOH
CH3
CH3OH
OCH3 CH3
• Strong acid: Mycophenolic acid• Weak acid: Alternariol monomethylether
NO
O
CH3
CH3
CH3
HHy• Base - amine: Roquefortine A
NH
• Water solubility changes with pH– Fungal hot-spot, pH = 7
H
– Unspoiled silage pH = 3-4 pH buffered extraction
National Food Institute, Technical University of Denmark
Detection – sample extraction
Sample preparation
Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) method1
1st e t action
Sample preparationHomogenisation with liquid nitrogen
1st extractionWater 1% Acetic acid in ACN Na-Acetate
2 min shake
2nd extractionAnhydrous MgSO4
1 min shake
Centrifugation 10 min
Preparation for LC-MS/MSDecant ACN phase Filter in vials
National Food Institute, Technical University of Denmark1) Anastassiades et al. (2003) J. AOAC Int. 86, 412-431
Detection – LC-MS/MS validationSpiked on 3 levels, repeated 3 days
Compound ESI MS-MS RECmean RSDr RSDIR LODspikeM » D1, D2 % % % ug/kg
Mycophenlic acid - 319 1 » 190 8 178 8 90 11 13 7Mycophenlic acid - 319.1 » 190.8, 178.8 90 11 13 7Zearalenone - 317.1 » 130.8, 174.7 90 12 16 9Alternariol - 257 » 214.9, 146.8 78 9 14 10Cyclopiazonic acid + 337.2 » 195.9, (181.9) 63 22 35 15Enniatin B + 657 4 » 314 527 60 21 24 24Enniatin B + 657.4 » 314, 527 60 21 24 24Deoxynivalenol - 341.1 » 265, (295) 83 17 18 739Marcfortine B + 464 » 436, 419 61 9 9 -…Citrinin + 251 1 » 233 190 9 Unstable LC-MS-MS sensitivityCitrinin + 251.1 » 233, 190.9 Unstable LC MS MS sensitivityFumonisin B1 + 722.5 » 334, 528 6 13 18 544
LOD (1-739 μg/kg)
Reproducibility (7–35% RSD)
National Food Institute, Technical University of Denmark
Summery - Detection
h d h f l b l• Both pre- and post-harvest fungal metabolites
• One extract
• No clean-up
• Buffering of pH
• Quick Easy Cheap Effective Rugged Safe (QuEChERS) method• Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) method
• LC-MS/MS
• 27 validated: LOD (1-739 μg/kg) & Reproducibility (7–35% RSD)
h C b l f• Rinsing the HPLC system between samples ensures performance
National Food Institute, Technical University of Denmark
Experiments mycotoxins and other Experiments – mycotoxins and other fungal metabolites
Cytotoxicity Detection Occurrence
Comparison of Method 27 fungal
• Mycotoxinsdevelopment
&metabolites
&• Fungal agar• Maize silage
validation performance
• Silage extraction• LC-MS/MS
• Hot-spots• Fresh & ensiled
maize silage
National Food Institute, Technical University of Denmark
Occurrence – maize silage
• Visibly mouldy maize silage from all over Denmark10 hot-spots infected withM b A f i t B i P d P f tiM. ruber, A. fumigatus, B. nivea, P. paneum and P. roqueforti
• Unspoiled fresh and ensilage maize collected 2007 – 2009• Unspoiled fresh and ensilage maize collected 2007 2009from all over Denmark
17 whole fresh maize plants taken at field level
82 ensiled maize samples collected• from the cutting face of the silage stack• with a silage drill approximately 1 meter behind the cutting face
of the silage stackof the silage stack
National Food Institute, Technical University of Denmark
Occurrence - visible mouldy silageFungal hot-spotsFungal hot spots
(n=10)Analyte npos max (µg/kg)Roquefortine C 3 33 662Roquefortine C 3 33,662Andrastin A 6 8,811Mycophenolic acid 6 1,646Gliotoxin 2 906
PenicillumPenicillum
B. B. niveaniveaA. A. fumigatusfumigatus
Quantitative
Gliotoxin 2 906Alternariol 1 236Alternariol monomethyl ether 1 51Nivalenol 2 142
A. A. fumigatusfumigatus
AlternariaAlternaria
FusariumFusariumDeoxynivalenol 2 1,092Zearalenone 4 156Enniatin B 3 200
FusariumFusarium
Roquefortine A 3Marcfortine B 1Marcfortine A 3
PenicillumPenicillum
Marcfortine A 3Citreoisocoumarin 5Fumigaclavine A 1
PhomaPhomaA. A. fumigatusfumigatus
National Food Institute, Technical University of Denmark
Occurrence – 99 unspoiled silages
µg/kg fresh weight
Analytenpos
µg/kg fresh weight
meanpos maxRoquefortine C 2 173 189
PenicillumPenicillum Andrastin A 15 169 691Mycophenolic acid 2 43 52
PenicillumPenicillumB. B. niveanivea
Alternariol 2 18 24Alternariol monomethyl ether 3 9 11Nivalenol 16 263 758
AlternariaAlternaria
FusariumFusarium
Quantitative
Deoxynivalenol 7 1841 2974Zearalenone 34 71 666Enniatin B 28 75 365
FusariumFusarium
Roquefortine A 9Marcfortine B 1Marcfortine A 6
PenicillumPenicillum
Qualitative
Marcfortine A 6Citreoisocoumarin 8PhomaPhoma
National Food Institute, Technical University of Denmark
Occurrence – 99 unspoiled silages
µg/kg fresh weight
Analytenpos
µg/kg fresh weight
meanpos maxRoquefortine C 2 173 189
PenicillumPenicillum Andrastin A 15 169 691Mycophenolic acid 2 43 52Gliotoxin 0
PenicillumPenicillumB. B. niveanivea
A. A. fumigatusfumigatus
Alternariol 2 18 24Alternariol monomethyl ether 3 9 11Nivalenol 16 263 758
AlternariaAlternaria
FusariumFusarium
Quantitative
Deoxynivalenol 7 1841 2974Zearalenone 34 71 666Enniatin B 28 75 365
FusariumFusarium
Qualitative
Roquefortine A 9Marcfortine B 1Marcfortine A 6
PenicillumPenicillum
Qualitative Marcfortine A 6Citreoisocoumarin 8Fumigaclavine A 0
PhomaPhomaA. A. fumigatusfumigatus
National Food Institute, Technical University of DenmarkOnly in hot-spots
Occurrence – 99 unspoiled silages
30
3535 samples was without detectable residues
25
30
ple
s
30 samples contained only one fungal metabolite
15
20
f sa
mp
10
15
No
. o
8 l t i d 4 diff t l t
0
5 8 samples contained 4 different analytes
0 1 2 3 4 5 6 7
No. of analytes detected in samples
National Food Institute, Technical University of Denmark
Summery – mycotoxin occurrence
• Low levels of multiple secondary metabolites is common
• High occurrence of Fusarium toxinsHigh occurrence of Fusarium toxins
• Fusarium toxins < values recommended by the European Commission (growth season 2006-2008)Commission (growth season 2006 2008)
• High content of fungal metabolites with visible fungal growth
PR i d li (hi hl i ) d d i • PR-toxin and patulin (highly cytotoxic) were not detected in field samples
National Food Institute, Technical University of Denmark
Conclusion
CytotoxicityCytotoxicity• The assay applicable to standards and fungal agar extracts – not silage
• 8 fungal species associated with maize and silage produce cytotoxic metabolites
• Fungal toxicity depends on growth media, species, isolate
• Interactions or other metabolites than tested here often contributed to cytotoxicity
• PR-toxin an important toxic metabolites from P roqueforti• PR toxin an important toxic metabolites from P. roqueforti
LC-MS/MS detection method
• 27 metabolites in maize silage QuEChERS extraction successfully validated• 27 metabolites in maize silage, QuEChERS extraction successfully validated
• Rinsing the HPLC system between samples ensures performance
Occurrences of fungal secondary metabolitesOccurrences of fungal secondary metabolites
• 15 metabolites detected
• High content of fungal metabolites with visible fungal growthHigh content of fungal metabolites with visible fungal growth
• Low level of multiple-mycotoxins is common in unspoiled silage
• Fusarium toxins dominated, < levels recommended by the European Commission
National Food Institute, Technical University of Denmark
Perspectives – mycotoxin occurrence and toxcityy y
• No indication that mycotoxins in maize silage have caused the general health problems observed at Danish dairy cattle farms
• Immunosuppresive (e.g. DON, NIV, GLI, MPA)
– Continuous exposure to low levels ? infectious diseases ?Continuous exposure to low levels ? infectious diseases ?– Long term in vivo studies are sparse
• Human exposure transport to milk• Human exposure, transport to milk
– Grain and not animal products a significant source for the pre-harvest toxins deoxynivalenol zearalenone and fumonisinsharvest toxins deoxynivalenol, zearalenone and fumonisins Genotoxic Alternaria toxins might be relevant
National Food Institute, Technical University of Denmark
Thank you to…Thank you to…
SupervisorsPeter H RasmussenMona Lise Binderup
External collaboration partnersStig PurupNiels B KristensenMona-Lise Binderup
Thomas O LarsenNiels B KristensenYvonne SimonsenAnita Iversen
Fellow PhDsIda MLD StormJens L Sørensen
Technical assistanceVivian JørgensenJens L Sørensen
Inspiration and advice
Vivian JørgensenTanja Thorskov BladtHanne Jakobsen
Ulf ThraneKristian F NielsenJens C Frisvad
All colleagues fromDiv. Food Chemistry
Birgitte AndersenJørn Smedsgaard
yDiv. of Toxicology and Risk AssessmentDep. of Systems Biology
National Food Institute, Technical University of Denmark
FundingThe Directorate for Food, Fisheries, and Agri Business