Reactivity of lipophilic diSchiff-Base coordinated copper in rat hepatocytes

The membrane permeability and intracellular fate of ([N,N'-bis(2 pyridyl-phenyl-methylene)-1,4-butanediamine](N,N ',N",N"')-copper(II))-diperchlorate (CuPuPhePy), a copper-diSchiff-base complex of superoxide dismutase(SOD)-mimetic activity surviving biochelation, were examined using rat hepatocytes. Lipophilicity was quantified by determining the octanol/water partition coefficients (K(p)) employing PBS as the aqueous phase. K(p)(octanol/water) was close to 1 (0.7 +/- 0.31) for Cu-PuPhePy. The complex associates with phosphatidylcholine liposomes, as deduced from ultracentrifugation and gel filtration experiments. The ability of the complex to permeate cellular membranes was proven by correlating copper release and viability of rat hepatocytes preincubated with CuPuPhePy and treated with digitonin and diethylmaleate (DEM), respectively. The toxicity and reactivity of CuPuPhePy (LD (50) approximately 10 muM for rat hepatocytes under the given conditions) were higher than those of CuSO (4)(LD(50) approximately 16 mu M) and CuZn-SOD (no toxicity in the tested range of concentration). Unlike CuSO(4) and CuZn-SOD, the toxicity and reactivity of the diSchiff-base complex were increased (LD(50) approximately 5 muM) when the concentration of intracellular glutathione was reduced to 16% of the initial content, by preincubating the cells with DEM. The toxicity of Cu-PuPhePy paralleled lipid peroxidation. This phenomenon was strongly enhanced when Cu-PuPhePy and cumene hydroperoxide (CumOOH) were simultaneously allowed to react with rat hepatocytes. This effect was intensified following preincubation with DEM. A decline in Cu(II)-EPR signals was indicative of the reduction of CuPuPhePy by GSH and liver extract, respectively. The concomitant formation of the Cu(I)-GSH complex during this reduction was monitored by the formation of luminescent Cu(I)-thiolate chromophores.