Superoxide free radicals are produced in glyoxysomes

The production of superoxide free radicals in pellet and supernatant fractions of glyoxysomes, specialized plant peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons, was investigated. Upon inhibition of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of O₂⁻ radicals and this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated the generation of superoxide radicals. Superoxide production by purines was due to xanthine oxidase, which was found predominantly in the matrix of glyoxysomes. The generation of O₂⁻ radicals in glyoxysomes by endogenous metabolites suggests new active oxygen-related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism.     

Neutron-induced free radicals in oriented DNA

Samples of oriented DNA containing 30 per cent water were irradiated with neutrons at 77 K. The electron spin resonance (e.s.r.) spectra obtained from these irradiated DNA samples show that the formation of radicals is different when the incident neutrons are parallel or perpendicular to the DNA helix. When the incident neutrons are perpendicular to the DNA helix the e.s.r. spectra of thymine and guanine ionic radicals (T-., G+.) are observed. An additional e.s.r. spectrum corresponding to the hydrogen addition radical on thymine (TH.) is observed when the incident neutrons are parallel to DNA helix. The TH. radical appears to be formed by protonation of T-. .     

Thymine lesions produced by ionizing radiation in double-stranded DNA

A DNA glycosylase which catalyzes the release of thymine residues damaged by ring saturation, fragmentation, or ring contraction from double-stranded DNA has been used to characterize such base derivatives in gamma-irradiated DNA. It is shown by chromatographic analysis that irradiation of DNA in neutral solution generates the ring-saturated forms cis-thymine glycol, trans-thymine glycol, and a monohydroxydihydrothymine, probably 6-hydroxy-5,6-dihydrothymine. The latter compound is only observed after irradiation under hypoxic conditions. The ring-contracted thymine derivative 5-hydroxy-5-methylhydantoin is also formed, and it is the major lesion after irradiation of DNA under O2. Ring-fragmented products such as methyltartronylurea were only generated in small quantities. Isolation and analysis of the DNA from gamma-irradiated human cells also revealed the formation of ring-saturated thymine derivatives, but 5-hydroxy-5-methylhydantoin was not found in this case.     

Double lesions are produced in DNA oligomer by ionizing radiation and by metal-catalyzed H2O2 reactions

It was demonstrated previously that double lesions are produced in DNA by ionizing radiation. These double lesions consist of adjacent nucleotides each bearing a modified base. The goal of the present investigation was to determine whether Fenton chemistry can generate the same kind of lesions. DNA oligomers were exposed to metal-catalyzed H(2)O(2) reactions, and the products were characterized by chromatography and by mass spectrometry. Double lesions are produced by this treatment in which deoxyguanosine is oxidized to 8-oxo-7,8-dihydrodeoxyguanosine and an adjacent pyrimidine nucleoside is degraded to a formamido remnant.     

Modification of DNA bases in mammalian chromatin by radiation-generated free radicals

Modification of DNA bases in mammalian chromatin in aqueous suspension by ionizing radiation generated free radicals was investigated. Argon, air, N2O, and N2O/O2 were used for saturation of the aqueous system in order to provide different radical environments. Radiation doses ranging from 20 to 200 Gy (J.kg-1) were used. Thirteen products resulting from radical interactions with pyrimidines and purines in chromatin were identified and quantitated by using the technique of gas chromatography/mass spectrometry with selected-ion monitoring after acidic hydrolysis and trimethylsilylation of chromatin. The methodology used permitted analysis of the modified bases directly in chromatin without the necessity of isolation of DNA from chromatin first. The results indicate that the radical environment provided by the presence of different gases in the system had a substantial effect on the types of products and their quantities. Some products were produced only in the presence of oxygen, whereas other products were detected only in the absence of oxygen. Products produced under all four gaseous conditions were also observed. Generally, the presence of oxygen in the system increased the yields of the products with the exception of formamidopyrimidines. Superoxide radical formed in the presence of air, and to a lesser extent in the presence of N2O/O2, had no effect on product formation. The presence of oxygen dramatically increased the yields of 8-hydroxypurines, whereas the yields of formamidopyrimidines were not affected by oxygen, although these products result from respective oxidation and reduction of the same hydroxyl-adduct radicals of purines. The yields of the products were much lower than those observed previously with DNA.     

Effects by doxorubicin on the myocardium are mediated by oxygen free radicals

We hypothesized that doxorubicin (DOX) induces cardiotoxicity of myocardium via oxygen radicals. The present study is aimed at examining the membrane alterations by oxygen radicals generated by DOX in adult rats and cultured neonatal myocytes. Our results showed that DOX 1) decreased beta-adrenoceptor (BAR) density in the cell membrane, 2) increased the membrane permeability of cultured neonatal rat myocytes and 3) altered the ultrastructure of myofibrils and subplasmalemmal actin networks. These effects were reproducible by exogenous hydrogen peroxide. The antioxidant melatonin (MLT) inhibited enzyme leakage and peroxidation in a concentration-dependent manner. It is concluded that DOX induces cardiotoxicity through lipid peroxidation and melatonin is an effective antioxidant against the reactive oxygen intermediates generated by DOX.     

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH⁻ substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.     

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH- substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.     

Substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae: excision of guanine lesions produced in DNA by ionizing radiation- or hydrogen peroxide/metal ion-generated free radicals.

We have investigated the substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae (yOgg1 protein) for excision of modified DNA bases from oxidatively damaged DNA substrates using gas chromatography/isotope dilution mass spectrometry. Four DNA substrates prepared by treatment with H2O2/Fe(III)-EDTA/ascorbic acid, H2O2/Cu(II) and gamma-irradiation under N2O or air were used. The results showed that 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were efficiently excised from DNA exposed to ionizing radiation in the presence of N2O or air. On the other hand, 8-OH-Gua and FapyGua were not excised from H2O2/Fe(III)-EDTA/ascorbic acid-treated and H2O2/Cu(II)-treated DNA respectively. Fourteen other lesions, including the adenine lesions 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine, were not excised from any of the DNA substrates. Kinetics of excision significantly depended on the nature of the damaged DNA substrates. The findings suggest that, in addition to 8-OH-Gua, FapyGua may also be a primary substrate of yOgg1 in cells. The results also show significant differences between the substrate specificities of yOgg1 protein and its functional analog Fpg protein in Escherichia coli.     

Characterization of DNA lesions produced by HgCl2 in cell culture systems

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.     

Radiation-induced free radicals in DNA, DNA level in tissues and tissue radiosensitivity

Radiochemical yields (G-values) of H-adducts of thymine bases of DNA (TH) in frozen (77 K) gamma-irradiated mouse and rat tissues were measured. The content of DNA and the number of TH radicals formed in DNA mass of 10(12)D at a dose of 1 Gy (beta parameter) were determined for each of the studied tissue. It was shown that beta parameter, which indicated DNA in situ radiosensitivity, was different for different tissues: it was higher for radiosensitive tissues. The possible causes of the effect observed are discussed.     

Role of oxygen-derived free radicals in hemorrhagic shock-induced gastric lesions of rats

This study was designed to determine whether oxygen-derived free radicals play a role in the pathogenesis of gastric lesions produced by hemorrhagic shock in the rat. Allopurinol (Zyloric), an inhibitor of xanthine oxidase (responsible for the formation of superoxide radicals) and MTDQ-DA (Kontrad), a synthetic antioxidant of dihydroquinoline type were used. In the anesthetized rat 0.1 N HCl was instilled into the stomach and the rat was bled to reduce the blood pressure to 30 mmHg for 20 min. The blood shed was retransfused. Twenty min later the stomach was removed. The area of gastric mucosal lesions were measured, the activity of endogenous peroxidase was examined histochemically and a histological grading was made. Both allopurinol and MTDQ-DA significantly protected against hemorrhagic shock-induced gastric lesions and peroxidation. These results suggest that oxygen-derived free radicals play an important role in the formation of gastric lesions produced by ischemia plus 0.1 N HCl.     

Proteins are major initial cell targets of hydroxyl free radicals

The principal aim of the current study was to identify the initial cell targets of hydroxyl free radicals. Our recent report showed that proteins were oxidized before lipids in U937 cells exposed to peroxyl radicals. Extending this finding, we investigated whether a similar oxidation sequence occurs in other lines of cells, whether hydroxyl radicals can also initiate cell protein oxidation, and whether DNA fragmentation is an early event in radical-induced cell damage. Mouse myeloma Sp2/0-Ag14 and U937 cells were exposed to hydroxyl radicals generated in solution by gamma irradiation and the formation of protein peroxides measured by a ferric-xylenol orange assay. No lipid peroxidation or DNA damage was evident by the time of significant formation of protein peroxides. DNA fragmentation was detectable after prolonged incubation at 37 degrees C and was characteristic of enzymatic action rather than of random scission by the radicals. Yields of protein hydroperoxides in the irradiated cells were independent of composition of the medium, suggesting that only the radicals produced within the cells or immediately near the cell surface were effective in oxidizing the cell proteins. The results are consistent with the hypothesis that proteins are major initial targets of free radicals in cells and suggest that treatments leading to the prevention of protein oxidation or to harmless reduction of protein peroxides is likely to result in alleviation of radical-induced biological damage.     

Cell damage by oxygen free radicals

The exposure of isolated and cultured cells to oxygen free radicals generated extracellularly or intracellularly during the metabolism of foreing compounds results in the development of damage that eventually lead to cell death. Multiple mechanisms are involved in these cytopathological processes, including direct attack of free radicals to macromolecules essential for cell life, as well as indirect activation of catabolic processes such as proteases, endonucleases and phospholipases. A key role in triggering these indirect events is played by Ca²⁺ whose cytosolic concentration during oxidative stress raises well above the physiological limits.