Visualization and Computer-Assisted Quantification of DNA Modifications in Individual Cells

  • L. den Engelse
  • J. S. Ploem
  • C. P. Wild
  • E. Scherer


Modification of cellular DNA has been linked with diverging biological effects, such as cell death, cytostatic effects, mutagenesis and carcinogenesis. Important parameters are the pattern and extent of DNA modification, the proliferative activity of the exposed cell, and its capacity to repair relevant DNA damage. The present paper reviews a specific, sensitive, and quantitative immunocytochemical method for the analysis of DNA damage at the single cell level. A number of existing and potential applications in the field of chemical carcinogenesis, chemical mutagenesis, experimental chemotherapy and molecular epidemiology will be discussed.


Immunocytochemical Analysis Buccal Cell Adduct Level Chemical Carcinogenesis Buccal Epithelial Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Appleton,M.S., Goetchius,M.P., and Campbell,T.C. (1982) Linear dose-response curve for the hepatic macromolecular binding of aflatoxin B, in rats at very low doses. Cancer Res., 42, 3659–3662.PubMedGoogle Scholar
  2. 2.
    Bases,R., Rubinstein,A., Kadish,A., Mendez,F., Wittner,D., Elequin,F. and Liebeskind,D. (1979) Mutagen-induced disturbances in the DNA of human lymphocytes detected by antinucleoside antibodies. Cancer Res., 39, 3524–3530.Google Scholar
  3. 3.
    Boven,E., Van der Vijgh,W.J.F., Nauta,M.M., Schlüper,H.M.M. and Pinedo,H.M. (1985) Comparative activity and distribution studies of five platinum analogues in nude mice bearing human ovarian carcinoma xenografts. Cancer Res., 45, 86–90.Google Scholar
  4. 4.
    Cremer,C., Cremer,T., Hens,L., Baumann,H. Cornelis,J.J. and Nakanishi,K. (1983) UV micro-irradiation of the Chinese hamster cell nucleus and caffeine post-treatment. Immunocytochemical localization of DNA photolesions in cells with partial and generalized chromosome shattering. Mutat. Res., 107, 465–476.Google Scholar
  5. 5.
    Croy,R.G., Essigman,J.M., Reinhold,V.N. and Wogan,G.N. (1978) Identification of the principal aflatoxin B,-DNA adduct formed in vivo in rat liver. Proc. Natl. Acad. Sci., 75, 1745–1749.Google Scholar
  6. 6.
    De Munter,H.K., Den Engelse,L. and Emmelot,P. (1979) Studies on lung tumours. IV. Correlation between [3H]thymidine labelling of lung and liver cells and tumour formation in GRS/A and C3Hf/A male mice following administration of dimethylnitrosamine. Chem.-Biol. Interactions, 24, 299–316.Google Scholar
  7. 7.
    Eggset,G., Volden,G. and Krokan,H. (1983) UV-induced DNA damage and its repair in human skin in vivo studied by sensitive immunohistochemical methods. Carcinogenesis, 4, 745–750.PubMedCrossRefGoogle Scholar
  8. 8.
    Fichtinger-Schepman,A.M.J., Van Oosterom,A.T., Lohman,P.H.M. and Berends,F. (1987) Cis-Diamminedichloroplatinum(II)-induced DNA adducts in peripheral leukocytes from seven cancer patients: quantitative immunochemical detection of the adduct induction and removal after a single dose of cis-diamminedichloroplatinum(II). Cancer Res., 47, 3000–3004.PubMedGoogle Scholar
  9. 9.
    Heyting,C., Van Der Laken,C.J., Van Raamsdonk,W. and Pooi,C.W. (1983) Immunohistochemical detection of 06-ethyldeoxyguanosine in the rat brain after in vivo applications of N-ethyl-N-nitrosourea. Cancer Res., 43, 2935–2941.Google Scholar
  10. 10.
    Hochmann, J. (1983) Histoautoradiographic detection of DNA repair synthesis in mouse liver following dimethylnitrosamine treatment using whole-body application of labeled thymidine. Neoplasma, 30, 13–22.PubMedGoogle Scholar
  11. 11.
    Hodgson,R.M., Wiessler,M. and Kleihues,P. (1980) Preferential methylation of target organ DNA by the oesophageal carcinogen Nnitrosomethylbenzyl-amine. Carcinogenesis, 1, 861–866.PubMedCrossRefGoogle Scholar
  12. 12.
    Huitfeldt,H.S., Spangler,E.F., Hunt,J.M. and Poirier,M.C. (1986) Immunohistochemical localization of DNA adducts in rat liver tissue and phenotypically altered foci during oral administration of 2-acetylaminofluorene. Carcinogenesis, 7, 123–129.PubMedCrossRefGoogle Scholar
  13. 13.
    Johansson,E.B. and Tjglve,H. (1978) The distribution of [“C]dimethylnitrosamine in mice. Autoradiographic studies in mice with inhibited and non-inhibited dimethylnitrosamine metabolism and a comparison with the distribution of [” C]formaldehyde. Toxicol. Appl. Pharmacol., 45, 565–575.PubMedCrossRefGoogle Scholar
  14. 14.
    Kleihues,P., Veit,C., Wiessler,M. and Hodgson,R.M. (1981) DNA methylation by N-nitrosomethylbenzylamine in target and non-target tissues of NMRI mice. Carcinogenesis, 2, 897–899.PubMedCrossRefGoogle Scholar
  15. 15.
    Kouros,M., MSnch,W., Reiffer,F.J. and Dehnen,W. (1983) The influence of various factors on the methylation of DNA by the oesophageal carcinogen N-nitrosomethylbenzylamine. 1. The importance of alcohol. Carcinogenesis, 4, 1081–1084.Google Scholar
  16. 16.
    Kraft,P.L. and Tannenbaum,S.R. (1980) The distribution of N-nitrosomethylbenzylamine evaluated by whole-body radioautography and densitometry. Cancer Res., 40, 1921–1927.PubMedGoogle Scholar
  17. 17.
    Labuc,G.E. and Archer,M.C. (1982) Esophageal and hepatic microsomal metabolism of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in the rat. Cancer Res., 42, 3181–3186.PubMedGoogle Scholar
  18. 18.
    Lewis,J.G. and Swenberg,J.A. (1980) Differential repair of O’-methylguanine in DNA of rat hepatocytes and nonparenchymal cells. Nature, 288, 185–187.PubMedCrossRefGoogle Scholar
  19. 19.
    Lipps,H.J., Nordheim,A., Lafer,E.M., Ammermann,D., Stollar,B.D. and Rich,A. (1983) Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate Stylonychia mytilus. Cell, 32, 435–441.CrossRefGoogle Scholar
  20. 20.
    Litterst,C.L. (1984) Plasma pharmacokinetics, urinary excretion and tissue distribution of platinum following IV administration of cyclobutanedicarboxylatoplatinum-II and cis-platinum to rabbits. In Hacker,M.P., Douple,E.B. and Krakoff,I.H. (eds), Platinum coordination complexes in cancer chemotherapy. Martinus Nijhoff Publishing Boston/The Hague/Dordrecht/Lancaster, pp. 71–81.Google Scholar
  21. 21.
    Magee,P.N. (1976) Some illustrative systems of chemical carcinogenesis: (3) nitrosamines. In Symington,T. and Carter,R.L. (eds), Scientific Foundations of Oncology. William Heinemann Medical Books, Kingswood, England, pp. 292–301.Google Scholar
  22. 22.
    Menkveld,G.J., Van Der Laken,C.J., Hermsen,T., Kriek,E., Scherer,E. and Den Engelse,L. (1985) Immunohistochemical localization of O’-ethyldeoxyguanosine and deoxyguanosin-8-yl-(acetyl)aminofluorene in liver sections of rats treated with diethylnitrosamine, ethylnitrosourea or N-acetylaminofluorene. Carcinogenesis, 6, 263–270.Google Scholar
  23. 23.
    Muysken-Schoen,M.A., Baan,R.A. and Lohman,P.H.M. (1985) Detection of DNA adducts in N-acetoxy-2-acetylaminofluorene-treated human fibroblasts by means of immunofluorescence microscopy and quantitative immunoautoradiography. Carcinogenesis, 6, 999–1004.PubMedCrossRefGoogle Scholar
  24. 24.
    Poirier,M.C., Stanley,J.R., Beckwith,J.B., Weinstein,I.B. and Yuspa,S.H. (1982) Indirect immunofluorescent localization of benzo(a)pyrene adducted to nucleic acids in cultured mouse keratinocyte nuclei. Carcinogenesis, 3, 345–348.Google Scholar
  25. 25.
    Reed,E., Ozols,R.F., Tarone,R., Yuspa,S.H. and Poirier,M.C. (1987) Platinum-DNA adducts in leukocyte DNA correlate with disease response in ovarian cancer patients receiving platinum-based chemotherapy. Proc. Natl. Acad. Sci., 84, 5024–5028.Google Scholar
  26. 26.
    Safirstein,R., Winston,J., Moel,D., Dikman,S. and Guttenplan,J. (1987) Cisplatin nephrotoxicity: insights into mechanism. Int. J. Androl., 10, 325–346.Google Scholar
  27. 27.
    Sage,E., Gabelman,N., Mendez,F. and Bases,R. (1981)Immunocytological detection of AAF-DNA adducts in HeLa cell nuclei. Cancer Lett., 14, 193–204.Google Scholar
  28. 28.
    Scherer,E., Jenner,A.A.J. and Den Engelse,L. (1987) Immunocytochemical studies on the formation and repair of 06-alkylguanine in rat tissues. In Bartsch,H., O’Neill,I., and Schulte-Hermann,R. (eds), The Relevance of N-Nitroso Compounds to Human Cancer Exposures and Mechanisms (IARC Scientific Publications No. 84). International Agency for Research on Cancer, Lyon, pp. 55–58.Google Scholar
  29. 29.
    Scherer,E., Van Benthem,J., Terheggen,P.M.A.B., Vermeulen,E., Winterwerp,H.H.K. and Den Engelse,L. (1988) Immunocytochemical analysis of DNA adducts at the single cell level: a new tool for experimental carcinogenesis, chemotherapy and molecular epidemiology. Proc: Detection methods for DNA-damaging agents in man, Helsinki, Sept. 1987, in press.Google Scholar
  30. 30.
    Sizaret,P., Malaveille,C., Montesano,R. and Frayssinet,C. (1982) Detection of aflatoxins and related metabolites by radioimmunoassay. J. Natl. Cancer Inst., 69, 1375–1381.Google Scholar
  31. 31.
    Sternson,L.A., Repta,A.J., Shih,H., Himmelstein,K.J. and Patton,T.F. (1984) Disposition of cisplatin vs total platinum in animals and man. In Hacker, M.P., Douple,E.B. and Krakoff,I.H. (eds), Platinum coordination complexes in cancer chemotherapy. Martinus Nijhoff Publishing Boston/The Hague/Dordrecht/Lancaster, pp. 126–137.Google Scholar
  32. 32.
    Stinson,S.F., Squire,R.A. and Sporn,M.B. (1978) Pathology of oesophageal neoplasms and associated proliferative lesions induced in rats by N-methyl-N-benzylnitrosamine. J. Natl. Cancer Inst., 61, 1471–1475.Google Scholar
  33. 33.
    Strickland,P.T. (1983) Detection of thymine dimers in DNA with monoclonal antibodies. In Milman,H.A. and Sell,S. (eds), Application of Biological Markers to Carcinogen Testing Plenum Publishing Co., New York and London, pp. 337–348.Google Scholar
  34. 34.
    Swenberg,J.A., Rickert,D.E., Baranyi,B.L. and Goodman,J.I. (1983) Cell specificity in DNA binding and repair of chemical carcinogens. Environ. Health Perspect., 49, 155–163.Google Scholar
  35. 35.
    Terheggen,P., Begg,A.C., Floot,B.G.J., Emondt,J. and Den Engelse,L. (1988b) Visualization of cisplatin-DNA and carboplatin-DNA adducts in tissues and cultured cells. In Proc. Fifth International Symposium on Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy. Padua, June 29-July 2, 1987, in press.Google Scholar
  36. 36.
    Terheggen,P.M.A.B., Floot,B.G.J., Scherer,E., Begg,A.C., FichtingerSchepman,A.M.J. and Den Engelse,L. (1988a) Immunocytochemical detection of interaction products of cis-diamminodichloroplatinum(II) and cisdiammine(1,1-cyclobutanedicarboxylato)platinum(II) with DNA in rodent tissue sections. Cancer Research, in press.Google Scholar
  37. 37.
    Van Benthem,J., Wild,C.P., Vermeulen,E., Winterwerp,H.H.K., Den Engelse,L. and Scherer,E. (1988) Immunocytochemical localization of DNA adducts in rat tissues following treatment with N-nitrosomethylbenzylamine (NMBzA). In Proc. Detection methods for DNA-damaging agents in man, Helsinki, Sept. 1987, in press.Google Scholar
  38. 38.
    Vanderlaan,M., Watkins, B.E., Hwang,M., Knize,M.G. and Felton,J.S. (1988) Monoclonal antibodies for the immunoassay of mutagenic compounds produced by cooking beef. Carcinogenesis, 9, 153–160.Google Scholar
  39. 39.
    Van Der Ploeg,M., Van Den Broek,K., Smulders,A.W.M., Vossepoel,A.M. and Van Duin,P. (1977) HIDACSYS: Computer programs for interactive scanning cytophotometry. Histochemistry, 54, 273–288.Google Scholar
  40. 40.
    Wild,C.P., Chapot,B., Scherer,E., Den Engelse,L. and Montesano,R. (1988) The application of antibody methodologies to the detection of aflatoxin in human body fluids. In Proc. Detection methods for DNA-damaging agents in man, Helsinki. Sept. 1987, in press.Google Scholar

Copyright information

© Springer Science+Business Media New York 1989

Authors and Affiliations

  • L. den Engelse
    • 1
  • J. S. Ploem
    • 2
  • C. P. Wild
    • 1
  • E. Scherer
    • 1
  1. 1.Division of Chemical CarcinogenesisThe Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis)AmsterdamThe Netherlands
  2. 2.Department of Histochemistry and CytochemistryState University of LeidenLeidenThe Netherlands

Personalised recommendations