Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis.

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Ozone exposure impairs antigen-specific immunity but activates IL-7-induced proliferation of CD4-CD8- thymocytes in BALB/c mice.

It is well known that ozone (O3), a potent reactive oxidant and air pollutant, induces respiratory inflammation and hyperresponsiveness upon inhalation. It was previously shown that O3 exposure (0.6 ppm, 10 h/day for 15 days) not only results in local bronchial inflammation, but also affects the nervous system and thymocyte proliferation, and places mice under oxidative stress. In the present study, data showed that O3 exposure could impair both the natural killer (NK) cell activity and the proliferation potential of spleen T cells to a specific antigen stimulus. Immunological function assays indicated that O3 exposure attenuated the proliferation of spleen mononuclear cells induced by concanavalin A and decreased CD4+ and CD28+ lymphocyte subsets. However, supplementation with natural antioxidants protected mice from O3-induced dysfunction of splenocyte proliferation. Meanwhile, O3 exposure resulted in a decline of mitogen-induced IL-2 production in splenocytes. It was also found that O3 exposure dramatically enhanced the proliferation of CD4-CD8- thymocytes stimulated by recombinant mouse interleukin-7 (rmIL-7), which is usually observed during the mammal aging process. Taken together, data conclude that short-term repetitive O3 exposure damages both innate and acquired immunity via altering the lymphocyte subset and cytokine profile, and via impact on thymocyte early development. O3-induced oxidative damage is one of the key factors leading to immune dysfunction in this mouse model.

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Antioxidative activities of fractions obtained from brewed coffee.

The antioxidative activity of column chromatographic fractions obtained from brewed coffee was investigated to find antioxidants and to assess the benefit of coffee drinking. The dichloromethane extract inhibited hexanal oxidation by 100 and 50% for 15 days and 30 days, respectively, at the level of 5 microg/mL. A GC/MS analysis of fractions, which exhibited oxidative activity, revealed the presence of antioxidative heterocyclic compounds including furans, pyrroles, and maltol. The residual aqueous solution exhibited slight antioxidative activity. The inhibitory activity (%) of the seven fractions from an aqueous solution toward malonaldehde formation from lipid oxidation ranged from 10 to 90 at a level of 300 microg/mL. The results indicate that brewed coffee contains many antioxidants and consumption of antioxidant-rich brewed coffee may inhibit diseases caused by oxidative damages.

Quantitative determination of urinary 8-hydroxy-2'-deoxyguanosine level in healthy Japanese volunteers.

8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.

Comparison of an oxidative stress biomarker "urinary8-hydroxy-2'-deoxyguanosine," between smokers and non-smokers.

A great deal of effort has been made on the effect of oxidative stress for smokers. What seems to be lacking, however, is its evidence. Analyzing 1076 participants (age 35.9 +/- 12.9, urinary8-OHdG Mean +/- S.D., 11.4 +/- 6.7, n = 1076), our study found the significant increase in a biomarker of DNA damage urinary 8-OHdG/creatinine among smokers (7.75 +/- 2.8 ng/ml x CRE (n = 154) and 7.36 +/- 2.5 ng/ml x CRE (n = 627) (p < 0.05), Relative Risk = 2.9 (1.4-6.2) sex and age +/- 2 matching 105 male smokers and non-smokers. There was no significance on the comparison between female smokers and non-smokers. Smokers have significantly decreased serum alpha-tocopherol (1012 +/- 455, 1152 +/- 857, p < 0.03). The amount of serum ascorbate did not change. Smokers lowered serum HDL-cholesterol compared to non-smokers (59.3 +/- 11.8, 63.9 +/- 13.3, p < 0.05). The result of oxidative stress profile (OSP) also indicated that the increase of oxidative stress to smokers (p < 0.05). The calculated value of oxygen radical absorbance capacity (ORAC) of the meal for subjects was 1600 ORAC units.

Antioxidative activities of volatile extracts from green tea, oolong tea, and black tea.

Antioxidative activities of volatile extracts from six teas (one green tea, one oolong tea, one roasted green tea, and three black teas) were investigated using an aldehyde/carboxylic acid assay and a conjugated diene assay. The samples were tested at levels of 20, 50, 100, and 200 micrograms/mL of dichloromethane. The results obtained from the two assays were consistent. All extracts except roasted green tea exhibited dose-dependent inhibitory activity in the aldehyde/carboxylic acid assay. A volatile extract from green tea exhibited the most potent activity in both assays among the six extracts. It inhibited hexanal oxidation by almost 100% over 40 days at the level of 200 micrograms/mL. The extract from oolong tea inhibited hexanal oxidation by 50% in 15 days. In the case of the extract from roasted green tea, the lowest antioxidative activity was obtained at the level of 200 micrograms/mL, suggesting that the extract from roasted green tea contained some pro-oxidants. The extracts from the three black teas showed slight anti- or proactivities in both assays. The major volatile constituents of green tea and roasted green tea extracts, which exhibited significant antioxidative activities, were analyzed using gas chromatography-mass spectrometry. The major volatile chemicals with possible antioxidative activity identified were alkyl compounds with double bond(s), such as 3,7-dimethyl-1,6-octadien-3-ol (8.04 mg/kg), in the extract from green tea and heterocyclic compounds, such as furfural (7.67 mg/kg), in the extract from roasted green tea. Benzyl alcohol, which was proved to be an antioxidant, was identified both in a green tea extract (4.67 mg/kg) and in a roasted tea extract (1.35 mg/kg).

FasL-induced downregulation of CD28 expression on jurkat cells in vitro is associated with activation of caspases.

Ligation of CD28, which is present on the majority of CD4(+)T cells, promotes proliferation and immune responses. However, expression of CD28 declines with aging, and apoptosis may contribute to this decline. We have investigated the molecular mechanism underlying the decrease in CD28 expression in Jurkat T cells cultured with FasL. FasL blocks expression of CD28 at the transcriptional level. This correlates with activation of caspase cascades: active caspase-3 can be detected and inhibitors of caspase-3 and caspase-8 increase CD28 promoter activity and CD28 expression. These findings are consistent with the hypothesis that apoptosis plays a key role in the age-related decline of CD28 expression and hence in immunosenescence.

Hydrogen peroxide induced down-regulation of CD28 expression of Jurkat cells is associated with a change of site alpha-specific nuclear factor binding activity and the activation of caspase-3.

CD28 is the requisite co-stimulatory molecule in the activation of T cells and in the generation of immune responses. But expression of CD28 declined and oxidants accumulated in the elderly. Although accumulation of reactive oxygen species (ROS) during senescence has been reported extensively, the effect of oxidants on CD28-expression remains totally unknown. In this study, we tried to address the molecular mechanism underlying the decrease in CD28-expression of Jurkat T cells cultured in H2O2. Our results indicate that H2O2 could partially block the expression of CD28. This correlates well with a change of nuclear protein binding activity to the motif of site alpha of the CD28 gene, while the site beta-binding activity remained unaltered. On the other hand, since caspase-3 is activated by H2O2, inhibitors of caspase-3 should increase the expression of CD28. What is more interesting is the fact that the site alpha-binding activity was mostly restored after caspase-3 inhibitors had being added. However, caspase-3 is not activated by caspase-8. Maybe it is activated by caspase-9, which is triggered by cytochrome c. We believe that the procaspase-3 is activated by ROS, and the active caspase-3 can induce the change of the site alpha-binding activity, causing a decrease in CD28 expression.

[Oxidative stress profile: OSP].

Oxidative stress is known to be related to various diseases such as inflammation, carcinogenesis, arteriosclerosis and ischemia-reperfusion injury, and is also a major cause of aging. For the prevention of diseases and control of aging, evaluation and control of oxidative stress in vivo may become essential. We have developed the new Oxidative Stress Profile(OSP), a total diagnostic system which provides information about oxidative stress inside human body. The OSP system consists of a number of biomakers including oxidative damage markers, prooxidant factors, antioxidants and life style-related markers. The result is shown in a two dimensional plot form. We measured a combination of biomarkers for oxidative damage of biological components, serum antioxidants and analyzed oxidative stress. The result show that oxidative stress was elevated in diabetic patients in comparison with normal controls. Oxidative stress is also elevated in smokers in comparison with non-smokers. It is also interesting to find that oxidative stress can be greatly reduced by the improvement of life style such as diet. Oxidative Stress Profile system may become a powerful tool for the evaluation of oxidative stress in vivo, and may be useful in prevention of diseases, "mi-byo"(possible cause of diseases)-diagnosis and the control of aging.

Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by Maillard reaction.

Typical heterocyclic compounds substituted with various functional groups found in Maillard reaction products were examined for antioxidant activity. Pyrroles exhibited the greatest antioxidant activity among all heterocyclic compounds tested. All pyrroles inhibited hexanal oxidation by almost 100% at a concentration of 50 microg/mL over 40 days. Addition of formyl and acetyl groups to a pyrrole ring enhanced antioxidative activity remarkably. Pyrrole-2-carboxaldehyde, 2-acetylpyrrole, 1-methyl-2-pyrrolecarboxaldehyde, and 2-acetyl-1-methylpyrrole inhibited hexanal oxidation by >80% at 10 microg/mL. Unsubstituted furan exhibited the greatest antioxidant activity among furans tested. Addition of all functional groups used in this study to furan decreased antioxidative activity. The antioxidant activity of thiophene increased with the addition of methyl and ethyl groups, but the addition of formyl or acetyl groups to thiophene decreased antioxidant activity. Thiazoles and pyrazines were ineffective antioxidants at all concentrations tested. Reaction of all heterocyclic compounds with hydrogen peroxide resulted in the formation of various oxidized products.