Fig 1.
Cyclophosphamide metabolic pathways.
Cyclophosphamide (CY) is metabolized to 4-hydroxy-cyclophosphamide (HCY) in the hepatic cytochrome P-450 enzyme (CYP) system (CYP2B6 and/or CYP2C19). HCY enters cells as tautomer aldocyclophosphamide (AldoCY). Through β-elimination, AldoCY can be converted to phosphoramide mustard (PM) and acrolein. Alternatively, AldoCY can also be oxidized to the inactive metabolite o-carboxyethylphosphoramide mustard (CEPM) by aldehyde dehydrogenase 1 (ALDH1). Other metabolites include chloroacetaldehyde (CAA), deschloroethyl-cyclophosphamide (DCCY), 4-keto-cyclophosphamide (KetoCY), hydroxypropyl-phosphoramide mustard (HPPM), imino-cyclophosphamide (IminoCY), and glutathionyl-cyclophosphamide (GSCY).
Fig 2.
Cytotoxicity of N-acetylcysteine on H9c2 cells.
H9c2 cell viability after 24-hour exposure to N-acetylcysteine (NAC) was assessed by MTT assay (mean + standard deviation (SD) from 4 independent experiments). *p < 0.05 compared with control.
Fig 3.
Pharmacokinetics of high-dose cyclophosphamide in patients.
We measured CY and CY metabolites in blood plasma samples from patients receiving high-dose CY and here present the concentration–time profiles obtained from all three patients for (A) CY, (B) HCY, and (C) CEPM. Underlying diseases were acute mixed lineage leukemia (male, 8 y.o.; open square) and granulocytic sarcoma (male, 17 y.o.; open circle), and acute myeloid leukemia (male, 1 y.o.; open triangle).
Fig 4.
Myocardial cytotoxicity induced by CY metabolized by S9 mix.
H9c2 cell viability after (A) 24-hour and (B) 48-hour exposure to CY alone and CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9) was assessed by MTT assay (mean + SD from 3 independent experiments). (C) The changes of CY and its metabolites HCY and CEPM concentration in H9c2 cell culture media exposed to CYS9 (mean + SD from 3 independent experiments). (D) Fluorescence intensities, corresponding to levels of H2O2, in control samples or cells exposed to 250 μM CY, S9, CYS9 for 1 hour (mean + SD from 3 independent experiments). Fluorescence intensity is shown in arbitrary units. *p < 0.05 compared with control.
Fig 5.
Inhibition of CYS9-induced cell cytotoxicity by candidate cardioprotectant agents.
(A) The effect of candidate cardioprotectant agents (NAC, isorhamnetin (ISO), and β-ionone (BIO)) on cytotoxicity of CYS9 in H9c2 cells after 24-hour exposure. (mean + SD from 2 independent experiments conducted in duplicate). *p < 0.05 compared with CYS9 group. (B) The effects of candidate cardioprotectant agents against LDH release from H9c2 cells exposed to CYS9 for 2 hours. (mean + SD from 3 independent experiments). *p < 0.05 compared with CYS9 group.
Fig 6.
CY and CY metabolites results from LC/MS/MS assays of culture supernatants of CYS9 with and without candidate cardioprotectant agents.
H9c2 cells were exposed to CYS9 for 1 or 2 hours with and without candidate cardioprotectant agents (NAC, ISO, and BIO). Changes in concentration in H9c2 cell culture media of (A) CY and its metabolites (B) HCY and (C) CEPM was evaluated using LC/MS/MS. (mean + SD from 3 independent experiments). *p < 0.05 compared with CYS9 group.
Fig 7.
The concentration of acrolein in cell culture media and ROS generation in H9c2 cells after exposure to CYS9 with and without NAC.
(A) H9c2 cells were exposed for 1 and 2 hours to CYS9 with and without NAC. The changes of acrolein in culture media was measured using HPLC. (mean + SD from 3 independent experiments). Effect of NAC on ROS generated by CYS9, as shown by fluorescence intensity of (B) DCFH, (C) APF, and (D) HPF in cells exposed for 1 hour to CYS9 or CYS9 plus NAC. Fluorescence intensity is shown in arbitrary units. (mean + SD from 3 independent experiments). *p < 0.05 compared with CYS9 group.
Fig 8.
Optical and fluorescence images of H9c2 cells exposed to CY, CYS9, and CYS9 plus NAC.
A, B, C, D: Optical images at 24-hour exposure. (A) Control (unexposed H9c2 cells), (B) H9c2 cells exposed to 250 μM CY, (C) H9c2 cells exposed to CYS9, and (D) H9c2 cells exposed to CYS9 presence of 1 mM NAC. Magnification, 100×. Bar = 200 μm. E, F, G, H: Induction of apoptosis in H9c2 cells by CYS9 with or without NAC. Living cell nucleii stained by Hoechst 33342 are blue. Apoptotic cells stained by FITC-conjugated probes are green. (E) Control (unexposed H9c2 cells), (F) H9c2 cells exposed for 2 hours to 250 μM CY, (G) H9c2 cells exposed to CYS9—green dots indicate apoptotic cells. (H) H9c2 cells exposed to CYS9 with 1 mM of NAC. Magnification, 100×. Bar = 200 μm.
Fig 9.
Reduced glutathione levels in H9c2 cells after treatment with CYS9 in presence or absence of NAC.
Effects of NAC on reduced glutathione (GSH) levels in H9c2 cells exposed to CYS9 for 2 hours. (mean + SD from 3 independent experiments). *p < 0.01 compared with control group; §p < 0.01 compared with CYS9 group.
Fig 10.
Myocardial cytotoxicity induced by acrolein.
H9c2 cell viability after (A) 24-hour and (B) 48-hour exposure to acrolein (Acr) with or without NAC was assessed by MTT assay (mean + SD from 2 independent experiments conducted in duplicate). *p < 0.05 compared with control group. †p < 0.05 compared with acrolein 100 μM group.
Fig 11.
CYS9-induced cytotoxicity in HL-60 cells with candidate cardioprotectant agents.
Cell viability was assessed by MTT assay after HL-60 cells were exposed to CYS9 with and without NAC or ISO or BIO: results show cell viability (mean + SD from 3 or 4 independent experiments) after exposure to CYS9 for 24 hours (A, C, E) or 48 hours (B, D, F). *p < 0.05 compared with CYS9 group.