Aflatoxin

1. Mycotoxins = Toxic metabolites of fungi.
Mycotoxinscontaminatethewidevarietyoffood
asaresultoffungalinfectionincrops,during
growthorinstorage.
Mycotoxins
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2. Mycotoxins Main Producing Fungi
Aflatoxins B1, B2, G1, G2Aspergillusflavus, A.
parasiticus, A. nomius
OchratoxinA Penicilliumverrucosum, A.
alutaceus, A.carbonarius
Patulin P. expansum, A. clavatus,
Byssochlamysnivea
Fumonisins Fusariummoniliforme, F.
proliferatum
Deoxynivalenol
(trichothecenes)
F. graminearum, F. culmorum,
F. crookwellense
Zearalenone F. graminearum, F. culmorum,
F. crookwellense
In summary: 6 major chemical types of mycotoxins
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3. The name Aflatoxincomes from
A (Aspergillus)
FLA (flavus)
toxin.
Aflatoxins
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4. Aflatoxinsareagroupofstructurallyrelatedtoxic
secondarymetabolitesproducedbythreespecies:
Aspergillusflavus,Aspergillusparasiticusandtherare
speciesA.nomius(KurtzmanetaL.,1987)andknownto
behighlytoxicandpotentialcarcinogens.
Aflatoxinswerefirstidentifiedin1961inanimalfeed
contaminatedbyAspergillusparasiticus(Sargeantetal.,
1961).
Aflatoxins
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5. The things which evoke scientist to study Aflatoxin
Consequently,morethanfivebillionpeoplein
developingcountryworldwideareatriskofchronic
exposuretoAflatoxinsthroughcontaminatedfoods
andmedicinalplants.Aflatoxin-associatedhealth
effectspervadethedevelopingworld.
Aflatoxinsaretheonlymycotoxinscurrently
regulatedbytheU.S.FoodandDrugAdministration.
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6. AccordingtoInternationalAgencyforResearchon
Cancer(IARC),Evidenceofacuteaflatoxicosisin
humanshasbeenreportedfrommanypartsofthe
world,namelytheThirdWorldCountries,likeTaiwan,
Ouganda,India,andmanyothers.AndIn1988,the
IARCplacedaflatoxinB1onthelistofhuman
carcinogens.
Thisissupportedbyanumberofepidemiological
studiesdoneinAsiaandAfricathathavedemonstrated
apositiveassociationbetweendietaryaflatoxinsand
LiverCellCancer(LCC).
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7. Studieshaveshownthatconcurrentinfectionwiththe
HepatitisBvirus(HBV)duringaflatoxinexposure
increasestheriskofhepatocellularcarcinoma(HCC).
AsHBVinterfereswiththeabilityofhepatocytesto
metabolizeaflatoxins,anaflatoxinM1-DNAconjugate
existsforalongerperiodoftimeintheliver,increasing
theprobabilityofdamagetotumorsuppressorgenes
suchasp53.
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8. FAOestimates,25%oftheworldfoodcropsare
affectedbymycotoxinseachyear.
CroplossduetoaflatoxinscontaminationcostsUS
producersmorethan$100millionperyearonaverage
including$26millionstopeanuts($69.34/ha).
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9. Food products contaminated with aflatoxinsinclude
Cereal (maize, sorghum, pearl millet, rice, wheat),
Oil seeds (groundnut, soybean, sunflower, cotton),
Spices (chillies, black pepper, coriander, turmeric,
zinger),
Tree nuts (almonds, pistachio, walnuts, coconut)
Natural occurrence of Aflatoxin
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10. Dairy products (Milk, Cheese, Fluid milk)
Green coffee
Dry fruits
Human biological fluids (human urine, milk
and blood samples.)
Eggs
Liver
Medicinal plant
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11. Corn
Peanuts
Cottonseed
Brazil nuts
Pistachio nuts
Copra
Figs
Almonds
Pecans
Walnuts
Sultanas
Spices
Soybeans
Pulses
Sorghum
Millet
Wheat
Oats
Barley
Rice
Natural occurrence and Aflatoxin risk
High risk
Moderate risk
Low risk
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12. Theaflatoxin-producingAspergillusspecies,and
consequentlydietaryaflatoxincontamination,are
ubiquitousinareasoftheworldwithhot,humid
climates,includingsub-SaharanAfricaand
SoutheastAsia.Exposureinthosecountriesresults
fromcontaminationofdietarystaplesandis
thereforelikelytobechronic.
Geographical Occurrence
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13. Origin No. of lots Lots %
Determinable > 26µ g/kg
China 2585 15 2.5
India 1453 92 58.0
Sudan 932 94 78.0
Argentina 446 40 4.0
South Africa112 41 95.0
Malawi 80 60 2.0
FAO/WO/UNEP Monitoring Program: Afatoxinsin raw,
shelled groundnuts imported into the USA, 1981
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14. Itisprobablynotpossibletoeliminatecompletely
exposureofhumanstoaflatoxins.
In1987,atleast50countrieshadexistingor
proposedregulationsforaflatoxinsinfoodstuffs.And
themaximumlimitsrangefromnonedetectableto50
µg/kgoffoodforeitherthesumofAflatoxinsB1,B2,
G1andG2orforAflatoxinB1alone;5µg/kgisthe
commonestmaximallimit.
Regulations and guidelines
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15. ln1987,aflatoxinM1levelsindairyproductswere
regulatedin14countries.Thetolerancesininfants'and
children'sfoodwere0.05-0.5µg/kgmilk.
AflatoxinswerereviewedbyajointFAO/WHO
ExpertCommitteeonFoodAdditivesin1987(WHO,
1987).
Noacceptabledailyintakewasgiven;itwas
recommendedthathumanintakebereducedtothe
lowestpracticablelevel.
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16. 20 ppb For corn and other grains intended for immature
animals (including immature poultry) and for dairy
animals, or when its destination is not known;
20 ppb For animal feeds, other than corn or cottonseed
meal;
100 ppb For corn and other grains intended for breeding
beef cattle, breeding swine, or mature poultry;
200 ppb For corn and other grains intended for finishing
swine of 100 pounds or greater;
200 ppb For corn and other grains intended for finishing (i.e.
feedlot) beef cattle and for cottonseed meal
intended for beef cattle, swine or poultry.
The FDA will consider action if Aflatoxin levels exceed
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17. Aflatoxinsarenormallyreferstothegroupof
difuranocoumarinsandclassifiedintwobroadgroups
accordingtotheirchemicalstructure.
A.Difurocoumarocyclopentenoneseries(AFB1,AFB2,
AFB2A,AFM1,AFM2,AFM2Aandaflatoxicol)
B.Difurocoumarolactoneseries(AFG1,AFG2,AFG2A,
AFGM1,AFGM2,AFGM2AandAFB3).
Aflatoxins and its Physio-chemical dimensions
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18. Main type
AflatoxinB1
AflatoxinB2
AflatoxinG1
AflatoxinG2
Major metabolites of Aflatoxin B1
AflatoxinM1
AflatoxinD1
AflatoxinP1
AflatoxinQ1
AflatoxinM2
AflatoxinB2a
Aflatoxicol
AflatoxicolH1
AflatoxcolM1
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19. The relative proportions of Aflatoxin B1, Aflatoxin G1,
Aflatoxin B2 and Aflatoxin G2 on crops depend on the
particular Aspergillusspecies present.
A. flavusproduces aflatoxins B1 and B2, whereas
A. parasiticusproduces aflatoxins B1, B2, G1 and G2
(Dorneret al., 1984).
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20. ELISA technique (Enzyme linked immunosorbentassay)
Fluorometry
TLC (Thin Layer Chromatography)
HPLC -FLD (High Performance Liquid Chromatography
–Fluorescence Detector)
HPTLC (High Performance Thin Layer Chromatography)
LC-MS
Method of analysis
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21. Colourlessto pale-yellow crystals.
Intensely fluorescent in ultraviolet light.
Aflatoxins B1 and B1 = blue fluorescence
Aflatoxins G1, G1 = yellow-green fluorescence
Aflatoxin M1 = blue-violet fluorescence
Description
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22. Aflatoxin Melting-point (ºC)
B1 268-269
B2 287-289
G1 244-246
G2 237-239
M1 299
Melting-point
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23. Veryslightlysolubleinwater(10-30µg/ml)
Insolubleinnon-polarsolvents
Freelysolubleinmoderatelypolarorganicsolvents
(e.g.,chloroformandmethanol)
Especiallyindimethylsulfoxide.
Unstabletoultravioletlightinthepresenceofoxygen,
toextremesofpH(<3,>10)andtooxidizingagents.
Solubility
Stability
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24. Thelactoneringissusceptibletoalkalinehydrolysis.
Aflatoxinsarealsodegradedbyreactionwithammonia
orsodiumhypochlorite.
Reactivity
Structurallythedihydrofuranmoiety,containingdouble
bond,andtheconstituentslikedtothecoumarinmoiety
areofimportanceinproducingbiologicaleffects.
Biological effects
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25. “Noanimalspeciesisresistanttotheacutetoxiceffects
ofaflatoxins;henceitislogicaltoassumethathumans
maybesimilarlyaffected.”
Theaflatoxinsdisplaypotencyoftoxicity,
carcinogenicity,mutagenicityintheorderofAFB1>
AFG1>AFB2>AFG2asillustratedbytheirLD50
valuesforday-oldducklings.
Life-threatening effect of Aflatoxins and its mechanism
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26. 1. Liver damage 2. Liver necrosis
3. Liver cirrhosis4. Fever
5. Progressive jaundice 6. Limb swelling
7. Pain Vomiting 8. Enlarged liver
Symptoms of Aflatoxin B1 exposure
Aflatoxicosisin humans
Thesyndromeischaracterizedbyvomiting,abdominal
pain,pulmonaryedema,convulsions,coma,anddeath
withcerebraledemaandfattyinvolvementoftheliver,
kidneys,andheart.
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27. Schematic representation of AFB1 metabolism highlighting the
formation of its critical product AFB1-exo-8,9-epoxide, its DNA-
and protein adducts and major urinary metabolites.
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28. Farming Storage
Processing
Transport
Distribution
Retail
Consumer
ENVIRONMENT
Natural Toxins
Mycotoxins
Veterinary
drugs
Mycotoxins
/ Aflatoxin
In situ formation
due to heat, pH,
etc.
Migration
from
packaging
Heat-induced
carcinogens
e.g. heterocyclic
aromatic amines,
acrylamide
Routes of Aflatoxin contamination
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29. Pre-Harvest
Harvesting
Storage
Preventing or Reducing Aflatoxin Exposure
1. Pre-Harvest
2. Post-Harvest: Drying & Storage
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30. Timing of planting;
Crop planted;
Genotype of seed planted;
Irrigation;
Insecticides;
Competitive exclusion;
Timing of harvest;
Pre-Harvest
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31. Hand sorting
Drying on mats
Sun drying
Rodent control
Storing bags on
wooden pallets or
elevated off ground
Insecticides
Post-Harvest: Drying & Storage
Hand sorting
Winnowing
Washing
Nixtamalization
Acidification
Chemoprotectant
Enterosorption
Crushing and dehulling
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32. Thanks for your attention
Analytical Diligence Services
[email protected]
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Special thanks
to
Mr. Rajesh Garg, Mrs. AnurekhaJain and all BRNCOP family
B.R. NahataCollege of Pharmacy, Mandsaur (M.P) 458001
&
AshwagandhaTechnology Development & Extension Center
National Medicinal Plant Board, Department of AYUSH, GOI