Enzymatic toxicogenation of "activated" cyclophosphamide by 3'-5' exonucleases

3'-5' Exonucleases from various sources were found to toxicogenate 4-hydroxycyclophosphamide ("activated" cyclophosphamide) by splitting the oxazaphosphorinane ring and releasing an alkylating moiety and acrolein. Neither cyclophosphamide (CP) nor the deactivated metabolites of CP, 4-keto-CP and carboxyphosphamide nor 4-(S-ethanol)-sulfido-CP were attacked by 3'-5' exonucleases. DNA polymerases with proofreading activity, such as DNA polymerase I from E. coli or DNA polymerase delta from rabbit bone marrow, exhibited a tenfold higher specific activity with "activated" CP than "plain" 3'-5' phosphodiesterases such as snake venom phosphodiesterase or 3',5'cyclic AMP phosphodiesterase from bovine heart tissue. High levels of toxicogenating activity were estimated in peripheric human lymphocytes and tissues of lymphatic origin, suggesting that enzymatic toxicogenation plays a key role with respect to the cytotoxic specificity of "activated" CP.