Evaluation of the Daily Intake of Carcinogenic mycotoxins - Molecular evidence of synergistic effect

Transcription

1 Evaluation of the Daily Intake of Carcinogenic mycotoxins - Molecular evidence of synergistic effect Annie Pfohl-Leszkowicz 1 UMR CNRS/INPT/UPS 5503, ENSA Toulouse 1 avenue agrobiopole Auzeville-Tolosane, France leszkowicz@ensat.fr

2 INTRODUCTION : Fusarium, Aspergillus,Penicillium Mycotoxins in modly food and feed ingestion Risk for health Meat, dairy product Animal feeds

3 Toxic effects of OTA, CIT, AFB1, FB1, ZEA AFB1 Hepatotoxic; Hepatocarcinogen OTA Nephrotoxic, teratogen, renalcarcinogen Citrinine = CIT Nephrotoxic FB1 Leuco encephalomalacia (equine), porcine pulmonary oedema, Nephrotoxic Carcinogen Zéaralénone =ZEA Endocrine disruptor oestrogenic-like

4 Mycotoxins analysis in several Food and Feed Olives Wine Rice AFB1, OTA, CIT, FB1, ZEA Beer Nuts Coffee cereals Breakfast cereal Spices 4

5 METHODS : Extraction of mycotoxins :The ground sample (20 g) were stirred in presence of 100 ml of acetonitrile 4% aqueous solution of potassium chloride (9:1). Sample Grinding Mixture Solvent of extraction ph < 4 agitation filtration Purification : The purification of mycotoxin can be of two types, according to the mycotoxins and the matrices: by partition and the immunoaffinity

6 Presence of OTA & CIT in french wheat Sampling 1 Harvest Storage in coop 3 2 Sale Storage at farm 1,2,3 Samples analysed in the laboratory 1: after harvesting 2 : after farm storage 3 : after coop storage

7 Influence of type of storage on amount of OTA &CIT Farm +/-coop n=14 OTA CIT OTA 5 µg/kg 3 µg/kg OTA< 5 µg/kg OTA<3µg/kg CIT<5µg/kg CIT 5 µg/kg 22% 22% 56% Coop n=41 14% 86% At Harvest n=28 OTA CIT OTA CIT 33% 13% 65% 22% 10% 90% 100% No contamination OTA CIT 66%

8 Influence of weather before harvest (rain) 61,4% n=51 38,6% n=32 avant pluie après pluie Range of OTA contaminations Exposition at rain Range of CIT contaminations proportion of contaminated samples increases Range of OTA contaminations Range of CIT contaminations 4,5 9 4,5% 9% Exposition to rain 25% 23,5% 10% Amounts: OTA higher/ CIT similar 29,4% 86,4% 75% 47% 90%

9 Occurrence of AFB1, CIT & OTA in all rice samples (100) collected in Vietnam 60 51% Samples (%) % 35% 0 No detectable AFB1 CIT OTA Contamination rate AFB µg/kg 2 µg/kg EU legislation CIT OTA µg/kg EU legislation

10 REPARTITION OF COCONTAMINATION IN RICE - 75 % of samples contain at least one mycotoxin 4% OTA/AF/CIT - 20 % of samples contain more than one mycotoxin - CIT is never alone

11 Repartition of AF, OTA, CIT in the five regions of Vietnam Dry season Raining season Dry season Raining season -AF & CIT contaminations are more frequent during raining period -OTA contamination is more frequent in dry season AFB1 legislation No. of samples over norms OMS : 30 ng/g 0 legislation CE 1998 : 2ng/g 10 legislation CE 2007 : 8 ng/g 1

12 Intake AFB1 & OTA via Rice in Asia Daily consumption of rice by the average adult Vietnamese is estimated to be 500 g. For the highest AFB1-contaminated sample (29.8 ng/g), the amount of AFB1 ingested was up to 15 µg/person/day =>intake of AFB1 for a 60 kg person would be 296 ng/kg b.w./day. Consumption of the most OTA contaminated rice corresponds to 1390 ng/day => 23 ng/kgbw/day This is five times higher than the EU recommendation (5ng/kg/day) and 1.6 times higher than JECFA recommendation (35 ng/kg/day).

13 Olives Wheat Maize OTA &FB1 FB1+FB2 Morocco origin Czech republic Czech republic OTA AFB1 µg/kg OTA 5 µg/kg 1.37 CIT FB FB2 4mg/kg µg/kg

14 Intake AFB1 & OTA via Olive Considering the four highest OTA contamination samples and the mean daily consumption of 60 g of olive without stone per person, the OTA intake via consumption range ng/day 1ng/kg/day For AFB1 the intake range between ng/day => 5ng/kg/day

15 Overall assessment on the contaminations of the cereals of breakfast s1, A, 0%, % Breakfast cereals OTA 3 µg/kg Series1, FB, 25.00%, 25% Series1, 4TOX, 3.33%, 3% OTA/CIT /FB 56% Series1, NC, Series1, 1TOX, 3.33%, 3% 13.33%, 14% Series1, 2TOX, 50.00%, 50% OTA/CI T/ZEA 11% CIT/FB/ ZEA 11% OTA/FB/ ZEA 22% Series1, 3TOX, 30.00%, 30% CIT/FB 6% FB/ZEA 40% OTA/FB 27% OTA/CIT 27% CIT ZEA 100 µg/kg FB1 800 µg/kg

16 FB1 has been detected not only in corn flakes, but also in samples containing oat or rice. Cereals of breakfast Samples without maize (9) contaminated by FB Serie s1, 2, 9, Serie s1, 1, 12, Samples with maize (12) contaminated by FB Why the legislation takes into account only in CORN?!

17 Intake FBs & OTA via Breakfast cereals Considering the highest OTA contamination and the advice provided by the suppliers in terms of daily consumption of such products (30 g), the consumption OTA would be 269 ng per day 13.5 ng/kg bw/day (child 20kg!) For FBs consumption would be 33 µg/kg => 1.6 µg/kg bw/day

18 Coffee analysis -Evaluate occurence of OTA in commercial ground coffee -Compare three methods of OTA extraction -Estimate the passage of OTA through coffee beverage -Compare two methods of coffee preparation Filtration (electric) Brewing (piston)

19 OTA : < LOQ to 11.9 µg/kg Coffee The proportion of OTA passing through the beverage ranged from 20 to 140% : Presence of OTB which cross-reacts with OTAantibodies Alkalinisation of OTA converting its in open ring OTA (OP-OA) More the coffee is roasted, more isomerization of OTA occurred leadin to underestimation.

20 OTA intake via most contaminated Coffee The highest amount of OTA found in ground coffee was 11.9 µg/kg. Assuming a total transfer of OTA from ground coffee to beverage - a bowl of 300 ml of coffee made with 7 g will contain 250 ng of OTA - a cup of expresso (85 ml) will contain 70 ng. Thus, the intake of a high coffee consumer who drinks 640 ml of coffee (1 bowl of 300 ml at breakfast; 1 cup of 85 ml at 10 o clock; 1 cup after the lunch; 1 cup in the afternoon; 1 cup after the dinner) will reach 530 ng. => 8.3 ng/kg bw/day (person 60kg)

21 Ingredients: Breakfastcereals (30g), salad containing olives (60g), rice dish (100g), wholewheat bread (100g), cakes with the multicereals, wine (2glasses), coffee (150 ml) Amount of mycotoxins ingested/day (average value) TDI Based on carcinogenicity (weight 60kg) on estrogenic activity The legislation is overpassed AF OTA FB ZEA 3756 ng 648 ng ng 95.1 ng (1.6ng/kg/d) (5 ng/kg/d) (300ng/kg/d) 96ng 300ng 18000ng 1200ng 39 x 2.5 x 2.9 x 0.7

22 One month-food and urine analyses in depth of three Serbian families of BEN area % of OTA / CIT contaminated samples BEN 1 BEN 2 NON BEN BEN 1 : FOUR persons ( years old); has ONE direct progenitor who died from BEN BEN 2 : FOUR persons ( years old); has TWO direct progenitors who died from BEN NON-BEN : FOUR members ( years old) no record of BEN cases in the ancestry Higher amount of OTA in BEN 1&2 ; Never CIT in NON-BEN NO ARISTOLOCHIC ACID whatever the families!

23 Comparative intake of each members of the three families ng/kgbw/day Father Mother Boy Girl Father Mother boy 1 boy 2 Father Mother boy 1 BEN 1 BEN 2 Non BEN boy 2 EU limit based carcinogenicity SF 5000 VSD JECFA limit 100 ng/kg bw/week (35 ng/kg bw/day) EU limit 5 ng/kg bw/day; VSD virtual safe dose 1.5 ng/kg bw/day

24 Viability of HK2 (human renal cell) exposed to OTA; CIT; FB alone or combined HK248h OTA 48h FB1 48h (OTA+FB1) 48h (OTA+FB1+CIT)48H FB1 48H (FB1+CIT) 48H HK2 48H CIT 48H (OTA+FB1+CIT) 48H % survival survival % concentration concentration in µm OTA or FB alone => cytotoxicity Combined => proliferation FB alone => cytotoxicity CIT alone => proliferation Combined => proliferation equivalent to CIT alone for dose < 5µM

25 Kidney genotoxicity of rat fed OTA and/or CIT Control Control CIT OTA OTA + CIT Confirm genotoxicity of CIT Exposition OTA + CIT : specific adduct of CIT and OTA with amplification of OTA adduct

26 OTA-DNA adduct in kidney of rat fed OTA and FB 5ng/kg OTA 5ng/kg OTA 200 ng/kgfb 5ng/kg OTA 50µg/kg FB 50µg/kg OTA 50µg/kg OTA 50µg/kg FB Exposure to low dose of OTA induced more adducts than high dose OTA genotoxicity is increased by simultaneous presence of FB

27 Kidney DNA adduct of Pig fed OTA or FB alone or simultaneously Control pig FB OTA FB + OTA Croatian farmer -Fumonisin B1 (FB) does not induce any DNA adduct -Ochratoxin A (OTA induce several DNA adduct) -FB in presence of OTA increase OTA-DNA adduct (mainly C-C8dG and OTHQ related adduct) -DNA adduct pattern of a croatian farmer has a similar profil

28 Malformation of cat Abdominal malformations Cleft lip Scattered fur

29 Analysis of mycotoxins (aflatoxin, citrinin, fumonisin, ochratoxin, zearalenone, DON) in cat feed and tissues of dead kittens, and blood of mother after specific extraction of each mycotoxin and HPLC with fluorimetric detection blood : 2 Tissues kidney: 2 M :5 O : 2 P : 2 AFB1, OTA, CIT, FB1, ZEA liver: 2 PETFOOD from 3 different providers: M, O, P intestine:1 brain: 1 29

30 Sampling and numbering Cat feeds Mothers Kittens Sample M1, M2, M3 Mother 1 kitten 1 blood Liver, kidney Breeding 1 O1 CAT x blood O2 Mother 3 blood, plasma kitten 3 Liver, kidney, placenta Breeding 2 PA, PB kitten 2 Liver, kidney, brain, intestine

31 Mycotoxin in petfood of mother 1 Sample ZEA Toxins fromfusarium FB 1 FB2 FB1+ FB2 DON Toxines from Aspergillus et Penicillium CIT AFB 1 OTA Autres AF M ND G2, G1, B2 M G2, G1, B2 M ND 0.92 G2, G1, B2 Mycotoxin in organ of dead kitten 1 Toxins from Fusarium Toxins from Aspergillus et Penicillium Sample ZEA FB 1 OTA CIT AFB 1 other AF LIVER kitten 1 ND ND ND B2 KIDNEY kitten 1 ND ND ND G2, B2

32 Mycotoxins occurrence in new petfood Toxins from Fusarium Sample ZEA µg/kg FB 1 µg/kg FB2 µg/kg DON µg/kg O1 Dérivé N.A 19.3 <LOQ Toxins from Aspergillus & Penicillium OTA CIT AFB 1 Other µg/kg µg/kg µg/kg AF G2, G1, B2 Mycotoxins occurrence in blood of cat eating this petfood Toxines de champs produites par Fusarium ZEA FB 1 Toxines de stockage produites par Aspergillus et Pénicillium Sample OTA CIT AFB 1 other AF Cat blood1(mother) ND ND ND ND ND B2 Cat blood X ND ND ND ND ND B2 (mother)

33 Mycotoxins occurrence in same petfood but other lot => death Toxins from Fusarium Toxins from Aspergillus & Penicillium sample ZEA FB 1 FB2 DON OTA CIT AFB 1 other µg/kg µg/kg µg/kg µg/kg µg/kg µg/kg µg/kg AFB O N.A G2, B2 Mycotoxins occurrence in blood of mother Toxins from Fusarium Toxins from Aspergillus & Penicillium Sample ZEA FB 1 OTA CIT AFB 1 other AFB Cat blood 3(mother) B2 Plasma cat3 (mother) B2 Mycotoxins occurrence in organ of kitten Toxines from Fusarium Toxines from Aspergillus & Penicillium Sample ZEA FB 1 OTA CIT AFB 1 Autres AFB Liver kitten ND ND G2, B2 Kidney kitten ND ND B2 Placenta kitten ND ND B2

34 Mycotoxins in petfood of second breding with teratogenesis Toxins from Fusarium Toxins from Aspergillus & Penicillium Sample ZEA FB 1 FB2 DON OTA CIT AFB 1 other AFB M A N.A ND G2, G1, B2 M B N.A <LOD <LOD G2, G1, Mycotoxins in organ of the dead kitten Toxins from Toxins from Aspergillus & Penicillium Fusarium Sample ZEA FB 1 OTA CIT AFB 1 Autres AFB Liver kitten ND ND ND ND B1, B2 Kidney kitten ND ND B2 brain kitten , ND 0.77 B2 Intestine kitten ND 0.63 ND B2

35 CONCLUSIONS : The contamination of rice was at alarming rate for AFB1 (30µg/kg). Amount of these toxins in rice was greatly affected by the season of the year. 75% of breakfast cereals are contaminated by OTA ( LOQ to 12.7µg/kg). All samples of ground coffee contain OTA (LOQ to 11.9 µg/kg). OTA, AFB and FB were also detected in maize and wheat above acceptable EU limit. All food are contaminated by several mycotoxins In the family non-affected by BEN, the OTA intake is low, and the food is not contaminated by CIT whereas OTA and CIT are often found in BEN families Based on a typical menu, the daily intake (TDI) was respectively 39-fold; 2.5-fold and 3-fold over the virtual safety dose (VSD) etablished for AFB1, OTA and FB. Synergystic toxic effect could be observed, even with concentrations considered as safe

36 ACKNOWLEDGEMENTS Mariana Tozlovanu (Moldavia) Kheira Hadjeba-Medjdoub Tri Nguyen (Vietnam) Chakib El Adlouni (Maroc) PhD students UMR CNRS 5503 Toulouse Ivana Polisenska Brno, Czech Republic Vladisav Stefanovic Nis, Serbia Maja Peraica, Zagreb, Croatia Richard Manderville, Guelph, Canada Annie PFOHL-LESZKOWICZ phone/fax ; leszkowicz@ensat.fr