Evidence for peroxidase-mediated metabolism of cyclophosphamide.
Lung and bladder injury are side-effects of chemotherapy with cyclophosphamide (CP). Although the mixed-function oxidases are known to metabolize CP to reactive species, inhibitors of these enzymes have no significant effect on therapeutic activity or lung toxicity in vivo. In contrast, inhibitors of prostaglandin H synthase (PHS) can significantly decrease CP-induced lung injury. The current studies examined whether peroxidases can bioactivate CP in vitro. A mixture of 14C (ring) and 3H (side chain)-labeled CP (0.25-2 mM) was incubated with fresh lung and liver microsomes, horseradish peroxidase, or purified PHS using H2O2 or arachidonate as cosubstrates. All systems showed irreversible binding of CP-derived radioactivity to protein, increases in oxygen consumption, and generation of polar metabolites, including acrolein. Arachidonate-catalyzed binding of CP-derived radioactivity to fresh lung or liver microsomes, as well as H2O2 or arachidonate-supported generation of polar metabolites by purified PHS, was maximal by 2 min. In contrast, NADPH-catalyzed microsomal binding was linear for 30 min. This is consistent with the rapid self-inactivation of PHS in vitro. Arachidonate-catalyzed binding of CP-derived radioactivity to pulmonary microsomes was greater than liver, whereas the converse was true with NADPH. Incubation of 0.5 to 4 mM CP with PHS and arachidonate resulted in concentration-dependent increases in prostaglandin E2 synthesis. One mM H2O2 generated more polar metabolites from CP than 0.3 mM arachidonate when incubated with 100 units of purified PHS (10 +/- 2 and 4 +/- 1.5 nmol in 2 min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)