Mycotoxins in maize silage

Transcription

1 Mycotoxins in maize silage Detection of toxins and toxicological aspects PhD defense by Rie Romme Rasmussen September 20 th 2010

2 utline Introduction Aims of the study Experiments and Results: Cytotoxicity of fungal metabolites Detection of myctoxins ccurrence in maize silage Conclusions Perspectives

3 Maize silage a feed product Natural fermentation of maize plants by lactic acid bacteria low ph = 3-4 low 2 < 2% high C % Stored for up to 14 months

4 Maize silage a feed product Tonnes 6,000,000 DK production Dairy cow ~ 40 kg silage pr. day 50-75% of the daily diet 5,000,000 Unexplained health problems 4,000,000 Decrease in milk yield 3,000,000 2,000,000 1,000, Year Illness, death Mycotoxins from fungi H H H? N H C H 3 H H S N S CH 3 CH 3 H CH 3

5 Maize silage a feed product Tonnes 6,000,000 DK production Dairy cow ~ 40 kg silage pr. day 50-75% of the daily diet 5,000,000 Unexplained health problems 4,000,000 Decrease in milk yield 3,000,000 2,000,000 1,000, Year Illness, death Mycotoxins from fungi H H H? N H C H 3 H H S N S CH 3 CH 3 H CH 3

6 Mycotoxins Toxic secondary metabolites from filamentous fungi Cancer, hormonal imbalance, liver and kidney damage or reduced immune defence Production type of fungi (species) Fungi growth conditions Mycotoxins Growth conditions

7 Maize silage pre-harvest fungi Pr re-harvest fung gi Fusarium infection Photo: Sørensen JL Photo o: Fangeat L

8 Maize silage pre-harvest fungi gi re-harvest fung Pr Fusarium Alternaria Epicoccum Phoma Aspergillus flavus Aspergillus parasiticus DK

9 Maize silage post-harvest fungi Po ost-harv vest fun ngi Penicillium infection alkrup H Photo: Ka

10 Maize silage post-harvest fungi Po ost-harv vest fun ngi Penicillium roqueforti Penicillium paneum Zygomycetes (Mucor and Rhizopus) Aspergillus fumigatus Byssochlamys nivea Monascus ruber

11 Maize silage post-harvest fungi Penicillium roqueforti / Penicillium paneum Very common fungal contaminant ptimal ph: 4-5 Growth at 0.3% 2, 25% C 2 Growth at 5 o C Secondary metabolites PR-toxin (acute toxic) Roquefortine C (neurotoxic) Mycophenolic acid (immunosuppressive) Patulin (genotoxic, immunosuppressive) Marcfortines (no adverse effects described) Andrastines and more

12 Joint project ( ) Mycotoxin carry-over from maize silage via cattle into dairy products + Preharvest fungi and their mycotoxins in maize, Sørensen, JL (2009) Post-harvest fungal spoilage of maize silage, Storm IMLD (2009) Ph oto: Storm IML LD Pho oto: Shutterstock.com

13 Joint project ( ) Mycotoxin carry-over from maize silage via cattle into dairy products + Preharvest fungi and their mycotoxins in maize, Sørensen, JL (2009) Aim Post-harvest fungal spoilage of maize silage, Storm IMLD (2009) Mycotoxin hazard for cattle feeding on maize silage evaluated by chemical and cyto-toxicity test methods Ph oto: Storm IML LD Pho oto: Shuttersto ock.com

14 Experiments mycotoxins and other fungal metabolites Cytotoxicity Detection ccurrence Comparison of Method 27 fungal development metabolites Mycotoxins Fungal agar Maize silage & & validation performance Silage extraction LC-MS/MS Hot-spots Fresh & ensiled maize silage

15 Cytotoxicity assay Cell viability assay => model system for acute toxicity Limit: Absorption, distribution, metabolism, excretion Dairy cows Rumen fermentation by bacteria, protozoa and fungi Drawings: The Department of Animal Science at Texas A&M University

16 Cytotoxicity assay Caco-2 = human colon cancer cell line Alamar Blue/resazurin assay A ,000 cells/well B C D growth medium 5% C 2, 37ºC E F 48 h toxin exposure G H Viable cell Resazurin Reduction by mitochondria i cell enzyme Resorufin, flurescent

17 Cytotoxicity of mycotoxin standards Concentration-response response graph Viability (%) h exposure Two distinct test days % IC 50 = 0.6 g/ml 0 IC Patulin ( g/ml)

18 Cytotoxicity of mycotoxin standards Viability (%) IC 50 > 50 g/ml 60 IC 50 = 48 g/ml Andrastine A ( g/ml) Roquefortine C ( g/ml) Viability (%) 100 IC 50 > 100 g/ml IC 50 = 0.6 g/ml 50 g 50% IC Patulin ( g/ml) Mycophenolic acid ( g/ml)

19 Cytotoxicity of mycotoxin standards Caco-2 cells, IC 50 mean Toxin, 48 h (µg/ml) SD T-2 toxin / Gliotoxin / Deoxynivalenol / Patulin / Roquefortine C 48 +/- 2 Zearalenone 58 +/- 6 Citrinin 83 +/- 32 N6-formyl-roquefortin-C >46 Andrastin A >50 Mycophenolic acid >100 1-hydroyeremophil-7(11),9(10)-dien-8-one >280 IC 50 = 50% inhibit concentration SD = sample standard deviation of 2 independent experiments

20 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 days old cultures, 25º C YES Yeast extract sucrose agar PDA Potato Dextrose agar CYA Czapek yeast extract agar SA Maize silage agar 15 cm 2 plugs with fungal growth extracted with EtAc:CH 3 Cl 2 :MeH (3:2:1), 1% (v/v) formic acid Re-dissolved in 2 ml MeH

21 Cytotoxicity of Penicillium paneum Viability % Mean of 2 tests YES CYA PDA SA fungal extract of IBT number Patulin >2.9 µg/ml

22 Cytotoxicity of Penicillium paneum Major metabolites: YES, CYA, PDA and SA agar patulin marcfortine A > IC 50 citreoiscoumarin roquefortine C andrastin A < IC 50 < IC 50 Cytotoxic Non-toxic extracts

23 Cytotoxicity Fungal agar and silage extracts Fungi Sec. metabolites detected in fungal agar extracts Penicillium paneum patulin, roquefortine C, andrastin A, citreoisocoumarin, macfortine A, Penicillium roqueforti (mycophenolic acid), roquefortine C, andrastine A, Interactions or ther; PR-toxin, roquefortine A, Aspergillus fumigatus gliotoxin, + ther; verrucologen, fumitremorgin A & B, fumigaclavine C,... Byssochlamys nivea patulin, mycophenolic acid, byssochlamic acid,... Monascus ruber Fusarium graminearum Citrinin, ther; monacolin K, ankaflavin, Zearalenone, deoxynivalenol, Interactions or ther; Aurofusarin, fusarin C, rubrofusarin, Red = present in cytotoxic concentrations Gray = present in non-toxic concentrations Black = present

24 Cytotoxicity of Penicillium roqueforti Major cytotoxic metabolite in fungal YES extracts? #300 P roq raw TTB diluted RIR_0929s24 3: Diode Array Hydroxyeremophil-7(11),9(10)-dien-8-one Range: 1.256e+1 Respon nse, DAD D nm AU 1.2e e+1 1.1e e+1 1.0e PR-toxin & mycophenolic acid N6-formylroquefortin-C e1 5.0e Roquefortine C PR-toxin analogues (?) Andrastin A Fractions by RT time targeting peaks PR-toxin H 3 C CH 3 CH 3 CH 3 H Time min

25 Cytotoxicity of maize silage Viability (%) 100 Healthy silage Inoculated silage Byssochlamys nivea Photo: Birgitte ML Raun QuEChERS extract of maize silage Evaporated and re-dissolved in MeH 80 Secondary metabolites + Byssochlamic acid 60 + Mycophenolic acid B. nivea + ther undescribed metabolites - Patulin maize silage (mg/ml) No significant cytotoxicity Penicillium roqueforti (+ PR-toxin) Penicillium paneum (- patulin) Aspergillus fumigatus (- gliotoxin) Monascus ruber (+ citrinin) i i

26 Summery of cytotoxicy Caco-2 resazurin assay sensitivty as other in vitro assays Applications: fungal agar extracts, screening fraction of fungal agar extracts PR-toxic an important toxic metabolite from P. roqueforti silage extracts Cytotoxic pre- and post-harvest species Alternaria 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

27 Experiments mycotoxins and other fungal metabolites Cytotoxicity Detection ccurrence Comparison of Method 27 fungal development metabolites Mycotoxins Fungal agar Maize silage & & validation performance Silage extraction LC-MS/MS Hot-spots Fresh & ensiled maize silage

28 Detection techniques High performance liquid chromatograph (separation of analytes) Mass spectrometer (detector) Photo: agile ent.com

29 LC-MS/MS Electrospray Quadrupole 1 Quadrupole 2 Quadrupole 3 (ionization) (mass filter) (Collision cell) (mass filter) Capillary, high voltage Cone Detector Sample,, HPLC eluent

30 LC-MS Electrospray Quadrupole 1 Quadrupole 2 Quadrupole 3 (ionization) (mass filter) (Collision cell) (mass filter) Capillary, high voltage Cone Detector Sample,, HPLC eluent Mass filter Photo: waters.com

31 Detection - Multi-mycotoxin analysis 32 fungal metabolites Size: Da H CH 3 CH 3 H H H CH 3 CH 3 CH 3 Strong acid: Mycophenolic acid Weak acid: Alternariol monomethylether Base - amine: Roquefortine A C H 3 H 3 C H N H CH 3 Water solubility changes with ph Fungal hot-spot, ph = 7 Unspoiled silage ph = 3-4 ph buffered extraction N H

32 Detection sample extraction Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) method 1 Sample preparation Homogenisation with liquid nitrogen 1 st extraction Water 1% Acetic acid in ACN Na-Acetate 2 min shake 2 nd extraction 1 min shake Anhydrous MgS 4 Centrifugation 10 min Decant ACN phase Preparation for LC-MS/MS Filter in vials 1) Anastassiades National Food Institute, et al. Technical (2003) University J. AAC of Int. Denmark 86,

33 Detection LC-MS/MS validation Spiked on 3 levels, repeated 3 days Compound ESI MS-MS REC mean RSD r RSD IR LD spike M» D1, D2 % % % ug/kg Mycophenlic acid » 190.8, Zearalenone » 130.8, Alternariol - 257» 214.9, Cyclopiazonic acid » 195.9, (181.9) Enniatin B » 314, Deoxynivalenol » 265, (295) Marcfortine B + 464» 436, Citrinin » 233, Unstable LC-MS-MS sensitivity Fumonisin B » 334, LD (1-739 μg/kg) Reproducibility (7 35% RSD)

34 Summery - Detection Both pre- and post-harvest fungal metabolites ne extract No clean-up Buffering of ph Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) method LC-MS/MS 27 validated: LD (1-739 μg/kg) & Reproducibility (7 35% RSD) Rinsing the HPLC system between samples ensures performance

35 Experiments mycotoxins and other fungal metabolites Cytotoxicity Detection ccurrence Comparison of Method 27 fungal development metabolites Mycotoxins Fungal agar Maize silage & & validation performance Silage extraction LC-MS/MS Hot-spots Fresh & ensiled maize silage

36 ccurrence maize silage Visibly mouldy maize silage from all over Denmark 10 hot-spots infected with M. ruber, A. fumigatus, B. nivea, P. paneum and P. roqueforti Unspoiled fresh and ensilage maize collected from 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 stack

37 ccurrence - visible mouldy silage Penicillum B. nivea A. fumigatus Quantitative Alternaria Fusarium Penicillum Phoma A. fumigatus Fungal hot-spots (n=10) Analyte n pos max (µg/kg) Roquefortine C 3 33,662 Andrastin A 6 8,811 Mycophenolic acid 6 1,646 Gliotoxin Alternariol Alternariol monomethyl ether 1 51 Nivalenol Deoxynivalenol 2 1,092 Zearalenone Enniatin B Roquefortine A 3 Marcfortine B 1 Marcfortine A 3 Citreoisocoumarin 5 Fumigaclavine A 1

38 ccurrence 99 unspoiled silages Penicillum B. nivea Alternaria Fusarium Quantitative Penicillum Phoma Qualitative n pos µg/kg fresh weight Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid Alternariol Alternariol monomethyl ether Nivalenol Deoxynivalenol Zearalenone Enniatin B Roquefortine A 9 Marcfortine B 1 Marcfortine A 6 Citreoisocoumarin 8

39 ccurrence 99 unspoiled silages µg/kg fresh weight Analyte mean pos max Roquefortine C Penicillum Andrastin A B. nivea Mycophenolic acid A. fumigatus Gliotoxin 0 Alternaria Alternariol Alternariol monomethyl ether Fusarium Nivalenol Deoxynivalenol Quantitative Zearalenone Enniatin B Penicillum Roquefortine A 9 Marcfortine B 1 Qualitative Marcfortine A 6 Phoma Citreoisocoumarin 8 A. fumigatus Fumigaclavine A 0 n pos nly in hot-spots

40 ccurrence 99 unspoiled silages f samp ples No. o samples was without detectable residues 30 samples contained only one fungal metabolite 8 samples contained 4 different analytes No. of analytes detected in samples

41 Summery mycotoxin occurrence Low levels of multiple secondary metabolites is common High occurrence of Fusarium toxins Fusarium toxins < values recommended by the European Commission (growth season ) 2008) High content of fungal metabolites with visible fungal growth PR-toxin and patulin (highly hl cytotoxic) were not detected d in field samples

42 Conclusion Cytotoxicity 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 LC-MS/MS detection method 27 metabolites in maize silage, QuEChERS extraction successfully validated Rinsing the HPLC system between samples ensures performance ccurrences of fungal secondary metabolites 15 metabolites detected High 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

43 Perspectives mycotoxin occurrence and toxcity No indication that mycotoxins in maize silage have caused the general health problems observed at Danish dairy cattle farms Immunosuppresive (e.g. DN, NIV, GLI, MPA) Continuous exposure to low levels? infectious diseases? Long term in vivo studies are sparse Human exposure, transport to milk Grain and not animal products a significant source for the pre- harvest toxins deoxynivalenol, zearalenone and fumonisins Genotoxic Alternaria toxins might be relevant

44 Thank you to Supervisors Peter H Rasmussen Mona-Lise Binderup Thomas Larsen Fellow PhDs Ida MLD Storm Jens L Sørensen Inspiration and advice Ulf Thrane Kristian F Nielsen Jens C Frisvad Birgitte Andersen Jørn Smedsgaard External collaboration partners Stig Purup Niels B Kristensen Yvonne Simonsen Anita Iversen Technical assistance Vivian Jørgensen Tanja Thorskov Bladt Hanne Jakobsen All colleagues from Div. Food Chemistry Div. of Toxicology and Risk Assessment Dep. of Systems Biology Funding The Directorate for Food, Fisheries, and Agri Business