Cooxidation of Cyclophosphamide As an Alternative Pathway for Its Bioactivation and Lung Toxicity1
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[CANCER RESEARCH 51. 542-548. Januar> 15. 1991] Cooxidation of Cyclophosphamide as an Alternative Pathway for Its Bioactivation and Lung Toxicity1 Robin D. Smith2 and James P. Kehrer' Division of Pharmacology and Toxicology, College of Pharmacy, The I'nirersily of Texas at Austin, Austin, Texas 78712-1074 ABSTRACT actions, has been reported after single doses in experimental animals and humans (2, 3). In rats, the damage appears as a A single i.p. dose of cyclophosphamidc produces lung cell injury and fibrosi* in mice. Although cyclophosphamidc is activated by the cyto- focal sclerosing pneumonitis at the alveolar level, with injury chrome P-450 mixed function oxidase (MKO) system, a role for this to type I cells, abnormal type II cells, a gradually increasing system in the development of lung injury has not been established. The content of collagen characteristic of fibrosis, and areas of alveo involvement of other metabolic pathways, such as cooxidation via pros- lar collapse (4). The lungs of mice given a single injection of taglandin II synthase, in the toxicity of Cyclophosphamide has not been Cyclophosphamide showed similar histopathology (5, 6). The studied. The objectives of the current study were to assess the effects of time course of the acute lung lesion in mice is relatively rapid, various inhibitors of MIO and prostaglandin II synthase activity on the with the alveolar labeling index being maximal after 5 days and development of cyclophosphamide-induced lung damage and fibrosis in pulmonary thymidine incorporation reaching a peak about 1 mice, to determine whether arachidonic acid as well as NADPII could week after treatment with a single dose (7, 8). support the activation of Cyclophosphamide to an alkylating metabolite, The mechanism of cyclophosphamide-induced lung damage and to assess the capacity of Cyclophosphamide to serve as a reducing has not been established, although the well described hepatic cosubstrate. In addition, the ability of a low dose of Cyclophosphamide metabolism of this drug by MFO4 enzymes to alkylating and to prevent the lung injury from a later higher dose was determined. Treatment with SKK 525A, piperonyl butoxide, or 1-benzylimidazole, oxidizing metabolites (9-11) has led to the assumption that this followed by a single 200 mg/kg dose of Cyclophosphamide, did not enzyme system is required for both the therapeutic and toxic diminish pulmonary thymidine incorporation (an index of cell division effects of Cyclophosphamide. Hyperoxia aggravates the cyclo after injury) or hydroxyproline content (an indicator of fibrosis), com phosphamide-induced lung lesion (7) and Patel (12) has sug pared to mice treated with Cyclophosphamide alone. Pretreatment with gested that oxidative injury is involved in the damaging process. l-aminobenzotriazole reduced the incorporation of thymidine into lung However, although metabolic activation must occur before cy- DNA on days 3 and 10, but not on day 7, and also reduced lung totoxic effects develop and MFOs can metabolize Cyclophos hydroxyproline accumulation. Treatment with indomethacin, nordihydro- phamide to alkylating metabolites in vitro, it is unclear what guiaretic acid, or aspirin prior to Cyclophosphamide greatly reduced levels role this particular enzyme system plays in the in vivo effects of of pulmonary thymidine incorporation and/or hydroxyproline content, compared to Cyclophosphamide alone. Low dose pretreatment with Cyclo this drug. MFO-mediated metabolism is an important, but not exclu phosphamide did not prevent the lung injury or fibrosis from a subsequent higher dose. NADPII supported greater production of alkylating metab sive, pathway to bioactivate various xenobiotics. This is illus olites in liver than in lung microsomes. In contrast, the arachidonic acid- trated by the increasing recognition of the role hydroperoxide- dependent oxidation (termed "cooxidation") plays in xenobiotic supported production of alkylating metabolites was greater in lung mi crosomes. No NADPII- or arachidonate-supported alkylating activity metabolism and subsequent carcinogenesis or tissue injury (13). was evident in lung or liver cytosol. SKI 525A and 1-aminobcnzotriazole The oxidation of various chemicals during the metabolism of inhibited the NADPII-supported reaction in liver, but not lung, while arachidonic acid by PHS was first shown in 1975 (14). This indomethacin and nordihydroguiaretic acid inhibited the arachidonic acid- was subsequently determined to involve the peroxidase portion supported reaction in lung but not liver. Cyclophosphamide was a mod erately active reducing cosubstrate for 5-phenyl-4-pentenyl hydroperox- of PHS, which reduces the prostaglandin G? formed by the cyclooxygenase reaction to the alcohol prostaglandin H2 (15). ide in both lung and liver microsomes. These results demonstrate that Numerous compounds which are structurally unrelated to fatty- pathways in lung tissue unrelated to MKOs can metabolize Cyclophos phamide to an alkylating compound and that MFO-mediated activation acid endoperoxides can be oxidized during this reduction step of Cyclophosphamide may not be essential for the development of the (13). Additional studies have shown that similar reactions can pulmonary toxicity associated with this drug. be catalyzed by lipoxygenase activities. Evidence is available supporting the concept that cooxidation INTRODUCTION by PHS, together with metabolism by pulmonary MFO sys tems, mediates the lung toxicity of 3-methylindole (16). Lung A variety of chemical compounds, including numerous cyto- tissue is capable of metabolizing Cyclophosphamide to alkylat toxic drugs, are known to damage lung tissue following systemic ing species (17), and similar metabolic pathways could be administration (1). Lung damage due to antineoplastic drugs involved in the pulmonary toxicity of this drug. Interestingly, can be produced by a number of factors, including direct drug in contrast to MFOs, which are found in the highest concentra action or the synergistic or antagonistic action of two drugs or tions in the liver, PHS and lipoxygenase activities are relatively of the same drug with itself. The lung damage produced by high in lung and bladder (13), sites of the major cyclophospha Cyclophosphamide, although possibly modified by such inter- mide-induced toxicities. The present study provides evidence Received8/6/90;accepted10/25/90. that the lung damage and fibrosis due to Cyclophosphamide is The costs of publication of this article were defrayed in part by the payment not decreased by most inhibitors of MFO activity or by a of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4The abbreviations used are: MFO. mixed function oxidase; PHS. prostaglan 1This work was supported by Grant HL 35689 from the National Heart. Lung din H synthase; ABT. l-aminoben/otria/ole: PB\. piperonyl butoxide: Bl. 1- and Blood Institute. ben/ylimidazole: NDGA. nordihydroguiaretic acid; BUT. butylaled hydroxyto- 2Current address: CH2M Hill. 625 Herndon Parkway. Herndon, VA. luene; BUA. butylated hydroxyanisole; NBP. 4-(/>-nitroben*yl)pyridine: PPHP. ' To whom requests for reprints should be addressed. 5-phenyl-4-pentcnyl hydroperoxide: PPA. 5-phenyl-4-pentenyl alcohol. 542 Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 1991 American Association for Cancer Research. COOXIDATION AND LUNG TOXICITY OF CYCLOPHOSPHAMIDE preliminary dose of this drug, which previous work suggested resultant supernatants at 100,000 x g for 60 min. The final supernal ani s decreased the lung toxicity of a subsequent higher dose (5). In were saved on ice and the microsomal pellets were washed in 0.1 M contrast, lung cell damage and pulmonary fibrosis are signifi potassium pyrophosphate, pH 7.4, containing 1 misi EDTA and 20 MM cantly reduced by inhibitors of PHS. The in vitro production of BHT. The final microsomal pellet was resuspended in 1.15% KCl. All alkylating metabolites was primarily NADPH dependent in microsomal and cytosolic fractions were prepared immediately before liver microsomes and primarily arachidonic acid dependent in their use to avoid potential problems with enzyme stability. Protein determinations were by the microbiuret method (25). lung microsomes. It was also found that cyclophosphamide could function in the presence of lung or liver microsomes, as Measurements of Alkylating Metabolites. The conversion of cyclo phosphamide to metabolites capable of alkylating NBP was determined a moderately active reducing cosubstrate. in both microsomes and cytosol. The alkylation of NBP by nitrogen mustards results in a compound which forms a chromophore under MATERIALS AND METHODS alkaline conditions, with an absorbance maximum of 540 nm (26). The standard reaction mixture contained 9 /jmol cyclophosphamide, 2 ^mol Animals and Treatments. Male ICR mice (24-32 g, 6-IO weeks of NADPH, 20 /¿molglucose-6-phosphate, 200 MHIO!K:HPO4 (pH 7.4), age) were used for all studies. The animals were obtained from HarÃan 10 /¿molMgCb, 2 mg microsomal or cytosolic protein, and sufficient Sprague Dawley (Houston. TX) and had free access to standard lab 1.15% KCl to make the final volume 3 ml. The mixture was preincu- chow and water. Cyclophosphamide (50 or 100 mg/kg), SKF 525A (10 bated at 37°Cfor 5 min and the reaction was started by adding 2 units or 50 mg/kg), ABT (50 mg/kg), or aspirin (50 mg/kg) were adminis glucose-6-phosphate dehydrogenase. In other studies, the NADPH- tered i.p. as saline solutions. PBX (400 mg/kg) was given as a single generating system was replaced by 300 nmol arachidonic acid, which i.p. dose in corn oil. BI (25 mg/kg) was administered as a single i.p. was added to initiate the reaction. Inhibitors were added to the reaction dose in 95% ethanol. Inhibitors of MFO activity and aspirin were given mixture (100 MM)and allowed to incubate for 5 min prior to the as single i.p. injections 2 h prior to cyclophosphamide. Indomethacin addition of cyclophosphamide. (1.5 mg/kg in corn oil) was administered twice daily for 2 days and The metabolism of cyclophosphamide was terminated after 15 min again 2 h prior to cyclophosphamide.