Cyclophosphamide (CP) is an orally active alkylating agent which must be bioactivated to exert its therapeutic and toxic effects. Although CP can be activated in vitro via mixed function oxidase (MFO-mediated 4- hydroxylation, a role for this system in the production of the active and toxic species in vivo has not been firmly established. The overall objectives of this proposal are to assess the hypothesis that alternative oxidation pathways, primarily cooxidation via prostaglandin H synthase (PHS), can also produce the 4-hydroxylated CP metabolite which is unstable and spontaneously breaks down to alkylating species, and to determine the importance of such metabolism in the lung and bladder toxicity of this drug. Preliminary studies have shown that inhibitors of PHS, but not MFO, activity prevents the development of lung damage in CP-treated mice. It was also found that arachidonic acid, as well as NADPH, could support the microsomal activation of CP to alkylating metabolite(s), and that CP could serve as a reducing cosubstrate in both lung and liver microsomes. Purified horseradish peroxidase, soybean lipoxygenase and ram seminal vesicle PHS, in the presence of arachidonic acid or H2O2, will be used as model systems to assess the ability of cooxidation reactions to metabolize CP. Analyses will include the loss of CP, the formation of water soluble species including acrolein, and covalent binding of 14C (ring) and 3H (chloroethyl side chain) labelled CP (which break down to labeled acrolein and phosphoramide mustard, respectively), to heat-inactivated microsomal protein. The metabolism and binding of CP by pure enzymes will be compared with metabolism and binding in tissues from ICR mice (sensitive to lung and bladder injury). Analyses will be done in vivo, in hepatic, kidney cortex and pulmonary microsomes, and in kidney papillae homogenates. In vivo studies will administer 200 mg/kg dual labelled CP ip. In vitro studies will initiate metabolism with NADPH, arachidonic acid or prostaglandin G2. Additional studies will assess the effect of specific cyclooxygenase, peroxidase, MFO and 5-lipogenase inhibitors on metabolism in vivo and on toxicity in vivo. Toxicity will be measured as changes in DNA synthesis and hydroxyproline content (lung) or blood content and gross morphology (bladder). The metabolism, cooxidation potential (using a synthetic hydroperoxide) and covalent binding of CP in tissues from ICR mice will be compared with that in C57 mice (resistant to lung and bladder injury) to obtain information on which factors are important for CP toxicity. Besides animal studies, experiments on the toxicity of CP to human lung cancer cell lines high or low in PHS activity will be done. CP is not directly toxic to cultured cells since it requires activation. The addition of arachidonic acid to cells high in PHS activity results in the activation of other agents. Since the same cell lines high in PHS also contain MFO activity, analyses will be performed with and without exogenous arachidonic acid and in the presence and absence of MFO and PHS inhibitors. The metabolism and covalent binding of CP in the presence and absence of these substances will be determined and correlated with cytotoxicity. Lastly, the effect of PHS inhibitors on the therapeutic activity of CP against tumor cell lines in mice will be assessed. The results of these studies will reveal the relative importance of cooxidation pathways for mediating the toxic and therapeutic effects of CP and could potentially lead to better targeting of tumors for CP therapy.
Showing the most recent 10 out of 15 publications
