H2O2-driven reduction of the Fe3+-quin2 chelate and the subsequent formation of oxidizing species.

The objective of this study was to compare effects of quin2 and EDTA in iron-driven Fenton-type reactions. Seven different assays for detection of strong oxidants were used: the DMSO, deoxyribose, benzoate hydroxylation, and plasmid DNA strand breakage assays, detection of 8-oxo-deoxyguanosine in deoxyguanosine mononucleosides and calf thymus DNA, and electron spin resonance with the spin-trap (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in the presence of ethanol or DMSO. With H2O2 and Fe3+, quin2 generally strongly increased the formation of reactive species in all assays, whereas with EDTA the results varied between the assays from barely detectable to highly significant increases compared to H2O2 and unchelated Fe3+. We found that the species produced in the reaction between Fe3+-quin2 and H2O2 behaved like the hydroxyl radical in all assays, whereas with Fe3+-EDTA no clear conclusion could be drawn about the nature of the oxidant. The effect of quin2 on the formation of oxidants on Fe2+ autoxidation, varied from generally inhibiting to slightly promoting, depending on the assay used. EDTA had a promoting effect on the amount of oxidant detected by all but one assay. None of the autoxidation systems produced DMSO or ethanol radical adducts with 4-POBN. In the presence of either chelator, H2O2, and Fe2+ DMSO and ethanol radical adducts of 4-POBN were produced. Using the Fe2+ indicator ferrozine, evidence for direct reduction of Fe3+-quin2 by H2O2 was found. Superoxide anion radical appeared to be less efficient than H2O2 as reductant of Fe3+-quin2 as addition of superoxide dismutase in the ferrozine experiments only decreased the amount of Fe2+ available for Fenton reaction by 10-20%. The main conclusions from our study are that the reduction of Fe3+-quin2 can be driven by H2O2 and that Fe2+ in the following oxidation step produces a species indistinguishable from free hydroxyl radical.

Effects of quin2 acetoxymethyl ester on H2O2-induced DNA single-strand breakage in mammalian cells: H2O2-concentration-dependent inhibition of damage and additive protective effect with the hydroxyl-radical scavenger dimethyl sulphoxide.

The cell-membrane-permeable calcium probe quin2 acetoxymethyl ester (quin2 AM) was ineffective, in comparison with o-phenanthroline, in protecting cells against H2O2-induced DNA single-strand breakage at H2O2 concentrations of about, and higher than, 0.5 mM. The present study shows that quin2 actually potentiated intracellular DNA damage at high H2O2 concentrations. H2O2-induced DNA breakage appeared within 5 min after exposure, and quin2 affected the induction of DNA breaks at both 0 degree C and 37 degrees C. Aurintricarboxylic acid, an endonuclease inhibitor, or a decrease in extracellular Ca2+, did not reduce DNA damage. These facts strongly suggest that the breaks were not produced by a Ca(2+)-dependent nuclease. We showed previously that, in the presence of Fe3+ and H2O2, quin2 strongly potentiated the formation of oxidizing species as well as plasmid DNA breakage, and, as could be expected for a transition-metal chelator, quin2 inhibited the Fenton reaction when Cu2+ was tested instead of Fe3+ [Sandström, Granström and Marklund (1994) Free Radicals Biol. Med. 16, 177-185]. In the present work with cultured cells, titration with quin2 AM showed that, despite the fact that Cu2+ has a three-to-four-orders-of-magnitude higher affinity for quin2 than has Fe3+, both inhibition and potentiation of H2O2-induced DNA damage occurred at quin2 AM concentrations of about 100 nM. Thus inhibition appeared not to involve Cu2+. The combination of quin2 AM and dimethyl sulphoxide (DMSO) gave an additive effect on H2O2-induced DNA damage compared with the effect of quin2 AM or DMSO alone, whereas the combination of o-phenanthroline and DMSO gave about the same effect as o-phenanthroline alone. In conclusion, our results do not support a role for Ca2+ in the inhibiting effect of quin2 on H2O2-induced DNA damage. Instead, it is likely that inhibition and potentiation by quin2 involves interaction with Fe ions.

A comparison of four assays detecting oxidizing species. Correlated reactivity of Fe(III)-quin2, but not Fe(III)-EDTA, with hydrogen peroxide.

Quin2, a fluorescent calcium probe, has a low affinity for calcium in comparison to its affinities for transition metal ions. Chelation of ferric ion with quin2 strongly enhanced the formation of oxidizing species in the presence of bolus H2O2 as detected with four assays, electron spin resonance with the spin-trap DMPO, the deoxyribose assay, the DMSO assay, and plasmid DNA strand breakage. In comparison, Fe(III)-EDTA reacted with bolus H2O2 only as detected with electron spin resonance and deoxyribose assay, but not as detected with the two latter assays. The addition of reductants, like ascorbate or superoxide generated by hypoxanthine/xanthine oxidase, to Fe(III)-EDTA in the presence of H2O2 produced plasmid DNA strand breakage and strong reactivity in both the DMSO and the deoxyribose assays. Our findings suggest that the main oxidizing species produced in Fenton-type reactions is hydroxyl radical. However, the reaction between Fe(III)-EDTA and bolus H2O2 appears to be exceptional and dominated by a nonhydroxyl radical species.

New roles for quin2: powerful transition-metal ion chelator that inhibits copper-, but potentiates iron-driven, Fenton-type reactions.

The objective of this study was to investigate whether quin2, through its metal chelating properties, could affect copper- or iron-driven Fenton reactions. Chelation of ferric ion with quin2 uniformly strongly enhanced the formation of oxidizing species, detected with the DMSO and deoxyribose assays, both by H2O2 and a mixture of superoxide/hydrogen peroxide produced by hypoxanthine/xanthine oxidase. Fe(3+)-EDTA gave the same effects, but lacked reactivity with bolus H2O2 as detected with the DMSO assay. Whereas the formation of oxidizing species with Fe(3+)-EDTA and ferric ions alone were strongly inhibited by superoxide dismutase both in the bolus H2O2 and hypoxanthine/xanthine oxidase systems, such formation in the presence of Fe(3+)-quin2 either did not decrease or decreased only moderately. Fe(3+)-quin2 also strongly enhanced plasmid DNA strand breakage in the presence of H2O2. Our findings suggest that quin2 as chelator of ferric ion may be a more powerful enhancer of oxidant formation than other chelators so far tested. The formation of oxidizing species from copper ions and bolus H2O2 was found to be fundamentally dependent on the choice of buffer system. We could only detect significant amounts of oxidants in both assays in Hepes buffer, but not in the phosphate, cacodylate or unbuffered systems, which all gave low reactivity in the DMSO assay compared to the deoxyribose assay. Quin2 chelation of cupric ion effectively inhibited the formation of oxidants as well as plasmid DNA strand breakage.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction and rejoining of DNA single-strand breaks in relation to cellular growth in human cells exposed to three hydroperoxides at 0 degrees C and 37 degrees C.

There was a 5-fold increase in cytotoxicity for cumene hydroperoxide, 10-fold for tert-butyl hydroperoxide and 25-fold for hydrogen peroxide, under metabolizing conditions (37 degrees C) in comparison to nonmetabolizing conditions (0 degrees C), when human P31 cells were exposed for 60 min. The induction of DNA single-strand breaks correlated poorly with cytotoxicity. Hydrogen peroxide was by far the most effective agent inducing single-strand breaks irrespective of temperature. Cumene hydroperoxide produced fewer strand breaks than tert-butyl hydroperoxide despite its greater cytotoxicity at either 37 degrees C or at 0 degrees C. The pattern of single-strand break induction did not change with temperature. The number of breaks, however, increased when the cells were exposed at 37 degrees C. The pattern of rejoining was similar for hydrogen peroxide- and tert-butyl hydroperoxide-induced breaks at both temperatures whereas the rejoining of cumene hydroperoxide-induced breaks deviated somewhat from this pattern. The results indicate that there is no clear-cut relationship between induction of DNA single-strand breaks and cytotoxicity after hydroperoxide exposure.

Effects of variation in glutathione peroxidase activity on DNA damage and cell survival in human cells exposed to hydrogen peroxide and t-butyl hydroperoxide.

The selenium-dependent glutathione peroxidase activities of two human cell lines, the colon carcinoma HT29 and the mesothelioma P31, cultured in medium containing 2% serum, increased from 195 to 541 and from 94 to 361 units/mg of protein respectively after supplementation with 100 nM-selenite. The catalase activity remained unchanged by this treatment. The effects of the obtained variation in glutathione peroxidase activities were investigated by exposing cells to H2O2 and t-butyl hydroperoxide. Selenite supplementation resulted in a decrease in H2O2-induced DNA single-strand breaks in both HT29 and P31 cells. A small, but significant, decrease in the number of DNA single-strand breaks for low doses (10-50 microM) of t-butyl hydroperoxide was found only in P31 cells and not in HT29 cells. We could detect neither induction of double-strand breaks (detection limit approx. 1000 breaks per cell) nor DNA-protein cross-links after exposing the cells to the two peroxides. In spite of the apparent protective effect of increased glutathione peroxidase activity on DNA single-strand break formation, there were no differences between selenite-supplemented and non-supplemented cells in cell survival after exposure to peroxide.

"The derivative assay"--an analysis of two fast components of DNA rejoining kinetics.

The DNA rejoining kinetics of human U-118 MG cells were studied after gamma-irradiation with 4 Gy. The analysis of the sealing rate of the induced DNA strand breaks was made with a modification of the DNA unwinding technique. The modification meant that rather than just monitoring the number of existing breaks at each time of analysis, the velocity, at which the rejoining process proceeded, was determined. Two apparent first-order components of single-strand break repair could be identified during the 25 min of analysis. The half-times for the two components were 1.9 and 16 min, respectively.

Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage.

The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or [3H]-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.

Variations among cultured cells in glutathione peroxidase activity in response to selenite supplementation.

The aim of this study was to devise conditions for manipulation of the activity of selenium-dependent glutathione peroxidase in cell lines by means of variation in culture medium contents of selenite and fetal calf serum. Nine different cell lines were studied. A low glutathione peroxidase activity was, in most cases, obtained by the use of a medium with a low (2%) serum content. Selenite induced in most of the cell lines an increase in glutathione peroxidase activity, with a plateau ranging from 10 nM to 300-1000 nM. Growth-retarding effects of selenite became apparent at 300-2000 nM, showing a large cell line variation. Supplementation with 50-100 nM selenite for 1 week should generally be suitable for maximal glutathione peroxidase induction. The selenium contents of serum batches were highly variable, pointing to the importance of using only one well-defined, preferably low-selenium, batch. The glutathione peroxidase activities varied considerably between cell lines and the selenite-induced increases ranged from negligible to more than 10-fold. The availability of cell lines with such variable responses should be valuable for experiments aimed at evaluating the importance of glutathione peroxidase and selenium compounds independently of glutathione peroxidase for the protection against oxidative insult.

A direct assay for detection of chemically induced changes in the rejoining kinetics of radiation induced DNA strand breaks.

A simple and sensitive procedure for testing various chemicals affecting DNA repair is presented. Cells, either labelled with [3H]thymidine or [14C]thymidine, were drug-treated or used as references cells. Both cell populations were irradiated with 5 Gy. The number of DNA breaks were determined, after mixing of drug-treated and reference cells of different labelling, at various intervals by the DNA unwinding technique and the drug-dependent DNA breaks were calculated. The drugs benzamide, 3-aminobenzamide, novobiocin and 9-beta-D-arabinofuranosyladenine (araA), all known to affect DNA repair, were used to study their effect on the number of DNA strand breaks with the presented technique. It was found that the assay improved the accuracy in determining the influence of DNA repair inhibitors compared to indirect measurements.