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Blood-Based Detection of Carcinogenic Environmental Exposures Stephen G. Grant

Center for Environmental Oncology University of Pittsburgh Cancer Institute. Blood-Based Detection of Carcinogenic Environmental Exposures Stephen G. Grant. Tobacco comprises: 1. Cigarette constituents Organic matter Nicotinic alkyloids Additives 2. Pyrolysis products Carbon dioxide

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Blood-Based Detection of Carcinogenic Environmental Exposures Stephen G. Grant

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  1. Center for Environmental Oncology University of Pittsburgh Cancer Institute Blood-Based Detection of Carcinogenic Environmental Exposures Stephen G. Grant

  2. Tobacco comprises: • 1. Cigarette constituents • Organic matter • Nicotinic alkyloids • Additives • 2. Pyrolysis products • Carbon dioxide • Carbon monoxide • Tar Filter traps some particulates Smoke production by pyrolysis (1600 - 1800°F) • To lungs, where • absorption occurs • Absorption factors: • Inhalation amount • Inhalation depth • Inhalation duration • pH of smoke • Absorption characteristics • of individual constituents Air dilution and cooling via porous paper Production and fate of cigarette smoke constitutents from Henningfield, 1984

  3. Molecular epidemiological paradigm - development of intermediate biomarkers of the disease process Markers of Markers of Clinical Internal Biologically General Specific DoseEffectiveBiological Biological Disease Dose Effect Effect Exposure Effect Susceptibility from Grant (2001) J. Environ. Pathol. Toxicol. Oncol. 20: 237-253.

  4. Biologically Effective Dose DNA adductsProtein adductsAlbuminHemoglobin

  5. General Biological Effect MutationsGPAHPRTCytogenetic abnormalitiesdicentrics (transient)translocations (persistent)Micronuclei(Sister Chromatid Exchange)

  6. Mutation frequency critically affects cancer: Incidence Age of Onset Mutation frequency affected by: Genotoxic exposure Genetic predisposition

  7. Advantages of the Human in VivoHPRT SomaticMutation Assay inT-lymphocytes • Well established assay, with extensive normals database • Applicable to all individuals • Variant phenotype can be confirmed at the DNA level - allows for the generation of “molecular spectra” • Issue of variant clonality (stem cell derivation) can be addressed by rearrangement at TCR • Established cell culture and animal methods Adapted from Grant and Jensen (1993) in Immunobiology of Transfusion Medicine, Marcel Dekker, pp. 299-323.

  8. Disadvantages of the Human in VivoHPRT Somatic Mutation Assay inT-lymphocytes • ~20 ml of fresh blood required • Expensive and labor intensive • Evidence for in vivo selection against variants • X-linked locus insensitive to chromosomal mechanisms • Vagaries of T-cell biology Adapted from Grant and Jensen (1993) in Immunobiology of Transfusion Medicine, Marcel Dekker, pp. 299-323.

  9. Biomarkers of PAH Exposure in Foundry Workers Perera et al. (1993) Carcinogenesis 14: 969-973

  10. Toxic Chemicals in Tobacco Smoke

  11. Effects of maternal smoking on fetal mutagenesis at the HPRT locus 2 HPRT mutation frequencies (X 10-6) 1 0 unexposed passively exposed active smokers quitters from Grant (2005) BMC Pediatrics 5: 20

  12. Effects of maternal smoking on fetal mutagenesis at the HPRT locus from Grant (2005) BMC Pediatrics 5: 20

  13. Mutational Spectra • based on concept that every mutagen will produce a distinctive pattern of mutations • usually considered to consist of locations and types of point mutations • large databases compiled on mutational spectra in the HPRT reporter gene, the ras oncogene and the p53 tumor suppressor gene • may have more power if used to delineate entirely different types of mutations, for example, point mutations vs. chromosomal rearrangements

  14. VDJ Recombination • directed DNA splicing used to generate immunologic diversity in B- and T-lymphocytes • most genetic rearrangements occurring in leukemia and lymphoma caused by “illegitimate” VDJ recombination • the HPRT reporter gene contains several cryptic recombination sites that result in a distinctive deletion of exons 2 and 3

  15. Cryptic VDJ Recombination Signals in the Human HPRT Gene from Finette (2006) DNA Repair 5: 1049-1064

  16. 6 5 4 Mothers Non-smokers, no passive exposure 3 Non-smokers, with passive exposure 2 Active smokers 1 0 Types of HPRT mutations observed in cord bloods HPRT mutation frequencies (X 10-7) small mutations deletions, rearrangements VDJ recombinant deletions from Grant (2005) BMC Pediatrics 5: 20

  17. Age Distribution of HPRT Mutation Frequencies from Evdokimova, Babra and Grant (2004) Environmental and Molecular Mutagenesis 44: 198

  18. Incidence of childhood lymphocytic leukemia and VDJ Recombinase-associated mutations at the HPRT locus from Finette (2006) DNA Repair 5: 1049-1064.

  19. Conclusions • active maternal smoking results in a significant increase in the somatic mutational burden in the fetus • chronic maternal secondary exposure to environmental tobacco smoke results in the same increase in mutational burden and the same spectrum of mutations • quitting smoking during pregnancy has no protective effect on the fetus if there is continued exposure to ETS

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