title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention.
abstract: Observed only after administration of high doses, cardiotoxicity is the dose-limiting effect of cyclophosphamide (CY). We investigated the poorly understood cardiotoxic mechanisms of high-dose CY. A rat cardiac myocardial cell line, H9c2, was exposed to CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9). Cytotoxicity was then evaluated by 3-(4,5-dimethyl-2-thiazolyl)¬2,5-diphenyl¬2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase release, production of reactive oxygen species (ROS), and incidence of apoptosis. We also investigated how the myocardial cellular effects of CYS9 were modified by acrolein scavenger N-acetylcysteine (NAC), antioxidant isorhamnetin (ISO), and CYP inhibitor β-ionone (BIO). Quantifying CY and CY metabolites by means of liquid chromatography coupled with electrospray tandem mass spectrometry, we assayed culture supernatants of CYS9 with and without candidate cardioprotectant agents. Assay results for MTT showed that treatment with CY (125-500 μM) did not induce cytotoxicity. CYS9, however, exhibited myocardial cytotoxicity when CY concentration was 250 μM or more. After 250 μM of CY was metabolized in S9 mix for 2 h, the concentration of CY was 73.6 ± 8.0 μM, 4-hydroxy-cyclophosphamide (HCY) 17.6 ± 4.3, o-carboxyethyl-phosphoramide (CEPM) 26.6 ± 5.3 μM, and acrolein 26.7 ± 2.5 μM. Inhibition of CYS9-induced cytotoxicity occurred with NAC, ISO, and BIO. When treated with ISO or BIO, metabolism of CY was significantly inhibited. Pre-treatment with NAC, however, did not inhibit the metabolism of CY: compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein. Furthermore, NAC pre-treatment did not affect intracellular amounts of ROS produced by CYS9. Since acrolein seems to be heavily implicated in the onset of cardiotoxicity, any competitive metabolic processing of CY that reduces its transformation to acrolein is likely to be an important mechanism for preventing cardiotoxicity.
authors.affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Nishikawa
authors.affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Miyahara
authors.affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kurauchi
authors.affiliation: Department of Clinical Pharmacy and Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Watanabe
authors.affiliation: Department of Clinical Pharmacotherapy, Hiroshima University, Hiroshima, Japan.
authors.forename: Kazuro
authors.initials: K
authors.lastname: Ikawa
authors.affiliation: Clinical Development Dept. 1, Clinical Development Division, EPS Corporation, Saitama, Japan.
authors.forename: Kousuke
authors.initials: K
authors.lastname: Asaba
authors.affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Tanabe
authors.affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Okamoto
authors.affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Kawano
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D054715:Cardiotoxins / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology; D002460:Cell Line; D003520:Cyclophosphamide / Q000009:adverse effects / Q000493:pharmacokinetics / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D008297:Male; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D054715:Cardiotoxins; D017382:Reactive Oxygen Species; D003520:Cyclophosphamide