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1、Acrylamide,David Kitts,Food Science,Food Nutrition and Health,UBC.,Overview,IntroductionRisk of Exposure and Formation in FoodKineticsMetabolites and BiomarkersToxicologyDietary IntakeNeutralizationStudy Urinary MetabolitesObjective CorrelationsMethods,ResultsCritique,What is Acrylamide?,Industrial
2、UsesFlocculant for wastewater treatmentCosmetics,Paper&TextilesSoil conditionerSDS-Page gel,Industrially produced vinyl compound,commonly polymerized,1994 deemed a“probable carcinogen”International Agency for Cancer Research viewed acrylamide only as a synthetic chemicalExposure thought to be airbor
3、ne and limited to the workplace,Evidence of Risk,1997 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 assessment
4、,Mechanism of Formation in Food,Multi-step pathwayPyrolysis 180 C,Factors affecting formation,Aspargine+Reducing sugar contentCooking methodDeep fryingPan fryingMicrowavingCooking temperatureFormation increases with temperature,Effect of Temperature,(Tareke 2002),Levels in Food,(JECFA 2005),Toxicoki
5、netics of acrylamide(AA),Absorption,Accumulation,Excretion,Metabolism,Absorption,Routes of exposure:Oral ingestion,inhalationPost administration:GI tract blood stream-Small vinyl monomer,readily pass through mucosal membranes-Biological barriers:ineffective,Absorption,Metabolism,Phase I reactionOxid
6、ization of acrylamide Catalized by cytochrome P450 2E1Yields glycidamide&further glyceramide,Metabolism,Phase II reactionConjugation of acrylamide and glycidamide-conjugated with glutathione-GST(glutathione-S-transferase)Yield AAMA(acrylamide mercapturic acid conjugates)and GAMA(glycidamide mercaptu
7、ric acid conjugates),Metabolism,Metabolites of acrylamideGlycidamide(GA):Evidenced by produced N-acetyl-s-(2-carbamoyl ethyl)cysteineSub-metabolites derived from glycidamid:-N-acetyl-s-(2-hydroxy-2-carbamoylethyl)cysteine-N-acetyl-s-(1-carbamoyl-2-hydroxyethyl)cysteine,Metabolism,Pathways,Parent com
8、pound,Metabolites,Accumulation,Generic model:Bloodbody compartmentssites of action,Accumulation,Widely distributed in tissues after absorptionShort half-life t1/2=1.4-3hrsMajor depot=RBCSmall amount detected in tissues after weeksTwo types of adducts contribute to accumulationHemoglobin adductsDNA a
9、dducts,Hemoglobin adducts,Acrylamide with Cysteine&valine residues-Internal dose biomarker for neurotoxicity-Use 14C-acrylamideDetection of cysteine adduct-Acid hydrolysis of protein isolate cysteine adducts-GC/MS analysisDetection of valine adducts-Adduct to N terminal valine in Hb-GC/MS-MS analysi
10、s,DNA adducts,Glycidamide adduct with 2-deoxynuleoside of DNA-Slow reaction(40 dayspH=7.0,37C)-Internal dose biomarker for genotoxicity-detect N7-(2-carbamoyl-2-hydroxyethyl)guanine-LC-MS/MS analysis,Excretion,First order removal from tissue,t1/2=1.4-3hrsParental compound and metabolites in urine-Ac
11、rylamide:2%in rat-N-acetyl-s-(2-carbamoyl-ethyl)cysteine:-Glycidamide:-readily detected in rat-reported in some human study,Toxicity,NeurotoxicityReproductive toxicity and genotoxicityCarcinogenicity(JECFA,2005),Toxicity-Neurotoxicity,Neurotoxicity:nervous system:a principal site for AA acute toxic
12、actionsSymptoms(Airborne Exposure):Tingling or numbness in hands or feet Weak legs,loss of toe reflexes and sensations,and numbness preceded by skin peelingCerebral dysfunction(Friedman,2003),Dose-Response:,Acute Dose-response:100 mg/kg bw:Single oral dose for acute neurotoxic effects 150 mg/kg bw:r
13、eported LD50 Chronic Dose-response:0.2 mg/kg bw/d,90 d:NOEL in morphological change in nervesAccumulative effects:LOEL: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 neurot
14、ransmitter receptors Alter the expression of genes governing the synthesis of brain proteins Formation between acrylamide 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)(Fri
15、edman,2003),Reproductive toxicity and genotoxicity,At neurotoxic doses,acrylamide acts as reproductive toxicantSymptoms:-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,200
16、3),Mechanism:,Related to neurotoxicity:similar dose-response and symptoms in offspringDNA-Bonding:GA 100-1000 times more reactive than AA Ames Samonella Assay:only GA showed mutation Both GA and AA had more A-G transitions and G-C transsitions than control(Friedman,2003),Carcinogenicity,Brain and re
17、productive system tumorsGenotoxicity:alkylation(adducts)of DNA with AA/GALower glutathione concentration-weaken detoxification system(JECFA,2005),Critique on Animal studies,Potentially different impact of AA among species From rodents to primates?Potentially chronic exposure dose to humans Low dose
18、design,Toxicity,From acute to chronicGeneral mechanism:Alkylation(adducts)of DNA,functional proteins and enzymesCarcinogenicity proven in animal models,but extrapolation to human model still being investigated,Dietary Intake,The average intake of the general population is 0.001 mg acrylamide/kg bw/d
19、high intake consumers 0.004 mg acrylamide/kg bw/d,JECFA 2005,Acrylamide levels in selected Canadian foodsTable 2.Acrylamide levels in some cereals and potato chip products sampled between 2002 and 2006.,Health Canada,CONCLUSION FROM FAO/WHO EXPERTS,No adverse effects for the average intake consumers
20、May have morphological changes in nerves for high intake consumers.Investigations on reducing acrylamide in food should continue.,Challenges,Data for Asian and African countries are lackingOverall acrylamide exposure from food cooked at home is unavailable.Difficult to estimate the overall acrlyamid
21、e daily intake worldwide.,NEUTRALIZATION,Removal of asparagine by asparaginaseEffective in potato chips and french fries2.Control the food suppliesselection of cultivars,storage temp,blanching,NEUTRALIZATION,3.Addition of amino acidLysine4.Lowering the frying temperature175 Celsius degree5.Oil type
22、and additivesCorn oil better than olive oil,rosemary,Bacalski et al 2003,Cons and pros,Klaveren J.v.2007 and Summa et al.2006,Methods on the industrial scale not available in the short term.,Challenges,Published December 2006 in Toxicology LettersPurposeAre there alternate methods to estimate acryla
23、mide exposure?Specifically:metabolites in urine?,What is excreted in urine?,Unchanged AA-short half-life 1.3-3hGlycidamide(GA)half-life 2.3-3.9hMercapturic conjugates longer half-livesAAMA 18hGAMA 25h,Correlations,Significant goal is to establish correlations,Serum(blood)levelsOr adduct levels,Previ
24、ous Study,Impact,Could predict adduct levels via urine analysisCould predict internal exposure to Acrylamide Glycidamide,Normalizing dietary administration dose,Actual AA doses delivered through diet Rats:0.06-0.16mg/kg bw Mice:0.150.65mg/kg bwNormalizing concentration of urinary metabolites followi
25、ng dietary administration of AA to 0.1mg/kg if the measured dose administered in feed to an individual rodent was 0.15mg/kg,each urinary metabolite concentration was divided by 1.5.,Analysis of AA/GA and AAMA/GAMA in urine,High throughput LC-ES/MS/MS methodsValiated with blank rat/mouse urinePrecisi
26、on 2-24%;accuracy 110-112%;limit of detection 0.01 uM for AA and 0.1 um for GA10 ul per urine sample for AA/GA with internal standards-200 ul total volume with water400-800 ul per urine sample for AAMA/GAMAWith internal standards-4ml total volume with materPurified through solid phase extractionMult
27、iple reaction monitoring mode:LC-ES/MS/MS,Results,Measured:AA,GA,AAMA,and GAMA by LC-EC/MS/MSPercentage of total dose excreted over 24hIntravenous-649%Gavage-4914%Dietary administration 4029%Formation of GA derived speciesOral route IV route Unchanged AA excretion 5%of total dose,Results mouse urine
28、,Molar percentages of the total dose administered represented by acrylamide and metabolites in 24h mouse urine,aThis ratio represents GA+GAMA/AA+AAMA.bThis parameter is the total molar percentage of the dose administered represented by the sum of AA+AAMA+GA+GAMA excreted in urine.,Results rat urine,
29、aThis ratio represents GA+GAMA/AA+AAMA.bThis parameter is the total molar percentage of the dose administered represented by the sum of AA+AAMA+GA+GAMA excreted in urine.cThe urine volume collected from one rat was anomalously low so the total metabolites excreted were 1%of the dose administered and
30、 those values were therefore excluded from consideration.,Correlation between metabolites,AA correlated significantly to AAMA GA correlated significantly to GAMA,Open symbols=glycidamide;filled symbols=acrylamide;diamonds=gavage;squares=IV;triangles=diet;x=controls.Rat plot:AA vs.AAMA,r2=0.78,p0.001
31、;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.,2006,Correlations between urinary metabolites with the respective average serum AUC in rats,Open symbols=glycidamide;filled symbols=acrylamide;diamonds=gavage;squares=IV;triangles=diet;x=controls
32、.Top plot:AUCAA vs.AAMA,r2=0.83,p0.001;AUC-GA vs.GAMA,r2=0.32,p0.02.Bottom plot:AUC-AA vs.AA,r2=0.74,p0.001;AUC-GA vs.GA,r2=0.53,p0.001.,Correlations between urinary metabolites with the respective average serum AUC in mice,Open symbols=glycidamide;filled symbols=acrylamide;diamonds=gavage;squares=I
33、V;triangles=diet;x=controls.Top plot:AUC-AA vs.AAMA,r2=0.41,p0.01;AUC-GA vs.GAMA,r2=0.56,p0.0001.Bottom plot:AUC-AA vs.AA,r2=0.41,p0.011;AUC-GA vs.GA,r2=0.34,p0.022.,Correlation between urinary metabolites and other biomarkers,Hb-adductsInconsistent data for mice and rats DNA-adductsRats&mice-signif
34、icant linear correlation AA with GA-DNA adduct levels(p=0.005)AAMA with GA-DNA adduct levels(p=0.004)Rats-significant linear correlation GA with average DNA adducts(p=0.001)GAMA with average DNA adducts(p=0.02)Mice-only for GAMA(p=0.03),not for GA(p=0.2),Challenges,Extrapolating results to humans di
35、fferent metabolism(ratio of AA to GA)Dose dependency of urinary metabolites over 24h,Formation of GA=mouse rats humansDoerge et al.,2007,Challenges,Variability 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,
