HumanEPIDataWhere the Question of Health Risk is RaisedResponseLog DosePaustenbach (1995)AnimalDataIssues Related to Uncertainty in Risk Assessment• High to low dose extrapolation• Species to species extrapolation• Mechanism of carcinogenesis• Interindividual differences• Chemicals that are carcinogenic in animals are expected to be carcinogenic in humans• Humans are assumed to be as sensitive as the most sensitive animal• The dose-response is assumed to be linearMajor Default Assumptions in Cancer Risk AssessmentEmerging Issues in Biologically-based Risk Assessment• Incorporation of PBPK models• Use of molecular dosimetry as a surrogate of exposure• Mode of action information– Role of cell proliferation– Mutagenicity• Life stage differences in susceptibilityPotential of Molecular Dosimetry in Risk Assessment• High to low dose extrapolation– Saturation of metabolic activation– Saturation of detoxication– Saturation of DNA repair• Route to route differences• Species to species differencesChemical Exposure (air, water, food, etc.)Internal ExposureMetabolic ActivationMacromolecular Binding DetoxicationDNA RNA Protein Biologically Effective DoseXEfficiency of MispairingXCell ProliferationBiomarkers of ExposureMutation/InitiationProgression CancerBiomarkers of EffectcabIncreasing External ExposureIncreasing Adduct ConcentrationSublinearSupralinearSOURCES OF MUTATIONSENDOGENOUS DNA DAMAGE EXOGENOUS DNA DAMAGEDepurinationDNA REPAIRMUTATIONLifeStylesEnvironmentalAgentsFreeRadicalsPolymeraseErrorsCELL REPLICATIONInitiatingEventCell Proliferation(clonal expansion)ProgressionCell ProliferationCell ProliferationMalignancySecond Mutating EventThird Mutating EventDDSSSSIIIIMμ1μ2β2First EventSecond EventA Moolgavkar Representation of Multistage CarcinogenesisRole of Increased Cell Proliferation in Carcinogenesis• Decreases time available for DNA repair• Converts repairable DNA damage into nonrepairable mutations• Necessary for chromosomal aberrations, insertions, deletions and gene amplification• Clonally expands existing cell populations8-oxo-G, FapyGuaLipid PeroxidationLipid PeroxideMDA, 4-HNEAP SitesDNA Base ModificationDNA Base adductROSSugarDamageDNA Base adductM1G, edG, edAOxidative Stress Induced-DNA DamageGlycosylasehOGG1GlycosylaseMPGBase PropenalNon-smoker Lymphocytes0.001.002.003.004.005.006.007.008.009.00123456789108OHdG/dG (10e-6)Pentachlorophenol Used as a Pesticide and Wood Preservatives Introduction to Humans: Air, Food and Drinking water Mutagen, Rodent CarcinogenOHClClClClClOHClClOHClClOClClOClClOClClOHClClO2O2-H2O2OHInduced Oxidative StressCalf Thymus DNA Exposed to TCHQCalf Thymus DNA Exposed to TCHQ010203040500.1 1 10 100 1000TCHQ (uM)8OHdG/dG (10e-6)Aldehydic DNA lesions (ADL) in HeLa cells exposed to H2O2(0.06-20 mM) for 15 min05101520250 5 10 15 20H2O2 (mM)ADL/1,000,000 ntd010203040500.01 0.1 1 10 100H2O2 (mM)Increased ADLs/H2O2 concentrationEfficiency of Low Doses of H2O2DNA Alkylation7%2%7%14%ENUDEN0.4%0.1%7%70%MNUDMN--0.3%85%MMSO2 Alkyl ThymineO4 Alkyl ThymineO6 AkylGuanineN7AlkylGuanineN-7-MethylguanineO -Methylguanine10000 1000 100 10 1 0.1 0.01 0.001 0.001 0.01 0.1 1 10 10040302010 032106DMN (mg/kg)Alkylations/10 guaninesAlkylations/10 guanines66AB0.00E+002.00E-064.00E-066.00E-068.00E-061.00E-051.20E-050 1020304050607080Duration of DEN exposure, daysMolar ratio in DNAO4-EtdThdO6-EtdGuo020 40 60 80 100020406080100020406080100Dose (ppm DEN) ET (pM)/dT (μM)0 5 10 15 20051015051015O2-ET O4-ETMolecular Dosimetry of DENVinyl Chloride• Vinyl chloride is a known human and animal carcinogen that induces hepatic angiosarcomas• Carcinogenic response is associated with high exposure (>50 ppm)• To date, 197 VC workers have developed hepatic angiosarcomas. All of them started work prior to lowering the occupational exposure 1 ppm• Vinyl chloride is present in many Superfund sites and some public drinking water in ppb amountsVinyl Chloride MetabolismClOClClOP-450Epoxide HydrolaseHOOClOHAlcohol dehydrogenaseGlutathioneDetoxicationDNA AdductsExposure-Response for Vinyl Chloride Metabolism and Carcinogenicity05000100000 1000 2000 3000 4000VC Exposure (ppm)ν (μg / 6 hr)(Gehring et al, 1978)0.00.10.20 2000 4000 6000VC Exposure (ppm)ASL Incidence(Maltoni et al, 1981)Formation of [13C2]-DNA Adducts by Vinyl ChlorideCH2ClOClNNHNNNOOHdRibNNNNdRibNNNNdRibONHNH2NNNHOdRibONHNNNNOdRibCYP450 2E1vinyl chloridechloroethylene oxideDNAHO-ethanodeoxyguanosine1,N6-ethenodeoxyadenosine 3,N4-ethenodeoxycytidine7-(2-oxoethyl)-deoxyguanosineN2,3-ethenodeoxyguanosine**************Immunoaffinity/GC-HRMS Method for N2,3-Ethenoguanine0204060801009.5 9.6 9.7 9.8 9.9 10 10.1 10.2Retention Time (min)Relative Response (%)4.6 fmol N2,3-εGua (m/z=354.0413)204 fmol Internal Standard (m/z=360.0498)Molecular Dosimetry of N2,3-Ethenoguanine in Adult Rats Exposed to Vinyl ChlorideHEP048120 255075100051015200 200 400 600 800 1000VC Concentration (ppm)mol N2,3 G / 107 mol G04812160 200 400 600 800 1000VC Concentration (ppm)mol N2,3- G / 107 mol G024680 25 50 75 100NPCVinyl Chloride Exposure-Response Relationship Between Endogenous and Exogenous N2,3-Ethenoguanine130 ± 5099 ± 2516.2 ± 0.53.5 ± 1.0εG/108G4 wk371671 ± 101100298.238 ± 41004.72.29.9 ± 6.510----4.6 ± 3.50Fold-IncreaseFold-IncreaseεG/108Gppm VC1 wk* Whole-body inhalation (6 hr/d; 5 d/wk)Formation of Endogenous εGua Adducts from Lipid PeroxidationHNNNNOH2NOHOOHHNNNNONOROHHOHOHOOHHNNNNONOHOOHHHNNNNONOHOOH++CH3(CH2)4CH CH CH CHOOOHFormation of [13C2]-EG in Hepatocyte DNA by Vinyl Chloride0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10Retention Time (min)Relative Intensity0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10Retention Time (min)Relative Intensity0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10.0Retention Time (min)Relative IntensityABCm/z=354N2,3-εG(Endogenous)m/z=356[13C2]N2,3-εG(VC-Derived)m/z=360[13C4,15N2]N2,3-εG(Internal Standard)0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10Retention Time (min)Relative Intensity0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10Retention Time (min)Relative Intensity0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10.0Retention Time (min)Relative IntensityABC0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10Retention Time (min)Relative Intensity0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10Retention Time (min)Relative Intensity0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10.0Retention Time (min)Relative Intensity0.00.51.09.4 9.5 9.6 9.7 9.8 9.9 10Retention Time (min)Relative Intensity0.00.51.09.4 9.5
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