Limits...
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

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482695&req=5

pone.0131394.g011: 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.

Mentions: To determine whether NAC or ISO or BIO affected the antitumor activity of CYS9, tests were carried out on cells from cancer cell line HL-60. As Fig 11 shows, CYS9 pre-treated with NAC produced as great a decrease in cancer cell viability as CYS9 used alone. However, CYS9 pre-treated with ISO for 24 hours and 48 hours or co-incubated with BIO for 24 hours statistically significantly (p < 0.05) inhibited the antitumor activity of CYS9. This finding confirms that of the three candidates, only NAC protects against the cardiotoxicity of CYS9 while not inhibiting its antitumor activity.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131394.g011: 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.
Mentions: To determine whether NAC or ISO or BIO affected the antitumor activity of CYS9, tests were carried out on cells from cancer cell line HL-60. As Fig 11 shows, CYS9 pre-treated with NAC produced as great a decrease in cancer cell viability as CYS9 used alone. However, CYS9 pre-treated with ISO for 24 hours and 48 hours or co-incubated with BIO for 24 hours statistically significantly (p < 0.05) inhibited the antitumor activity of CYS9. This finding confirms that of the three candidates, only NAC protects against the cardiotoxicity of CYS9 while not inhibiting its antitumor activity.

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