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Adducts of Mitomycin C and DNA in EMT6 Mouse Mammary Tumor Cells: Effects of Hypoxia and Dicumarol on Adduct Patterns¹

Department of Chemistry, Hunter College, City University of New York, New York, New York 10021 [R. B., D. C., M. T.]; Departments of Therapeutic Radiology [C. S. H., S. R.] and Pharmacology [A. C. S.], Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510; and Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Company, 1188 Shimotogari, Nagaizumi, Sunto, Shizuoka 411, Japan [H. A., M. K.]

6-CH₃-³H-Mitomycin C (MC) was used to identify MC-DNA adducts formed in EMT6 mouse mammary tumor cells. DNA was isolated from cells treated with ³H-MC. The DNA was enzymatically digested, and the digest was analyzed for ³H-labeled adducts by high performance liquid chromatography. All four major adducts previously isolated and characterized in cell-free systems were detected: two different monoadducts and two bisadducts forming DNA-interstrand and DNA-intrastrand cross-links, respectively. No MC-DNA adducts other than the DNA interstrand cross-link had been shown previously to be formed in living cells. A MC-deoxyguanosine adduct of unknown structure was also detected in DNA from EMT6 cells; this adduct was also formed with purified EMT6 DNA. High performance liquid chromatography analysis was further applied to study the relationship between DNA adducts and cytotoxicity. The number of adducts increased with the concentration of MC in both aerobic and hypoxic cells. At a constant drug level, more adducts were observed in cells treated under hypoxic conditions than in cells treated aerobically; at 2 µM MC, 4.8 ¢ 10^–7 and 3.1 x 10^–7 adducts/nucleotide were observed under hypoxic and aerobic conditions, respectively. The increased adduct frequency under hypoxia correlates with the known increased cytotoxicity of MC to EMT6 cells under hypoxic conditions. In addition, a higher ratio of cross-linked adducts to monoadducts was observed in hypoxic cells. The high performance liquid chromatography techniques were also used to examine the effects of dicumarol (DIC) on adduct patterns in cells treated simultaneously with ³H-MC. The MC-DNA adduct frequencies in DIC-treated cells were increased 1.5-fold under hypoxia and decreased 1.6-fold under aerobic conditions from those observed without DIC. This finding correlates with the known DIC-induced increase and decrease in the cytotoxicity of MC in hypoxic and aerobic EMT6 cells, respectively. The monoadduct resulting from monofunctionally activated MC was suppressed by DIC under both hypoxic and aerobic conditions. In addition, DIC induced the selective formation of an unknown DNA-associated radiolabeled substance in hypoxic cells; this is hypothesized to be a cytotoxic DNA lesion produced by a DIC-stimulated oxido-reductase. The methodology developed to measure MC adduct patterns may be useful as an indicator of distinct enzymatic activation processes for this drug.

¹ Supported by USPHS Grant CA-28681 (M. T.), Training Grant CA-09259 (C. S. H.), Grants DHP-35 (S. R.) and CH-530 (A. C. S.) from the American Cancer Society, a "Research Centers in Minority Institutions" award RRO-3037 from the Division of Research Resources, NIH (M. T.), and a Postdoctoral Fellowship from the Deutsche Forschungsgemeinschaft (R. B.). A preliminary report of this work has been presented (45).

² Present address: Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232.

³ Present address: Department of Microbiology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103.

⁴ Present address: College of Veterinary Medicine, Room H114, 4700 Hillsborough St., North Carolina State University, Raleigh, NC 27606.

⁵ To whom requests for reprints should be addressed.

Received 5/12/93. Accepted 8/25/93.

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