Enhancement of cytotoxicity of bleomycin by dithiocarbamates: formation of bis(dithiocarbamato) cu(II).

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.

Properties of the initial reaction of bleomycin and several of its metal complexes with Ehrlich cells.

Characteristics of the reaction of bleomycin-A2 (BLM) and several of its metal complexes with Ehrlich cells in culture are described. Short incubation of BLM and Fe(III)-, Cu-, Zn-, and CdBLM with Ehrlich cells effectively inhibits cell proliferation. There is a sharp break at 30 min in the dependence of cytotoxicity upon time of exposure of cells to these forms of the drug. Qualitatively, the same curve can be generated by sequential additions of CoCl2 to cells during their first hour of incubation with BLM or Fe(III)BLM. The cobalt salt has less effect on CuBLM. The kinetics of initiation of the effect are directly correlated with the rapid kinetics of uptake of [3H]BLM by cells. Measurements of the initial rate of association of drug with cells as a function of extracellular BLM concentration suggest that a binding step is involved, for the rate of association approaches a maximal velocity at large concentrations of BLM. Uptake leads to both specific and nonspecific binding of tritium label; however, very little BLM gets into these cells. The internal concentration is estimated to be less than that in the external medium. BLM and its Fe(III) and copper complexes are taken up by Ehrlich cells to the same general extent after 60 min incubation; the cellular uptake of CoBLM is 25-50 times higher. Even the distributions of Fe(III)-, Cu-, and metal-free BLM within cytosol are comparable. A fraction binds to macromolecules; the rest appears unbound in low molecular weight fractions. The binding of [3H]BLM to cells cannot be reversed by incubation of labeled cells in drug-free medium or in media containing large concentrations of cold BLM.

Host zinc metabolism and the Ehrlich ascites tumour. Zinc redistribution during tumour-related stress.

Zinc redistribution between plasma and liver has been examined in mice injected with Ehrlich-ascites-tumour cells. Within 24 h of injection plasma Zn levels decrease and Zn appears in newly synthesized liver metallothionein. This response is dependent upon the number of tumour cells injected into the host. Uptake of Zn into liver and its specific accumulation in a Zn-binding protein, identified as metallothionein, continues for a number of days and reaches a plateau as tumour growth ceases. Over this time period, plasma copper rises. This redistribution also occurs in mice pretreated with cadmium in their drinking water for 1 month at levels of 20, 50, and 100 micrograms/ml. However, in each case there is a lag of 3 days before Zn increases in the livers of these animals which already contain substantial amounts of Cd/Zn-metallothionein. When Ehrlich cells are injected into mice previously placed on a Zn-deficient diet for several days, plasma Zn is already low and no net uptake of Zn into liver metallothionein is apparent. Finally, it is shown that ascites fluid can itself stimulate a transient shift of host of Zn into liver. Heat-inactivated fluid loses this property. It is suggested that, in the peritoneum, tumour cells initiate a stress response mediated by an ascites-fluid factor.