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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

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.
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pone.0131394.g007: 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.

Mentions: In testing for MTT and release of LDH, assay results showed that NAC, ISO, and BIO all inhibited CYS9-induced cytotoxicity (Fig 5A and 5B). While metabolism of CY was statistically significantly inhibited by treatment with ISO or BIO, pre-treatment with NAC did not result in similar inhibition (Fig 6A–6C). Compared with control results, we observed no difference in HCY, statistically significantly more CEPM (Fig 6B and 6C), and statistically significantly less acrolein (Fig 7A). Furthermore, NAC pre-treatment did not affect intracellular ROS levels, including superoxide, H2O2, HOCl and the •OH, produced by CYS9 (Fig 7B–7D).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482695&req=5

pone.0131394.g007: 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.
Mentions: In testing for MTT and release of LDH, assay results showed that NAC, ISO, and BIO all inhibited CYS9-induced cytotoxicity (Fig 5A and 5B). While metabolism of CY was statistically significantly inhibited by treatment with ISO or BIO, pre-treatment with NAC did not result in similar inhibition (Fig 6A–6C). Compared with control results, we observed no difference in HCY, statistically significantly more CEPM (Fig 6B and 6C), and statistically significantly less acrolein (Fig 7A). Furthermore, NAC pre-treatment did not affect intracellular ROS levels, including superoxide, H2O2, HOCl and the •OH, produced by CYS9 (Fig 7B–7D).

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