Cross-talk between microRNAs, nuclear factor E2-related factor 2, and heme oxygenase-1 in ochratoxin A-induced toxic effects in renal proximal tubular epithelial cells.

SCOPE: Ochratoxin A (OTA) is a mycotoxin exhibiting nephrotoxic and potential carcinogenic activity. We investigated the cross-talk between microRNAs, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in ochratoxin A-mediated effects. METHODS AND RESULTS: In porcine renal proximal tubular cells, OTA increased expression of profibrotic transforming growth factors ß (TGFß) while concomitantly decreasing expression of Nrf2, HO-1, and erythropoietin. Adenoviral overexpression of Nrf2 counteracted OTA-mediated reduction in HO-1 and erythropoietin expression and cell proliferation as well as increase in reactive oxygen species (ROS) generation and TGFß expression. Additionally, inhibition of HO activity enhanced whereas adenoviral overexpression of HO-1 reduced expression of TGFß. Moreover, antioxidants, N-acetyl-cysteine and desferioxamine, prevented OTA-mediated enhancement of ROS generation, and TGFß expression. Finally, OTA modulated microRNA processing by upregulating LINeage protein 28 and DiGeorge syndrome critical region-8, increasing the total pool of cellular microRNAs and elevating the expression of miR-132 and miR-200c. Inhibition of miR-132 by specific antagomir restored the OTA-driven reduction in Nrf2 expression. Moreover, anti-miR-132 and anti-miR-200c counteracted OTA-mediated decrease in HO-1 levels as well as increase in ROS production and TGFß expression. CONCLUSION: We showed that attenuation of Nrf2 and HO-1 expression through induction of miR-132 and miR-200c by OTA elevates ROS levels and profibrotic TGFß expression.

Effect of quercetin on inflammatory gene expression in mice liver in vivo - role of redox factor 1, miRNA-122 and miRNA-125b.

The anti-inflammatory properties of the flavonol quercetin have been intensively investigated using in vitro cell systems and are to a great extent reflected by changes in the expression of inflammatory markers. However, information relating to the degree at which quercetin affects inflammatory gene expression in vivo is limited. Recently, micro RNAs (miRNAs) have been identified as powerful post-transcriptional gene regulators. The effect of quercetin on miRNA regulation in vivo is largely unknown. Laboratory mice were fed for six weeks with control or quercetin enriched high fat diets and biomarkers of inflammation as well as hepatic levels of miRNAs previously involved in inflammation (miR-125b) and lipid metabolism (miR-122) were determined. We found lower mRNA steady state levels of the inflammatory genes interleukin 6, C-reactive protein, monocyte chemoattractant protein 1, and acyloxyacyl hydrolase in quercetin fed mice. In addition we found evidence for an involvement of redox factor 1, a modulator of nuclear factor κB signalling, on the attenuation of inflammatory gene expression mediated by dietary quercetin. Furthermore, the results demonstrate that hepatic miR-122 and miR-125b concentrations were increased by dietary quercetin supplementation and may therefore contribute to the gene-regulatory activity of quercetin in vivo.

Interaction between marginal zinc and high fat supply on lipid metabolism and growth of weanling rats.

The impact of a moderate Zn deficiency on growth and plasma and liver lipids was investigated in two 4-week experiments with male weanling rats fed fat-enriched diets. Semisynthetic, approximately isocaloric diets containing 3% soybean oil were supplemented with either 7 or 100 mg Zn/kg diet and with 22% beef tallow (BT) or sunflower oil (SF). In Experiment 1, which compared the dietary fat level and the fat source in a factorial design of treatments, all diets were fed ad libitum to 6 × 8 animals, whereas intake of the high-Zn BT and SF diets was restricted in Experiment 2 (5 × 6 rats) to the level of intake of the respective low-Zn diets. The low-Zn SF diet consistently depressed food intake and final live weights of the animals to a greater extent than the other low-Zn diets, while intake and growth were comparable among the animals fed the high-Zn diets. The marginal Zn deficit per se did not alter plasma triglyceride and cholesterol concentrations nor hepatic concentrations of triglyceride, cholesterol and phospholipids. The fatty acid pattern of liver phospholipids did not indicate that chain elongation and desaturation of fatty acids was impaired by a lack of zinc. It was concluded that dietary energy and fat intake, and fat source have a greater effect on plasma and liver lipids than a moderate Zn deficiency. Marginally Zn-deficient diets enriched with sunflower oil as a major energy source cause a greater growth retardation than diets rich in carbohydrates or beef tallow.

Importance of ß,ß-carotene 15,15'-monooxygenase 1 (BCMO1) and ß,ß-carotene 9',10'-dioxygenase 2 (BCDO2) in nutrition and health.

In humans, varying amounts of absorbed ß-carotene are oxidatively cleaved by the enzyme ß,ß-carotene 15,15'-monooxygenase 1 (BCMO1) into two molecules of all-trans-retinal. The other carotenoid cleavage enzyme ß,ß-carotene 9',10'-dioxygenase (BCDO2) cleaves ß-carotene at the 9',10' double bond forming ß-apo-10'-carotenal and ß-ionone. Although the contribution of BCDO2 to vitamin A formation has long been debated, BCMO1 is currently considered the key enzyme for retinoid metabolism. Furthermore, BCMO1 has limited enzyme activity towards carotenoids other than provitamin A carotenoids, whereas BCDO2 exhibits a broader specificity. Both enzymes are located at different sites within the cell, with BCMO1 being a cytosolic protein and BCDO2 being located in the mitochondria. Expression of BCMO1 in tissues other than the intestine has recently revealed its function for tissue-specific retinoid metabolism with importance in embryogenesis and lipid metabolism. On the other hand, biological activity of BCDO2 metabolites has been shown to be important in protecting against carotenoid-induced mitochondrial dysfunction. Single-nucleotide polymorphisms (SNPs) such as R267S and A379V in BCMO1 can partly explain inter-individual variations observed in carotenoid metabolism. Advancing knowledge about the physiological role of these two enzymes will contribute to understanding the importance of carotenoids in health and disease.

Anti-inflammatory potential of allyl-isothiocyanate--role of Nrf2, NF-(κ) B and microRNA-155.

In this study, the underlying mechanisms of the potential anti-inflammatory properties of allyl-isothiocyanate (AITC) were analysed in vitro and in vivo. Murine RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) were supplemented with increasing concentrations of AITC. In addition, C57BL/6 mice (n= 10 per group) were fed a pro-inflammatory high-fat diet and AITC was administered orally via gavage for 7 days. Biomarkers of inflammation were determined both in cultured cells and in mice. AITC significantly decreased tumour necrosis factor α mRNA levels and its secretion in LPS stimulated RAW264.7 macrophages. Furthermore, gene expression of other pro-inflammatory markers including interleukin-1ß and inducible nitric oxide synthase were down-regulated following AITC treatment. AITC decreased nuclear p65 protein levels, a subunit of the transcription factor NF-κB. Importantly, our data indicate that AITC significantly attenuated microRNA-155 levels in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner. The anti-inflammatory effects of AITC were accompanied by an increase in Nrf2 nuclear translocation and consequently by an increase of mRNA and protein levels of the Nrf2 target gene heme-oxygenase 1. AITC was slightly less potent than sulforaphane (used as a positive control) in down-regulating inflammation in LPS-stimulated macrophages. A significant increase in nuclear Nrf2 and heme-oxygenase 1 gene expression and only a moderate down-regulation of interleukin-1ß and microRNA-155 levels due to AITC was found in mouse liver. Present data suggest that AITC exhibits potent anti-inflammatory activity in cultured macrophages in vitro but has only little anti-inflammatory activity in mice in vivo.

Nrf2-dependent gene expression is affected by the proatherogenic apoE4 genotype-studies in targeted gene replacement mice.

An apoE4 genotype is an important risk factor for cardiovascular and other chronic diseases. The higher cardiovascular disease risk of apoE4 carriers as compared to the apoE3 genotype has been mainly attributed to the differences in blood lipids between the two genotype subgroups. Recently, a potential protective role of the transcription factor Nrf2 in cardiovascular disease prevention has been suggested. In this study, we show that Nrf2-dependent gene expression is affected by the apoE genotype. ApoE4 vs. apoE3 mice exhibited lower hepatic Nrf2 nuclear protein levels. Furthermore, mRNA and protein levels of Nrf2 target genes including glutathione-S-transferase, heme oxygenase-1 and NAD(P)H dehydrogenase, quinone 1 were significantly lower in apoE4 as compared to apoE3 mice. Lower hepatic mRNA levels of phase II enzymes, as observed in apoE4 vs. apoE3 mice, were accompanied by higher mRNA levels of phase I enzymes including Cyp26a1 and Cyp3a16. Furthermore, miRNA-144, miRNA-125b, and miRNA-29a involved in Nrf2 signaling, inflammation, and regulation of phase I enzyme gene expression were affected by the apoE genotype. We provide first evidence that Nrf2 is differentially regulated in response to the apoE genotype.

APOE ε4 is associated with higher vitamin D levels in targeted replacement mice and humans.

The allele ε4 of apolipoprotein E (APOE), which is a key regulator of lipid metabolism, represents a risk factor for cardiovascular diseases and Alzheimer's disease. Despite its adverse effects, the allele is common and shows a nonrandom global distribution that is thought to be the result of evolutionary adaptation. One hypothesis proposes that the APOE ε4 allele protects against vitamin D deficiency. Here we present, for the first time, experimental and epidemiological evidence that the APOE ε4 allele is indeed associated with higher serum vitamin D [25(OH)D] levels. In APOE4 targeted replacement mice, significantly higher 25(OH)D levels were found compared with those in APOE2 and APOE3 mice (70.9 vs. 41.8 and 27.8 nM, P<0.05). Furthermore, multivariate adjusted models show a positive association of the APOE ε4 allele with 25(OH)D levels in a small collective of human subjects (n=93; P=0.072) and a general population sample (n=699; P=0.003). The novel link suggests ε4 as a modulator of vitamin D status. Although this result agrees well with evolutionary aspects, it appears contradictory with regard to chronic diseases, especially cardiovascular disease. Large prospective cohort studies are now needed to investigate the potential implications of this finding for chronic disease risks.

Paraoxonase 1 polymorphism Q192R affects the pro-inflammatory cytokine TNF-alpha in healthy males.

BACKGROUND: Human paraoxonase 1 (PON1) is an HDL-associated enzyme with anti-oxidant/anti-inflammatory properties that has been suggested to play an important protective role against coronary heart diseases and underlying atherogenesis. The common PON1 Q192R polymorphism (rs662, A>G), a glutamine to arginine substitution at amino acid residue 192, has been analyzed in numerous association studies as a genetic marker for coronary heart diseases, however, with controversial results. FINDINGS: To get a better understanding about the pathophysiological function of PON1, we analyzed the relationships between the Q192R polymorphism, serum paraoxonase activity and serum biomarkers important for atherogenesis. Genotyping a cohort of 49 healthy German males for the Q192R polymorphism revealed an allele distribution of 0.74 and 0.26 for the Q and R allele, respectively, typical for Caucasian populations. Presence of the R192 allele was found to be associated with a significantly increased paraoxonase enzyme activity of 187.8 ± 11.4 U/l in comparison to the QQ192 genotype with 60.5 ± 4.9 U/l. No significant differences among the genotypes were found for blood pressure, asymmetric dimethylarginine, LDL, HDL, triglycerides, and cholesterol. As expected, MIP-2 alpha a cytokine rather not related to atherosclerosis is not affected by the PON1 polymorphism. In contrast to that, the pro-inflammatory cytokine TNF-alpha is enhanced in R192 carriers (163.8 ± 24.7 pg/ml vs 94.7 ± 3.2 pg/ml in QQ192 carriers). CONCLUSIONS: Our findings support the hypothesis that the common PON1 R192 allele may be a genetic risk factor for atherogenesis by inducing chronic low-grade inflammation.

Stress resistance and longevity are not directly linked to levels of enzymatic antioxidants in the ponerine ant Harpegnathos saltator.

BACKGROUND: The molecular mechanisms of variations in individual longevity are not well understood, even though longevity can be increased substantially by means of diverse experimental manipulations. One of the factors supposed to be involved in the increase of longevity is a higher stress resistance. To test this hypothesis in a natural system, eusocial insects such as bees or ants are ideally suited. In contrast to most other eusocial insects, ponerine ants show a peculiar life history that comprises the possibility to switch during adult life from a normal worker to a reproductive gamergate, therewith increasing their life expectancy significantly. RESULTS: We show that increased resistance against major stressors, such as reactive oxygen species and infection accompanies the switch from a life-history trait with normal lifespan to one with a longer life expectancy. A short period of social isolation was sufficient to enhance stress resistance of workers from the ponerine ant species Harpegnathos saltator significantly. All ant groups with increased stress resistances (reproducing gamergates and socially isolated workers) have lower catalase activities and glutathione levels than normal workers. Therewith, these ants resemble the characteristics of the youngest ants in the colony. CONCLUSIONS: Social insects with their specific life history including a switch from normal workers to reproducing gamergates during adult life are well suited for ageing research. The regulation of stress resistance in gamergates seemed to be modified compared to foraging workers in an economic way. Interestingly, a switch towards more stress resistant animals can also be induced by a brief period of social isolation, which may already be associated with a shift to a reproductive trajectory. In Harpegnathos saltator, stress resistances are differently and potentially more economically regulated in reproductive individuals, highlighting the significance of reproduction for an increase in longevity in social insects. As already shown for other organisms with a long lifespan, this trait is not directly coupled to higher levels of enzymatic and non-enzymatic antioxidants.

Ascorbic acid partly antagonizes resveratrol mediated heme oxygenase-1 but not paraoxonase-1 induction in cultured hepatocytes - role of the redox-regulated transcription factor Nrf2.

BACKGROUND: Both resveratrol and vitamin C (ascorbic acid) are frequently used in complementary and alternative medicine. However, little is known about the underlying mechanisms for potential health benefits of resveratrol and its interactions with ascorbic acid. METHODS: The antioxidant enzymes heme oxygenase-1 and paraoxonase-1 were analysed for their mRNA and protein levels in HUH7 liver cells treated with 10 and 25 µmol/l resveratrol in the absence and presence of 100 and 1000 µmol/l ascorbic acid. Additionally the transactivation of the transcription factor Nrf2 and paraoxonase-1 were determined by reporter gene assays. RESULTS: Here, we demonstrate that resveratrol induces the antioxidant enzymes heme oxygenase-1 and paraoxonase-1 in cultured hepatocytes. Heme oxygenase-1 induction by resveratrol was accompanied by an increase in Nrf2 transactivation. Resveratrol mediated Nrf2 transactivation as well as heme oxygenase-1 induction were partly antagonized by 1000 µmol/l ascorbic acid. CONCLUSIONS: Unlike heme oxygenase-1 (which is highly regulated by Nrf2) paraoxonase-1 (which exhibits fewer ARE/Nrf2 binding sites in its promoter) induction by resveratrol was not counteracted by ascorbic acid. Addition of resveratrol to the cell culture medium produced relatively low levels of hydrogen peroxide which may be a positive hormetic redox-signal for Nrf2 dependent gene expression thereby driving heme oxygenase-1 induction. However, high concentrations of ascorbic acid manifold increased hydrogen peroxide production in the cell culture medium which may be a stress signal thereby disrupting the Nrf2 signalling pathway.

Comparison of tetrahydrofuran, fetal calf serum, and Tween 40 for the delivery of astaxanthin and canthaxanthin to HepG2 cells.

The present investigation aimed to compare fetal calf serum (FCS) and Tween 40 with the commonly employed tetrahydrofuran (THF) with respect to cytotoxicity, stability of the solubilized carotenoids, and uptake and accumulation of the xanthophylls astaxanthin (AX) and canthaxanthin (CX) in cultured human liver cells (HepG2). Incubation of HepG2 cells for 24 h with THF (≥1.25%) or FCS (≥11.25%) with or without AX (≥25 µmol/L) or CX (≥25 µmol/L) did not affect cell viability. Tween 40 (0.25-1.25% in medium) reduced cell viability by 75-99%. The stabilities of AX and CX in cell-free RPMI 1640 medium for ≤24 h were higher when delivered with THF instead of FCS. The dose- and time-dependent accumulations of AX and CX (1-10 µmol/L) in HepG2 cells were higher when carotenoids were delivered with FCS compared to THF. In conclusion, FCS and THF, but not Tween 40, were suitable solvent systems for the delivery of AX and CX to HepG2 cells. In our experiments FCS was superior with regard to the uptake and accumulation of both carotenoids.

Effect of quercetin and its metabolites isorhamnetin and quercetin-3-glucuronide on inflammatory gene expression: role of miR-155.

In the present study the effect of quercetin and its major metabolites quercetin-3-glucuronide (Q3G) and isorhamnetin on inflammatory gene expression was determined in murine RAW264.7 macrophages stimulated with lipopolysaccharide. Quercetin and isorhamnetin but not Q3G significantly decreased mRNA and protein levels of tumor necrosis factor alpha. Furthermore a significant decrease in mRNA levels of interleukin 1ß, interleukin 6, macrophage inflammatory protein 1α and inducible nitric oxide synthase was evident in response to the quercetin treatment. However Q3G did not affect inflammatory gene expression. Anti-inflammatory properties of quercetin and isorhamnetin were accompanied by an increase in heme oxygenase 1 protein levels, a downstream target of the transcription factor Nrf2, known to antagonize chronic inflammation. Furthermore, proinflammatory microRNA-155 was down-regulated by quercetin and isorhamnetin but not by Q3G. Finally, anti-inflammatory properties of quercetin were confirmed in vivo in mice fed quercetin-enriched diets (0.1 mg quercetin/g diet) over 6 weeks.

Determinants of paraoxonase activity in healthy adults.

SCOPE: Paraoxonase-1 (PON-1), associated with HDL, is regarded as anti-atherogenic, attributed to its ability to hydrolyze oxidized lipids. Here, the impact of PON and apolipoprotein E genotypes, age, alcohol and HDL-cholesterol (HDL-C) on PON activity is examined. METHODS AND RESULTS: In total, 104 healthy UK adults participated in the study, with basal (PONA) and stimulated (PONB) PON-1 activities and arylesterase activity determined in these individuals. In univariate and correlation analysis age, HDL-C, alcohol intake and both PON genotypes were significantly associated with PONA and PONB activities (p<0.05). However, in the standard linear regression model, which explained 69% of the variability in both PONA (p<0.001) and PONB activities (p<0.001) only PON Q192R genotype emerged as a significant independent determinant, with four to fivefold higher levels in the RR versus wild-type QQ genotype groups. For PON arylesterase, HDL-C (p=0.030), apolipoprotein E (p=0.023) and PON Q192R (p=0.002) and PON L55M (p=0.002) genotypes collectively explained 14% of the total variability in the regression model. CONCLUSION: Our results indicate that PON genotypes are stronger determinants of PON activity relative to the other potential modulators assessed. The relative impact of dietary components on PON activities remains to be established.

Impact of apolipoprotein E genotype and dietary quercetin on paraoxonase 1 status in apoE3 and apoE4 transgenic mice.

OBJECTIVES: The aim of the present study was to determine hepatic paraoxonase 1 (PON1) status in response to apoE genotype and dietary quercetin supplementation in mice. METHODS AND RESULTS: ApoE3 and apoE4 transgenic mice were fed semi-synthetic diets without (controls) and with quercetin (2 mg/g diet) for 6 weeks. Hepatic mRNA and protein levels of PON1 were significantly lower in apoE4 as compared to apoE3 mice. Feeding quercetin-enriched diets induced hepatic PON1 gene expression with a tendency for greater induction in apoE3 as compared to apoE4 mice. Furthermore, hepatic mRNA and protein levels of beta-glucuronidase and sulfatase, both enzymes centrally involved in the deconjugation of quercetin conjugates, were lower in apoE4 vs. apoE3 mice. PPARgamma (which partly controls PON1 gene expression) mRNA levels were lower in apoE4 vs. apoE3 mice. CONCLUSION: We provide first evidence that PON1 is differentially regulated in response to apoE genotype.

Effect of dietary quercetin on brain quercetin levels and the expression of antioxidant and Alzheimer's disease relevant genes in mice.

The aim of the present feeding trial was to investigate the effect of the dietary flavonol quercetin on brain quercetin concentration and the expression of antioxidant and Alzheimer's disease relevant genes in mouse brain. Laboratory mice were fed control and quercetin-enriched diets (2 mg/g diet) for 6 weeks. Dietary quercetin supplementation significantly increased the levels of quercetin and its methylated metabolite isorhamnetin in plasma and brain. However, quercetin and isorhamnetin levels in the brain were manifold lower as compared to the plasma. Both mRNA and activity levels of alpha- and beta-secretase in cortex remained unchanged by the dietary quercetin supplementation. Furthermore dietary quercetin did not affect brain mRNA levels of neprilysin, heme oxygenase-1 and gamma-glutamylcysteine synthetase. Taken together, a short term dietary treatment with quercetin increased quercetin and isorhamnetin levels in the brain but had no effect on mRNA levels of antioxidant and Alzheimer's disease relevant genes in mouse brain.

Serum lipid and blood pressure responses to quercetin vary in overweight patients by apolipoprotein E genotype.

Our objective was to examine the effect of a quercetin supplementation on blood pressure, lipid metabolism, markers of oxidative stress, inflammation, and body composition in an at-risk population of 93 overweight-obese volunteers aged 25-65 y with metabolic syndrome traits in relation to apolipoprotein (apo) E genotype. Participants were randomized to receive 150 mg/d quercetin in a double-blinded, placebo-controlled, crossover trial with 6-wk treatment periods separated by a 5-wk washout period. Retrospectively, 5 apoE genotype variants were found (epsilon2/epsilon3, n = 3; epsilon3/epsilon3, n = 60; epsilon3/epsilon4, n = 23; epsilon2/epsilon4, n = 4; and epsilon4/epsilon4, n = 3). Participants were classified into the following 3 apoE phenotypes: apoE2 (n = 3), apoE3 (n = 60), and apoE4 (n = 26). Data were analyzed for apoE3 and apoE4 subgroups. Quercetin decreased systolic blood pressure by 3.4 mm Hg (P < 0.01) in the apoE3 group, whereas no significant effect was observed in the apoE4 group. Quercetin decreased serum HDL cholesterol (P < 0.01) and apoA1 (P < 0.01) and increased the LDL:HDL cholesterol ratio (P < 0.05) in the apoE4 subgroup, whereas the apoE3 subgroup had no significant changes in these variables. Quercetin significantly decreased plasma oxidized LDL and tumor necrosis factor-alpha in the apoE3 and apoE4 groups, whereas no significant inter-group differences were found. Serum C-reactive protein and nutritional status (body weight, waist circumference, fat mass, fat-free mass) were unaffected compared with placebo. In conclusion, quercetin exhibited blood pressure-lowering effects in overweight-obese carriers of the apo epsilon3/epsilon3 genotype but not in carriers of the epsilon4 allele. Furthermore, quercetin supplementation resulted in a reduction in HDL cholesterol and apoA1 in apo epsilon4 carriers.

Effects of quercetin and catechin on hepatic glutathione-S transferase (GST), NAD(P)H quinone oxidoreductase 1 (NQO1), and antioxidant enzyme activity levels in rats.

Cell culture data indicate that quercetin and catechin may affect the activity of phase II and antioxidant enzymes. However, little is known about the impact of dietary flavonoids in vivo. Therefore, the present study aimed to investigate the in vivo effects of the flavonoids quercetin and catechin on mRNA and activity levels of phase II enzymes glutathione-S transferase (GST) and NAD(P)H quinone oxidoreductase-1 (NQO1) in rat liver. Furthermore, the activity of the hepatic antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) was determined. Feeding male Wistar rats (3 x 6 animals) over 3 wk with semisynthetic diets enriched with quercetin and catechin (2 g/kg diet) did not affect liver enzyme activity of CAT, GPx, and SOD as well lipid peroxidation and glutathione levels. Dietary quercetin significantly decreased activity of hepatic GST (24%), whereas dietary catechin significantly decreased NQO1 activity (26%) compared to controls. Changes in GST and NQO1 activity were partly reflected on mRNA levels. Current data indicate that dietary flavonoids have little effects on liver oxidant/antioxidant status but do significantly affect the phase II enzymes GST and NQO1 in rat liver. This in turn may affect the ability of the organism to detoxify endogenous and exogenous xenobiotics.

Effect of quercetin on paraoxonase 2 levels in RAW264.7 macrophages and in human monocytes--role of quercetin metabolism.

There is increasing evidence that the intracellular antioxidant enzyme paraoxonase 2 (PON2) may have a protective function in the prevention of atherogenesis. An enhancement of PON2 activity by dietary factors including flavonoids is therefore of interest. In the present study we determined the effect of quercetin on paraoxonase 2 levels in cultured murine macrophages in vitro and in overweight subjects with a high cardiovascular risk phenotype supplemented with 150 mg quercetin/day for 42 days in vivo. Supplementation of murine RAW264.7 macrophages in culture with increasing concentrations of quercetin (1, 10, 20 micromol/L) resulted in a significant increase in PON2 mRNA and protein levels, as compared to untreated controls. Unlike quercetin, its glucuronidated metabolite quercetin-3-glucuronide did not affect PON2 gene expression in cultured macrophages. However the methylated quercetin derivative isorhamnetin enhanced PON2 gene expression in RAW264.7 cells to similar extent like quercetin. Although supplementing human volunteers with quercetin was accompanied by a significant increase in plasma quercetin concentration, dietary quercetin supplementation did not change PON2 mRNA levels in human monocytes in vivo. Current data indicate that quercetin supplementation increases PON2 levels in cultured monocytes in vitro but not in human volunteers in vivo.

Effect of dietary genistein on Phase II and antioxidant enzymes in rat liver.

Isoflavones are thought to be biologically active components in soy that play a role in the prevention of chronic diseases including cancer. How isoflavones may mediate their beneficial effects has not yet been fully established. Potential mechanisms of cellular action of isoflavones may include their ability to modulate gene expression and the activity levels of enzymes involved in antioxidant defence and the metabolism of xenobiotics including NAD(P)H (Nicotinamide-adenine-dinucleotide-phosphate) quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST). Although there is increasing evidence from cell culture studies that genistein, the major isoflavone present in soy, may regulate the expression of genes encoding for phase II and antioxidant enzymes, little is known about its effect in vivo. Feeding rats over 3 weeks with semisynthetic diets enriched with genistein (2 g/kg) significantly increased both the hepatic mRNA and activity levels of NQO1. The total GST activity did not change in response to dietary genistein supplementation, whereas the mRNA levels of individual GST isoenzymes were differentially modulated. The hepatic mRNA level of Gsta2 (class alpha 2) was significantly increased whereas the mRNA levels of Gstm2 (class mu 2) and Gstp1 (class pi 1) were significantly lowered due to genistein supplementation. The protein level of Nrf2 (Nuclear factor E2-related factor 2), a transcription factor involved in the regulation of phase II enzymes, was not altered by dietary genistein. Furthermore, genistein did not affect the hepatic enzyme activity of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) or liver lipid peroxidation and glutathione levels. The induction of NQO1 may be one mechanism by which dietary genistein improves the capacity of the liver to detoxify carcinogens.

Dietary vitamin E, brain redox status and expression of Alzheimer's disease-relevant genes in rats.

Oxidative stress is one of the major pathological features of Alzheimer's disease (AD). Here, we investigated whether dietary vitamin E (VE) depletion may induce adverse effects and supplementation with alpha-tocopherol (alphaT) may result in beneficial effects on redox status and the regulation of genes relevant in the pathogenesis of AD in healthy rats. Three groups of eight male rats each were fed diets with deficient ( < 1 mg alphaT equivalents/kg diet), marginal (9 mg alphaT equivalents/kg diet) or sufficient (18 mg alphaT equivalents/kg diet) concentrations of natural-source VE for 6 months; a fourth group was fed the VE-sufficient diet fortified with alphaT (total VE, 146 mg alphaT equivalents/kg diet). Feeding of the experimental diets dose dependently altered alphaT concentrations in the cortex and plasma. No significant changes in F2-isoprostane concentrations, activities of antioxidative enzymes (total superoxide dismutase, Se-dependent glutathione peroxidase) and concentrations of glutathione or the expression of AD-relevant genes were observed. In this non-AD model, depletion of VE did not induce adverse effects and supplementation of alphaT did not induce positive effects on the parameters related to the progression of AD.