title: Evidence for a role of chloroethylaziridine in the cytotoxicity of cyclophosphamide.
abstract: A number of investigators have observed that the use of 4-hydroperoxycyclophosphamide (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 particular, as the active species.
  
  
  OBJECTIVE
  The purpose of this study was to elucidate the species responsible for the airborne cytotoxicity of 4-HC, and to devise ways to minimize such effects in chemosensitivity assays.
  
  
  METHODS
  To this end, analogues of 4-HC were synthesized to identify the contributions of individual cyclophosphamide metabolites to cytotoxicity. The analogues were then tested for activity against three human breast tumor cell lines (including a line resistant to 4-HC), and one non-small-cell lung carcinoma line. Cytotoxicity was evaluated by assays that quantitate cellular metabolism and nucleic acid content.
  
  
  RESULTS
  Didechloro-4-hydroperoxycyclophosphamide, a compound that generates acrolein and a nontoxic analogue of phosphoramide 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 authentic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide. Increasing the buffering capacity of the growth medium and sealing the microtiter plates prevented airborne cytotoxicity.
  
  
  CONCLUSIONS
  Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity. 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 contribute to the toxicity of cyclophosphamide in vivo.
authors.affiliation: Department of Medicine and Duke Comprehensive Cancer Center, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Flowers
authors.forename: S M
authors.initials: SM
authors.lastname: Ludeman
authors.forename: M P
authors.initials: MP
authors.lastname: Gamcsik
authors.forename: O M
authors.initials: OM
authors.lastname: Colvin
authors.forename: K L
authors.initials: KL
authors.lastname: Shao
authors.forename: J H
authors.initials: JH
authors.lastname: Boal
authors.forename: J B
authors.initials: JB
authors.lastname: Springer
authors.forename: D J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000171:Acrolein / Q000737:chemistry; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry / Q000633:toxicity*; D001388:Aziridines / Q000737:chemistry / Q000633:toxicity*; D001943:Breast Neoplasms / Q000473:pathology; D002452:Cell Count / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003520:Cyclophosphamide / Q000031:analogs & derivatives / Q000737:chemistry / Q000633:toxicity*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D014407:Tumor Cells, Cultured
chemical_list: D018906:Antineoplastic Agents, Alkylating; D001388:Aziridines; D007202:Indicators and Reagents; C103230:N-(2-chloroethyl)aziridine; D000171:Acrolein; D003520:Cyclophosphamide; C011272:perfosfamide