Detection of toxins and toxicological aspects PhD defense

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



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


Year


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


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


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

:


Maize silage pre-harvest fungigi

est

fung

re-h

arv

Pr

Fusarium

Alternaria

Epicoccum

Fusarium

DKEpicoccum

Phoma

Aspergillus flavusAspergillus flavus

Aspergillus parasiticus


Maize silage post-harvest fungi

ngi

vest

fun

ost

-harv

Penicillium infection

Po

Penicillium infectional

krup H

Photo

: Ka



ngi

Maize silage

vest

fun

ost

-harv

Po

Penicillium roqueforti

Penicillium paneumPenicillium paneum

Zygomycetes (Mucor and Rhizopus)

Aspergillus fumigatusAspergillus fumigatus

Byssochlamys nivea

Monascus ruber



• 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


– 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


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


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


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)


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


. Mycophenolic acid (g/mL)Patulin (g/mL)


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


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


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


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


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


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


. 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 )


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






&metabolites





maize silage


Detection techniques

High performance liquid chromatograph

(separation of analytes)

Mass spectrometer

(detector)(detector)

ent.

com

Photo

: ag

ile


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 ,


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


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


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)


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






&metabolites





maize silage


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


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


Marcfortine A 3Citreoisocoumarin 5Fumigaclavine A 1

PhomaPhomaA. A. fumigatusfumigatus


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


FusariumFusarium

Quantitative

Deoxynivalenol 7 1841 2974Zearalenone 34 71 666Enniatin B 28 75 365

FusariumFusarium



Qualitative

Marcfortine A 6Citreoisocoumarin 8PhomaPhoma



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


FusariumFusarium

Quantitative

Deoxynivalenol 7 1841 2974Zearalenone 34 71 666Enniatin B 28 75 365

FusariumFusarium

Qualitative



Qualitative Marcfortine A 6Citreoisocoumarin 8Fumigaclavine A 0

PhomaPhomaA. A. fumigatusfumigatus

National Food Institute, Technical University of DenmarkOnly in hot-spots


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


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


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


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


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


FundingThe Directorate for Food, Fisheries, and Agri Business

Documents

Detection of toxins and toxicological aspects PhD defense