Evidence for a role of chloroethylaziridine in the cytotoxicity of cyclophosphamide

A number of investigators have observed that the use of 4-hydroperoxycyclop hosphamide (4-HC) in multiwell plate cytotoxicity assays can be associated with toxicity to cells in wells that contain no drug. Previous reports have implicated diffusion of 4-HC decomposition products, and acrolein in parti cular, as the active species. Purpose: The purpose of this study was to elu cidate the species responsible for the airborne cytotoxicity of 4-HC, and t o devise ways to minimize such effects in chemosensitivity assays. Methods: To this end, analogues of 4-HC were synthesized to identify the contributi ons of individual cyclophosphamide metabolites to cytotoxicity. The analogu es were then tested for activity against three human breast tumor cell line s (including a line resistant to 4-HC), and one non-small-cell lung carcino ma line. Cytotoxicity was evaluated by assays that quantitate cellular meta bolism and nucleic acid content. Results: Didechloro-4-hydroperoxycyclophos phamide, a compound that generates acrolein and a nontoxic analogue of phos phoramide mustard, gave no cross-well toxicity. In contrast, a significant neighboring well effect was observed with phenylketophosphamide, a compound that generates phosphoramide mustard but not acrolein. Addition of authent ic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide. Increasing the buffering capacity of th e growth medium and sealing the microtiter plates prevented airborne cytoto xicity. Conclusions: Since it is unlikely that phosphoramide mustard is vol atile, these findings implicate chloroethylaziridine rather than acrolein a s the volatile metabolite of 4-HC that is responsible for airborne cytotoxi city. The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro. Furthermore, these findings suggest that chloroethylaziridine may also cont ribute to the toxicity of cyclophosphamide in vivo.