Ionizing radiation induces chronic metabolic oxidative stress and a mutator phenotype in hamster fibroblasts that is mediated by H 2 O 2 , but the intracellular source of H 2 O 2 is not well defined. To determine the role of mitochondria in the radiation-induced mutator phenotype, end points of mitochondrial function were determined in unstable (CS-9 and LS-12) and stable (114) hamster fibroblast cell lines derived from GM10115 cells exposed to 10 Gy X rays. Cell lines isolated after irradiation demonstrated a 20-40% loss of mitochondrial membrane potential and an increase in mitochondrial content compared to the parental cell line GM10115. Surprisingly, no differences were observed in steady-state levels of ATP (P > 0.05). Unstable clones demonstrated increased oxygen consumption (two-to threefold; CS-9) and/or increased mitochondrial electron transport chain (ETC) complex II activity (twofold; LS-12). Using Western blot analysis and Blue Native gel electrophoresis, a significant increase in complex II subunit B protein levels was observed in LS-12 cells. Furthermore, immunoprecipitation assays revealed evidence of abnormal complex II assembly in LS-12 cells. Treatment of LS-12 cells with an inhibitor of ETC complex II (thenoyltrifluoroacetone) resulted in significant decreases in the steady-state levels of H 2 O 2 and a 50% reduction in mutation frequency as well as a 16% reduction in CAD gene amplification frequency. These data show that radiation-induced genomic instability was accompanied by evidence of mitochondrial dysfunction leading to increased steady-state levels of H 2 O 2 that contributed to increased mutation frequency and gene amplification. These results support the hypothesis that mitochondrial dysfunction originating from complex II can contribute to radiation-induced genomic instability by increasing steady-state levels of reactive oxygen species.

Chronic oxidative stress has been associated with genomic instability following exposure to ionizing radiation (IR). However, data showing direct causal linkages between specific reactive oxygen species and the IR-induced mutator phenotype are lacking. The current study demonstrates that IR-induced genomically unstable cells (characterized by chromosomal instability and increased mutation and gene amplification frequencies) show a 3-fold increase in steady-state levels of hydrogen peroxide, but not superoxide. Furthermore, stable clones isolated from parallel studies showed significant increases in catalase and glutathione-peroxidase activity. Treatment of unstable cells with polyethylene glycol-conjugated catalase [PEG-CAT] reduced the mutation frequency and mutation rate in a dose dependent fashion. In addition, inhibiting catalase activity in the stable clones using 3-aminotriazole increased mutation frequency and rate. These results clearly demonstrate the causal relationship between chronic oxidative stress mediated by hydrogen peroxide and the mutator phenotype that persists many generations following exposure of mammalian cells to IR.

Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), [Zn(GTSC)]₃, complex. As compared with the well-studied zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM), complex, which was used as a reference, [Zn(GTSC)]₃ had 2.5-fold higher fluorescence. When cellular fluorescence was measured using flow cytometry, we observed that [Zn(GTSC)]₃ had 3.4-fold to 12-fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy results showed that [Zn(GTSC)]₃ appeared to have a nuclear localization within 30 min of addition to MCF7 cells. Moreover, [Zn(GTSC)]₃ showed minimal cytotoxicity compared with Zn(ATSM), suggesting that [Zn(GTSC)]₃ may be less deleterious to cells when used as an imaging agent. Our data suggest that the novel [Zn(GTSC)]₃ complex can potentially serve as a biocompatible fluorescent imaging agent for live cells.

SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Over expression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (p < 0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (p < 0.0005). Finally, 95% depletion of glutathione using L-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (p < 0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.

Oxidative stress and mitochondrial dysfunction in cancer cells represent features that may be exploited therapeutically. We determined if agents which induce mitochondrial dysfunction such as zidovudine (AZT) and cisplatin (CIS) could enhance killing of human head and neck cancer cells via oxidative stress. AZT and/or CIS-induced cytotoxicity was determined using clonogenic survival, mitochondrial membrane potential was analyzed to investigate mitochondrial function, and glutathione was measured to determine thiol metabolism perturbations. AZT+CIS significantly increased toxicity and reduced mitochondrial membrane potential in FaDu, Cal-27 and SQ20B head and neck cancer cells while increasing % glutathione disulfide (GSSG). Treatment with the thiol antioxidant, N-acetylcysteine (NAC), reversed the loss of mitochondrial membrane potential and the increase in %GSSG, and partially protected FaDu and Cal-27 cells from AZT+CIS. Finally, an inhibitor of glutathione synthesis, L-buthionine-[S,R]-sulfoximine, sensitized the cells to AZT+CIS-induced cytotoxicity which was partially reversed by NAC. These results suggest that exposure of cancer cells to agents which induce mitochondrial dysfunction such as AZT cause significant sensitization to CIS-induced toxicity via disruptions in thiol metabolism and oxidative stress. These findings provide a biochemical rationale for evaluating agents which induce mitochondrial dysfunction in combination with chemotherapy and inhibitors of glutathione metabolism in head and neck cancer.