Gamma-tocopherol prevents airway eosinophilia and mucous cell hyperplasia in experimentally induced allergic rhinitis and asthma.

BACKGROUND: Traditional therapies for asthma and allergic rhinitis (AR) such as corticosteroids and antihistamines are not without limitations and side effects. The use of complementary and alternative approaches to treat allergic airways disease, including the use of herbal and dietary supplements, is increasing but their efficacy and safety are relatively understudied. Previously, we have demonstrated that gamma-tocopherol (gammaT), the primary form of dietary vitamin E, is more effective than alpha-tocopherol, the primary form found in supplements and tissue, in reducing systemic inflammation induced by non-immunogenic stimuli. OBJECTIVE: We used allergic Brown Norway rats to test the hypothesis that a dietary supplement with gammaT would protect from adverse nasal and pulmonary responses to airway allergen provocation. METHODS: Ovalbumin (OVA)-sensitized Brown Norway rats were treated orally with gammaT before intranasal provocation with OVA. Twenty-four hours after two challenges, histopathological changes in the nose, sinus and pulmonary airways were compared with gene expression and cytokine production in bronchoalveolar lavage fluid and plasma. RESULTS: We found that acute dosing for 4 days with gammaT was sufficient to provide broad protection from inflammatory cell recruitment and epithelial cell alterations induced by allergen challenge. Eosinophil infiltration into airspaces and tissues of the lung, nose, sinus and nasolacrimal duct was blocked in allergic rats treated with gammaT. Pulmonary production of soluble mediators PGE(2), LTB(4) and cysteinyl leukotrienes, and nasal expression of IL-4, -5, -13 and IFN-gamma were also inhibited by gammaT. Mucous cell metaplasia, the increase in the number of goblet cells and amounts of intraepithelial mucus storage, was induced by allergen in both pulmonary and nasal airways and decreased by treatment with gammaT. CONCLUSIONS: Acute treatment with gammaT inhibits important inflammatory pathways that underlie the pathogenesis of both AR and asthma. Supplementation with gammaT may be a novel complementary therapy for allergic airways disease.

Acetyl-L-carnitine and alpha-lipoic acid supplementation of aged beagle dogs improves learning in two landmark discrimination tests.

Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC) over approximately 2 months made significantly fewer errors in reaching the learning criterion on two landmark discrimination tasks compared to controls administered a methylcellulose placebo. Testing started after a 5 day wash-in. The dogs were also tested on a variable delay version of a previously acquired spatial memory task; results were not significant. The improved performance on the landmark task of dogs supplemented with LA + ALC provides evidence of the effectiveness of this supplement in improving discrimination and allocentric spatial learning. We suggest that long-term maintenance on LA and ALC may be effective in attenuating age-associated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.

Pre-treatment with R-lipoic acid alleviates the effects of GSH depletion in PC12 cells: implications for Parkinson's disease therapy.

Oxidative stress is believed to play a key role in the degeneration of dopaminergic neurons in the substantia nigra (SN) of Parkinson's disease (PD) patients. An important biochemical feature of PD is a significant early depletion in levels of the thiol antioxidant compound glutathione (GSH) which may lead to the generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately to subsequent neuronal cell death. In earlier work from our laboratory, we demonstrated that depletion of GSH in dopaminergic PC12 cells affects mitochondrial integrity and specifically impairs the activity of mitochondrial complex I. Here we report that pre-treatment of PC12 cells with R-lipoic acid acts to prevent depletion of GSH content and preserves the mitochondrial complex I activity which normally is impaired as a consequence of GSH loss.

Oxidative damage increases with age in a canine model of human brain aging.

We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) activity and both oxidized and reduced glutathione to study the link between oxidative damage, aging and beta-amyloid (Abeta) in the canine brain. The aged canine brain, a model of human brain aging, naturally develops extensive diffuse deposits of human-type Abeta. Abeta was measured in immunostained prefrontal cortex from 19 beagle dogs (4-15 years). Increased malondialdehyde (MDA), which indicates increased lipid peroxidation, was observed in the prefrontal cortex and serum but not in cerebrospinal fluid (CSF). Oxidative damage to proteins (carbonyl formation) also increased in brain. An age-dependent decline in GS activity, an enzyme vulnerable to oxidative damage, and in the level of glutathione (GSH) was observed in the prefrontal cortex. MDA level in serum correlated with MDA accumulation in the prefrontal cortex. Although 11/19 animals exhibited Abeta, the extent of deposition did not correlate with any of the oxidative damage measures, suggesting that each form of neuropathology accumulates in parallel with age. This evidence of widespread oxidative damage and Abeta deposition is further justification for using the canine model for studying human brain aging and neurodegenerative diseases.

Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC.

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.

gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention.

gamma-tocopherol is the major form of vitamin E in many plant seeds and in the US diet, but has drawn little attention compared with alpha-tocopherol, the predominant form of vitamin E in tissues and the primary form in supplements. However, recent studies indicate that gamma-tocopherol may be important to human health and that it possesses unique features that distinguish it from alpha-tocopherol. gamma-Tocopherol appears to be a more effective trap for lipophilic electrophiles than is alpha-tocopherol. gamma-Tocopherol is well absorbed and accumulates to a significant degree in some human tissues; it is metabolized, however, largely to 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), which is mainly excreted in the urine. gamma-CEHC, but not the corresponding metabolite derived from alpha-tocopherol, has natriuretic activity that may be of physiologic importance. Both gamma-tocopherol and gamma-CEHC, but not alpha-tocopherol, inhibit cyclooxygenase activity and, thus, possess antiinflammatory properties. Some human and animal studies indicate that plasma concentrations of gamma-tocopherol are inversely associated with the incidence of cardiovascular disease and prostate cancer. These distinguishing features of gamma-tocopherol and its metabolite suggest that gamma-tocopherol may contribute significantly to human health in ways not recognized previously. This possibility should be further evaluated, especially considering that high doses of alpha-tocopherol deplete plasma and tissue gamma-tocopherol, in contrast with supplementation with gamma-tocopherol, which increases both. We review current information on the bioavailability, metabolism, chemistry, and nonantioxidant activities of gamma-tocopherol and epidemiologic data concerning the relation between gamma-tocopherol and cardiovascular disease and cancer.

Heme deficiency selectively interrupts assembly of mitochondrial complex IV in human fibroblasts: revelance to aging.

Heme deficiency was studied in young and old normal human fibroblasts (IMR90). Regardless of age, heme deficiency increased the steady-state level of oxidants and lipid peroxidation and sensitized the cells to fluctuations in intracellular Ca(2+). Heme deficiency selectively decreased the activity and protein content of mitochondrial complex IV (cytochrome c oxidase) by 95%, indicating a decrease in successful assembly. Complexes I-III and catalase remained intact under conditions of heme deficiency, whereas ferrochelatase was up-regulated. Complex IV is the only hemeprotein in the cell that contains heme a, which may account for its susceptibility. The rate of removal and assembly of complex IV declines with age. These findings are relevant to worldwide iron deficiency in women and children and to an age-related decline in complex IV in Alzheimer's disease patients.

The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro.

We performed a comprehensive study on the genotoxic and cytotoxic effects of in vitro folic acid deficiency on primary human lymphocytes. Lymphocytes were cultured in medium containing 12-120 nM folic acid for 9 days in a novel cytokinesis-block micronucleus (CBMN) assay system (n = 20). Besides identifying optimal folic acid concentrations for in vitro genomic stability, we tested the hypothesis that lymphocytes from individuals homozygous for the C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism (TTs, n = 10) are protected against chromosome damage relative to controls (CCs, n = 10) under conditions of folic acid deficiency. This hypothesis is based on the assumption that reduced MTHFR activity in TT lymphocytes causes a diversion of 5,10-methylene tetrahydrofolate toward thymidine synthesis, which minimizes uracil-induced double-stranded DNA breakage. Cells were scored for micronuclei, apoptosis, necrosis, nucleoplasmic bridges, and nuclear budding. The latter two endpoints are indicative of chromosome rearrangements and gene amplification, respectively, and to the best of our knowledge, this is the first report of their association with folic acid concentration. Folic acid concentration correlated significantly (P < 0.0001) and negatively (r, -0.63 to -0.74) with all markers of chromosome damage, which were minimized at 60-120 nM folic acid, much greater than concentrations assumed "normal," but not necessarily optimal in plasma. Two-way ANOVA revealed no effect of the MTHFR genotype on any of the endpoints. Results show that the C677T polymorphism does not affect the ability of a cell to resist chromosome damage induced by folic acid deficiency in this in vitro system.

N-t-Butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro.

N-t-butyl hydroxylamine (NtBHA) delays senescence-dependent changes in human lung fibroblasts (IMR90) (Atamna et al., J. Biol. Chem. 275, 6741-6748). The current study examines the effect of NtBHA on mitochondria in old and young rats and human primary fibroblasts (IMR90). In NtBHA-treated rats, the age-dependent decline in food consumption and ambulatory activity was reversed without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding NtBHA. These findings suggest that NtBHA improved mitochondrial function in vivo. The age-dependent increase in proteins with thiol-mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats; no significant effect was observed in young rats. In IMR90 cells, NtBHA delayed senescence-associated changes in mitochondria and cellular senescence induced by maintaining the cells under suboptimal levels of growth factors. Proteasomal activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates in cells 10- to 15-fold the extracellular concentration and is maintained by mitochondrial NADH. NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radical-induced toxicity to mitochondria.

Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro.

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme which converts 5,10-methylene tetrahydrofolate (5,10-MnTHF) to 5-methyl tetrahydrofolate. A common C to T transition (C677T) in the MTHFR gene is reported to reduce the risk for colorectal cancer and acute lymphocytic leukemia in homozygotes (TTs). It is hypothesized that because TTs have reduced MTHFR activity, more 5,10-MnTHF is available to provide methyl groups for the conversion of uracil to thymidine. Folic acid deficiency causes the intracellular accumulation of dUMP and the subsequent incorporation of uracil into DNA. The removal of uracil from DNA may result in double-stranded DNA breaks, the accumulation of which is a putative risk factor for cancer. We tested whether human lymphocytes taken from TTs (n = 10) were more able to resist uracil incorporation into DNA than controls (n = 14 CCs and 6 CTs) when cultured in medium containing 12-120 nM folic acid for 9 days. DNA uracil content of these lymphocytes was measured by CG-MS. TTs and controls showed a dose-dependent increase in DNA uracil content during folic acid deficiency (P < 0.0001, R2 = 0.23 for TTs and P < 0.0001, R2 = 0.19 for controls). DNA uracil content was not different between the two groups at any of the folic acid concentrations (two-way ANOVA: media [folic acid], P < 0.0001; genotype, P = 0.4). The results show that, in this in vitro system, the MTHFR C677T polymorphism does not affect the cell's ability to resist uracil incorporation into DNA. Chromosome breakage, as measured by micronuclei, was also shown to correlate with folic acid concentration in a preliminary experiment (P < 0.0001). Although the results appear not to support the hypothesis that a reduced risk for certain cancers in TTs is due to diversion of folic acid to thymidine synthesis, differences between the in vivo and in vitro situation make this conclusion not definitive.

DNA damage from micronutrient deficiencies is likely to be a major cause of cancer.

A deficiency of any of the micronutrients: folic acid, Vitamin B12, Vitamin B6, niacin, Vitamin C, Vitamin E, iron, or zinc, mimics radiation in damaging DNA by causing single- and double-strand breaks, oxidative lesions, or both. For example, the percentage of the US population that has a low intake (<50% of the RDA) for each of these eight micronutrients ranges from 2 to >20%. A level of folate deficiency causing chromosome breaks was present in approximately 10% of the US population, and in a much higher percentage of the poor. Folate deficiency causes extensive incorporation of uracil into human DNA (4 million/cell), leading to chromosomal breaks. This mechanism is the likely cause of the increased colon cancer risk associated with low folate intake. Some evidence, and mechanistic considerations, suggest that Vitamin B12 (14% US elderly) and B6 (10% of US) deficiencies also cause high uracil and chromosome breaks. Micronutrient deficiency may explain, in good part, why the quarter of the population that eats the fewest fruits and vegetables (five portions a day is advised) has about double the cancer rate for most types of cancer when compared to the quarter with the highest intake. For example, 80% of American children and adolescents and 68% of adults do not eat five portions a day. Common micronutrient deficiencies are likely to damage DNA by the same mechanism as radiation and many chemicals, appear to be orders of magnitude more important, and should be compared for perspective. Remedying micronutrient deficiencies should lead to a major improvement in health and an increase in longevity at low cost.

Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers.

OBJECTIVE: To measure folate levels in seminal plasma from smokers and nonsmokers and to evaluate relationships between seminal plasma folate levels and both folate nutriture and semen quality measures. DESIGN: Observational study. SETTING: United States Department of Agriculture, Western Human Nutrition Research Center, Presidio of San Francisco, San Francisco, California. PATIENT(S): Healthy male smokers (n=24) and nonsmokers (n=24). MAIN OUTCOME MEASURE(S): Blood levels of plasma folate and homocysteine, seminal plasma total, non-methyl- and 5-methyltetrahydrofolate concentrations, and total sperm count and density. RESULTS: Total seminal plasma folate concentrations were on average 1.5 times higher than blood plasma folate concentrations in all men. Seminal plasma folates contained 5-methyltetrahyrdofolate (74% of total) and non-methyltetrahydrofolates (26% of total); all samples had less than four glutamyl residues. Total and 5-methyltetrahydrofolate concentrations correlated significantly with blood plasma folate and homocysteine concentrations. Seminal plasma non-methyltetrahydrofolate levels correlated significantly with sperm density and total sperm count. Seminal plasma of smokers contained a proportionally lower concentration of non-methyltetrahydrofolates compared with nonsmokers. CONCLUSION(S): Seminal plasma total folate and 5-methyltetrahydrofolate concentrations reflect folate nutriture. The non-methyltetrahydrofolate fraction of seminal plasma may be important for male reproductive function.

A simpler, more robust method for the analysis of 8-oxoguanine in DNA.

The oxidized DNA base 8-oxoguanine has been commonly measured by enzymatic digestion of DNA to nucleosides followed by high-performance liquid chromatography (HPLC) separation of the adduct 8-oxodeoxyguanosine. There has recently been an enormous debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-oxoguanine can occur at numerous stages in sample preparation. Hence, we have designed an alternative protocol to traditional enzymatic digestion of DNA which (i) limits the potential for artifactual oxidation, (ii) speeds up the assay markedly, (iii) increases the assay's sensitivity moderately, and (iv) addresses criticisms that have been raised concerning the efficiency of DNA digestion by nucleases. In short, we use the Escherichia coli repair enzyme formamidopyrimidine (Fapy) glycosylase to release the base 8-oxoguanine from full-length DNA, then separate 8-oxoguanine from high molecular weight molecules by ultrafiltration (10,000 Da exclusion) and analyze the base adduct by reverse-phase HPLC. Benefits of this approach include (i) rapid removal of the roughly million-fold molar excess of unaltered bases from the sample, (ii) reduction in the length of enzymatic incubations and the number of steps, (iii) elimination of high temperature incubation, (iv) a very clean chromatographic separation, and (v) rapid elution of the analyte and correspondingly greater throughput. Using this improved method, we have followed the induction of 8-oxoguanine in the DNA of peroxide-treated HeLa cells, an experiment that had proved cumbersome with traditional methods.

Supplementation of postmenopausal women with fish oil rich in eicosapentaenoic acid and docosahexaenoic acid is not associated with greater in vivo lipid peroxidation compared with oils rich in oleate and linoleate as assessed by plasma malondialdehyde and F(2)-isoprostanes.

BACKGROUND: Although the replacement of dietary saturated fat with unsaturated fat has been advocated to reduce the risk of cardiovascular disease, diets high in polyunsaturated fatty acids (PUFAs) could increase lipid peroxidation, potentially contributing to the pathology of atherosclerosis. OBJECTIVE: The objective of this study was to examine indexes of in vivo lipid peroxidation, including free F(2)-isoprostanes, malondialdehyde (MDA), and thiobarbituric acid reacting substances (TBARS), in the plasma of postmenopausal women taking dietary oil supplements rich in oleate, linoleate, and both eicosapentaenoic acid and docosahexaenoic acid. DESIGN: Fifteen postmenopausal women took 15 g sunflower oil/d, providing 12.3 g oleate/d; safflower oil, providing 10.5 g linoleate/d; and fish oil, providing 2.0 g EPA/d and 1.4 g DHA/d in a 3-treatment crossover trial. RESULTS: Plasma free F(2)-isoprostane concentrations were lower after fish-oil supplementation than after sunflower-oil supplementation (P: = 0.003). When plasma free F(2)-isoprostane concentrations were normalized to plasma arachidonic acid concentrations, significant differences among the supplements were eliminated. Plasma MDA concentrations were lower after fish-oil supplementation than after sunflower-oil supplementation (P: = 0.04), whereas plasma TBARS were higher after fish-oil supplementation than after sunflower oil (P: = 0.003) and safflower oil (P: = 0.001) supplementation. When plasma MDA concentrations were normalized to plasma PUFA concentrations, significant differences were eliminated, but TBARS remained higher after fish-oil supplementation than after sunflower oil (P: = 0.01) and safflower-oil (P: = 0.0003) supplementation. CONCLUSIONS: With fish-oil supplementation, there was no evidence of increased lipid peroxidation when assessed by plasma F(2)-isoprostanes and MDA, although plasma TBARS was higher than with sunflower-oil and safflower-oil supplementation.

gamma-tocopherol and its major metabolite, in contrast to alpha-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells.

Cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E(2) (PGE(2)) plays a key role in inflammation and its associated diseases, such as cancer and vascular heart disease. Here we report that gamma-tocopherol (gammaT) reduced PGE(2) synthesis in both lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and IL-1beta-treated A549 human epithelial cells with an apparent IC(50) of 7.5 and 4 microM, respectively. The major metabolite of dietary gammaT, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), also exhibited an inhibitory effect, with an IC(50) of approximately 30 microM in these cells. In contrast, alpha-tocopherol at 50 microM slightly reduced (25%) PGE(2) formation in macrophages, but had no effect in epithelial cells. The inhibitory effects of gammaT and gamma-CEHC stemmed from their inhibition of COX-2 activity, rather than affecting protein expression or substrate availability, and appeared to be independent of antioxidant activity. gamma-CEHC also inhibited PGE(2) synthesis when exposed for 1 h to COX-2-preinduced cells followed by the addition of arachidonic acid (AA), whereas under similar conditions, gammaT required an 8- to 24-h incubation period to cause the inhibition. The inhibitory potency of gammaT and gamma-CEHC was diminished by an increase in AA concentration, suggesting that they might compete with AA at the active site of COX-2. We also observed a moderate reduction of nitrite accumulation and suppression of inducible nitric oxide synthase expression by gammaT in lipopolysaccharide-treated macrophages. These findings indicate that gammaT and its major metabolite possess anti-inflammatory activity and that gammaT at physiological concentrations may be important in human disease prevention.

Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants.

The responses to oxidative stress induced by chronic exercise (8-wk treadmill running) or acute exercise (treadmill running to exhaustion) were investigated in the brain, liver, heart, kidney, and muscles of rats. Various biomarkers of oxidative stress were measured, namely, lipid peroxidation [malondialdehyde (MDA)], protein oxidation (protein carbonyl levels and glutamine synthetase activity), oxidative DNA damage (8-hydroxy-2'-deoxyguanosine), and endogenous antioxidants (ascorbic acid, alpha-tocopherol, glutathione, ubiquinone, ubiquinol, and cysteine). The predominant changes are in MDA, ascorbic acid, glutathione, cysteine, and cystine. The mitochondrial fraction of brain and liver showed oxidative changes as assayed by MDA similar to those of the tissue homogenate. Our results show that the responses of the brain to oxidative stress by acute or chronic exercise are quite different from those in the liver, heart, fast muscle, and slow muscle; oxidative stress by acute or chronic exercise elicits different responses depending on the organ tissue type and its endogenous antioxidant levels.

Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy.

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.