Flow cytometric measurement of benzo[a]pyrene-diol-epoxide-DNA adducts in normal human peripheral lymphocytes and cultured human lung cancer cells.
DNA adducts are mainly detected by 32P-postlabeling and enzyme-linked immunosorbent assays. We have established a method for detection of benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts by flow cytometry, and have clarified the effects of the DNA adducts on cell-cycle progression and the relationship between cell-cycle phases and DNA adduct formation, using human peripheral lymphocytes and three human lung cancer cell lines. We measured the BPDE-DNA adduct levels in both lymphocytes and cancer cells by isolating nuclei, using a nuclear isolation buffer containing Triton X-100 and staining with a BPDE-DNA-specific monoclonal antibody and biotin-streptavidin fluorescein conjugates. BPDE did not affect cell-cycle progression in human peripheral lymphocytes. However, in human lung cancer cells exposed to > 1 microg/ml BPDE, accumulation of cells in the S phase was seen. Cells with DNA content greater than G2M (aneuploid cells) or cells with less than G1 DNA content (apoptotic cells) increased gradually with exposure to increasing BPDE concentrations, suggesting that BPDE may affect cell-cycle progression through binding to DNA. Thus, the measurement of DNA adducts by flow cytometry may provide new insights into carcinogenesis.
Shinozaki
R
R
Department of Environmental Medicine and Informatics, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
Inoue
S
S
Choi
K S
KS
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis* / Q000633:toxicity; D000328:Adult; D002273:Carcinogens / Q000633:toxicity; D002453:Cell Cycle / Q000187:drug effects; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D018736:DNA Adducts / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry* / Q000473:pathology; D008214:Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D008297:Male; D014407:Tumor Cells, Cultured
D002273:Carcinogens; D018736:DNA Adducts; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide