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research-article |
Indirect immunofluorescent localization of benzo[a]pyrene adducted to nucleic acids in cultured mouse keratinocyte nuclei
1In Vitro Pathogenesis Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH Bethesda, MD 20205
2Department of Dermatology, Uniformed Services University of the Health Sciences 4301 Jones Bridge Rd., Bethesda, MD 20814
3Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, NIH Bethesda, MD 20205
4Columbia University College of Physicians and Surgeons, Health Science Center 701-W. 168th St., New York, NY 10032, USA
The localization of benzo[a]pyrene-deoxyguanosine adducts was studied by indirect immunofluorescence in cultured BALB/c epidermal cells exposed to (±)7
,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (the antiisomer) utilizing an antiserum specific for the major benzo[a]pyrene-deoxyguanosine adduct in DNA. This antiserum does not cross-react with benzo[a]pyrene or DNA alone. When cultured keratinocytes were incubated with the carcinogen for 1 h, the immunofluorescence was localized in the nucleus as intense spots on a background of diffuse fluorescence. Fluorescence was absent from cells not exposed to carcinogen and from carcinogen-exposed cells incubated with normal rabbit serum in place of the antiserum. Fluorescence was abolished when the specific antiserum was absorbed with the immunogen DNA prior to incubation with cells, and substantially diminished when exposed cells were preincubated with deoxyribonuclease before the application of the specific antiserum. Incubation of exposed cells with ribonuclease prior to incubation with the specific antiserum removed the bright fluorescent spots and resulted in fluorescent nuclei containing dark spots in similar frequency. Dose-response studies in which benzo[a]pyrene-deoxyguanosine adducts were quantified by enzyme-linked immunosorbent assay and compared with intensity of immunofluorescence demonstrated that decreasing doses of the carcinogen resulted in fewer numbers of adducts as well as proportionally less fluorescence. When cells were exposed to non-toxic doses of the activated carcinogen for 1 h, nuclear fluorescence was detectable in immediately-fixed cells but faded to non-detectable levels when cells were washed and cultured for an additional 24–48 h before fixation.