Benzene Metabolite 1,2,4-Benzenetriol Induces Halogenated DNA and Tyrosines Representing Halogenative Stress in the HL-60 Human Myeloid Cell Line

Background: Although benzene is known to be myelotoxic and to cause myeloid leukemia in humans, the mechanism has not been elucidated

Role of metabolites of cyclophosphamide in cardiotoxicity

Abstract Background The dose-limiting toxic effect of cyclophosphamide (CY) is cardiotoxicity. The pathogenesis of myocardial damage is poorly understood, and there is no established means of prevention. In previous studies, we suggested that for CY-induced cardiotoxicity, whereas acrolein is the key toxic metabolite, carboxyethylphosphoramide mustard (CEPM) is protective. We sought to verify that acrolein is the main cause of cardiotoxicity and to investigate whether aldehyde dehydrogenase (ALDH), which is associated with greater CEPM production, is involved in the protective effect for cardiotoxicity. We also evaluated the protective effect of N-acetylcysteine (NAC), an amino acid with antioxidant activity and a known acrolein scavenger. Methods H9c2 cells were exposed to CY metabolites HCY (4-hydroxy-cyclophosphamide), acrolein or CEPM. The degree of cytotoxicity was evaluated by MTT assay, lactate dehydrogenase (LDH) release, and the production of reactive oxygen species (ROS). We also investigated how the myocardial cellular protective effects of CY metabolites were modified by NAC. To quantify acrolein levels, we measured the culture supernatants using high performance liquid chromatography. We measured ALDH activity after exposure to HCY or acrolein and the same with pre-treatment with NAC. Results Exposure of H9c2 cells to CEPM did not cause cytotoxicity. Increased ROS levels and myocardial cytotoxicity, however, were induced by HCY and acrolein. In cell cultures, HCY was metabolized to acrolein. Less ALDH activity was observed after exposure to HCY or acrolein. Treatment with NAC reduced acrolein concentrations. Conclusions Increased ROS generation and decreased ALDH activity confirmed that CY metabolites HCY and acrolein are strongly implicated in cardiotoxicity. By inhibiting ROS generation, increasing ALDH activity and decreasing the presence of acrolein, NAC has the potential to prevent CY-induced cardiotoxicity

Dose-response effect of charged carbon beam on normal rat retina assessed by electroretinography

Purpose: To compare the effects of carbon beam irradiation with those of proton beam irradiation on the physiology of the retina of rats. Methods and Materials: Eight-week-old Wister rats were used. The right eyes were irradiated with carbon beam (1, 2, 4, 8, and 16 Gy) or proton beam (4, 8, 16, and 24 Gy) with the rats under general anesthesia. Electroretino- grams were recorded 1, 3, 6, and 12 months after the irradiation, and the amplitudes of the a and b waves were compared with those of control rats.Results: The amplitude of b waves was reduced more than that of a waves at lower irradiation doses with both types of irradiation. With carbon ion irradiation, the amplitudes of the b wave were significantly reduced after radiation doses of 8 and 16 Gy at 6 months and by radiation doses of 4, 8, and 16 Gy at 12 months. With proton beam irradiation, the b-wave amplitudes were significantly reduced after 16 and 24 Gy at 6 months and with doses of 8 Gy or greater at 12 months. For the maximum b-wave amplitude, a significant difference was observed in rats irradiated with carbon beams of 4 Gy or more and with proton beams of 8 Gy or more at 12 months after irradiation.Conclusions: These results indicate that carbon beam irradiation is about two times more damaging than proton beam irradiation on the rat retina at the same dose

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

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

Inhibition of CYS9-induced cell cytotoxicity by candidate cardioprotectant agents.

<p>(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). *<i>p</i> < 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). *<i>p</i> < 0.05 compared with CYS9 group.</p

Myocardial cytotoxicity induced by CY metabolized by S9 mix.

<p>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. *<i>p</i> < 0.05 compared with control.</p

CYS9-induced cytotoxicity in HL-60 cells with candidate cardioprotectant agents.

<p>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). *<i>p</i> < 0.05 compared with CYS9 group.</p

Cytotoxicity of N-acetylcysteine on H9c2 cells.

<p>H9c2 cell viability after 24-hour exposure to N-acetylcysteine (NAC) was assessed by MTT assay (mean + standard deviation (SD) from 4 independent experiments). *<i>p</i> < 0.05 compared with control.</p