Cooxidation of cyclophosphamide as an alternative pathway for its bioactivation and lung toxicity.

A single i.p. dose of cyclophosphamide produces lung cell injury and fibrosis in mice. Although cyclophosphamide is activated by the cytochrome P-450 mixed function oxidase (MFO) system, a role for this system in the development of lung injury has not been established. The involvement of other metabolic pathways, such as cooxidation via prostaglandin H synthase, in the toxicity of cyclophosphamide has not been studied. The objectives of the current study were to assess the effects of various inhibitors of MFO and prostaglandin H synthase activity on the development of cyclophosphamide-induced lung damage and fibrosis in mice, to determine whether arachidonic acid as well as NADPH could support the activation of cyclophosphamide to an alkylating metabolite, and to assess the capacity of cyclophosphamide to serve as a reducing cosubstrate. In addition, the ability of a low dose of cyclophosphamide to prevent the lung injury from a later higher dose was determined. Treatment with SKF 525A, piperonyl butoxide, or 1-benzylimidazole, followed by a single 200 mg/kg dose of cyclophosphamide, did not diminish pulmonary thymidine incorporation (an index of cell division after injury) or hydroxyproline content (an indicator of fibrosis), compared to mice treated with cyclophosphamide alone. Pretreatment with 1-aminobenzotriazole reduced the incorporation of thymidine into lung DNA on days 3 and 10, but not on day 7, and also reduced lung hydroxyproline accumulation. Treatment with indomethacin, nordihydroguiaretic acid, or aspirin prior to cyclophosphamide greatly reduced levels of pulmonary thymidine incorporation and/or hydroxyproline content, compared to cyclophosphamide alone. Low dose pretreatment with cyclophosphamide did not prevent the lung injury or fibrosis from a subsequent higher dose. NADPH supported greater production of alkylating metabolites in liver than in lung microsomes. In contrast, the arachidonic acid-supported production of alkylating metabolites was greater in lung microsomes. No NADPH- or arachidonate-supported alkylating activity was evident in lung or liver cytosol. SKF 525A and 1-aminobenzotriazole inhibited the NADPH-supported reaction in liver, but not lung, while indomethacin and nordihydroguiaretic acid inhibited the arachidonic acid-supported reaction in lung but not liver. Cyclophosphamide was a moderately active reducing cosubstrate for 5-phenyl-4-pentenyl hydroperoxide in both lung and liver microsomes. These results demonstrate that pathways in lung tissue unrelated to MFOs can metabolize cyclophosphamide to an alkylating compound and that MFO-mediated activation of cyclophosphamide may not be essential for the development of the pulmonary toxicity associated with this drug.