Cyclophosphamide (CP) is a widely used alkylating agent which must be bioactivated to exert its therapeutic and toxic effects. There is a dose- limiting toxicity to the bladder and lung that may be caused by acrolein formed via a elimination reaction following an initial 4-hydroxylation of CP. Because metabolism occurs mainly in the liver, the mechanism by which this reactive aldehyde reaches the sites of injury is unknown. The overall hypothesis to be tested is that glutathione (GSH) conjugates or mercapturic acid metabolites of acrolein represent transport forms to deliver toxic CP metabolites to the sites of injury. Further processing may occur in the target organs prior to toxicity, or the conjugates themselves may be toxic. A secondary, but related, hypothesis is that nitrogen mustard metabolites of CP enhance the toxicity of these thiol metabolites in bladder.
The specific aims to investigate these hypotheses are: 1) to assess the production of acrolein-glutathione metabolites in rats treated with CP. 2) to determine whether cells known to metabolize CP (hepatocytes), a tissue that is a target for CP toxicity (lung), and a lung cell line (A549) can generate toxic mercapturate(s) given the presumed precursor materials (acrolein or 3-oxopropylGSH). 3) to assess the general toxicity of 3-oxopropylGSH, 3-oxopropyl mercapturic acid and its S-oxide, and the di-acid forms [S-3-proprionic acid mercapturic acid 3-PrAMCA and its S-oxide] to these same systems as well as to isolated bladder tissue. 4) to determine the mechanism of toxicity of the various thiol metabolites. Whether it is the parent molecule, a breakdown product or a metabolite resulting from the oxidation of the aldehyde functionality to its acid will be determined. 5) to assess the mechanism of toxicity of CP as related to acrolein glutathione conjugates in vivo. Studies will be done in mice treated with CP or the glutathione metabolites it generates. Both lung and bladder toxicity will be evaluated with and without pretreatment with acivicin (gamma-glutamyl transpeptidase inhibitor), probenecid (organic anion transport inhibitor), hexahydrocoumarin (aldehyde oxidase inhibitor), and aminooxyacetic acid (Beta-lyase inhibitor). 6) to determine if and how nor-HN2 can enhance the bladder toxicity induced by these conjugates. The results of these studies may help devise new strategies to minimize the toxic effects of this drug.
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