metallothionein and copper-bleomycin

Bleomycin-Cu(II) [BLM-Cu(II)], which does not cause DNA strand breaks in vitro, exhibits antitumor activity in vivo. The copper in BLM-Cu(II) is reductively removed in vivo, and the liberated Cu(I) is trapped by intracellular thiol-proteins, thus yielding metal-free BLM. The bioactive species of BLM appears to be the iron-complex. For characterization of the thiol-proteins, thiol-compounds such as dithiothreitol (DTT), cysteine, metallothionein (MT) and alcohol dehydrogenase (ADH) were examined for their ability to trap Cu(I) liberated from BLM-Cu(II). BLM-Cu(II) was incubated at 37 degrees C with the thiol-compounds anaerobically for 1 hour followed by aerobic incubation for 10 minutes. Under these conditions DTT converted BLM-Cu(II) to metal-free BLM, but cysteine did not. However, in the presence of MT, cysteine gave metal-free BLM. The Cu(I) liberated from BLM-Cu(II) by cysteine was trapped by MT with accompanying liberation of Zn(II) and Cd(II) from MT. A part of the liberated Zn(II) formed the complex with the metal-free BLM. Metal-free BLM was also formed from BLM-Cu(II) by combined treatment with cysteine and ADH. BLM-Cu(II) was aerobically incubated with cytosol of MT-induced rat liver at 37 degrees C for 1 hour, and the mixture was analyzed with Sephadex G-75 column chromatography. The amount of copper in the MT fraction was increased concomitant with decrease of the Zn(II) and Cd(II). These results suggest that MT, ADH and other thiol-compounds, which have polythiol ligands, act as Cu(I)-trapping agents to yield metal-free BLM in cells.

Topics: Alcohol Oxidoreductases; Animals; Bleomycin; Chromatography, High Pressure Liquid; Copper; Cysteine; Dithiothreitol; Female; Liver; Metallothionein; Rats; Sulfhydryl Compounds; Zinc