Differential toxicities of cyclophosphamide and its glutathione metabolites to A549 cells

Cyclophosphamide (CP), a widely used antineoplastic agent, is metabolized to species responsible for both the therapeutic and toxic effects of this drug. Acrolein is believed to be the primary toxic metabolite. This alpha,beta-unsaturated aldehyde reacts rapidly with glutathione (GSH) and can then be further metabolized to the mercapturic acid derivatives. The toxicities of the acrolein-glutathione adduct, 3-oxopropyl glutathione (oxoPrGSH) and the acrolein mercapturic acid derivatives S-3 oxopropyl N-acetylcysteine (oxoPrMCA) and S-3 hydroxypropyl N-acetylcysteine (hydroxyPrMCA) have not been fully tested. OxoPrMCA, hydroxyPrMCA and oxoPrGSH were synthesized. The toxicities of these compounds, along with those of CP and acrolein, were assessed by measuring their effects on the growth of human type II A549 lung carcinoma cells using the alamarBlue assay. Each compound was incubated with A549 cells under serum-free conditions for 2 hr, followed by 94 hr more growth in the presence of fresh medium with serum. A 50% reduction in cell growth 72 hr after treatment was achieved with 83 muM oxoPrMCA or 4 muM acrolein. No significant toxicity was seen with hydroxyPrMCA (10 mM) or oxoPrGSH (5 mm). CP (5 mM) also had no effect on the growth of A549 cells under these conditions. This latter finding is consistent with previous evidence that CP requires metabolic activation to exert its toxicity. When present during xenobiotic exposure, GSH (2 mm) almost completely protected against the growth inhibition caused by 1 mM oxoPrMCA or 10 mum acrolein. N-Acetylcysteine (1 mM) also prevented the toxicity caused by 1 mM oxoPrMCA and provided significant protection against the growth inhibition induced by 10 muM acrolein. These data support the concept that toxicity from oxoPrMCA may be due to the release of acrolein.