Enhanced gamma-glutamyl transpeptidase expression and superoxide production in Mpv17-/- glomerulosclerosis mice.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.

Chemical studies of proanthocyanidins and hydrolyzable tannins.

We investigated a number of natural polyphenols representing flavan-3-ols, gallotannins, and ellagitannins with regard to their antioxidant potential. For this purpose we used pulse radiolysis to determine scavenging rate constants with hydroxyl radicals and decay rates of the respective aroxyl radicals and EPR spectroscopy to identify the radicals after in situ oxidation. Using NMR spectroscopy, we could confirm phenolic coupling reactions of epigallocatechin gallate and pentagalloyl glucose after radical-induced oxidation.

Electron paramagnetic resonance studies of radical species of proanthocyanidins and gallate esters.

The polyphenols present in green tea or red wine comprise both regular flavon(ol)s and proanthocyanidins, i.e., derivatives of flavan-3-ols, whose distinct antioxidative potential is of great importance for explaining the beneficial effects of these nutrient beverages. Using EPR spectroscopy, we investigated radical structures obtained after oxidation of the parent compounds either by horseradish peroxidase/hydrogen peroxide or after autoxidation in moderately alkaline solutions. Both proanthocyanidins (monomers of condensed tannins, e.g., (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate, Pycnogenol) and gallate esters (hydrolyzable tannins, e.g., propylgallate, beta-glucogallin, pentagalloyl glucose and tannic acid) yielded predominantly semiquinone structures derived from the parent catechol or pyrogallol moieties. Evidence for a time-dependent oligomerization was obtained for (-)-epigallocatechin gallate, supporting our hypothesis that o-quinones formed from the initial semiquinone disproportionation are prone to nucleophilic addition reactions. Such phenolic coupling reactions would retain the numbers of reactive catechol/pyrogallol structures and thus the antioxidative potential. We therefore propose that proanthocyanidins are superior antioxidants as compared to flavon(ol)s proper, whose quinones are more likely to redox-cycle and act as prooxidants.

Radiation chemistry of physiological saline reinvestigated: evidence that chloride-derived intermediates play a key role in cytotoxicity.

Contrary to common belief, hydrogen peroxide (H2O2) and hypochlorite (HOCl) are not produced continuously and independently during the irradiation of buffer solution containing chloride. Different buildup and decay reactions are involved in a complex interaction of these substances during irradiation. Which of the species predominates is determined by the parameters of the solution. The amount of either compound detectable after irradiation depends on the dissolved gas (O2, N2O or N2), on the pH value and to some extent on the presence of catalytic metals: Under slightly acidic conditions, low oxygen content and high generation rates of OH radicals, the only detectable species is hypochlorite; at high oxygen content and at pH values in the physiological range, hydrogen peroxide is the main detectable product. However, H2O2 and HOCl react with each other in a pH-dependent way, yielding the stable products O2 and Cl-. This reaction limits the expected lifetime of both species in aqueous solution to some tens of seconds. Therefore, analysis of the sample solution after irradiation determines only the substance that was present in greater relative concentration at the termination of irradiation. Such analysis, however, does not allow conclusions about the processes that occurred during irradiation. We have investigated the decay and formation reactions of H2O2 and HOCl under all relevant irradiation conditions and found evidence that the formation and further reaction of HOCl-, the precursor of HOCl, is of central importance even in cases where no significant amounts of H2O2 or HOCl are detectable after irradiation. We discuss the consequences of these results for the cytotoxicity observed after irradiation of cells suspended in physiological saline and conclude that analogous processes must also be relevant for irradiations under in vivo conditions.

Radiation-induced cell killing is highly dependent upon buffer treatment (filtration compared to autoclaving) due to metal-catalyzed formation of hypochlorite: a cautionary note.

Buffer solutions used in experiments in radiation biology may be sterilized by either autoclaving or filtration. We show here that for phosphate-buffered saline such differences in buffer treatment may result in widely differing dose-effect curves for cell killing. The temperature-dependent transformation of monophosphate ions into di- or polyphosphate evidently proceeds to an appreciable extent upon autoclaving the buffers at 120 degrees C for 10 to 20 min. This increases the capability of the buffer to chelate spurious metal contaminations and, as a consequence, to reduce the amount of cytotoxic hypochlorite being produced. Depending on conditions of buffer treatment we have observed dose modification factors for the colony-forming ability of yeast cells up to the order of 3. Thus effects due to buffer treatment might easily outweigh the effect which the experiment was originally designed to determine. We strongly advise, therefore, that results of parallel sets of experiments in which different methods of buffer sterilization have been used should not be compared directly.

Marchantins and related polyphenols from liverwort: physico-chemical studies of their radical-scavenging properties.

Structurally unique macrocyclic phenols from liverwort, i.e., marchantins and related substances, were studied for their antioxidative potential using pulse-radiolytic and EPR-spectroscopic techniques. The generally diffusion-controlled rate constants for scavenging of azide radicals as a model electrophilic species and the sufficiently slow bimolecular decay confirm their antioxidative potential. Transient spectra after pulse radiolysis and the EPR spectra both demonstrate the internal strain of the macrocyclic ring. One compound, Perrottetin D, furthermore gave proof to the hitherto only kinetically verifyable superior radical-scavenging capability of the aroxyl radical derived from a phenolic antioxidant.

Lack of an association between serum vitamin E and myocardial infarction in a population with high vitamin E levels.

The antioxidant effects of vitamin E may protect low density lipoproteins from peroxidation and thus inhibit the development of arteriosclerosis. Inverse associations between vitamin E levels and coronary heart disease have been reported from cross-sectional and ecologic studies. In the population-based MONICA Augsburg cohort (2023 men, 1999 women, age 25-64 years at baseline in 1984, 93% of whom were reexamined in 1987/88) we investigated the relationship between serum vitamin E concentrations and the risk of subsequent myocardial infarction (MI). Between 1984 and 1991, 46 cases of fatal and non-fatal myocardial infarction from this cohort were recruited for a nested case-control study. Four controls were sampled from the cohort for each case of MI with matching for age, sex, and total cholesterol. There were no marked differences between cases and their matched controls in the means of vitamin E concentrations (33.9 mumol/l vs. 32.8 mumol/l, P = 0.37) or in the mean vitamin E/total cholesterol ratios (4.89 mumol/mmol vs. 4.82 mumol/mmol, P = 0.75). The covariate adjusted relative risk (RR) for fatal plus non-fatal MI in the lowest tertile of vitamin E relative to the upper two tertiles was 0.72 (90% confidence interval: 0.33-1.57). Likewise, for the lowest tertile of the ratio (vitamin E/total cholesterol) the RR was 0.81 (0.42-1.56). The association was not modified by history of previous coronary heart disease, fatality of MI, temporal distance of MI onset from vitamin E determinations, or season.(ABSTRACT TRUNCATED AT 250 WORDS)

Radical intermediates during the oxidation of nitropropanes. The formation of NO2 from 2-nitropropane, its reactivity with nucleosides, and implications for the genotoxicity of 2-nitropropane.

The chemistry of the nonenzymatic oxidation of the rat liver carcinogen, 2-nitropropane, and its anionic form, propane-2-nitronate, was investigated using pulse radiolysis and EPR/spin trapping with 3,5-dibromo-4-nitrosobenzenesulfonic acid as the trapping agent. The results suggest that, following initial oxidation to a secondary alkyl radical, propane-2-nitronate is effectively degraded in a peroxidative chain reaction with the intermediary formation of peroxyl and NO2.radicals. The latter radical was shown to react appreciably fast with ribonucleosides, deoxyribonucleosides, and guanosine nucleotides. It is proposed that nonenzymatic formation of NO2.radicals after enzymatic oxidation of propane-2-nitronate to the corresponding secondary alkyl radical accounts for the induction of DNA damage observed after exposure of rats to 2-nitropropane.

Characterization of Cat-2t, a radiation-induced dominant cataract mutation in mice.

A dominant cataract mutation was detected recently among the offspring of x-ray-irradiated male mice. The mutation, which causes total lens opacity, has provisionally been designated by the gene symbol Cat-2t. In the lenses of heterozygous and homozygous Cat-2t mutants, the epithelial and fiber cells were swollen and the lens capsule was ruptured. The histologic analysis demonstrated a complete destruction of the cellular organization of the lens, which might be caused by its altered developmental processes. The data derived from biochemical investigations indicate that biochemistry of the cataractous Cat-2t lenses is affected: the osmotic state as indicated by the increased water content and increased Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity; the energy state as indicated by the decreased adenosine triphosphate (ATP) concentration; and the redox state as indicated by the enhanced content of oxidized glutathione. Additionally, the lenticular protein composition is altered because of the presence of vimentin in the water-soluble fraction. This cannot be explained by the enhanced crosslinking activity of transglutaminase. The changes of the osmotic, energy, and redox states are considered to be secondary in relation to the altered lenticular development. In contrast, the variations concerning vimentin and transglutaminase might be a biochemical indication of the changed development. Possible similarities to other dominantly expressed murine cataract mutants are discussed.

Biochemical analysis of young rats homozygous for the cataract mutation cat.

Cataractogenesis was studied in young rats homozygous for the radiation-induced recessive cataract mutation cat. Homozygous cat/cat rats have reduced body weight (about two-thirds of the wild type) when 3 weeks old. The litter size is also diminished to about two-thirds of the wild type. For lens-specific parameters, as compared with homozygous wild type, the wet weight of the cataractous lenses is reduced, although the concentration of water-soluble lens proteins per wet weight is the same. No major alterations could be detected in the pattern of the water-soluble lens proteins separated by isoelectric focusing or gel electrophoresis run with or without mercaptoethanol. Additionally, no statistically significant alterations could be detected in the biochemical parameters of the lens used as indicators for osmotic stress (water content of the lens and the Na+-K+-dependent ATPase), for the energy state (ATP) and for the redox state (oxidized glutathione and superoxide dismutase). In contrast, the activity of transglutaminase is significantly enhanced in lenses as well as in the liver of young cat-rats, which might be understood as a biochemical marker for alterations in the developmental program. Cataractogenesis in the cat-rat is, therefore, suggested to be part of a syndrome including dwarfism and reduced litter size.

Oxidative stress and inherited cataracts in mice.

To determine whether an unbalanced redox state might accompany the development of particular inherited mouse cataracts, the lenticular content of oxidized glutathione (GSSG) and the activity of superoxide dismutase (SOD) were chosen as markers. For wild-type lenses, an enhanced GSSG content could be observed in females as compared to males. Such a sex effect could not be detected for the SOD activity. In the mutants, GSSG content in cataractous lenses was found to be enhanced in 2 of 7 cases; the increases in other mutants were not significant. Changes of the SOD activity were even less consistent and only a random correlation of GSSG content and SOD activity with cataractogenesis could be deduced.

Determination of sulfite radical (SO.3-) reaction rate constants by means of competition kinetics.

The sulfite radical anion (SO.3-) was found to react rapidly with the flavonoid quercetin (k = 2.5 x 10(8) dm3mol-1s-1) and the carotenoids crocin (k = 1.0 x 10(9) dm3mol-1s-1) and crocetin (k = 1.5 x 10(9) dm3mol-1s-1). The reactions can easily be monitored due to the strong absorptions of the substrates and, in the case of quercetin, the formation of a strongly absorbing transient species. Using these substances, we determined by means of competition kinetics rate constants of SO.3- reactions with nucleic acid components, polyunsaturated fatty acids, and glutathione.

Formation of ethylene from methionine. Reactivity of radiolytically produced oxygen radicals and effect of substrate activation.

Ethylene was determined by gas chromatography after reaction of radiolytically produced OH and O2- radicals with methionine, methionine + pyridoxal phosphate and S-adenosyl-methionine (SAM). Both oxygen radicals, alone or in combination, liberate ethylene from methionine and methionine/pyridoxal phosphate. From SAM ethylene was primarily produced by the combined attack of OH nad H2O2 or O2-.

Oxygen activation in isolated chloroplasts. Mechanism of ferredoxin-dependent ethylene formation from methionine.

Low-potential electron acceptors of photosystem I of chloroplast lamellae produce superoxide anions (0-2) and hydrogen peroxide by autoxidation, but have no effect on ethylene formation from methionine; equimolar amounts of ferredoxin are less active in photosynthetic O-2 and H2O2 production but strongly stimulate ethylene production from methionine. 2. Ten to fifty units of superoxide dismutase inhibit fifty to two hundred units of superoxide dismutase stimulate ethylene formation from methionine by chloroplast lamellae in the presence of ferredoxin. This stimulation is stronger at pH 7.0 than at pH 7.8. Catalase inhibits ethylene formation from methionine. 3. Pulse-radiolytic production of nitrite (NO-2) from hydroxylamine, initiated by hydroxyl radicals (.OH) or O-2, shows no difference in the presence or absence of ferredoxin, nor do the decay kinetics of O2. 4. From the above observations and from model reactions (xanthine/xanthine oxidase; iron salts in the presence of H2O2), it is concluded that reduced ferredoxin in the presence of H2O2 forms a Fenton-type oxidizing species for methionine, generating ethylene in the presence of pyridoxal phosphate. 5. Inhibitory effects of both superoxide dismutase and catalase in oxygen-dependent reactions need not necessarily indicate the participation of the 'Haber-Weiss' reaction.