Reevaluation of the 2-deoxyribose assay for determination of free radical formation

Background

The 2-deoxyribose (2-DR) degradation assay is a widely used test for determining anti/pro-oxidant properties of molecules and plant extracts. Most reports use reaction blanks omitting 2-DR or thiobarbituric acid (TBA). However, when studying Fe(II)-mediated reactions, we verified that these blanks are not appropriate. Fe(III) – a product of these reactions – causes a relevant artifact in the assay, where 2-DR is oxidized by Fe(III).

Method

2-DR degradation was determined at 532 nm as TBA-reactive substances.

Results and conclusion

HPLC determinations indicated that Fe(III) added after or before TBA generates considerable amounts of malondialdehyde (2-DR degradation product) in comparison with assays employing Fenton reagents or Fe(II) autoxidation. Addition of catalase and thiourea has no effect on Fe(III)-induced 2-DR degradation indicating lack of ROS involvement. This Fe(III)-mediated 2-DR damage is dependent on iron and 2-DR concentrations, but not on H₂O₂, buffer composition or iron-chelators. Depending on the assay conditions Fe(III)-interference accounts for 20% to 90% of 2-DR degradation mediated by Fe(II).

Significance

A new reaction blank is proposed herein–based on the use of Fe(III)–for the assay. The lack of such correction has caused the underestimation of antioxidant capacity of various compounds in many studies in the last 2 decades.     

The effect of electromagnetic field exposure on the formation of DNA lesions

Abstract

In an attempt to determine whether electromagnetic field (EMF) exposure might lead to DNA damage, we exposed SnCl₂-treated pBR322 plasmids to EMF and analysed the resulting conformational changes using agarose gel electrophoresis. An EMF-dependent potentiation of DNA scission (i.e. the appearance of relaxed plasmids) was observed. In confirmation of this, plasmids pre-exposed to EMF also were less capable of transforming Escherichia coli. The results indicate that EMF, in the presence of a transition metal, is capable of causing DNA damage. These observations support the idea that EMF, probably through secondary generation of reactive oxygen species, can be clastogenic and provide a possible explanation for the observed correlation between EMF exposure and the frequency of certain types of cancers in humans.     

The effect of electromagnetic field exposure on the formation of DNA lesions

In an attempt to determine whether electromagnetic field (EMF) exposure might lead to DNA damage, we exposed SnCl2-treated pBR322 plasmids to EMF and analysed the resulting conformational changes using agarose gel electrophoresis. An EMF-dependent potentiation of DNA scission (i.e. the appearance of relaxed plasmids) was observed. In confirmation of this, plasmids pre-exposed to EMF also were less capable of transforming Escherichia coli. The results indicate that EMF, in the presence of a transition metal, is capable of causing DNA damage. These observations support the idea that EMF, probably through secondary generation of reactive oxygen species, can be clastogenic and provide a possible explanation for the observed correlation between EMF exposure and the frequency of certain types of cancers in humans.     

The effect of free radical derived hydroxyicosatetraenoic acids on hexose transport in the human polymorphonuclear leukocyte

The following racemic hydroxyicosatetraenoic acids were prepared and assayed for their ability to stimulate hexose transport in human polymorphonuclear leukocytes: 15-, 12-, 11-, 9-, 8-, and 5-hydroxyicosatetraenoic acids. The compounds were isolated from reduced, autoxidized arachidonic. The results demonstrate that only the 12-, and 5-hydroxyicosatetraenoic acids are biologically active, inducing half-maximal responses at 820 and 176 nM, respectively. Thus, the bioactions of hydroxicosatetraenoates are crucially dependent upon the position of the hydroxy residue. Response to both hydroxyicosatetraenoates was effectively blocked by two inhibitors of arachidonic acid metabolism: nordihydroguaiaretic acid and indomethacin. A third arachidonic acid antimetabolite, 5,8,11,14-eicosatetraynoic acid, completely inhibited the response to 12-HETE but caused only partial inhibition of the response to 5-HETE.     

The crypto-OH radical in the damage of DNA by bleomycin-Fe2+?

1. Effects of various OH scavengers, superoxide dismutase and catalase on the formation of malondialdehyde-like products from DNA by bleomycin-Fe2+ were studied. In no case was a protective effect observed. 2. These results can be interpreted on the basis that a crypto-OH radical mediates the damage to DNA by bleomycin-Fe2+.     

Oxidative damage to fibronectin. II. The effect of H2O2 and the hydroxyl radical.

The effect of H2O2 and the hydroxyl radical (.OH) on fibronectin was investigated. .OH was generated in three ways: (i) by radiolysis with 60Co under N2O, or by the Fenton system using either (ii) equimolar Fe(2+)-EDTA and H2O2 or (iii) H2O2 and catalytic amounts of Fe(2+)-EDTA recycled with ascorbate. Each system had a different effect. H2O2 alone caused no changes, even at an 800-fold molar excess. Radiolytic .OH caused a rapid loss of tryptophan fluorescence, an increase in bityrosine fluorescence, and extensive crosslinking. The Fenton system using Fe-EDTA, H2O2, and ascorbate caused a loss in tryptophan fluorescence, a smaller increase in bityrosine than was seen with radiolytic .OH, and a threefold increase in carbonyl groups. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis fragmentation of fibronectin was seen. In contrast, when .OH was generated with equimolar Fe-EDTA and H2O2, the only change was a small increase in bityrosine fluorescence at the highest dose of oxidant. None of the systems used affected cysteine. All the changes except the loss of tryptophan by radiolytic .OH were completely inhibited with mannitol. The differences seen with radiolytic .OH and the Fe-EDTA, H2O2, ascorbate system were not solely due to O2 in the latter system since similar results were obtained under N2. The differences between radiolytic .OH and the Fenton systems could be partly due to the components of the latter systems reacting with .OH and thus competing with fibronectin. Our results demonstrate that the extent and type of fibronectin damage by .OH is dependent on the mode of radical generation.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

DNA alterations induced by the carbon-centered radical derived from the oxidation of 2-phenylethylhydrazine

The possible significance of carbon-centered radicals in hydrazine-induced carcinogenesis is explored by studies of the interaction between the 2-phenylethyl radical and DNA. The radical is efficiently generated during oxidation of phenelzine (2-phenylethylhydrazine) promoted by oxyhemoglobin or ferricyanide, as demonstrated by spin-trapping experiments and analysis of the reaction products. In the ferricyanide promoted oxidation, ethylbenzene formation accounts for about 40% of the initial drug concentration, from 5 to 100 mM phenelzine. By contrast, product formation in the presence of oxyhemoglobin depends on the enzyme concentration due to the fact that the prosthetic heme is destroyed during catalytic turnover. Covalent binding of the 2-phenylethyl radical to oxyhemoglobin is demonstrated by experiments with 2-[3H]phenelzine, where tritium incorporation to the protein is inhibited by the spin-trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. The 2-phenylethyl radical is also able to alkylate DNA as suggested by electrophoretic studies with plasmid DNA, and proved by experiments with 2-[3H]-phenelzine. The carbon-centered radical has a preference for attacking guanine residues as demonstrated by the use of sequencing techniques with 32P-DNA probes. The results indicate that the 2-phenylethyl radical is an important product of phenelzine oxidation and that this species can directly damage protein and DNA.     

The effect of exercise training modality on serum brain derived neurotrophic factor levels in individuals with type 2 diabetes

Introduction

Brain derived neurotrophic factor (BDNF) has been implicated in memory, learning, and neurodegenerative diseases. However, the relationship of BDNF with cardiometabolic risk factors is unclear, and the effect of exercise training on BDNF has not been previously explored in individuals with type 2 diabetes.

Methods

Men and women (N = 150) with type 2 diabetes were randomized to an aerobic exercise (aerobic), resistance exercise (resistance), or a combination of both (combination) for 9 months. Serum BDNF levels were evaluated at baseline and follow-up from archived blood samples.

Results

Baseline serum BDNF was not associated with fitness, body composition, anthropometry, glucose control, or strength measures (all, p>0.05). Similarly, no significant change in serum BDNF levels was observed following exercise training in the aerobic (−1649.4 pg/ml, CI: −4768.9 to 1470.2), resistance (−2351.2 pg/ml, CI:−5290.7 to 588.3), or combination groups (−827.4 pg/ml, CI: −3533.3 to1878.5) compared to the control group (−2320.0 pg/ml, CI: −5750.8 to 1110.8). However, reductions in waist circumference were directly associated with changes in serum BDNF following training (r = 0.25, p = 0.005).

Conclusions

Serum BDNF was not associated with fitness, body composition, anthropometry, glucose control, or strength measures at baseline. Likewise, serum BDNF measures were not altered by 9 months of aerobic, resistance, or combination training. However, reductions in waist circumference were associated with decreased serum BDNF levels. Future studies should investigate the relevance of BDNF with measures of cognitive function specifically in individuals with type-2 diabetes.     

Generation of 5-(2'-deoxycytidyl)methyl radical and the formation of intrastrand cross-link lesions in oligodeoxyribonucleotides

Hydroxyl radical is one of the major reactive oxygen species (ROS) formed from γ-radiolysis of water or Fenton reaction, and it can abstract one hydrogen atom from the methyl carbon atom of thymine and 5-methylcytosine to give the 5-methyl radical of the pyrimidine bases. The latter radical can also be induced from Type-I photo-oxidation process. Here, we examined the reactivity of the independently generated 5-(2′-deoxycytidyl)methyl radical (I) in single- and double-stranded oligodeoxyribonucleotides (ODNs). It was found that an intrastrand cross-link lesion, in which the methyl carbon atom of 5-methylcytosine and the C8 carbon atom of guanine are covalently bonded, could be formed from the independently generated radical at both GmC and mCG sites, with the yield being much higher at the former site. We also showed by LC-MS/MS that the same cross-link lesions were formed in mC-containing duplex ODNs upon γ irradiation under both aerobic and anaerobic conditions, and the yield was ~10-fold higher under the latter conditions. The independently generated radical allows for the availability of pure, sufficient and well-characterized intrastrand cross-link lesion-bearing ODN substrates for future biochemical and biophysical characterizations. This was also the first demonstration that the coupling of radical I with its 5′ neighboring guanine can occur in the presence of molecular oxygen, suggesting that the formation of this and other types of intrastrand cross-link lesions might have important implications in the cytotoxic effects of ROS.     

The effect of human serum transferrin and milk lactoferrin on hydroxyl radical formation from superoxide and hydrogen peroxide

The effect of transferrins on hydroxyl radical formation from the superoxide anion and hydrogen peroxide generated by the xanthine-xanthine oxidase system has been studied by EPR using 5,5-dimethyl-1-pyrroline N-oxide as a spin trap. Neither diferriclactoferrin nor diferrictransferrin were found capable of promoting hydroxyl radical formation via the Haber-Weiss reaction even in the presence of EDTA in concentrations up to 1 mM. Activity observed by other authors may have been due to the presence of extraneous iron or an active protein impurity. Partially saturated transferrin and lactoferrin present in normal subjects may protect cells from damage by binding iron that might catalyze hydroxyl radical formation from superoxide and hydrogen peroxide. In any event, the hydroxyl radical formation observed in active neutrophils during phagocytosis cannot be associated with lactoferrin activity.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Microbial production of 2-deoxyribose 5-phosphate from acetaldehyde and triosephosphate for the synthesis of 2'-deoxyribonucleosides

2-Deoxyribose 5-phosphate was produced from acetaldehyde and dihydroxyacetone phosphate via D-glyceraldehyde 3-phosphate by Klebsiella pneumoniae B-4-4 through deoxyriboaldolase- and triosephosphate isomerase-catalyzing reactions. Under the optimum conditions, 98.7 mM 2-deoxyribose 5-phosphate was produced from 200 mM acetaldehyde and 117 mM dihydroxyacetone phosphate in 2 h with a molar yield of 84%. The 2-deoxyriobse 5-phosphate produced was directly transformed to 2'-deoxyribonucleoside by phosphopentomutase- and nucleoside phosphorylase-catalyzing reactions.     

The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells

Background. Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. Methods. Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. Results. DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5–75.8), n = 18 and 21% (11.9–29.8), n = 65, respectively, p < .001]. Intermediate levels were found in reactive gastritis [55.5% (41.3–71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3–60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3–51.0), p = .007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = –.92 and –.88 for normal mucosa and H. pylori gastritis, respectively). Conclusions. Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.     

Hydroxyl radical formation from the auto-reduction of a ferric citrate complex.

When a ferric citrate complex is prepared from citric acid and ferric chloride, and the pH value left unchanged, a reduction of the iron moiety takes place. Within several hours a substantial yield of ferrous ions can be detected in the solution. When placed in a phosphate buffer pH 7.0 with a suitable detector molecule, oxidative damage to the detector molecule can be observed. Thus, deoxyribose is degraded with the release of thiobarbituric acid-reactive material and benzoate is hydroxylated to form fluorescent dihydroxy products. Damage can be prevented by scavengers of the hydroxyl radical such as mannitol, formate the thiourea, by catalase and by the protein caeruloplasmin, suggesting that Fenton chemistry occurs leading to the formation of hydroxyl radicals.     

The formation of chromosome aberrations from single-strand damage in irradiated DNA

Consideration is given to molecular structures by which chromosome aberrations can arise from single-strand damage in irradiated DNA.