title: Genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells exposed to peroxynitrite generated from SIN-1.
abstract: SIN-1 (3-morpholinosydnonimine), the active metabolite of the vasodilator drug molsidomine, decomposes spontaneously in solution. In the presence of oxygen, NO* and O(2)(*-) are released, generating peroxynitrite, a potent oxidizing agent, at a constant rate over a 2 h period. We utilized this system to investigate mechanisms of peroxynitrite-induced cytotoxicity, genotoxicity, apoptosis, and mitochondrial damage in two human lymphoblastoid cell lines carrying either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) genes. Treatment of TK6 cells with 5 mM SIN-1 for 1.5 h resulted in 28 +/- 6% survival 24 h later. Exposure in the presence of different radical scavengers significantly increased survival, as follows: cytochrome c, 96 +/- 3%; Tiron, 69 +/- 0%; SOD plus catalase, 83 +/- 5%; carboxy-PTIO, 87 +/- 3%; and uric acid, 87 +/- 2%. D-mannitol was ineffective in reducing lethality, as were SOD and catalase when added individually or in heat-inactivated form. Spontaneous as well as SIN-1-induced mutant fractions (MF) in both HPRT and TK genes were significantly higher in WTK-1 cells than in TK6 cells (p < 0.05-0.01). Exposure to 2.5 mM SIN-1 induced time-dependent apoptosis in TK6 cells, but not in WTK-1 cells. Mitochondrial membrane depolarization was also observed in both cell lines after SIN-1 treatment. Neutral comet assay demonstrated that SIN-1 treatment resulted in higher levels of DNA double-strand breaks in TK6 cells than in WTK-1 cells. Collectively, these data show that SIN-1 can be used as an effective peroxynitrite generator in cell culture experiments under these experimental conditions, in which it induced a greater apoptotic response but was less potent as a mutagen in TK6 cells compared with WTK-1 cells. Thus, p53 status was an important determinant of SIN-1 induced mutagenesis and apoptosis in these two human lymphoblastoid cell lines.
authors.affiliation: Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Chun-Qi
authors.initials: CQ
authors.lastname: Li
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Trudel
authors.forename: Gerald N
authors.initials: GN
authors.lastname: Wogan
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D020552:Comet Assay; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D004336:Drug Antagonism; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008928:Mitochondria / Q000187:drug effects*; D008981:Molsidomine / Q000031:analogs & derivatives / Q000378:metabolism / Q000633:toxicity*; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity*; D030421:Peroxynitrous Acid / Q000378:metabolism / Q000633:toxicity*; D013937:Thymidine Kinase / Q000235:genetics; D014665:Vasodilator Agents / Q000378:metabolism / Q000633:toxicity*
chemical_list: D016166:Free Radical Scavengers; D009153:Mutagens; D014665:Vasodilator Agents; D030421:Peroxynitrous Acid; C002385:linsidomine; D004247:DNA; D008981:Molsidomine; D007041:Hypoxanthine Phosphoribosyltransferase; D013937:Thymidine Kinase