32P-Postlabeling for Detection of DNA Adducts

  • R. C. Gupta


It is generally believed that the formation of DNA adducts by covalent interaction of electrophilic species of carcinogens (ultimate carcinogens) with macromolecules, particularly DNA, is an essential first step in the multistage process of carcinogenesis (Miller and Miller, 1981). Until the early 1980s, use of radiolabeled carcinogens was the main method to determine the binding of chemical carcinogens to DNA. Since then several methods have been utilized for nonradio-labeled carcinogens. The assays that are practiced currently are based on specific antibodies, fluorescence properties of adducts, gas chromatography/mass spectrometry, and 32P-postlabeling. These assays require a few micrograms to several hundred micrograms of DNA, with a detection limit of 1 adduct per 106 to 1010 nucleotides, depending on the method (Beach and Gupta, 1992). The 32P-postlabeling assay has emerged as the major tool for measuring DNA adducts because of its ultrasensitivity and applicability to theoretically any DNA-damaging agent, irrespective of its chemical nature, including unknowns. Over 100 individual agents or complex mixtures have been tested in rodents and aquatic systems in vivo and rodent and human cells in vitro (Beach and Gupta, 1992).


Ammonium Hydroxide Ammonium Formate Adduct Level Micrococcal Nuclease Major Spot 
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  1. Beach, A. C., and Gupta, R. C. (1992). 32P-postlabeling assay and human biomonitoring. Carcinogenesis 13:1053–1074.PubMedCrossRefGoogle Scholar
  2. Beach, A. C., and Gupta, R. C. (1994). DNA adducts induced in vivo by the ubiquitous environmental contaminant cyclopenta[cdpyrene. Carcinogenesis 15:1065–1072.PubMedCrossRefGoogle Scholar
  3. Chacko, M., and Gupta, R. C. (1988). Evaluation of DNA damage in the oral mucosa of tobacco users and nonusers. Carcinogenesis 9:2309–2314.PubMedCrossRefGoogle Scholar
  4. Dunn, B. P., and San, R. H. C. (1988). HPLC enrichment of hydrophobic DNA-carcinogen adducts for enhanced sensitivity of 32P-postlabeling analysis. Carcinogenesis 9:1055–1060.PubMedCrossRefGoogle Scholar
  5. Gallagher, J. E., Jackson, M. A., George, M. H., Lewtas, J., and Robertson, I. G. C. (1989). Differences in detection of DNA adducts in the 32P-postlabeling assay after either 1-butanol extraction or nuclease P1 treatment. Cancer Lett. 45:7–12.PubMedCrossRefGoogle Scholar
  6. Gupta, R. C. (1984). Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. USA 81:6943–6947.PubMedCrossRefGoogle Scholar
  7. Gupta, R. C. (1985). Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen-DNA adducts. Cancer Res. 45:5656–5662.PubMedGoogle Scholar
  8. Gupta, R. C. (1993). 32P-postlabeling of bulky aromatic adducts, IARC Publication No. 124, pp. 11-24.Google Scholar
  9. Gupta, R. C., and Earley, K. (1988). 32P-adduct assay: Recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis 9:1687–1693.PubMedCrossRefGoogle Scholar
  10. Gupta, R. C, and Randerath, K. (1988). Analysis of DNA adducts by 32P-labeling and thin-layer chromatography, in:DNA Repair, Volume 3 (E. C. Friedberg and P. C. Hanawalt, eds.), Dekker, New York, pp. 401–420.Google Scholar
  11. Gupta, R. C., Reddy, M. V., and Randerath, K. (1982). 32P-postlabeling analysis of nonradioactive aromatic carcinogen DNA adducts. Carcinogenesis 3:1081–1092.PubMedCrossRefGoogle Scholar
  12. Gupta, R. C, Earley, K., and Sharma, S. (1988). Use of human peripheral blood lymphocytes to measure DNA binding capacity of chemical carcinogens. Proc. Natl. Acad. Sci. USA. 85:3513–3517.PubMedCrossRefGoogle Scholar
  13. Johnson, R. A., and Walseth, T. F. (1979). The enzymatic preparation of [α-32P]ATP, [α-32P]GTP, [32P]cAMP, and [32P]cGMP, and their use in assay of adenylate and guanylate cyclases, and cyclic nucleotide phospho-diesterases. Adv. Cyclic Nucleotide Res. 10:135–167.PubMedGoogle Scholar
  14. Miller, E. C., and Miller, J. A. (1981). Mechanisms of chemical carcinogenesis. Cancer 47:1055–1064.PubMedCrossRefGoogle Scholar
  15. Randerath, K., and Randerath, E. (1967). Thin-layer separation methods for nucleic acid derivatives. Methods Enzymol. 12A:323–347.CrossRefGoogle Scholar
  16. Reddy, M. V., and Randerath, K. (1986). Nuclease P1-mediated enhancement of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7:1543–1551.PubMedCrossRefGoogle Scholar
  17. Reddy, M. V., Irvin, T. R., and Randerath, K. (1985). Formation and persistence of sterigmatocystin-DNA adducts in rat liver determined via 32P-postlabeling analysis. Mutat. Res. 152:85.PubMedCrossRefGoogle Scholar
  18. Spencer, G. G., and Gupta, R. C. (1992). A C18 TLC-mediated enhancemnent of 32P-postlabeling for detecting DNA adducts of varying degrees of lipophilicity. Proc. Am. Assoc. Cancer Res. 33, Abstr. 720.Google Scholar
  19. Spencer, G. G., Beach, A. C., and Gupta, R. C. (1993). Enhanced thin-layer Chromatographie separation of 32P-postlabeled DNA adducts. J. Chromatogr. 612:295–301.PubMedGoogle Scholar
  20. Spencer, G. G., Beach, A. C., and Gupta, R. C. (1996). High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts. J. Chromatogr. in press.Google Scholar
  21. Stansbury, K., Flesher, J. W., and Gupta, R. C. (1994). Mechanism of aralkyl-DNA adduct formation from benzo[a]pyrene in vivo. Chem. Res. Toxicol. 7:254–259.PubMedCrossRefGoogle Scholar
  22. Talaska, G., Al-Juburi, A. Z. S. S., and Kadlubar, F. F. (1991). Smoking-related carcinogen-DNA adducts in biopsy samples of human urinary bladder: Identification of N-(deoxyguanosine-8-yl)-4-aminobiphenyl as a major adduct. Proc. Natl. Acad. Sci. USA 88:5350–5354.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • R. C. Gupta
    • 1
  1. 1.Preventive Medicine and Environmental HealthUniversity of Kentucky Medical CenterLexingtonUSA

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