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Biomedical subjects

R A Floyd

Publications and source records attributed to R A Floyd.

At least 73 records · Page 4Linked to original sources
PubMed1996

Increased oxidative stress and decreased antioxidant defenses in mucosa of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation whose cellular components are capable of oxidative respiratory bursts that may result in tissue injury. Mucosal biopsies were analyzed for protein carbonyl content (POPs), DNA oxidation products [8-hydroxy-2'-deoxyguanosine (8-OHdG)], reactive oxygen intermediates (ROIs), trace metals (copper, zinc, and iron) and superoxide dismutase (Cu-Zn SOD). In Crohn's disease biopsies, there was an increase in ROIs, POPs, 8-OHdG, and iron, while decreased copper and Cu-Zn SOD activity were found in inflamed tissues compared to controls. For ulcerative colitis, there was an increase in ROIs, POPs, and iron in inflamed tissue compared to controls, while decreased zinc and copper were observed. An imbalance in the formation of reactive oxygen species and antioxidant micronutrients may be important in the pathogenesis and/or perpetuation of the tissue injury in IBD and may provide a rationale for therapeutic modulation with antioxidants.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1996

Association between reactive oxygen species and disease activity in chronic hepatitis C.

Reactive Oxygen Species (ROS) may be involved in the damage occurring in the course of chronic HCV infection. Individuals with chronic hepatitis C present increased hepatic levels of malondialdehyde (MDA) and reduced levels of glutathione. To determine whether these observations are associated with serological evidence for ROS injury, MDA and protein carbonyl content (PCC) of serum was determined in 20 HCV positive patients (14 chronic active hepatitis -- CAH and 6 cirrhosis) and 20 controls. Compared to controls, HCV positive subjects had increased levels of MDA (13.33 +/- 0.21 SE ng/ml vs. 9.90 +/- 0.65 P < .05) and PCC (4.74 +/- 0.21 mmol/mg vs 3.68 +/- 0.21, p < .02). Patients with CAH had higher levels than did cirrhotics. Both MDA and PCC correlated with serum ALT levels (r = .792 and r = .818 respectively, p < .001). A common origin for MDA and PCC found in patients with chronic hepatitis C was suggested by the correlation between the two measures (r = .741, p < .001). No correlation were found between MDA or PCC and the hepatic iron content. These data demonstrate that: (1) lipid and protein oxidation occur in chronic hepatitis C, (2) oxidative damage can be demonstrated as increased serum levels of MDA and PCC, and (3) both MDA and PCC levels correlate with disease activity.

Adult↗
PubMed1996

Preferential targeting of oxidative base damage to internucleosomal DNA.

The structure of nuclear chromatin may limit the accessibility of carcinogenic agents to DNA. In the case of oxidative DNA strand cleavage mediated by the physiologically relevant iron chelate, iron-ADP, histone-associated nucleosomal DNA is protected while internucleosomal DNA is susceptible to damage. We now find that the distribution of iron-ADP-generated 8-hydroxydeoxyguanosine, a potentially mutagenic oxidative base change, shows relative targeting to internucleosomal sites (3.5-fold increased oxidative modification of internucleosomal compared with nucleosomal DNA as the minimal degree of enrichment). In contrast, iron-EDTA, which generates hydroxyl radical in the 'fluid phase', does not target internucleosomal DNA. Thus, physiologic iron chelates may promote site-specific damage and thereby be relevant to mechanisms of iron-dependent oxidative mutagenesis and carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1996

Evaluation of nitrone spin-trapping agents as radioprotectors.

The focus of this investigation was to determine whether the nitrone spin-trapping compounds alpha-phenyl-N-tert-butylnitrone (PBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) are radioprotectors. Two methods were used to assess for radioprotection: measurement of oxidative damage to DNA bases and mammalian cell survival assays. Oxidative damage to DNA was quantified by measuring the relative amounts of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) produced by the reaction of hydroxyl radicals (OH.) with 2-deoxyguanosine (dG) after irradiation. PBN, DMPO and POBN, when dissolved in aqueous solutions of either dG or naked salmon sperm DNA, reduced the formation of 8-OH-dG by 137Cs gamma irradiation significantly. The spin-trapping agents, especially PBN at lower concentrations, were more effective in preventing radiation-induced formation of 8-OH-dG in naked DNA than in free dG. These data suggest that PBN, DMPO and POBN act as free radical scavengers which may associate with DNA and afford protection against gamma rays. However, no enhancement of survival was observed when Chinese hamster ovary (CHO) cells were exposed to high non-toxic concentrations of PBN or POBN prior to and during irradiation with 60Co gamma rays and scored for clonogenic survival. DMPO provided only minimal protection from radiation-induced cell killing.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1995

Cytotoxic effects of autoxidative glycation.

Incubation of the RNA phage Q beta at 37 degrees C with a mixture of 100 mM ribose and 10 microM CuSO4 resulted in a complete loss of viable phage after 20 min. This cytotoxic effect required both ribose and cupric ions. There was a direct correlation between the decrease in the percentage of phage survival and: (a) the length of incubation, and (b) the concentrations of both ribose and CuSO4. Addition of the strong chelator diethylenetriaminepentaacetic acid abolished the cytotoxic effect. These results are consistent with an initial production of superoxide free radicals by transition metal catalyzed autoxidation of ribose and Amadori products, followed by dismutation of superoxide to hydrogen peroxide and generation of lethal hydroxyl radicals by the Fenton reaction. RNA isolated from phage incubated with ribose and CuSO4 retained its infectivity, suggesting that the cytotoxic effect may be mediated by a free radical attack on proteinaceous components of the phage through a site specific generation of hydroxyl radicals on protein-bound transition metal ions.

Copper↗
PubMed1994

A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease.

beta-Amyloid is a 39- to 43-amino-acid neurotoxic peptide that aggregates to form the core of Alzheimer disease-associated senile (amyloid) plaques. No satisfactory hypothesis has yet been proposed to explain the mechanism of beta-amyloid aggregation and toxicity. We present mass spectrometric and electron paramagnetic resonance spin trapping evidence that beta-amyloid, in aqueous solution, fragments and generates free radical peptides. beta-Amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes. Also, salicylate hydroxylation assays indicate that reactive oxygen species are generated by the beta-amyloid-(25-35) fragment during cell-free incubation. These results are formulated into a free radical-based unifying hypothesis for neurotoxicity of beta-amyloid and are discussed with reference to membrane molecular alterations in Alzheimer disease.

Alzheimer Disease↗
PubMed1994

Generation of strand breaks and formation of 8-hydroxy-2'-deoxyguanosine in DNA by a Thiol/Fe3+/O2-catalyzed oxidation system.

Strand breaks are produced in pBluescript plasmid DNA by a metal-catalyzed oxidation system composed of Fe3+, O2, and a thiol as an electron donor. Formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in calf thymus DNA was also observed utilizing the same system. DNA damage was observed with the physiological thiols dihydrolipoic acid, glutathione, and cysteine as well as with the synthetic thiols dithiothreitol and 2-mercaptoethanol under a wide range of conditions. Both strand breaks and formation of 8-OH-dG in DNA were temperature dependent, occurring more rapidly at higher temperatures. Sodium azide and mannitol as well as a metal chelator, diethylenetriaminepentaactic acid, decreased strand breaks and 8-OH-dG formation in DNA. Superoxide dismutase did not block damage to DNA, whereas a thiol-dependent protector protein from Saccharomyces cerevisiae and catalase inhibited DNA damage in a concentration-dependent manner. The results of the present study indicate that H2O2 may be generated from a thiol/Fe3+/O2 system and that hydroxyl free radicals may be produced by metal-catalyzed Fenton reactions and may be the ultimate species mediating the DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agents.

Susceptibility of two key protective enzymes, glutathione peroxidase (GPX) and glutathione reductase (GR), to oxidative damage and the possible protective action of spin traps have been studied. Several oxidizing protocols including: (a) Fe(II) or Fe(III)/ascorbate, (b) a singlet oxygen producing system (methylene blue and visible light), (c) ozone, and (d) a hydroxyl radical-generating system (hydrogen peroxide/uv light) have been employed. Our results show that both enzymes are susceptible to oxidative modification and damage as indicated by the loss of activity and formation of carbonyl groups (in the case of GR). Treatment of GR with any of the mentioned oxidants resulted in formation of carbonyl groups and inactivation except when treated with iron, where the observed carbonyl formation was not accompanied with significant activity loss. GPX was inactivated to varying degrees when treated with the mentioned oxidants, but no carbonyls were detected. Ultraviolet exposure per se resulted in inactivation of both enzymes. Presence of the spin traps N-tert-butyl-alpha-phenylnitrone or 5,5'-dimethyl-1-pyroline-N-oxide was effective in protecting the enzymes against oxidation by uv, hydrogen peroxide/uv, and ozone as determined by the preservation of activity and decreased carbonyl content. The degree of protection, however, was found to be specific for each enzyme and for the employed oxidizing system.

Animals↗
PubMed1994

8-Hydroxydeoxyguanosine in DNA from TPA-stimulated human granulocytes.

8-Hydroxydeoxyguanosine (8-OHdG) is now widely used as a sensitive marker of oxidative damage to DNA. When human granulocytes are stimulated with TPA, they release a large quantity of reactive oxygen species (superoxide, hydrogen peroxide) which might be expected to generate hydroxyl radicals (OH.) which in turn could produce 8-OHdG in the DNA. There had been considerable debate as to whether OH. is detectable in stimulated granulocytes; most workers now agree that none can be detected, unless exogenous iron is added. An earlier report had described that 8-OHdG (a marker of OH.) was increased in the DNA of TPA-stimulated, compared to control, granulocytes. We have repeated this experiment and have been unable to reproduce this finding. We conclude that the amount of 8-OHdG produced in the DNA of TPA-stimulated human granulocytes is indistinguishable from that seen in control (unstimulated) cells (less than one 8-OHdG/10(5) dG).

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Intestinal ischemia: reperfusion-mediated increase in hydroxyl free radical formation as reported by salicylate hydroxylation.

Oxygen free radicals may play a pivotal role in the development of the shock-induced inflammatory response syndrome. Hydroxyl free radicals (.OH) react with salicylate (SA) to form 2,5- and 2,3-dihydroxybenzoic acid (DHBA) products. We utilized salicylate hydroxylation to investigate .OH formation during intestinal ischemia/reperfusion injury in male Sprague-Dawley rats. After administering salicylate, the superior mesenteric artery was occluded for 45 min and then allowed to reperfuse for 90 min after declamping. No significant changes in plasma 2,3- and 2,5-DHBA/SA ratios were observed in sham-operated or in animals given intestinal ischemia without reperfusion. A significant increase (p < .05) in arterial, venous, and portal venous 2,5-DHBA/SA ratios occurred 5 min after reperfusion. This increase was prevented by allopurinol as well as by dimethylthiourea (.OH scavenger) pretreatment. 2,3-DHBA was significantly increased (p < .05) in venous and portal venous blood after 30 min of reperfusion, but was not detectable in plasma of allopurinol- and dimethylthiourea-treated rats. These results indicate that hydroxyl free radical formation as reported by SA hydroxylation appears to be important in intestinal ischemia/reperfusion-related tissue injury.

Animals↗
PubMed1993

Methylene blue and rose bengal photoinactivation of RNA bacteriophages: comparative studies of 8-oxoguanine formation in isolated RNA.

Several reactive oxygen species, including singlet oxygen (1O2) and hydroxyl free radical (.OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB). Both 1O2 and .OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA. Evidence that MB-or RB-induced bacteriophage (R17 or Q beta) inactivation and 8-oxoGua formation in RNA result from 1O2 rather than .OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37 degrees C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a .OH generating system, the ultraviolet light irradiation of H2O2; and (iii) deuterium oxide (D2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA. The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation. The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O2-mediated lethal lesions in RNA bacteriophages.

Centrifugation, Density Gradient↗
PubMed1993

Anoxia/reoxygenation induces hydroxyl free radical formation in brain microvessels.

Isolated rat brain microvessels have been utilized to examine whether they produce hydroxyl free radicals if they are subjected to a 10- to 20-min anoxia period followed by a 40-min reoxygenation period. Hydroxyl free radical flux was assessed utilizing salicylate as a trap. The 2,3- and 2,5-dihydroxybenzoic acids (DHBA) products as well as salicylate in the microvessels were quantitated utilizing high-pressure liquid chromatography (HPLC) with electrochemical and fluorescence detection. The results show that a period of anoxia followed by reoxygenation resulted in an enhanced formation of DHBA compared to the normoxic control microvessels. Addition of superoxide dismutase (SOD) and catalase to the microvessels undergoing anoxia decreased the amount of hydroxyl free radicals trapped, suggesting that superoxide and hydrogen peroxide were produced and excreted from the endothelial cell surfaces and then unless quenched reentered the cells to form hydroxyl free radicals within. The amount of 2,5-DHBA formed closely correlated with the amount of 2,3-DHBA formed, indicating that either product can be used to assess hydroxyl free radical flux in brain microvessels.

Animals↗
PubMed1992

Mutations induced by methylene blue plus light in single-stranded M13mp2.

Reactive oxygen species are generated by a variety of cellular processes. These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases. We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response. Lesions produced block the progression of DNA synthesis one base preceding template guanines. In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZ alpha gene of M13mp2 DNA are single-base substitutions opposite template guanines. The most frequent mutations are G----C transversions. The G----T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency. Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E. coli. Instead, an as yet unidentified lesion generated by active oxygen species is a more potent mutagenic event.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Free radical damage to protein and DNA: mechanisms involved and relevant observations on brain undergoing oxidative stress.

Iron mediates damage to proteins and DNA. The mechanisms of damage not only involve iron but also oxygen free radical intermediates. Oxidative damage to DNA causes not only strand breaks, but also formation of specific base adducts, such as 8-hydroxy-2'-deoxyguanosine. Oxidative damage also inactivates certain enzymes such as glutamine synthetase. Novel methods of assessing oxidative damage to tissue, including quantitation of salicylate hydroxylation as an index of hydroxyl free radical flux as well as specific lesions to proteins and DNA, have yielded results that clearly show that ischemia/reperfusion injury to mongolian gerbil brain involves oxidatively damaging events. Aging in gerbil as well as human brain is also associated with increased oxidative damage. Recent novel observations have shown that the spin-trapping agent phenyl alpha-tert-butylnitrone (PBN) offers protection in gerbil brain during ischemia/reperfusion injury. We also show that oxidative damage to brain during aging is decreased by chronic administration of PBN. The mechanism of action of PBN may be related to its trapping of specific free radicals, which triggers a cascade of oxidative events that eventually lead to tissue injury.

Animals↗
PubMed1992

Protein modification in aging.

During aging a number of enzymes accumulate as catalytically inactive or less active forms. The age-related changes in catalytic activity are due in part to reactions of the protein with "active" oxygen species such as ozone, singlet oxygen, or with oxygen free radicals as are produced during exposure to ionizing radiation or to metal ion catalyzed oxidation (MCO) systems. The levels of oxidized proteins in cultured human fibroblasts from individuals of various ages and in liver and brain extracts of rats of different ages increase progressively with age, and in old rats can represent 30-50% of the total cellular protein. The age-related increase in oxidized protein in rat liver and brain tissue is accompanied by a loss of glutamine synthetase (GS) and glucose-6-P dehydrogenase (G-6-PDH) activities, and to a decrease in the level of cytosolic neutral protease activity which is responsible for the degradation of oxidized (denatured) protein. Of particular significance are the results of experiments showing that similar age-related changes occur in the gerbil brain and that these changes are accompanied by a loss of short-term memory as measured by the radial arm maze technique. Chronic treatment (intraperitoneal injections) of old animals with the free radical spin-trap reagent, N-tert-butyl-alpha-phenylnitrone (PBN) resulted in normalization of the several biochemical parameters to those characteristic of the young animals; coincidentally, the short-term memory index was restored to the young animal values. These results provide the first evidence that there is likely a linkage between the age-dependent accumulation of oxidized enzymes and the loss of physiological function.

Aging↗