Lidocaine potentiation of bleomycin A2 cytotoxicity and DNA strand breakage in L1210 and human A-253 cells.

The survival of cultured L1210 cells exposed to bleomycin A2 (BLM A2) was markedly decreased by coincubation with the local anesthetic lidocaine. The potentiation occurred with concentrations of lidocaine that were nontoxic and was dependent upon both the concentration of lidocaine and BLM A2. A 1000-fold decrease in survival was seen with a 1-h exposure to 8 mM lidocaine and 10 microM BLM A2 compared to incubation with 10 microM BLM A2 alone. Prior exposure to lidocaine did not markedly alter BLM A2 cytotoxicity, while treatment with lidocaine immediately after BLM A2 exposure did, suggesting that increased cellular content of BLM A2 was not the mechanism of enhancement. Furthermore, lidocaine reduced the total amount of cell-associated radioactivity seen after incubation with [3H]BLM A2. The enhancement in L1210 cell cytotoxicity with lidocaine was not specific for the C- and N-terminal moieties of the BLM molecule. Other DNA-interacting antitumor agents, such as etoposide and mitomycin C, did not exhibit biologically significant alterations in their cytotoxicity when coincubated with lidocaine, although cis-diamminedichloroplatinum was significantly more toxic in the presence of lidocaine. The potentiation of BLM A2 cytotoxicity was not unique to murine tumor cells, since it was also seen with cultured human head and neck carcinoma (A-253) cells. Lidocaine did not increase directly BLM A2-induced breakage of DNA in vitro as measured by loss of form I pAT 153 DNA, but it did increase BLM A2-induced DNA strand breaks in intact L1210 cells coincubated with lidocaine and BLM A2. Exposure of L1210 cells to lidocaine after BLM A2 treatment also greatly increased DNA breakage consistent with possible inhibition of DNA repair. In addition, a modest reduction in the in vitro inactivation of BLM A2 by BLM hydrolase was found with lidocaine. We propose that inhibition of BLM metabolism and repair of BLM-induced DNA damage by lidocaine may have a role in the enhanced cytotoxicity.

Bleomycin hydrolase activity in pulmonary cells.

The metabolism of bleomycin (BLM) A2 by BLM hydrolase in the 105,000 X g supernatant fraction of homogenates obtained from freshly isolated and cultured pulmonary cells was assayed by high-pressure liquid chromatography. BLM A2 was converted solely to the less toxic desamido metabolite by the cytosol from isolated rabbit and bovine alveolar and interstitial macrophages, cultured rabbit and bovine pulmonary fibroblasts and cultured rabbit pulmonary artery endothelial cells. The BLM hydrolase activity in the cytosol from cultured rabbit fibroblasts had an apparent Km of 700 microM and Vmax of 33 nmol/hr/mg protein. The rate of BLM dA2 formation found with the cytosol of cultured rabbit pulmonary artery endothelial cells and pulmonary fibroblasts was 3 to 5 times greater per cell than that from the cytosol of rabbit alveolar and interstitial macrophages. Freshly isolated rabbit type II pneumocytes and bovine pulmonary artery endothelial cells grown in culture had undetectable levels of this inactivating enzyme activity. The expression of BLM hydrolase activity in rabbit pulmonary fibroblasts was stable for at least five passages in culture and was not significantly different over wide cell densities in culture. These data suggest that heterogeneity in the cellular distribution of BLM hydrolase activity exists in lungs. High levels of BLM hydrolase activity in the pulmonary endothelium or fibroblasts of some species may have an important role in determining the toxicity of BLM to the lungs.

Pulmonary fate of [3H]bleomycin A2 in mice.

The pulmonary fate of radiolabeled bleomycin [S-methyl-3H]bleomycin A2 ( [3H]BLM A2) was studied in mice after intratracheal (i.t.) or s.c. injection. The loss of radioactivity from the lungs followed apparent first-order kinetics during the first 3 hr after i.t. drug instillation with a half-time of removal of 32 min. In addition, the initial pulmonary removal was linear with instilled doses ranging from 0.02 to 2.2 mg/kg. Radioactivity was detected in liver, kidneys, spleen and serum 1 hr after i.t. administration. Approximately 1% of the i.t. administered dose was detected in the lungs after 24 hr, indicating that the rate of removal of radioactivity slowed between 3 and 24 hr after i.t. drug instillation. Eighty percent of the radioactivity found in the lungs 1 hr after i.t. instillation was unmetabolized [3H]BLM A2, but by 24 hr less than 25% was unmetabolized drug and almost 40% was the nonfibrogenic metabolite, desamidobleomycin A2. After s.c. administration of 10 mg/kg of [3H]BLM A2, peak pulmonary levels were observed between 45 and 60 min and were less than 1% of the injected dose. Pulmonary levels comparable to those seen with a single fibrogenic i.t. dose (2.2 mg/kg) could not be obtained after a s.c. injection even with a toxic dose of the drug (100 mg/kg). In addition, twice weekly s.c. injections of unlabeled BLM A2 (10 mg/kg) for 5 weeks did not alter the amount of radioactivity seen in the lungs after a s.c. injection of [3H]BLM A2. Thus, the pulmonary fibrosis seen with i.t. BLM administration may reflect the high initial content of unmetabolized drug achieved in the lungs.