Effect of acrolein on phosphoramide mustard-induced sister chromatid exchanges in cultured human lymphocytes

Abstract

Although phosphoramide mustard (PM) is generally recognized as being the most genotoxic metabolite of cyclophosphamide (CP), the contribution of acrolein to the cytogenetic toxicity of CP is unclear. Besides covalently binding to DNA, acrolein can inactivate critical proteins necessary for replicative DNA synthesis, RNA transcription, cell membrane integrity, and metabolism of xenobiotic and endogenous substrates. Because enzymatic processes are involved in sister chromatid exchange (SCE) formation and DNA excision repair, we hypothesized that acrolein might modulate SCE induction by PM due to acrolein's high binding affinity for proteins and low molecular weight sulfhydryl compounds. Human mononuclear leukocytes were isolated on a Ficoll-Hypaque density gradient, and 10(6) cells were inoculated into 1.9 ml of complete medium. T-cells were stimulated to grow with 4 micrograms concanavalin A/ml, and 5-bromo-2'-deoxyuridine (5 microM) was added 24 h later. The cultures were then treated with PM (0.069 microM) in the absence or presence of diethyl-4'-hydroperoxy-CP (DEHP-CP), an activated acrolein-generating compound, at concentrations of 0.1, 1, or 10 microM for 48 h. Demecolcine (1.35 microM) was added for the final 4 h of culture. PM alone induced about a 2-fold increase in the SCE frequency (PM, 14.1 +/- 0.5 (SD) versus control, 7.7 +/- 0.4) without cell cycle inhibition or reduced mitotic activity. DEHP-CP induced a concentration-related increase in the SCE frequency of up to 1.6-fold without any significant cell cycle inhibition or lowered mitotic activity. When PM and DEHP-CP were combined, SCE induction was additive for all three DEHP-CP concentrations. Except at the highest molar ratio of DEHP-CP:PM (145:1), there was no evidence of cytotoxicity in the other treatment groups. These results suggest that acrolein has a diminished role in mediating the cytogenetic and cytotoxic effects of CP. In addition, enzymes associated with SCE formation and, by inference, DNA excision repair may not be particularly susceptible to acrolein-induced inactivation.