Kinetics of DMA Cross-Linking in Normal and Neoplastic Mouse Tissues following Treatment with Cfs-Diamminedichloroplatinum(ll) in Vivo1

The formation and repair of c/s-diamminedichloroplatinum(ll) (c/s-DDP)-induced DNA cross-links in cells from a number of different mouse tissues, both normal and neoplastia, were com pared in three different populations of animals, tumor-free mice and mice bearing a transplanted fibrosarcoma (either FSa or NFSa) in their thighs. Groups of mice were given i.v. injections of 4-12-mg/kg doses of c/s-DDP, and the amount of c/s-DDPinduced DNA cross-linking was determined at different times after injection using an adaptation of the alkaline elution tech nique. The degree of cross-linking in each tissue was linearly related to the dose of c/s-DDP at either 6 or 24 h after injection and varied significantly among the different tissues, with FSa, NFSa, kidney, and liver showing the highest level of cross-linking of the tissues studied. The relative contributions of DNA-interstrand and DNA-protein cross-links to the elution profiles were estimated by proteinase K (PK) digestion. At either 6 or 24 h after injection with c/s-DDP, the rate of elution of the DNA was substantially increased by PK, indicating a large contribution of DNA-protein cross-links. This effect was observed in all tissues studied, although the proportion of PK-resistant lesions appeared to vary from tissue to tissue, liver and spleen showing a signifi cantly lower proportion of DNA-interstrand to total cross-links than either of the tumors. For liver, virtually no interstrand cross links could be detected after PK treatment. The kinetics of the repair of c/s-DDP-induced DNA crosslinking in these tissues were also compared. In cells from tumorfree animals, the amount of total (DNA-interstrand plus DNAprotein) cross-linking increased gradually, reaching a maximum after about 6 h; however, little evidence of repair of these lesions was observed in any of these normal tissues. In fact, the degree of cross-linking tended to increase somewhat between 6 and 24 h after injection. The kinetics of cross-linking in cells isolated from the FSa tumor were very different; while there was an initial increase in cross-linking up to 6 h, these lesions were subse quently repaired, although at a somewhat slower rate than has been reported for cultured mammalian cells. In contrast, NFSa tumor cells behaved more like the normal tissues, with little evidence of any cross-link repair in the first 24 h. For each of the tissues studied, the proportion of DNA-interstrand to total cross links remained constant between 6 and 24 h. The data also suggest that the presence of the FSa tumor in an animal may have some effect on the kinetics of drug-induced lesions in the Received 1/11/85; revised 7/24/85; accepted 8/28/85. 1This investigation was supported by USPHS Grant CA 23270 awarded by the National Cancer Institute. Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture and Department of Health and Human Services, NIH. 2To whom requests for reprints should be addressed, at Department of Physics, Box 94, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Ave., Houston, TX 77030. normal tissues of that animal, the tissues from FSa-bearing animals generally showing some small capacity to remove cross links from their DNA. Investigations of this type may provide an easily characterizable and relevant pharmacological end point (DNA lesions) to indicate the distribution and modulation of chemotherapy agents that have DNA as their target.