Oxidative damage to DNA in diabetes mellitus.

BACKGROUND: Increased production of reactive oxygen species (ROS) and lipid peroxidation may contribute to vascular complications in diabetes. to test whether DNA is also oxidatively damaged in diabetes, we measured 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative damage of DNA, in mononuclear cells. METHODS: For this laboratory-based study, 12 patients with insulin-dependent diabetes mellitus (IDDM) and 15 patients with non-insulin-dependent diabetes mellitus (NIDDM) were matched by age with ten healthy volunteers each. DNA was extracted from mononuclear cells from whole blood. 8-OHdG was assayed by high-pressure liquid chromatography, and ROS were assayed by chemiluminescence. FINDINGS: IDDM and NIDDM patients had significantly higher median concentrations (p , 0.001, U test) of 8-OHdG in their mononuclear cells than their corresponding controls (in fmol/micrograms DNA): 128.2 (interquartile range 96.0-223.2) and 95.2 (64.0-133.5) vs 28.2 (21.7-43.4) and 21.9 (18.0-24.4), respectively. ROS generation by mononuclear cells was also significantly greater (p < 0.01) in diabetic patients than in their controls (in mV): 238.0 (107.0-243.0) and 101.3 (66.0-134.0) vs 69.5 (49.8-91.9) and 56.0 (38.8-62.5), respectively. INTERPRETATION: IDDM and NIDDM patients showed greater oxidative damage to DNA, with increased generation of ROS, than controls. Such changes might contribute to accelerated aging and atherogenesis in diabetes and to the microangiopathic complications of the disease.

Photosensitized formation of 8-hydroxy-2'-deoxyguanosine and DNA strand breakage by a cationic meso-substituted porphyrin.

Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA. In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, using a photoactivated cis-dicationic porphyrin. The data demonstrated a dose-dependent formation for each type of DNA damage. Inhibition of strand scission and 8-OHdG formation with the singlet oxygen scavenger 1,3-diphenylisobenzofuran and with MgCl2 and no apparent effect by D2O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage. However, a nearly complete inhibition of 8-hydroxy-2'-deoxyguanosine formation in 75% D2O and the substantial enhancement of 8-hydroxy-2'-deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process. These data indicate that distinct mechanisms lead to 8-OHdG formation and strand scission activity.

Differential proliferative responses of Syrian hamster embryo fibroblasts to paraquat-generated superoxide radicals depending on tumor suppressor gene function.

Oxygen radicals have been widely implicated in neoplastic transformation; however, little is known regarding their mode of action. In an attempt to delineate potential mechanisms of action, an analysis of superoxide effects on cell growth was studied in normal and two nontumorigenic, immortal cell lines derived from normal Syrian hamster embryo (SHE) fibroblasts. The two immortal cell lines differed in their ability to suppress tumorigenicity of tumor cells in cell hybrids. One cell line suppressed tumorigenicity (sup+), while a second clone was unable to suppress tumorigenicity (sup-). Paraquat was used to generate superoxide through its capacity to be reduced by NAD(P)H and to generate superoxide radicals. The growth response of the various cell types was measured by colony-forming ability as well as by tritiated thymidine incorporation using autoradiography. At low paraquat concentrations (25 microM), primary SHE cells and two sup+ clones showed up to a 40% enhancement in colony formation, while two sup- clones showed no increase. Toxicity was observed at high doses, starting at approximately 100 microM paraquat. Since oxygen radicals are also mutagenic, primary SHE cells were examined for chromosomal aberrations. Chromatid gaps and breaks were induced at all concentrations of paraquat used. Thus, superoxide not only causes cellular toxicity at high doses but at low doses enhances cell growth of certain cells (primary SHE cells and sup+ cells) but not others (sup- cells). Therefore, differing responses of cells at different stages of neoplastic progression must be considered in understanding oxygen radical effects in growth control and carcinogenesis.

Elevated production of active oxygen in Bloom's syndrome cell lines.

Based on our previous evidence indicating that the elevated sister chromatid exchange that characterizes Bloom syndrome (BS) cells may arise in response to elevated production of active oxygen, we have quantitated the levels of active oxygen in two control, two BS and one BS revertant cell lines. Luminol-dependent chemiluminescence was used as a measure of active oxygen production following treatment of the cells with the calcium ionophore A23187 or the chemotactic tripeptide N-formylmethionylleucylphenylalanine. A peptide factor present in plasma was required for priming the cells to undergo the oxidative response. As determined with A23187, active oxygen production was elevated in BS cell lines by 48.6% above control. Using N-formylmethionylleucylphenylalanine, active oxygen production was found to be increased by 250-314%. Chemiluminescence was inhibited in a dose-dependent manner by diphenylene iodonium, which specifically binds to and inhibits membrane-associated NADPH oxidase activity. This compound inhibited oxygen radical production nearly 3 times more effectively in control cells than in BS cells. The capacity to produce elevated levels of oxygen radicals may contribute to the spontaneous chromosomal instability of BS cells and to the associated high incidence of neoplasia in individuals with BS.

Molecular and biochemical aspects of Bloom's syndrome.

Bloom's syndrome (BS) is an autosomal recessive disorder, characterized by a high incidence of cancer at a young age. Cytogenetically, BS cells exhibit a high frequency of chromosomal damage and sister chromatid exchanges. Thus, BS provides one of the best correlations of a human genetic disorder exhibiting both chromosomal instability and a high incidence of cancer. It is increasingly evident that a spontaneous mutagenic event may be responsible for the inherent chromosomal instability. Oxidative stress is now shown to occur in BS cells and may be responsible for the observed chromosomal instability. Furthermore, treatment with antioxidants decreases the level of sister chromatid exchanges. The combination of a mutagenic event and an elevated rate of recombination could potentially lead to homozygosity of tumor suppressor gene function. Hypomethylation and expression of an activated c-myc gene are now demonstrated in BS lymphoblastoid cells. Identifying the mechanism(s) of the ongoing cellular and DNA damage is important in understanding the etiology of this complex disorder. This article reviews the recent biochemical and molecular advances in the study of BS.

Elevated superoxide dismutase in Bloom's syndrome: a genetic condition of oxidative stress.

We have found that Bloom's syndrome (BS) cells exhibit elevated levels of superoxide dismutase activity. Since SOD activity has been shown to reflect the intracellular superoxide (O2-) content, these results indicate that BS cells exhibit oxidative stress which ultimately results in DNA damage. Elevated sister chromatid exchange, the major cytological characteristic of BS, and superoxide dismutase induction were simulated in normal lymphoblastoid cells by treatment with compounds that increase the steady-state concentration of O2(-.). The sister chromatid exchange response of a BS lymphoid cell line was modulated through the control of the endogenous O2-. content. We therefore suggest that a major biochemical defect resulting from this genetic disorder is chronic over-production of the superoxide radical anion. The consequence of high O2-. levels concomitant with induced superoxide dismutase activity is the formation of enormous amounts of H2O2 which can apparently inactivate the enzymes responsible for its elimination. The inefficient removal of peroxide can result in high rates of sister chromatid exchange and chromosomal damage in BS cells and in normal cells treated with oxidation-reduction cycling compounds through the formation of highly reactive intermediary forms of active oxygen.

Cell kinetic evidence suggests elevated oxidative stress in cultured cells of Bloom's syndrome.

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the G1 phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.

Induction of superoxide dismutase, chromosomal aberrations and sister-chromatid exchanges by paraquat in Chinese hamster fibroblasts.

We have recently shown that Bloom syndrome fibroblasts have elevated levels of superoxide dismutase activity compared to those of normal fibroblasts. Based on this observation we decided to test whether an increased rate of superoxide radical production could be responsible for the induction of superoxide dismutase and of chromosomal aberrations and sister-chromatid exchanges characteristic of Bloom syndrome. Utilizing the superoxide-generating herbicide paraquat in Chinese hamster fibroblasts, we assayed the cells for dismutase activity, chromosomal aberrations and sister-chromatid exchanges. All 3 parameters investigated demonstrated a dose-dependent increase with paraquat and, consequently, with the superoxide produced. Since the induction of the enzyme is mediated by its substrate, the superoxide anion radical, we concluded that the increased dismutase activity (in Bloom syndrome and paraquat-treated cells) may be a secondary manifestation of an overall imbalance in oxygen metabolism and that this elevated enzymatic activity is insufficient to detoxify the high superoxide levels, which results in elevated levels of chromosomal damage.