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Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention.

Nishikawa T, Miyahara E, Kurauchi K, Watanabe E, Ikawa K, Asaba K, Tanabe T, Okamoto Y, Kawano Y - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0131394.g008: 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.

Mentions: Live-cell imaging also confirmed acute cytotoxicity in CYS9 samples, which was inhibited by NAC (Fig 8A–8D). In CYS9 samples without NAC, cells appeared either shrunken or irregularly shaped; but with NAC, the cells had a very similar appearance to those in control samples. The protective effect of NAC on H9c2 cells was corroborated using Hoechst 33342 staining and fluorescent assays of Caspase-3/7 activity (Fig 8E–8H). In Fig 8H, with round-shaped nuclei and homogeneous blue fluorescence intensity, the cells in CYS9 samples treated with NAC have a similar appearance to normal cells. As Fig 8G shows, caspase-3 and caspase-7 activity was greater in CYS9 (CY 250 μM) samples; such activity was suppressed in samples pre-treated for 2 hours with 1 mM NAC (Fig 8H).


Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention.

Nishikawa T, Miyahara E, Kurauchi K, Watanabe E, Ikawa K, Asaba K, Tanabe T, Okamoto Y, Kawano Y - PLoS ONE (2015)

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.
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pone.0131394.g008: 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.
Mentions: Live-cell imaging also confirmed acute cytotoxicity in CYS9 samples, which was inhibited by NAC (Fig 8A–8D). In CYS9 samples without NAC, cells appeared either shrunken or irregularly shaped; but with NAC, the cells had a very similar appearance to those in control samples. The protective effect of NAC on H9c2 cells was corroborated using Hoechst 33342 staining and fluorescent assays of Caspase-3/7 activity (Fig 8E–8H). In Fig 8H, with round-shaped nuclei and homogeneous blue fluorescence intensity, the cells in CYS9 samples treated with NAC have a similar appearance to normal cells. As Fig 8G shows, caspase-3 and caspase-7 activity was greater in CYS9 (CY 250 μM) samples; such activity was suppressed in samples pre-treated for 2 hours with 1 mM NAC (Fig 8H).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

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.

No MeSH data available.


Related in: MedlinePlus