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Benzo(a)pyrene:DNA Adduct Formation in Normal Human Mammary Epithelial Cell Cultures and the Human Mammary Carcinoma T47D Cell Line¹

Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907 [D. P-S., W. M. B.], and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 [A. N., B. A. M., J. K. S.]

The benzo(a)pyrene (BaP):DNA adducts formed in normal human mammary epithelial cell cultures and the human mammary carcinoma T47D cell line were analyzed by chromatography and acid hydrolysis of the BaP:deoxyribonucleoside adducts to BaP:purine adducts and BaP:tetraols. Human mammary epithelial cell cultures and human mammary carcinoma T47D cells were exposed to [³H]BaP for 24 h, and the levels of binding were 81 and 182 pmol BaP/mg DNA in normal and T47D cultures, respectively. Analysis of BaP:deoxyribonucleoside adducts resolved by immobilized boronate chromatography and reversephase high-performance liquid chromatography demonstrated the presence of three BaP:deoxyribonucleoside adducts in both cells: M2, MS1, and MS2 in a ratio of 1.6:1:14. Two adducts (MS1 and MS2) bound to the immobilized boronate column indicating the presence of cis-vicinal hydroxyl groups, a configuration which would result from reaction of 7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (anti-BaPDE) with DNA. MS2 was identified as (+)-anti-BaPDE:deoxyguanosine (dGuo) for it cochromatographed with a [¹⁴C]-(+)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS2 cochromatographed with [¹⁴C]-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (±)-anti-BaPDE:tetraols. MS1 was identified as (-)-anti-BaPDE:dGuo for MS1 eluted in the same relative position as a (-)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS1 cochromatographed with [¹⁴C]-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (±)-anti-BaPDE:tetraols. Thus, both adducts that bound to the immobilized boronate column were formed from (±)-anti-BaPDE. One major adduct that did not contain cis-vicinal hydroxy groups, M2, was detected in both cell types. M2 was formed from (±)-7ß,8-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydroBaP (syn-BaPDE) as M2 eluted in the same relative position as a syn-BaPDE:dGuo adduct marker and the tetraol hydrolysis products of M2 cochromatographed with tetraols formed from (±)-syn-BaPDE. The isolation of the individual BaP:DNA adducts followed by acid hydrolysis allowed the identification of the BaP:DNA adducts formed in human mammary cell cultures and demonstrated the presence of (-)-anti-BaPDE:dGuo. Thus, this work provides the first evidence, other than cochromatography, that (-)-anti-BaPDE is formed in cell systems and reacts with DNA in cells to form (-)-anti-BaPDE:dGuo. The similarity of the BaP:deoxyribonucleoside adduct profiles in normal mammary epithelial cells and the human mammary carcinoma T47D cell line suggests that the T47D cell line is an excellent model for studies on the metabolic activation of polycyclic aromatic hydrocarbons to DNA binding intermediates in human mammary cells.

¹ This investigation was supported by Grants CA-28825 and CA-40228 from the National Cancer Institute, Department of Health and Human Services, and by United States Department of Energy Contract DE-AC05-840R21400 with the Martin-Marietta Energy Systems, Inc.

² Recipient of a David Ross Research Fellowship from Purdue University.

³ To whom requests for reprints should be addressed.

⁴ Present address: Carcinogenesis and Toxicology Evaluation Branch, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.

Received 11/19/85. Revised 2/20/86. Accepted 2/27/86.

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