Mycotoxin Secondary metabolite of fungi Contaminating food and feeds Environmental contamination

Mycotoxin
Secondary metabolite of fungi
Contaminating food and feeds
Environmental contamination
By Susumu Kumagai
University of Tokyo
Research Centre for Food safety
Fumonisin 1-2 ppm
Aflatoxins
Pulmonary edema, Pig
By Kasetsart University
Cirrhosis, Duck
Zearalenone
Fusarium graminearum, F. Crookwellence etc
Corn, wheat, barley, sorghum
Hyperestrogenism, hepatotoxic, hepatocarcinogenic
-Zearalenol, -Zearalenol
PMTDI=0.5 g/kg bw (JECFA)
Trichothecenes
A group of mycotoxin having a common structure, T-2 toxin,
deoxynivalenol, nivalenol, etc.
Deoxynivalenol, Nivalenol
Fusarium graminearum etc
Wheat, barley, corn, processed foods derived from these
Vomit, feed refusal, diarrhea, decrease of leukocyte, depression of
immune function
DON, PMTDI=1 g/kg bw (JECFA)
Fumonisin
Fusarium moniliforme, F.proliferatum etc
Corn, processed food from corn
Human esophagal cancer, ELEM (equine leukoencephalomalacia),
PPE (porcine pulmonary edema), human neural tube defect
PMTDI=2 g/kg bw (JECFA)
Ochratoxin A
Penicilium verrucosum, Aspergillus ochraceus etc
Wheat, dried grape, corn, wine, beer, coffee, pork kidney
Human endemic nephropathy & urinary tract tumor, porcine
nephropathy, carcinogenic, nephrosis
PTWI = 100 ng/kg bw/week
Ergot alkaloids (ergotamine, ergotoxine, etc.)
Claviceps purpurea etc., Neotyphodium coenophialum etc.,
Rye, wheat, barley, rice, oats, corn, rye grass, tall fescue
Vasoconstriction, gangrene, neurotoxic symptoms, abortion in
human and farm animals
Limit for feed contamination in some countries.
Aflatoxin
• In 1960, Spring-Summer：Turkey-X disease in
England
• Causal feedstuff：peanut meal
• Food: nuts, spices, corn, cacao, coffee, rice, milk
• Producer：Aspergillus flavus, A. parasiticus, other
species
• Toxicity：
Acuteーhepatotoxic
ChronicーLiver cancer
Ochratoxin：Porcine nephropathy
Aflatoxin: Human lung cancer
Aflatoxin: Turkey X diseases
Ｔｒｉｃｈｏｔｈｅcenes：Pig acute disease
Zearalenone: Pig acute disease
Fumonisin:ELEM, PPE
Fumonisin:Neural Tube Defect
Trichothecenes: Human ATA
:
Fumonisin: Human esopgagal cancer
Ｔｒｉｃｈｏｔｈｅcenes： Human red mold disease
Ochratoxin：Human nephropathy
Ergot alkaloids: Human gangrene
Aflatoxin: Human acute disease
Aflatoxin: Human hepatic cancer
Aflatoxin: Human hepatic cancer
Aflatoxin: Human acute disease
Aflatoxin: Human hepatic cancer
Aflatoxin: Human hepatic cancer
Fumonisin: Human esopgagal cancer
ELEM
Main mycotoxicoses occurred through consumption
of naturally contaminated food and feed
Risk assessment and risk
management of mycotoxin in food
Animal study
Epidemiological study
Molecular mechanisms
Hazard Characterization
Carcinogenic
Genotoxic
Non-carcinogenic
Non-genotoxic
NOEL or NOAEL or LOAEL
Safety factor
Risk characterisation
Cancer risk
Margin of exposure
Exposure
assessment
PMTDI or PTWI
( Situation of mycotoxin intake
Contribution of food item
Risk management
(Establishment of regulatory limit）
The maximum level for mycotoxin in feed
Mycotoxin in feed
Consumption of
residue in edible tissues,
milk, and eggs
Adverse health effects
Animal health
Human health
The maximum level for feed should ensure both animal
health and human health.
Effects of feed mycotoxin on animal health can be
estimated based on data of animal experiments and natural
intoxication cases of animals.
Effects of feed mycotoxin on human health can be
estimated based on carry-over of mycotoxin from feed to
animal derived food.
Mycotoxin concentration in food =
Mycotoxin concentration in feed / Carry over (ratio of
mycotoxin concentration in feed to that in animal derived
food, or, relationship between intake by animal and
mycotoxin concentration in animal derived food)
Aflatoxin as an example
Typical aflatoxins in Food
O
O
O
O
O
O
O
O
O
AFB１
O
O
AFB2
AFG1
Total aflatoxin
O
OH
O
O
O
OCH3
OCH3
O
O
O
O
O
O
O
OCH3
O
OCH3
AFG2
All foods
O
O
O
O
aflatoxin
OCH3
M1
Milk and products
Acute toxicity in animals
Liver: Biliary proliferation, necrosis, jaundice
LD50 (mg kg bw) AFB1
Animal
Duckling
Rabbits
po
ip
0.0335
0.3
Cat
0.55
Dog
0.2
Guinea pig
1.4
Rat, neonate
Rat, adult
Hamster
0.56
2-30
Female>Male
10.2
13
AFB1 is metabolized in animal body to
AFM1 >>> Excreted into milk
AFP1
AFQ1
Aflatoxicol
AFB1-epoxide >>> AFB1-DNA >>> Cancer
AF-aldehyde >>> AFB1-protein >>> Acute
toxicity
AFB1-glutathione conjugate
AF-diol etc.
Metabolic pathway of Aflatoxin B1
OH
AFL
O
O
CYP 1A2
O
AFM1
O
O
O
O
O
feces,urine
milk
OCH 3
O
AFB1 O
O
Molecular mecahnisms
O
O
AFP1
feces,urine
OCH 3
O
O
OH
O
CYP 3A4
O
AFQ1
O
O
O
OH
O
O
O
Cellular
injury
AFB1-protein adduct
O
O
OCH 3
OCH3
glutathion-S-transferase
AFB1-exo8,9-epoxide
AF-glutathoin
conjugate
AFB1-DNA adduct
O
AFB1-lysin-adduct
O
carcinogenic
O
HO
O
(serum)
N
H 2N
N
N
feces,urine
N
O
OCH 3
AFB1-guanine （feces, urine)
feces,urine
Acute toxicity of AF metabolites
AFB1=Aflatoxicol > G1, P1, M1, Q1 > B2, G2
Major Acutely Affected Human Cases
India 1974.11-1975
397 Cases. 106 Deaths. Dogs also died.
Clinical symptoms: Ascites, jaundice, fever, legs edema, vomit.
Liver histopathology: Bile duct proliferation, gastrointestinal
hemorrhage, periductal fibrosis.
Moldy corn: 6.25-15.6 ppm afl.
Ingested amount: 0.055 mg/kg/day< for several wks.
Kenya 1982.4-5
20 Cases. 12 Deaths. Dogs also died.
Clinical symptoms: Same as India.
Pathology: Liver necrosis
Stored crops: 3.2-12 ppm afB1 (corn), 1.6 ppm afB1 (bean)
Ingested amount: 0.038 mg/kg/day< for some days.
Kenya 2004.1-6
397 Cases. 125 Deaths.
Clinical symptoms: Jaundice.
Corn: mean 354.53 ppb, max 20ppm afl.
Kenya 2005.4-6
76 Cases. 32 Deaths. Max 33 ppm afl.
Kenya 2006.4-6.
51 Cases. 21 Deaths. Higher than 1 ppm.
Human cancer
Epidemiological studies
The relationship between primary liver cancer and intake of aflatoxin
Area
Occurrence of
Primary liver cancer
（105 /year）
Kenya highland
Thailand-Song khla
Swaziland
Kenya midland
Swaziland- midland
Thailand-Ratchaburi
Swaziland- lowland
Intake of AF
（ng/kg body weight／day）
0.7
3.5
2.0
5.0
2.2
2.9
4.0
6.0
9.7
MozambiqueーInhambane 13.0
5.1
5.8
8.9
45.0
43.1
222.4
International risk
assessment of Aflatoxins
Carcinogenecity of AFB1（Rat）
Animal study
Strain dependent
Sex AFB1 in feed (ug/g) period（week） Occurrence of
liver cancer
♂
1
41
18/22
♀
1
64
4/4
♂
0.3
52
6/20
♀
0.3
70
11/11
♂
0.015
68
12/12
♀
0.015
82
13/13
Wogan,G.N. and Newberne, P.M., Cancer Res.27, 2370-2376, 1967
Aflatoxin is a potent carcinogenic &
genotoxic mycotoxin in human.
Tolerable intake has not been set
for human for regulation.
Regulation for food is based on
population risk.
Risk chracterization
Human HBsAg(0.001) AFM1
0.0
Syrian Hamster
Tree Shrew
Human HBsAg(0.01)
0.014
0.029
0.057
Monkey
Fischer
Rat
AFM1
Human HBsAg
status unknown
Human HBsAg+
(0.03) AFM1
Human HBsAg+
(0.3)
Wistar Rat
0.23
0.5
1.0
Fischer Rat
1.0
Potency estimates (cancers/year per 100,000 people per ng/kg bw per day)
for human liver cancer resulting from exposure to aflatoxin B1 or M1 derived from
epidemiological and toxicological studies.
( JECFA, 1997)
Aflatoxin Ｍ１
Metabolite of Aflatoxin B1
Excreted in Milk
Potency of AFM1 is approximately 1/10 times
that of AFB1
（from the data of animal experiment）
Worldwide limits for AFM1in milk
From “Worldwide regulations for
mycotoxins in food and feed in 2003”ｂｙ FAO Rome 2004
How much AFB1 level gives the limit levels
of AFM1 in milk?
Carry over from feed to cattle milk?
Carry-over of feed AFB1 to milk AFM1
Ratio of AFB1 level in feed to AFM1 level in milk
= 34:1 ~ 1600:1
From available data, 75:1 was proposed by Park &
Pohland (1986) as most likely ratio.
If this ratio is used,
3.75 µg AFB1 in 1 kg feed is converted to 0.05 µg
AFM1 in 1 kg milk (EU regulation)
37.5 µg AFB1 in 1 kg feed is converted to 0.5 µg
AFM1 in 1 kg milk (USA regulation)
AFM1(ng/kg of milk)=1.2 X AFB1 intake
（µg/ cow/day）＋1.9 （Veldman et al, 1992)
AFM1 (ng/kg milk) = 10.95 + 0.787 x AFB1 intake
(µg/cow/day) （Petterson 1998)
Worldwide limits for
AFB1 in feed for
dairy cattle
5 ppb can ensure 0.5 ppb
AFM1 in milk, but might not
0.05 ppb, under
the assumption.
Exposure assessment is
required based on data of
AFM1 concentration in
milk.
Worldwide limits for
Total AF in feed for
dairy cattle
From “Worldwide
regulations for
mycotoxins in food and
feed in 2003”ｂｙ
FAO Rome 2004
Carry-over from feed to eggs & edible tissue
Chicken egg
Ratio of AFB1 level in feed to AFB1 level in
chicken egg
= 250:1 ~ 55,000:1
Broiler liver
AFB1 250:1~ 4,000:1
Pig liver
AFB1 35:1 ~ 17,600:1
AFM1 286:1 ~ 35,200:1
Worldwide limits for
AFB1in food
Worldwide limits for
Total AF in food
From “Worldwide regulations for
mycotoxins in food and feed in
2003”ｂｙ FAO Rome 2004
Limit of feed contamination required for
regulatory limit for AFB1 in food
Pig liver
Assumed regulatory limit = 0.002 mg/kg food
Feed to tissue ratio (worst case) = 35
Limit of feed contamination = 0.070 mg/kg feed
Chicken egg
Assumed regulatory limit = 0.002 mg/kg food
Feed to tissue ratio (worst case) = 250
Limit of feed contamination = 0.5 mg/kg feed
The maximum levels for fumonisin, DON and zearalenone
in feed are regarded to be set based on
their adverse effects on animal health, because their
carry-over from feed to edible tissues, eggs and milk is
too small to affect human health.
However,
data of carry-over are not enough for many other
mycotoxins, and more research works are required.
Thank you for your attention!
Thank you
Carry-over from feed to eggs & edible tissue
Feed AFB1 to tissue AFB1 or AFM1 ratio
Chicken egg
AFB1 250 - 55,000:1
AFB1 2,200:1 (Park & Pohland, 1986)
Broiler liver
AFB1 250- 4,000:1
AFB1 2,200 (Park & Pohland, 1986)
Pig liver
AFB1 35 – 17,600:1 (Rodrocks & Stoloff 1977)
AFB1 800:1 (Park & Pohland, 1986)
AFM1 286 – 35,200:1 (Rodrocks & Stoloff 1977)
Transfer of Aflatoxin Ｍ１to cow milk
Feed AFB1 to milk AFM1 ratio
0.01 mg/kg feed, 7 days, 201:1 (Patterson, 1980)
0.02 mg/kg feed, 7 wks, 333:1 (Shreeve, 1979)
Various data, 34-1600:1, average 46:1 (Rodrick &
Stoloff, 1977)
Various data, 75:1 (Park & Pohland, 1986)
0.01 mg/kg feed
3.75 g/kg feed
37.5 g/kg feed
0.13 g/kg milk
0,05 g/kg milk (EU)
0,5 g/kg milk (USA)
AFM1(ng/kg of milk)=1.2 X AFB1 intake （g/cow/day）＋1.9
（Veldman et al, 1992)
AFM1 (ng/kg milk) = 10.95 + 0.787 x (g/cow/day) （Petterson 1998)
fusariumn
■Fusarium
mycotoxin
Red mold disease, wheat
F. graminearum
Fusarium mycotoxins
Trichothecenes
Deoxynivalenol (DON)
Nivalenol (NIV)
T-2 toxin
HT-2 toxin etc.
Zearalenone(ZER)
Fumonisin B1, B2, B3
Trichothecenes
T-2：R1=OAｃ、
HT-2：R1=OH
T-2 toxin
Deoxynivalenol
Contaminated food
•
•
•
•
Wheat
Barley
Corn
Processed foods derived from these
Trichothecenes LD50
LD50 (mg/kg bw)
Compounds Mice
ip
T-2
5.2
HT-2
9.0
DON
43-77
NIV
4.1
po
10.5
46-78
Rats
ip
po
5.2
Health hazard by trichothecenes
Acute & short term toxicity
• Vomit, Feed refusal, Diarrhea
• Hemorrhage, necrosis of skin
• Hemorrhage in GIT
• Inhibition of protein synthesis, Cytotoxicity
• Actively divided cells are more damaged (radiomimeric)
• Decrease of leukocyte, Depression of immune function
JECFA evaluation (2001)
DON:
non-carcinogene
PMTDI=1 micro g/kg bw
T-2 toxin, HT-2 toxin:
PMTDI=60 ng/kg bw as T-2+HT-2 toxins
DON Regulation
DON regulation
Country
Commodity
USA
Finished wheat products for food
Grain and grain byproducts for swine and other animals except cattle
and chicken, not exceed 20%
diet for swine (40% for other species)
Grain and grain byproducts for beef, feedlot cattle older than 104
months and chiken; not exceed
50% of the diet
EU
Cereal products as consumed and other products at retail stage
Flour used as raw material in food products
Japan
Wheat for foods
Feed for cattle and cow of or older than 3 months
Feed for other farm animals
mg/
kg
1
5
10
0.5
0.75
1.1
4.0
1.0
Zearalenone
Fusarium graminearum
F. Crookwellence etc
World-wide
Corn, wheat, barley, sorghum
ZEA toxicity
ZEA toxicity
Estrogennic action
Hyperestrogenism: swollen valva & mamma, enlargement
of uterus, infertility
Swine is the most sensitive: 5 mg/kg induces cycle failure
Hepatotoxic, hepatocarcinogenic
Estrogen activity :Alpha-ZEA (3-100 fold) > ZEA > beta-ZEA
LD５０
Mice M&F
po >2000 mg/kg bw
Rats M&F
po >4000
Guinea pigs F po >5000
JECFA Evaluation: PMTDI= 0.5 micro g/kg bw/day (1999)
ZEA regulation
Country
Commodity
USA
none
EU
Unprocessed cereals other than maize
Unprocessed maize
Cereal flour except maize four
Maize flour, meal, grits and refined maize oil
Bread, pastries, biscuits, other cereal snacks and breakfast cereals
Maize snacks and maize-based breakfast cereals
Processed maize-based foods for infants and young children
Other processed cereal-based foods for infants and young children
and baby food
Japan
Feed for farm animals
g/kg
100
200
75
200
50
50
20
20
1000
Fumonisin B1
shingosin
Fumonisin human epidemiology
Human
Human Esophagal cancer
Transki/South Africa,
Age standard incidence, 37-56/100,000
Fumonisin level in corn, 32-67 mg/kg (moldy corn),
2.0-2.1 mg/kg (healthy corn)
China,
Age standard incidence, 108/100,000 (Linxian),
140/100,000 (Hebei)
Fumonisin B1 level in corn, 74 mg/kg (moldy corn),
0.7-3.5 mg/kg (Healthy corn)
Animal
Animal epidemiology
Equine Leukoencephalomalacia (ELEM)
Fatal disease observed in USA and South Africa etc, in association
with feed contaminated by Fusarium moniliforme.
Clinical symptoms: ataxia, sweating, convulsion, death.
Pathology: necrosis of cerebral white matter.
Minimum oral dose for ELEM = 8-22 mg/kg feed, 0.18 mg/kg bw/day
for 180 days.
Porcine pulmonary edema
Outbreaks in USA in association with feed contaminated by
Fusarium verticillioides (FB1=20-360 mg/kg feed).
Clinical symptoms: dyspnoea, weakness, cyanosis.
Pathology: pulmonary edema, hydrothorax.
Tissue residue
Little evidence showing significant transfer
to milk. (JECFA 2001)
75mg/kg feed, <5 ng/ml milk, ratio >15,000,000
(Richard et al., 1996)
Pig 22 mg/kg feed, 5 mo (DeLiguoro et al., 2004)
Kidney, Feed to tissue ratio ＞4400:1
Neural tube defect
Effects of fumonisin B1
(FB1) in mouse embryonic
development.
A: Embryos after 24 h in
culture medium containing
none (right) or 50 mol/L
FB1 (left);
B: Embryos after 24 h in
medium containing 50
mol/L FB1 (top) or 50
mol/L FB1 plus 1 mol/L
folinic acid (bottom) (panel
B is reproduced from Fig. 3
of Ref. 62).
C: Fetus from pregnant
LMBc dam injected i.p. with
20 mg/kg body weight FB1
on
gestational d 7.5 and 8.5
and killed on d 17.5 (top)
versus normal fetus at this
gestational age (bottom).
Incidence of
neural tube
defects (NTD per
10,000 live
births) in
different regions
of Guatemala,
South Africa, and
China. Mean
incidence and
range in
incidence of
various locations
within the
regions or
countries are
shown;
the bar for
Limpopo
represents one
data point.
Incidence rates
for the United
States are similar
to those for
urban areas
in South Africa
Long term
Long term toxicity/carcinogenicity
B6C3F1 mice, 728 days
Male
Female
0
mg/kg
feed
Hepatocellular carcinoma,
4/47
5
3/47
15
4/48
50
3/48
150
2/48
0
0/47
5
0/48
15
0/48
50
10/47
80
9/45
Fischer rats, 728 days
Male
Female
0 mg/kg
feed
Kidney carcinoma, 0/48
5
0/40
15
0/48
50
4/48
150
6/48
0
No lesion
5
15
50
100
Fumonisin regulation
Count
ry
Commodity
USA
Foods
Degermed dry milled corn products
Whole or partially degenerated dry milled corn products
Dry milled corn bran
Cleaned corn intended for mass production
Cleaned corn intended for popcorn
Corn and corn byproducts for animals
Equids and rabbits
Swine and catfish
Breeding ruminants, poultry, mink, dairy cattle, laying hens
Ruminants >3month before slaughter and mink for pets
Poultry for slaughter
All other livestock and pet animal species
EU
Unprocessed maize
Maize grits, meal and flour
Maize-based food for direct consumption except maize grits, meal, flour
and processed maized-based foods for infants and young children and
baby food
Processed maize-based foods for infants and young children and baby food
mg/kg
2
4
4
4
3
5 <20% diet
20 <50% diet
30 <50% diet
60 <50% diet
100 <50% diet
10 <50% diet
2
1
0.4
0.2
Wheat etc
オクラトキシンＡ
Dried grape
Corn
Wine
Beer
Coffee
Meat products (Pork kidney）
• OA
OA producing mold
Penicillium Temperate & cool region
Aspergillus Topical
World wide contamination
OTA
Click on image to enlarge
Human cases
1) Endemic nephropathy
Balkan area (Bosnia and Herzegivina, Bulgaria, Croatia,
Romania, Servia)
Onset, 30-50 yr old.
Prevalence rate, 2-10%.
Female > Male cases.
OA contamination in foods, endemic > non-endemic region.
OA concentration in human blood, endemic > non-endemic
region.
2) Epithelial tumor of upper urinary tract.
Youger, Female, more frequently affected.
Bilateral tumor.
Cases of farm animals
Porcine nephropathy
Denmark, Sweden, Ireland
Feed contamination, > 200 ppb.
Acute toxicity-LD50 (mg/kg bw)
Hepatic, lymphoid necrosis, hemorrhage in various
organs, enteritis, nephrosis.
Mice
Rats
Neonate rats
Dog
Pig
Chicken
oral 46-58 ip 22-40 iv 26-34
oral 20-30 ip 13
iv 13
oral 3.9
oral 0.2
oral 1
oral 3.3
Long term toxicity
B6C3F1 mice, 0, 1, 40 mg/kg, 24 mo.
40 mg/kg, nephropathy/benign & malignant renal
tumor, male is more severe than female.
Fischer rats, 0, 0.021, 0.07, 0.21 mg/kg, 9 mo, 15
mo, 103 wk.
Renal adenoma, male:1/50, 1/51, 6/51, 10/50.
Renal carcinoma, male: 0/50, 0/51, 16/51, 30/50.
Female, lower than male.
OA residue in tissues
Pig 0.025-2.0 mg/kg feed (EFSA, 2004)
Liver 12600-130000:１
Laying hen: 2 mg/kg feed, 3 wks; (Denli et al., 2008)
Liver, 0.015 mg/kg; Feed to tissue ratio=133:1
Eggs, <0.00005 mg/kg
Hen (EFSA, 2004)
Liver, 0.5-10 mg/kg;
Feed to tissue ratio=19000 -1920000:1
Chicken, 0,05-2.0 mg/kg;
Feed to tissue ratio=10000 – 83000: 1
国際的動向（2）
n u m be r o f c o u n trie s
30
25
29
20
15
10
5
3
0
50
1
1
1
1
30
20
15
10
1
5
3
limits (g/kg)
Fig. Worldwide limits for ochratoxin A in cereals and cereal products
Worldwide regulations for mycotoxins in food and feed in 2003
FAO food and nutrution paper 81
JECFA evaluation
PTWＩ = 100 ng/kg bw/week
=14 ng/kg bw/day (LOEL:8 ug/kg bw/day)
BMD (rat )
BMDL 10
25 ug/kg bw/day
Regulatory limit for feed (mg/kg)
AF
Japan
USA
DON
Japan
USA
Canada
Netherland
ZEA
Japan
Canada
Ukraine
OA
Canada
Slovania
Fumonisin
USA
0.01-0.02 as B1
0.02-0.3 as Total
1-4
5-10
1-5
1-10
1
3
0.04-3
2
0.2-1
5-100
Tissue residue
Feed AFB1 to tissue AFB1 or AFM1 ratio
Chicken egg
0.5 mg/kg feed, 8 wks, AFB1 5,000:1 (Oliveira 2000)
3.3 mg/kg feed, 10 days, AFB1 55,000:1 (Wolzak 1985)
0.4 mg/kg feed, 15 days, AFB1 286:1 (Jacobson 1974)
8.0 mg/kg feed, 7 days, AFB1 3,800:1 (Jacobson 1974)
Broiler liver
2.1 mg/kg feed, 35 days, AFB1 12,100:1
AFM1 34,300:1 (Chen 1984)
2.0 mg/kg feed, 8 wks, AFB1 250:1 (Mintzlaff 1977)
0.05mg/kg feed, 36-64 days, AFB1 2500:1
AFL 45:1(Miragrin 1988)