Cytotoxicity of A^A^-Bis(2-chloroethyI)-A^nitrosourea in Hypoxie Rat Hepatocytes1

Incubation of rat hepatocytes with V. Y-bis(2-dili.rm-th> I)V-nitrn.smirea (IU AT. 10-100 MM)and 5% O caused a time-dependent loss of cell viability, whereas no cytotoxicity was observed when BCNU was incu bated with hepatocytes and 95% O2. BCNU (50-100 UM» reduced intracelluhv glutathione concentrations by 40 and 80% in hepatocytes incu bated in 95 and 5% <>., respectively. Intracellular glutatbione disulfide concentrations were not altered by 95 or 5% ()•or by the presence of BCNU. The extracellular glutathione disulfide content of cells exposed to BCNU and 95% O2, but not to BCNU and 5% O2, exhibited a 150% increase. Incubation of hepatocytes with 100 UM BCNU and 5% O2 reduced the cellular energy charge from 0.85 to 0.58; no effect on energy charge was observed in hepatocytes incubated with BCNU and 95% <>,. The decrease in energy charge was due to a decrease in cellular ATP content (66%) and increases in cellular ADP (180%) and AMP (50%) concentrations. The reduction in both cellular ATP and glutathione concentrations was paralleled by a rise in the activity of phosphorylase a, a sensitive indicator of cytosolic Ca2* content. These findings indicate that hepatocytes incubated in 5% O2 are more vulnerable to BCNUinduced cytotoxicity than are hepatocytes incubated in 95% O2 and that this vuInorahilit> is associated with the loss of both ATP and glutathione. This conclusion is supported by data showing (a) a similar hypoxiadependent pattern of cytotoxicity in hepatocytes exposed to the BCNU degradation products 2-chloroethyl isocyanate, 2-chloroethanol, and 2chloroethylamine and (h) little BCNU-induced cytotoxicity, no increase in phosphorylase a activity, and no loss of ATP with 5% <>• in the presence of adenosine (1 HIM).