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32P-Postlabeling Analysis of DNA Adducts

  • David H. PhillipsEmail author
  • Volker M. Arlt
Protocol
  • 172 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2102)

Abstract

32P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA, and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of 32P-orthophosphate from [γ-32P]ATP; chromatographic separation of labeled adducts, and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 109–1010 normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.

Key words

DNA adducts 32P-postlabeling T4 polynucleotide kinase Nuclease P1 Complex mixtures DNA repair Oxidative damage to DNA Carcinogens Mutagens Genotoxicity Environmental carcinogens Chemotherapeutic drugs HPLC DNA digestions 

References

  1. 1.
    Phillips DH (2002) The formation of DNA adducts. In: Allison MR (ed) The Cancer Handbook. Macmillan, London, pp 293–306Google Scholar
  2. 2.
    Strickland PT, Routledge MN, Dipple A (1993) Methodologies for measuring carcinogen adducts in humans. Cancer Epidemiol Biomarkers Prev 2:607–619PubMedGoogle Scholar
  3. 3.
    Poirier MC, Santella RM, Weston A (2000) Carcinogen macromolecular adducts and their measurement. Carcinogenesis 21:353–359CrossRefGoogle Scholar
  4. 4.
    Phillips, D.H. (2013) On the origins and development of the (32)P-postlabelling assay for carcinogen-DNA adducts. Cancer Lett, 334, 5–9CrossRefGoogle Scholar
  5. 5.
    Phillips DH, Arlt VM (2007) The 32P-postlabeling assay for DNA adducts. Nat Protoc 2:2772–2781CrossRefGoogle Scholar
  6. 6.
    Randerath K, Reddy MV, Gupta RC (1981) 32P-Labeling test for DNA damage. Proc Natl Acad Sci U S A 78:6126–6129CrossRefGoogle Scholar
  7. 7.
    Gupta RC, Reddy MV, Randerath K (1982) 32P-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis 3:1081–1092CrossRefGoogle Scholar
  8. 8.
    Reddy MV, Randerath K (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7:1543–1551CrossRefGoogle Scholar
  9. 9.
    Gupta RC (1985) Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts. Cancer Res 45:5656–5662PubMedGoogle Scholar
  10. 10.
    Randerath K, Randerath E, Danna TF, van Golen KL, Putman KL (1989) A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5′-monophosphates. Carcinogenesis 10:1231–1239CrossRefGoogle Scholar
  11. 11.
    Pfau W, Lecoq S, Hughes NC, Seidel A, Platt KL, Grover PL, Phillips DH (1993) Separation of 32P-labelled 3′,5′-bisphosphate adducts by HPLC. In: Phillips DH, Castegnaro M, Bartsch H (eds) Postlabelling Methods for Detection of DNA Adducts. IARC, Lyon, pp 233–242Google Scholar
  12. 12.
    Phillips DH, Hewer A, Horton MN, Cole KJ, Carmichael PL, Davis W, Osborne MR (1999) N-Demethylation accompanies α-hydroxylation in the metabolic activation of tamoxifen in rat liver cells. Carcinogenesis 20:2003–2009CrossRefGoogle Scholar
  13. 13.
    Nagy E, Cornelius MG, Moller L (2009) Accelerated (32)P-HPLC for bulky DNA adducts. Mutagenesis 24:183–189CrossRefGoogle Scholar
  14. 14.
    Jones GD, Dickinson L, Lunec J, Routledge MN (1999) SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during 32P-labeling. Carcinogenesis 20:503–507CrossRefGoogle Scholar
  15. 15.
    Beach AC, Gupta RC (1992) Human biomonitoring and the 32P-postlabelling assay. Carcinogenesis 13:1053–1074CrossRefGoogle Scholar
  16. 16.
    Phillips DH (1997) Detection of DNA modifications by the 32P-postlabelling assay. Mutat Res 378:1–12CrossRefGoogle Scholar
  17. 17.
    Phillips DH, Farmer PB, Beland FA, Nath RG, Poirier MC, Reddy MV, Turteltaub KW (2000) Methods of DNA adduct determination and their application to testing compounds for genotoxicity. Environ Mol Mutagen 35:222–233CrossRefGoogle Scholar
  18. 18.
    Phillips DH, Castegnaro M (1993) Results of an interlaboratory trial of 32P-postlabelling. In: Phillips DH, Castegnaro M, Bartsch H (eds) Postlabelling Methods for Detection of DNA Damage. IARC, Lyon, pp 35–49Google Scholar
  19. 19.
    Phillips DH, Castegnaro M (1999) Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols. Mutagenesis 14:301–315CrossRefGoogle Scholar
  20. 20.
    Beland FA, Doerge DR, Churchwell MI, Poirier MC, Schoket B, Marques MM (1999) Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard. Chem Res Toxicol 12:68–77CrossRefGoogle Scholar
  21. 21.
    Osborne MR, Phillips DH (2000) Preparation of a methylated DNA standard, and its stability on storage. Chem Res Toxicol 13:257–261CrossRefGoogle Scholar
  22. 22.
    Mourato LLG, Beland FA, Marques MM (1999) 32P-Postlabeling of N-(deoxyguanosin-8-yl)arylamine adducts: a comparative study of labeling efficiencies. Chem Res Toxicol 12:661–669CrossRefGoogle Scholar
  23. 23.
    Munnia A, Saletta F, Allione A, Piro S, Confortini M, Matullo G, Peluso M (2007) 32P-Post-labelling method improvements for aromatic compound-related molecular epidemiology studies. Mutagenesis 22:381–385CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Analytical, Environmental & Forensic Sciences, MRC-PHE Centre for Environmental & HealthKing’s College LondonLondonUK

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