Poly (ADP-ribose) polymerase-1-inhibiting flavonoids attenuate cytokine release in blood from male patients with chronic obstructive pulmonary disease or type 2 diabetes.

Recently, we identified several flavonoids as inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 in vitro and in vivo. PARP-1 is recognized as coactivator of nuclear factor-kappaB and plays a role in the pathophysiology of diseases with low-grade systemic inflammation, such as chronic obstructive pulmonary disease (COPD) and type 2 diabetes (T2D). In this study, we assessed the antiinflammatory effects of flavonoids with varying PARP-1-inhibiting effects in whole blood from male patients with COPD or T2D and healthy men. A total of 10 COPD, 10 T2D patients, and 10 healthy volunteers matched for age and BMI were recruited. Blood from each participant was exposed to 1 microg/L lipopolysaccharide (LPS) over 16 h with or without preincubation with 10 micromol/L of flavone, fisetin, morin, or tricetin. Concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, -8, and -10 were measured in the supernatant. Preincubation with fisetin and tricetin strongly attenuated LPS-induced increases in concentrations of TNFalpha in blood from COPD patients [mean (+/- SEM): -41 +/- 4% (fisetin) and -31 +/- 4% (tricetin); P < 0.001] and IL-6 in blood from T2D patients [-31 +/- 5% (fisetin) and -29 +/- 6% (tricetin); P < or = 0.001]. Moreover, LPS-induced changes in TNFalpha and IL-6 concentrations were positively correlated with the extent of reduction by fisetin and tricetin. The PARP-1-inhibiting flavonoids fisetin and tricetin were able to attenuate LPS-induced cytokine release from leukocytes of patients with chronic systemic inflammation, indicating a potential application as nutraceutical agents for these patient groups.

Telomere length assessment: biomarker of chronic oxidative stress?

Telomeres are nucleoprotein structures, located at the ends of chromosomes and are subject to shortening at each cycle of cell division. They prevent chromosomal ends from being recognized as double strand breaks and protect them from end to end fusion and degradation. Telomeres consist of stretches of repetitive DNA with a high G-C content and are reported to be highly sensitive to damage induced by oxidative stress. The resulting DNA strand breaks can be formed either directly or as an intermediate step during the repair of oxidative bases. In contrast to the majority of genomic DNA, there is evidence that telomeric DNA is deficient in the repair of single strand breaks. Since chronic oxidative stress plays a major role in the pathophysiology of several chronic inflammatory diseases, it is hypothesized that telomere length is reducing at a faster rate during oxidative stress. Therefore, assessment of telomere length might be a useful biomarker of disease progression. In this review several features of telomere length regulation, their relation with oxidative stress, and the potential application of measurement of telomere length as biomarker of chronic oxidative stress, will be discussed.

Flavone as PARP-1 inhibitor: its effect on lipopolysaccharide induced gene-expression.

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) which was initially known for its role in the repair of oxidative stress-induced DNA damage, has also been reported to play a mediating role in the inflammatory response. Studies with PARP-1 knockout models have shown that PARP-1 is a co-activator of Nuclear Factor-kappa B (NF-kappaB), although this appears not to require its enzyme activity. In addition, drug-induced inhibition of the enzyme activity of PARP-1 was observed to reduce the production of pro-inflammatory mediators. In this study, the flavonoid compound flavone was demonstrated to significantly inhibit the enzyme activity of PARP-1. Further evaluation of flavone in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated human pulmonary epithelial and vascular endothelial cells revealed that both the decrease in NAD(+) levels, as well as the formation of PAR-polymers was dose-dependently attenuated by flavone. In addition, flavone was found to reduce the lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in pulmonary epithelial cells, which was confirmed by transcription analysis. Furthermore, the transcription Inhibitor kappa B alpha (of IkappaBalpha) was significantly increased by flavone. The results of the present study indicate that the flavonoid flavone could be a potential candidate for application in treatment of chronic inflammatory diseases. PARP-1 inhibition could have beneficial effects in such diseases as Chronic Obstructive Pulmonary Disease (COPD) and diabetes, by preservation of cellular NAD(+) levels and attenuating inflammatory conditions.

Caffeine metabolites are inhibitors of the nuclear enzyme poly(ADP-ribose)polymerase-1 at physiological concentrations.

The activity of the nuclear enzyme poly(ADP-ribose)polymerase-1 (E.C.2.4.2.30), which is highly activated by DNA strand breaks, is associated with the pathophysiology of both acute as well as chronic inflammatory diseases. PARP-1 overactivation and the subsequent extensive turnover of its substrate NAD+ put a large demand on mitochondrial ATP-production. Furthermore, due to its reported role in NF-kappaB and AP-1 mediated production of pro-inflammatory cytokines, PARP-1 is considered an interesting target in the treatment of these diseases. In this study the PARP-1 inhibiting capacity of caffeine and several metabolites as well as other (methyl)xanthines was tested using an ELISA-assay with purified human PARP-1. Caffeine itself showed only weak PARP-1 inhibiting activity, whereas the caffeine metabolites 1,7-dimethylxanthine, 3-methylxanthine and 1-methylxanthine, as well as theobromine and theophylline showed significant PARP-1 inhibiting activity. Further evaluation of these compounds in H2O2-treated A549 lung epithelial and RF24 vascular endothelial cells revealed that the decrease in NAD+-levels as well as the formation of the poly(ADP-ribose)polymer was significantly prevented by the major caffeine metabolite 1,7-dimethylxanthine. Furthermore, H2O2-induced necrosis could be prevented by a high dose of 1,7-dimethylxanthine. Finally, antioxidant effects of the methylxanthines could be ruled out with ESR and measurement of the TEAC. Concluding, caffeine metabolites are inhibitors of PARP-1 and the major caffeine metabolite 1,7-dimethylxanthine has significant PARP-1 inhibiting activity in cultured epithelial and endothelial cells at physiological concentrations. This inhibition could have important implications for nutritional treatment of acute and chronic inflammatory pathologies, like prevention of ischemia-reperfusion injury or vascular complications in diabetes.

CYP1A2 and NAT2 genotype/phenotype relations and urinary excretion of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in a human dietary intervention study.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking, and is subsequently metabolically activated by cytochrome P4501A2 (CYP1A2) and N-acetyltransferase 2 (NAT2). Respective genes encoding for these enzymes, display polymorphic distribution in the human population and are thus believed to cause interindividual differences in cancer risk susceptibility. The present study investigated the influence of dietary exposure and CYP1A2 and NAT2 genotypes and phenotypes on differential urinary PhIP excretion levels in 71 human volunteers after consumption of either a high (7.4 ng/g) or low (1.7 ng/g) dose of PhIP. Urinary PhIP excretion levels were found to reflect recent dietary exposure levels, with average levels of 174% (high dose group) and 127% (low dose group), as compared to pre-feed levels. Urinary caffeine metabolite ratios were significantly different between the two NAT2 genotypes, whereas for CYP1A2, the apparent difference in metabolic ratios between the genotypes was statistically non-significant. Significant correlations were firstly found between the CYP1A2-164A-->C (CYP1A2*1F) polymorphism and differential urinary PhIP excretion levels. Although the found correlations are driven primarily by a small number of subjects possessing the homozygous variant constellation, the strong influence of this genotype indicates that the CYP1A2*1F polymorphism could play an important role in human cancer risk susceptibility.

Antioxidant status associated with inflammation in sarcoidosis: a potential role for antioxidants.

RATIONALE: Enhanced production of reactive oxygen species (ROS), capable of reducing endogenous defense levels and enhancing inflammation, is suggested to play a role in sarcoidosis. Antioxidant supplementation might offer protection against such ROS-mediated damage. A promising candidate for antioxidant supplementation is the flavonoid quercetin. AIM: To determine the antioxidant and inflammatory status in sarcoidosis. Furthermore, the potential of quercetin to mitigate the occurring inflammation will be assessed. METHODS: Non-smoking sarcoidosis patients and healthy controls matched for age, gender and dietary behavior were enrolled (NCT-00512967). Measurements included assessment of total plasma antioxidant capacity, vitamin C, uric acid, glutathione, basal and LPS-induced levels of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-8 and -10 as well as the effect of quercetin on these levels. RESULTS: Compared to their controls, the sarcoidosis patients displayed significantly lower total plasma antioxidant capacity, decreased levels of vitamin C, uric acid and glutathione and increased levels of basal TNFalpha and IL-8. Quercetin significantly decreased ex vivo LPS-induced TNFalpha- and IL-8 production in a concentration-dependent manner in both groups. Interestingly, this quercetin effect was more pronounced in sarcoidosis patients. DISCUSSION: The endogenous antioxidant defense was significantly reduced in sarcoidosis, indicating that oxidative stress underlies the pathology of this disease. Furthermore, the inflammatory status was significantly enhanced in sarcoidosis. Finally, our results regarding the effect of quercetin on cytokine production imply that sarcoidosis patients might benefit from antioxidant supplementation not only by empowering the relatively low protection against ROS but also by reducing inflammation.

Dietary flavones and flavonoles are inhibitors of poly(ADP-ribose)polymerase-1 in pulmonary epithelial cells.

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1), which was initially known to be highly activated by oxidative stress-induced DNA strand breaks, has been shown to be involved in the pathophysiology of acute and chronic inflammatory diseases. PARP-1 deficiency in mice led to the discovery of its coactivating function in the nuclear factor-kappa B-mediated gene expression and in addition, pharmaceutical inhibition of PARP-1 was shown to reduce the production of inflammatory mediators. In this study, the in vitro PARP-1-inhibiting effect of various flavonoids was investigated. The flavonoids myricetin, tricetin, gossypetin, delphinidin, quercetin, and fisetin were identified as significant inhibitors of the purified enzyme. Further evaluation of these compounds in N-methyl-N'-nitro-N-nitrosoguanidine-treated human pulmonary epithelial cells showed that the formation of the poly(ADP-ribose) polymers, as well as the decreased NAD(+) levels, was reduced by quercetin, fisetin, and tricetin. Finally, IL-8 production of LPS-stimulated human pulmonary epithelial cells could be significantly reduced by these flavonoids. The results of this study indicate that specific flavonoids have PARP-1-inhibiting activity in addition to the earlier described antioxidant effects. PARP-1 inhibition and preservation of cellular NAD(+) and energy production could play a role in the antiinflammatory activity of these specific flavonoids. In addition, these results indicate additional mechanisms by which flavonoids can exert antiinflammatory activity. Furthermore, these results indicate possibilities to use food-derived flavonoids in the treatment of chronic inflammatory diseases.

Theophylline prevents NAD+ depletion via PARP-1 inhibition in human pulmonary epithelial cells.

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD+, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD+ pool, and of NAD+-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD+ levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.

Generation of free radicals and induction of DNA adducts by activation of heterocyclic aromatic amines via different metabolic pathways in vitro.

Food-derived heterocyclic aromatic amines (HCAs) have proved to be carcinogenic in both rodents and nonhuman primates. Two different metabolic pathways are suggested for the metabolic activation of HCA. The hepatic pathway proceeds via a two-step process involving N-hydroxylation by cytochrome P4501A2 and subsequent O-acetylation by N-acetyltransferase-2. An alternative pathway may be of particular interest in extrahepatic tissues and proceeds via one-electron oxidation catalyzed by prostaglandin H synthase (PHS), rendering free-radical metabolites. In this study, we investigated the metabolic activation of two HCAs, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), by two different enzyme systems in vitro, generating different primary and secondary reactive metabolites. Rat liver S9 mix and PHS were used as the activating system and represent the hepatic and extrahepatic pathways, respectively. Electron-spin resonance spectroscopy showed that both IQ and PhIP exerted inhibiting effects on PHS-mediated formation of hydroxyl radicals during the conversion of arachidonic acid to prostaglandins. Evidence for the formation of HCA free radicals was presented in an indirect way by the formation of glutathione-derived thiyl radicals, with purified PHS as the activating system. Activation by S9 mix did not result in the formation of detectable radical metabolites, showing that the two metabolic routes primarily led to the formation of different metabolites. In all electron-spin resonance experiments, IQ appeared to be more effective than PhIP. In contrasts, DNA adduct analysis by means of (32)P-postlabeling showed similar adduct patterns for S9 and PHS in single-stranded and double-stranded salmon testes DNA after incubation with PhIP, indicating the ultimate formation of a common reactive intermediate. For IQ, activation by PHS led to an additional adduct spot that was not present after S9 activation. Furthermore, activation of IQ resulted in higher adduct levels compared with PhIP for both activation pathways. Overall, adduct levels were higher in single-stranded DNA than double-stranded DNA. Our results showed that the hepatic and extrahepatic pathways resulted in different primary metabolites, while the ultimate formation of a similar reactive intermediate for PhIP, possibly an arylnitrenium ion, suggested that both pathways could play an important role in the onset of carcinogenesis.

In vitro investigations into the interaction of beta-carotene with DNA: evidence for the role of carbon-centered free radicals.

Supplementation by beta-carotene has unexpectedly appeared to increase lung cancer risk among smokers. In order to explain this it has been suggested that at high serum levels of beta-carotene, prooxidant characteristics of beta-carotene may become manifest, yielding reactive oxygen species (ROS) and inducing oxidative DNA damage. It has further been hypothesized that cigarette smoke carcinogens such as benzo[a]pyrene (B[a]P) and/or B[a]P metabolites, may directly react with beta-carotene. Furthermore, beta-carotene oxidation products may have a role in the bioactivation of B[a]P analogous to the peroxide shunt pathway of cytochrome P450 supported by cumene hydroperoxide. The aim of this study was to assess the effects of beta-carotene on the formation of B[a]P-DNA adducts and oxidative DNA damage in vitro in isolated DNA, applying as metabolizing systems rat liver and lung metabolizing fractions and lung metabolizing fractions from smoking and non-smoking humans. We established that beta-carotene in the presence of various metabolizing systems was unable to induce oxidative DNA damage (8-oxo-dG), although beta-carotene is capable of generating ROS spontaneously in the absence of metabolizing fractions. We also could not find an effect of beta-carotene on DNA adduct formation induced by B[a]P upon metabolic activation. We could, however, provide evidence of the occurrence of a carbon-centered beta-carotene radical which was found to be able to interact with B[a]P and to intercalate in DNA.