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Immunohistochemical Quantitation of Polycyclic Aromatic Hydrocarbon-DNA Adducts in Human Lymphocytes¹

Department of Tumor Biology, Institute of Oncology, 44-100 Gliwice, Poland [G. M., E. M., E. G., M. C.], and Cancer Center/Division of Environmental Sciences, Columbia School of Public Health, New York, New York 10032 [Y-J. Z., F. P. P., R. M. S.]

The formation of polycyclic aromatic hydrocarbon-DNA adducts was studied in peripheral blood lymphocytes obtained from men with occupational and environmental exposure. Subjects included coke factory workers, residents from the vicinity of the cokery, and rural region inhabitants (16 individuals in each exposure group). Adducts were determined by immunohistochemical analysis using a polyclonal antiserum recognizing benzo-()pyrene and related polycyclic aromatic hydrocarbon diol epoxide-DNA adducts, a biotinylated secondary antiserum, and streptavidin-conjugated FITC. Propidium iodide was used to quantitate nuclear DNA. Dual fluorescence intensities were simultaneously measured with a Zeiss Axiovert microscope and a Bio-Rad MRC-600 argon laser scanning confocal attachment. Adducts were significantly elevated (P < 0.001) in both occupational and environmental groups, as compared to the rural control group by Mann-Whitney U test. The distribution of the data indicated the existence of cells with relatively higher adduct levels. The percentages of these so called "higher adduct-level cells" were 13.6, 11.5, and 3.7 in cokery workers, environmentally exposed individuals, and rural controls, respectively. The immunohistochemical method allows visualization and relative quantitation of polycyclic aromatic hydrocarbon-DNA adducts in individual lymphocytes. It requires a much smaller amount of blood than the previously used ³²P-postlabeling and ELISA methods, which used isolated bulk DNA. It can also be used for adduct quantitation in biopsy material. The results of this pilot study indicate that this technique is a promising addition to biomonitoring studies.

¹ This work was supported by Grants CA21111 and ES05249 from the NIH, Grant 6 P20706405p01 from the Committee for Scientific Research (Komitet Badan Navkowych) to M. C., and a fellowship to G. M. from the National Cancer Institute Short-Term Scientist Exchange Program.

² To whom requests for reprints should be addressed, at Columbia University, 701 West 168th St., New York, NY 10032.

Received 1/18/95. Accepted 2/20/95.

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