Per Leanderson

Cigarette smoke-induced DNA damage in cultured human lung cells: Role of hydroxyl radicals and endonuclease activation

Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.

Interleukin-6 levels in relation to psychosocial factors: Studies on serum, saliva, and in vitro production by blood mononuclear cells.

Psychosocial factors and interleukin-6 (IL-6) levels are both related to risk of morbidity and mortality. The aim of this study was to investigate how a broad range of psychosocial factors related to levels of IL-6 in different media. Fifty-nine men and women aged 30-65 were recruited from a larger study and selected to cover a broad range of psychosocial status. IL-6 levels were analyzed in serum, in saliva collected at home at three different time points during a day, and in the supernatant of cell cultures stimulated in vitro with lipopolysaccharide. After adjustments for age, gender, self-reported health problems, and lifestyle factors, IL-6-levels in serum were negatively correlated with coping and self-esteem, and positively correlated with cynicism, hostile affect, hopelessness, depression, and vital exhaustion. In saliva samples, at all time points, IL-6 levels were positively correlated to cynicism, and IL-6 levels 30 min after awakening were also positively correlated with hopelessness, depression, and vital exhaustion. After adjustment for age and gender, cynicism, depression, and vital exhaustion were negatively correlated to IL-6 levels in the supernatant of cell cultures stimulated in vitro with lipopolysaccharide, but this effect was lost after control for self-reported health problems and lifestyle factors. In conclusion, we found that IL-6 levels in serum and saliva were negatively related to psychosocial resources and positively related to psychosocial risk factors. These data strengthen the argument that IL-6 is involved in mediating the risk for disease development that has been associated with psychosocial factors.

Cigarette smoke-induced DNA-damage : role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8-hydroxydeoxyguanosine

This study demonstrates the ability of cigarette smoke condensate to generate hydrogen peroxide and to hydroxylate deoxyguanosine (dG) residues in isolated DNA to 8-hydroxydeoxyguanosine (8-OHdG). Both the formation of hydrogen peroxide and that of 8-OHdG in DNA was significantly decreased when catalase or tyrosinase was added to the smoke condensates, and this also occurred when pure hydroquinone or catechol, two major constitutes in cigarette smoke, was used instead of smoke condensate. Moreover, pure hydroquinone and catechol both caused dose-dependent formation of hydrogen peroxide and 8-OHdG, and there was good correlation between the amounts of hydrogen peroxide and 8-OHdG formed. These findings suggest that (i) hydroquinone and catechol may be responsible for the ability of cigarette smoke to cause 8-OHdG formation in DNA, (ii) this oxidative DNA-damage is due to the action of hydroxyl radicals formed during dissociation of hydrogen peroxide and (iii) the hydrogen peroxide in cigarette smoke is generated via autooxidation of hydroquinone and catechol.

Antioxidant intake, plasma antioxidants and oxidative stress in a randomized, controlled, parallel, Mediterranean dietary intervention study on patients with rheumatoid arthritis

BackgroundPreviously we have reported that patients with rheumatoid arthritis (RA) obtained a significant reduction in disease activity by adopting a Mediterranean-type diet. The present study was carried out to investigate the antioxidant intake, the plasma levels of antioxidants and a marker of oxidative stress (malondialdehyde) during the study presented earlier.MethodsRA patients randomized to either a Mediterranean type diet (MD group; n = 26) or a control diet (CD group; n = 25) were compared during a three month dietary intervention study. Their antioxidant intake was assessed by means of diet history interviews and their intake of antioxidant-rich foods by a self-administered questionnaire. The plasma levels of retinol, antioxidants (α- and γ-tocopherol, β-carotene, lycopene, vitamin C and uric acid) and urinary malondialdehyde (MDA), a marker for oxidative stress, were determined using high performance liquid chromatography. The Students t-test for independent samples and paired samples were used to test differences between and within groups. For variables with skewed distributions Mann-Whitney U-test and Wilcoxon signed ranks test were performed. To evaluate associations between dietary intake of antioxidants, as well as between disease activity, MDA and antioxidants we used Pearsons product moment correlation or Spearmans rank correlation.ResultsThe MD group had significantly higher intake frequencies of antioxidant-rich foods, and also higher intakes of vitamin C (p = 0.014), vitamin E (p = 0.007) and selenium (p = 0.004), and a lower intake of retinol (p = 0.049), compared to the CD group. However, the difference between the groups regarding vitamin C intake was not significant when under- and over-repoters were excluded (p = 0.066). There were no changes in urine MDA or in the plasma levels of antioxidants (after p-lipid adjustments of the tocopherol results), from baseline to the end of the study. The levels of retinol, vitamin C and uric acid were negatively correlated to disease activity variables. No correlation was found between antioxidant intake and the plasma levels of antioxidants.ConclusionsDespite an increase in reported consumption of antioxidant-rich foods during the Mediterranean diet intervention, the levels of plasma antioxidants and urine MDA did not change. However, the plasma levels of vitamin C, retinol and uric acid were inversely correlated to variables related to RA disease activity.

GREEN TEA POLYPHENOLS INHIBIT OXIDANT-INDUCED DNA STRAND BREAKAGE IN CULTURED LUNG CELLS

The influence of green tea polyphenols (GTP) on the formation of DNA strand breaks (DNA-SB) and lipid peroxidation products (LPP) in cultured human lung cells (A 549) exposed to different oxidants was investigated. Cells were pretreated with GTP for 2 h and then exposed to cigarette smoke solution, H2O2 or FeCl3 for 30 min. After exposure, the cells were analyzed for DNA-SB, LPP, and viability. In addition, the effects of GTP added directly to the incubation mixtures during exposure were examined, using the same end points. It appeared that pretreatment with GTP inhibited both cigarette smoke- and H2O2-induced DNA breakage; i.e., following exposure to cigarette smoke or H2O2, the fraction of DNA passing through a microfilter increased significantly in cells not subjected to GTP, but this effect was prevented or inhibited in GTP-treated cells. Pretreatment with GTP also reduced the overall toxicity of H2O2 as determined by cell growth after exposure. Moreover, addition of GTP during exposure reduced both cigarette smoke- and H2O2-induced DNA breakage as well as formation of LPP after exposure to Fe3+. These results indicate that GTP inhibit the formation of DNA-SB in cells exposed to oxidants. It is possible that this ability to GTP to inhibit DNA-SB formation might contribute to the antitumorogenic properties of green tea.

(1→3)-β-d-Glucan stimulates nitric oxide generation and cytokine mRNA expression in macrophages

Beta-glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms seen in both occupational and residential environments. Here, the ability of a (1→3)-β-d-glucan (Curdlan) to stimulate nitric oxide generation and cytokine mRNA expression in rat alveolar macrophages (AMs) and the murine monocyte/macrophage cell line, RAW 264.7 was investigated. Exposure to (1→3)-β-d-glucan (20, 100 and 500 μg/ml) induced a dose-dependent increase in the expression of inducible nitric oxide synthase mRNA and a release of nitric oxide into the culture medium in both rat AMs and RAW 264.7 cells. The mRNA expression of a number of other inflammatory mediators such as interleukin-1β, interleukin-6, tumor necrosis factor-α and cyclooxygenase-2 was also increased by the exposure to β-glucan. The capability of (1→3)-β-d-glucan (500 μg/ml) to induce mRNA synthesis of these various mediators were comparable to that of endotoxin (1 μg/ml). These results imply that (1→3)-β-d-glucan stimulates the generation of nitric oxide, cytokines and prostaglandins in macrophages and suggest the possibility that this may contribute to bioaerosol-induced respiratory symptoms seen in exposed individuals.

The periodontal pathogen Porphyromonas gingivalis cleaves apoB-100 and increases the expression of apoM in LDL in whole blood leading to cell proliferation

Objective.  Several studies support an association between periodontal disease and atherosclerosis with a crucial role for the pathogen Porphyromonas gingivalis. This study aims at investigating the proteolytic and oxidative activity of P. gingivalis on LDL in a whole blood system using a proteomic approach and analysing the effects of P. gingivalis‐modified LDL on cell proliferation.

Cytocidal effects of atheromatous plaque components: The death zone revisited

Objective: Earlier we suggested that atheroma lesions constitute a “death zone” containing toxic materials that may cause dysfunction and demise of invading macrophages to prevent the removal of plaque materials. Here we have assessed the cytotoxic effects of nonfractionated gruel and insoluble (ceroid‐like) material derived from advanced human atheroma. Methods and Results: The insoluble material within advanced atherosclerotic plaque was isolated following protease K digestion and extensive extraction with aqueous and organic solvents. FTIR, Raman, and atomic absorption spectroscopy suggested that, despite its fluorescent nature, this material closely resembled hydroxyapatite and dentin, but also contained a significant amount of iron and calcium. When added to J774 cells and human macrophages in culture, this insoluble substance was phagocytosed, and progressive cell death followed. However, an even more cytotoxic activity was found in the atheromatous “gruel” that contains abundant carbonyls/aldehydes. Cell death caused by both crude gruel and ceroid could be blocked by preincubating cells with the lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone, apoferritin, BAPTA/AM, or sodium borohydride, indicating that cellular iron, calcium, and reactive aldehyde(s) are responsible for the observed cytotoxicity. Conclusions: Toxic materials within atheromatous lesions include both ceroid and even more cytotoxic lipidaceous materials. The cytotoxic effects of these plaque components may help explain the persistence of atherosclerotic lesions.—Li, W., Östblom, M., Xu, L‐H., Hellsten, A., Leanderson, P., Liedberg, B., Brunk, U. T., Eaton, J. W., Yuan, X‐M. Cytocidal effects of atheromatous plaque components: the death zone revisited. FASEB J. 20, 2281–2290 (2006)

Determination of Urinary 8-Hydroxydeoxyguanosine by Coupled-Column High-Performance Liquid Chromatography with Electrochemical Detection: A Noninvasive Assay for in Vivo Oxidative DNA Damage in Humans

Oxidative damage to DNA has been suggested to contribute to a number of diseases including cancer and chronic inflammation. In order to study the relationship between oxidative damage to DNA and diseases, it is desirable to develop techniques that can be used for the analysis of DNA damage products in individuals. The present article describes a sensitive analytical method for determination of the oxidative DNA adduct, 8-hydroxydeoxyguanosine (80HdG) in human urine, based on coupled-column high-performance liquid chromatography with electrochemical detection. The method measures down to 2 nmol 80HdG/L urine, which is well below the levels of 80HdG in normal human urine (5–15 nmol/L). It is shown that smokers excrete slightly more 80HdG in their urine than nonsmokers and that patients undergoing radiotherapy or chemotherapy for different malignant diseases excrete significantly more than healthy individuals. The potential use of the method for detecting increased urinary 80HdG excretion and conditions asso...

Lower serum levels of beta-carotene in Lithuanian men are accompanied by higher urinary excretion of the oxidative DNA adduct, 8-hydroxydeoxyguanosine : The LiVicordia study.

OBJECTIVE In 1995, middle-aged Lithuanian men had a four-fold higher risk than Swedish men of dying from coronary heart disease. The cross-sectional LiVicordia study had reported significantly lower levels of the lipid-soluble antioxidants lycopene, beta-carotene, and gamma-tocopherol among Lithuanian men than among Swedish men. We examined whether there were differences in urinary 8-hydroxydeoxyguanosine (8OHdG), a marker of oxidative stress, between these groups of men. METHODS Using automated coupled column high-performance liquid chromatography with electrochemical detection, we examined 50-y-old men randomly sampled from Linköping, Sweden (n = 99) and Vilnius, Lithuania (n = 109) with regard to urinary concentrations of 8-OHdG. RESULTS Levels of 8-OHdG were higher in the Lithuanian men than in the Swedish men (20.9 +/- 0.91 versus 14.9 +/- 0.75 nM/L, P < 0.001), and this difference was evident in smokers (P < 0.01) and non-smokers (P < 0.001). Serum levels of alpha- and beta-carotene were inversely correlated to urinary 8-OHdG levels (P < 0.05 in both cases). Habitual smoking and low levels of beta-carotene contributed significantly to higher oxidative DNA damage expressed as urinary 8-OHdG. CONCLUSIONS These findings indicate that increased urinary 8-OHdG levels accompany lower serum levels of antioxidants in Lithuanian men. They supported previous suggestions that increased oxidative stress may be one factor behind the higher mortality in Lithuanian men.