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.
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.
The emergence of drug-resistant microorganisms is an important medical and social problem. Drug-resistant microorganisms are thought to grow selectively in the presence of antibiotics. Most clinically isolated drug-resistant microorganisms have mutations in the target genes for the drugs. While any of the many mutagens in the environment may cause such genetic mutations, no reports have yet described whether these mutagens can confer drug resistance to clinically important microorganisms. We investigated how environmental mutagens might be implicated in acquired resistance to antibiotics in clinically important microorganisms, which causes human diseases. We selected mutagens found in the environment, in cigarette smoke, or in drugs, and then exposed Pseudomonas aeruginosa to them. After exposure, the incidence of rifampicin- and ciprofloxacin-resistant P. aeruginosa strains markedly increased, and we found mutations in genes for the antibiotic-target molecule. These mutations were similar to those found in drug-resistant microorganisms isolated from clinical samples. Our findings show that environmental mutagens, and an anticancer drug, are capable of inducing drug-resistant P. aeruginosa similar to strains found in clinical settings.
Benzene Induces Cytotoxicity without Metabolic Activation: Takuro Nishikawa, et al.
Background Fatal adverse effects or relapse can occur with excessive or insufficient busulfan exposure in hematopoietic stem cell transplantation. Given that busulfan is mainly metabolized by glutathione S‐transferase (GST), we investigated the influence of GST polymorphisms on busulfan pharmacokinetics in Japanese pediatric patients. Methods Blood samples were taken from patients receiving high‐dose i.v. busulfan as the first dose. Plasma busulfan concentration was measured using high‐performance liquid chromatography. The area under the plasma busulfan concentration–time curve (AUC) was calculated. The genotype of GSTA1 was determined on polymerase chain reaction (PCR)‐restriction fragment length polymorphism. Multiplex PCR was used to detect the presence or absence of GSTM1 and GSTT1 in the genomic DNA samples. Results Twenty patients were consecutively enrolled. Phenotype prediction was defined as follows: poor metabolizer (n = 4), one or more GSTA1*B haplotype or GSTM1/GSTT1 double‐null genotypes; and extensive metabolizer (n = 16), other genotypes. GSTA1, M1, and T1 independently had no significant differences in AUC0‐∞, clearance or elimination rate constant. For the infant with unexpectedly high AUC0‐∞ (2,591 μmol/L min), the GSTA1, M1, and T1 polymorphisms were wild type. On further analysis, the poor metabolizer group had lower clearance and higher AUC0‐∞, except for the aforementioned patient, compared with the extensive metabolizer group (1,531 vs 1,010 μmol/L min; P < 0.01). Conclusions GST polymorphisms may have affected busulfan pharmacokinetics, but these effects were obscured by other factors, such as underlying disease, systemic conditions, treatment history, and race.
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