Cytotoxicity of N,N-bis(2-chloroethyl)-N-nitrosourea in hypoxic rat hepatocytes.
Incubation of rat hepatocytes with N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU, 10-100 microM) and 5% O2 caused a time-dependent loss of cell viability, whereas no cytotoxicity was observed when BCNU was incubated with hepatocytes and 95% O2. BCNU (50-100 microM) reduced intracellular glutathione concentrations by 40 and 80% in hepatocytes incubated in 95 and 5% O2, respectively. Intracellular glutathione disulfide concentrations were not altered by 95 or 5% O2 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 microM 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% O2. 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 BCNU-induced cytotoxicity than are hepatocytes incubated in 95% O2 and that this vulnerability is associated with the loss of both ATP and glutathione. This conclusion is supported by data showing (a) a similar hypoxia-dependent pattern of cytotoxicity in hepatocytes exposed to the BCNU degradation products 2-chloroethyl isocyanate, 2-chloroethanol, and 2-chloroethylamine and (b) little BCNU-induced cytotoxicity, no increase in phosphorylase a activity, and no loss of ATP with 5% O2 in the presence of adenosine (1 mM).
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002330:Carmustine / Q000378:metabolism / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D004734:Energy Metabolism / Q000187:drug effects; D005978:Glutathione / Q000032:analysis; D008099:Liver / Q000187:drug effects*; D008297:Male; D010100:Oxygen / Q000494:pharmacology*; D010762:Phosphorylase a / Q000032:analysis; D051381:Rats
D000255:Adenosine Triphosphate; D010762:Phosphorylase a; D005978:Glutathione; D000241:Adenosine; D010100:Oxygen; D002118:Calcium; D002330:Carmustine