MITOMYCIN C METABOLISM AND INTERACTION WITH SULFUR NUCLEOPHILES IN BONE MARROW, DNA, AND CLONOGENIC TUMOR CELLS (ANTICANCER, ANTIBIOTIC)

Abstract

A series of studies in mice were performed to determine the interaction of two sulfur nucleophiles, oral n-acetylcysteine (NAC) and intravenous sodium thiosulfate (Na₂S₂O₃) with the anticancer drug mitomycin C (MMC). Neither nucleophile reduced MMC lethality or hematopoietic toxicity. Both increased the antitumor activity of MMC in mice bearing P-388 and L-1210 leukemias. There was no nucleophile reduction of MMC effects on normal bone marrow stem cells (CFUs) using a murine spleen colony forming assay. In contrast, the nucleophiles significantly enhanced MMC bone marrow toxicity. Three clonogenic human tumor cell lines (HEC-1A endometrial, 8226 myeloma, WiDr colon) were relatively resistant to MMC and the nucleophiles did not increase activity. A human breast cancer cell line (MCF-7) was sensitive to MMC and this activity was blocked by glutathione. Oxygen free radical scavengers did not reduce MMC activity. A novel isocratic high performance liquid chromatography (HPLC) assay (48:52, methanol:0.01M phosphate buffer) using ultraviolet detection at 365 nm was used to characterize MMC-protein binding and murine pharmacokinetics. The k' for MMC was 7.91 and 9.86 for porfiromycin. Peaks were confirmed by mass spectroscopy. MMC was bound 30% to albumin and S-9 microsomal proteins and 60-70% to calf thymus DNA. MMC uptake into mouse bone marrow was enhanced by the nucleophiles and was rapidly cleared from the plasma (half-life 0.5 hours). In vitro MMC metabolism with rat liver S-9 microsomes demonstrated production of a polar eluting, putative MMC metabolite (K' = 4.486, lambda maximum 300 nm). This metabolite was inactive in the in vitro clonogenic tumor cell assay. Finally, molecular pharmacology studies using alkaline DNA elution showed that MMC causes both DNA-DNA and DNA-protein crosslinks. There was no evidence for free radical-induced DNA strand scission by MMC. There was also some evidence of moderate DNA protection with the sulfur nucleophiles.