ICANCERESEARCH 55. 1417—1422. AprilI, 19951 Advances in Brief Immunohistochemical Quantitation of Polycyclic Aromatic Hydrocarbon-DNA Adducts in Human Lymphocytes' Grazyna Motykiewicz,Ewa Matusecka, Ewa Grzybowska, MieczyslawChorazy, Yu-Jing Zhang, Frederica P. Perera, and Regina M. Santella2 Department of Tumor Biology. In.uitute of Oncology, 44-100 Gliwice, Poland (G. M., E. M., E. G., M. CI, and Cancer Center/Division of Environmental Sciences, Columbia School of Public Health, New York, New York 10032 (1'-). 1, F. P. P., R. M. S.) Refs. 2—5). They include 32P-postlabeling, gas chromatography/mass spectrometry, fluorescence, and immunoassays (ELISA) using spe cific antisera. Most methods are not applicable to small amounts of blood or tissue, a major limitation in biomonitoring studies. Antisera directed against PAH-DNA adducts can also be used for the immunocytochemical localization of adducts in individual cells. This approach has been successfully applied to mouse cells (6) and tissues (7, 8) as well as human WBC treated in vitro with B[a]P (8). Pilot immunofluorescence studies on bronchial cells from a smoker have also been reported (9). Application of immunoperoxidase tech niques have been applied to tissues and oral mucosa cells of smokers and nonsmokers (10, 11). Here we report on an immunofluorescence technique for visualiza tion and quantitation of PAH-DNA adducts in individual human lymphocytes. The formation of BPDE and related PAH-DNA adducts was studied in lymphocytes obtained from coke-factory workers in Silesia, Poland, residents from the vicinity of the cokery, and inhab itants of a rural region in Poland with PAH levels estimated to be 10-fold lower than in Silesia. This study builds upon our earlier results on aromatic adducts and PAH-DNA adducts measured on isolated blood DNA by 32P-postlabeling and ELISA (12). Both methods showed DNA adducts to be significantly elevated in the Silesian groups (cokery workers and subjects living in the surrounding area) compared to rural controls, with only a slight difference between cokery workers and local controls. In addition to DNA adducts, chromosomal aberrations and Sister chromatid exchanges were signif icantly elevated in occupationally and environmentally exposed residents of Silesia compared to controls (13, 14). Materials and Methods Chemicals. BPDE (444 mCi/mmol) was supplied by Chemsyn Science Laboratories (Lenexa, KS). RNase, proteinase K, P1, Histopaque-1077, and 1,4-phenylenediamine were purchased from Sigma Chemical Co. (St. Louis, MO).Biotinylatedgoatanti-rabbitlgGandfluorescein-conjugatedstreptavidin was from Boehringer Mannheim (Indianapolis, IN). FCS was obtained from HyCloneLaboratories, Inc. (Logan,UT),and DMEMmediumwaspurchased from GIBCO-BRL Life Technologies (Gaithersburg, MD). Tissue culture chambers were supplied by NUNC (Naperville, IL). Subjects and Blood Samples. Subjects included 16 healthy males from each exposure group as well as 16 rural controls. Donors were selected on the basis of their occupation; environmental and control groups did not include cokery workers or any other workers connected with the processing of coal. Additional information on cigarette smoking, diet, X-ray exposure, current medication, and family history of cancer was also collected. Samples were taken in September 1992 and 1993, which is considered the summer season. Therefore, the exposure was not influenced by the additional impact of PAH from domestic heating in the winter. Treatment of Cells with BPDE. The immunostaining method was estab lished, and the measurements were calibrated on cultured C3H lOT½cells treated with BPDE. Cells were plated in eight-chambered slides and main tamed in DMEM medium supplemented with 10% FCS. After 24 h, cells were 1417 Abstract The formation of polycydlic aromatic hydrocarbon-DNA adducts was studied in @pheral blood lymphocytes obtained from men with occupa fional and environmental exposure. Subjects Included coke factory workers, residents from the vicinity of the cokery, and niral region inhabitants (16 individuals in each exposure group). Adducts were determined by immuno histochemical analysis using a polyclonal antiserum recognizing benzo (a)pyrene and related polycydicaromatic hydrocarbon dial epoxide-DNA adducts, a biotinylated secondary antiserum, and @ptavidin-conjupted F1TC. Propidium iodide was used to quantitate nuclear DNA. Dual fluores cence intensifies were simultaneously measured with a Zeiss Axiovert micro scope and a Blo-Rad MRC-600 argon laser scanning confocal attachment. Adducts were significantly elevated (P < 0.001) in both occupational and envlromnental 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 Ievel& The percentages ofthese so called â€oehigh er adduct-level cells― were 13.6, 11.5, and 3.7 in cokery workers, environ mentally exposed Individuals, and rural controls, respectively. The immuno histochemical method allows visualization and relative quantitation of polycydlicaromatic hydrocarbon-DNA adducts In Individual lymphocytes. It requires a much smaller amount of blood than the previously used @ @bellngand ELISA methods, which used isolated bulk DNA. It can also be used for adduct quantitadon In biopsy materiaL The results of this pilot study indicate that this technique is a promising addition to biomonitoring sftidIe@ Introduction PAH3 are common environmental pollutants, and their uptake has been causally associated with the induction of cancer. As a result of coal burning and/or processing, PAH are among the most prevalent mutagenic air pollutants in Silesia, Poland (1). The highest levels are found in the occupational environment, i.e., coke-oven plants (0.12—89.58 @ of B[a}P/m3 of air). Chronic exposure to lower levels of PAH (1.0—55.3ng B[a]P/m3) occurs in the general environment of Silesia and mainly originates from coal-based heavy industry, power plants, and individual domestic heating, as well as automobile traffic. Among the known molecular effects of PAH is the ability to bind covalently to DNA, giving rise to PAH-DNA adducts. A number of methods have been developed for quantitation of adducts, resulting from occupational or environmental exposure to PAH (reviewed in Received 1/18/95; accepted 2/20/95. Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by Grants CA21 1 1 1 and ESO5249 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. 3 The abbreviations used are: PAH, polycycic aromatic hydrocarbons; B[aJP, benzo (a)pyrene;BPDE,(±)7,8-dihydroxy-9,lO-epoxy-7,8,9, lO-tetrahydrobenzo(a)pyrene (the anti isomer); P1, propidium iodide. Research. on February 24, 2015. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from