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.
Highlights
Cyclophosphamide (CY) is an alkylating agent that has both potent immunosuppressive properties and antineoplastic activity
We investigated how the myocardial cellular effects of CYS9 were modified by acrolein scavenger N-acetylcysteine (NAC), antioxidant isorhamnetin (ISO), and cytochrome P-450 (CYP) inhibitor β-ionone (BIO)
CY is activated by the hepatic cytochrome P-450 (CYP) enzyme system to form 4-hydroxycyclophosphamide (HCY), which is in equilibrium with aldocyclophosphamide (AldoCY)
Summary
Cyclophosphamide (CY) is an alkylating agent that has both potent immunosuppressive properties and antineoplastic activity. Administration of posttransplantation CY (PTCy) in high doses has been attracting attention as novel strategy for preventing graft-versus-host disease (GVHD). High-dose CY, when administered at the appropriate time after transplantation, depletes alloreactive T cells from the donor and host and can inhibit both GVHD and graft rejection [3, 4]. CY is activated by the hepatic cytochrome P-450 (CYP) enzyme system to form 4-hydroxycyclophosphamide (HCY), which is in equilibrium with aldocyclophosphamide (AldoCY). Depending on the type of cell, AldoCY may, through the chemical process of β-elimination, decompose to form cytotoxic phosphoramide mustard (PM) and byproduct acrolein, or may be oxidized by aldehyde dehydrogenase 1 (ALDH1) to the inactive metabolite o-carboxyethylphosphoramide mustard (CEPM) (Fig 1) [5, 6]
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