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1、AcrylamideDavid Kitts,Food Science,Food Nutrition and Health,UBC.OverviewIntroductionRisk of Exposure and Formation in FoodKineticsMetabolites and BiomarkersToxicologyDietary IntakeNeutralizationStudy Urinary MetabolitesObjective CorrelationsMethods, ResultsCritiqueWhat is Acrylamide?Industrial Uses
2、-Flocculant for wastewater treatment-Cosmetics, Paper & Textiles-Soil conditioner-SDS-Page gelIndustrially produced vinyl compound, commonly polymerized1994 deemed a “probable carcinogen”International Agency for Cancer Research viewed acrylamide only as a synthetic chemicalExposure thought to be air
3、borne and limited to the workplaceEvidence of Risk1997 Evidence of acrylamide exposure in control group (Bergmark, E. 1997)2002 Swedish study links acrylamide exposure to fried food (animal study) (Tareke, 2002)2002 presentMany studies on formation, food sources, metabolism, toxicokinetics and risk
4、assessmentMechanism of Formation in FoodMulti-steppathwayPyrolysis180CFactors affecting formationAspargine + Reducing sugar contentCooking methodDeep fryingPan fryingMicrowavingCooking temperatureFormation increases with temperatureEffect of Temperature(Tareke2002)Levels in FoodFood#samplesMeang/kgR
5、eportedMaxg/kgBakedProducts11,3273667,834PotatoChips3,5557524,080PotatoFrenchFry6,3093345,312Coffee(decaf)346685,399(JECFA2005)Toxicokinetics of acrylamide (AA)AbsorptionAccumulationExcretionMetabolismAbsorptionRoutes of exposure: Oral ingestion, inhalationPost administration: GI tract blood stream
6、-Small vinyl monomer, readily pass through mucosal membranes -Biological barriers: ineffective AbsorptionMetabolismPhase I reactionOxidization of acrylamide Catalized by cytochrome P450 2E1Yields glycidamide & further glyceramideMetabolismPhase II reactionConjugation of acrylamide and glycidamide -c
7、onjugated with glutathione -GST (glutathione-S-transferase)Yield AAMA (acrylamide mercapturic acid conjugates) and GAMA (glycidamide mercapturic acid conjugates)MetabolismMetabolites of acrylamideGlycidamide (GA):Evidenced by produced N-acetyl-s-(2-carbamoyl ethyl) cysteineSub-metabolites derived fr
8、om glycidamid: -N-acetyl-s-(2-hydroxy-2-carbamoylethyl) cysteine -N-acetyl-s-(1-carbamoyl-2-hydroxyethyl) cysteineMetabolismPathwaysParent compoundMetabolitesAccumulation Generic model: Bloodbody compartmentssites of action AccumulationWidely distributed in tissues after absorptionShort half-life t1
9、/2= 1.4-3hrsMajor depot = RBCSmall amount detected in tissues after weeksTwo types of adducts contribute to accumulationHemoglobin adductsDNA adductsHemoglobin adductsAcrylamide with Cysteine & valine residues -Internal dose biomarker for neurotoxicity -Use 14C-acrylamideDetection of cysteine adduct
10、 -Acid hydrolysis of protein isolate cysteine adducts -GC/MS analysisDetection of valine adducts -Adduct to N terminal valine in Hb -GC/MS-MS analysisDNA adductsGlycidamide adduct with 2-deoxynuleoside of DNA -Slow reaction (40 dayspH=7.0, 37C) -Internal dose biomarker for genotoxicity -detect N7-(2
11、-carbamoyl-2- hydroxyethyl) guanine -LC-MS/MS analysisExcretionFirst order removal from tissue, t1/2=1.4-3hrsParental compound and metabolites in urine -Acrylamide: 2% in rat -N-acetyl-s-(2-carbamoyl-ethyl)cysteine:-Glycidamide: -readily detected in rat -reported in some human studyToxicityNeurotoxi
12、cityReproductive toxicity and genotoxicityCarcinogenicity (JECFA,2005)Toxicity-Neurotoxicityl Neurotoxicity: nervous system: a principal site for AA acute toxic actionslSymptoms (Airborne Exposure):Tingling or numbness in hands or feet Weak legs, loss of toe reflexes and sensations, and numbness pre
13、ceded by skin peelingCerebral dysfunction (Friedman, 2003)Dose-Response:l Acute Dose-response:l100 mg/kg bw: Single oral dose for acute neurotoxic effects l150 mg/kg bw: reported LD50l Chronic Dose-response:l 0.2 mg/kg bw/d, 90 d: NOEL in morphological change in nerveslAccumulative effects: l LOEL:
14、for degenerative changes in nerves - 20 mg/kg bw, 90 days = 2 mg/kg bw, 2 years (JECFA, 2005)Mechanism:Hypothesis: Both effect neurofilaments and cause changes in the expression of neurotransmitter receptorsqAlter the expression of genes governing the synthesis of brain proteinsq Formation between a
15、crylamide and cysteine-rich terminal proteins that mediate fusion of membranes Inhibit the action of brain Glutathione-S-transferase (GST) and reduce the levels of brain glutathione (GSH)(Friedman, 2003)Reproductive toxicity and genotoxicityAt neurotoxic doses, acrylamide acts as reproductive toxica
16、ntSymptoms: - reduce fertility rates, increase resorptions of fetuses, reduce litter size in pregnant females, and formation of abnormal sperm - impact survival and health of offsprings(Friedman, 2003)Mechanism:Related to neurotoxicity: similar dose-response and symptoms in offspringDNA-Bonding: GA
17、100-1000 times more reactive than AA Ames Samonella Assay: only GAshowed mutation Both GA and AA had more A-G transitions and G-C transsitions than control (Friedman, 2003)CarcinogenicityBrain and reproductive system tumorsGenotoxicity: alkylation (adducts) of DNA with AA/GALower glutathione concent
18、ration-weaken detoxification system(JECFA,2005)Critique on Animal studieslPotentially different impact of AA among species From rodents to primates?lPotentially chronic exposure dose to humans Low dose designToxicityFrom acute to chronicGeneral mechanism: Alkylation (adducts) of DNA, functional prot
19、eins and enzymesCarcinogenicity proven in animal models, but extrapolation to human model still being investigatedDietary IntakeThe average intake of the general population is 0.001 mg acrylamide/kg bw/dhigh intake consumers 0.004 mg acrylamide/kg bw/dJECFA2005Acrylamide levels in selected Canadian
20、foodsTable 2. Acrylamide levels in some cereals and potato chip products sampled between 2002 and 2006. ProductOrigin, Type or BrandAcrylamide, ppbJun.02Sept.03Jul.04Jul.05Jul.06Potato chipsKettle chips, regular salted37002640200045502700Pringles, regular150013701040885955Ruffles, regular73031922135
21、3250Spudz, regulara55018802390-PC, Olive oil and rosemary5301150320650211Lays, baked, regular-16401480858397CerealsGM Oatmeal Crisp, Maple nut17011115986135Post - Shreddies, wheat120161149901501Kelloggs Rice Krispies1001622009188HealthCanadaCONCLUSION FROM FAO/WHO EXPERTSNo adverse effects for the a
22、verage intake consumersMay have morphological changes in nerves for high intake consumers.Investigations on reducing acrylamide in food should continue.ChallengesData for Asian and African countries are lackingOverall acrylamide exposure from food cooked at home is unavailable.Difficult to estimate
23、the overall acrlyamide daily intake worldwide.NEUTRALIZATION1.Removal of asparagine by asparaginaseEffective in potato chips and french fries2.Control the food suppliesselection of cultivars, storage temp, blanchingNEUTRALIZATION3. Addition of amino acidLysine4. Lowering the frying temperature200ult
24、otalvolumewithwater400-800 ul per urine sample for AAMA/GAMAWithinternalstandards-4mltotalvolumewithmaterPurified through solid phase extractionMultiple reaction monitoring mode: LC-ES/MS/MS ResultsMeasured: AA,GA, AAMA, and GAMA by LC-EC/MS/MSPercentage of total dose excreted over 24hIntravenous -
25、649%Gavage - 4914%Dietary administration 4029%Formation of GA derived speciesOral route IV route Unchanged AA excretion 5% of total doseResults mouse urineMolar percentages of the total dose administered represented by acrylamide and metabolites in 24h mouse urineSex/routeAAGAAAMAGAMAGA/AAaTotalbM-I
26、V351421285110130.516282F-IV122031131614252.53.26063M-gavage0.60.716185992244.83348F-gavage0.111228576123.85.12348M-diet019492031202122.360101F-diet01321610382.92139aThisratiorepresentsGA+GAMA/AA+AAMA.bThisparameteristhetotalmolarpercentageofthedoseadministeredrepresentedbythesumofAA+AAMA+GA+GAMAexcr
27、etedinurine.Results rat urineSex/routeAAGAAAMAGAMAGA/AAaTotalbM-IV24%34%4249%1017%0.30.45774%F-IV235323511180.40.75457M-gavage2631272911.16466F-gavage1247283020220.815357M-diet0.41.30.84.3494141.11.91028F-dietc0.45.7483.318aThisratiorepresentsGA+GAMA/AA+AAMA.bThisparameteristhetotalmolarpercentageof
28、thedoseadministeredrepresentedbythesumofAA+AAMA+GA+GAMAexcretedinurine.cTheurinevolumecollectedfromoneratwasanomalouslylowsothetotalmetabolitesexcretedwere1%ofthedoseadministeredandthosevalueswerethereforeexcludedfromconsideration.Correlation between metabolitesAA correlated significantly to AAMA GA
29、 correlated significantly to GAMAOpen symbols=glycidamide; filled symbols=acrylamide; diamonds=gavage; squares=IV; triangles=diet; x=controls. Rat plot: AA vs. AAMA, r2=0.78, p0.001; GA vs. GAMA, r2=0.81, p0.001. Mouse plot: AA vs. AAMA, r2=0.86, p0.001; GA vs. GAMA, r2=0.57, p0.001.Doergen et al.,
30、2006 Correlations between urinary metabolites with the respective average serum AUC in ratsOpensymbols=glycidamide;filledsymbols=acrylamide;diamonds=gavage;squares=IV;triangles=diet;x=controls.Topplot:AUCAAvs.AAMA,r2=0.83,p0.001;AUC-GAvs.GAMA,r2=0.32,p0.02.Bottomplot:AUC-AAvs.AA,r2=0.74,p0.001;AUC-G
31、Avs.GA,r2=0.53,p0.001.Correlations between urinary metabolites with the respective average serum AUC in miceOpen symbols=glycidamide; filled symbols=acrylamide; diamonds=gavage; squares=IV; triangles=diet; x=controls. Top plot: AUC-AA vs. AAMA, r2=0.41, p0.01; AUC-GA vs. GAMA, r2=0.56, p0.0001. Bott
32、om plot: AUC-AA vs. AA, r2=0.41, p0.011; AUC-GA vs. GA, r2=0.34, pmouseratshumansDoergeetal.,2007ChallengesVariability in GA/AA ratio biomarkers from both Hb and urine hard to assess exposure to genotoxic GASuggested strategy: for assessing cancer risk from dietary AA to predict target tissue GA-DNA adduct levels in humans and to compare them with those from rodent carcinogenicity bioassays = reduced error from inter-species extrapolationsData from only one biomarker deficientSuggested: integration of all types of data
