Phospho Tensin Homolog in Human and Lipid Peroxides in Peripheral Blood Mononuclear Cells Following Exposure to Flavonoids.

Objectives: Studies have shown that human and peripheral blood mononuclear cells (PBMCs) are mostly used for research purposes to study several biochemical endpoints. The effects of the flavonoids, genistein, kaempferol, and quercetin on phospho tensin homolog (PTEN) levels in cancer cells (i.e., breast [BT549], lung [A549]), human embryonic kidney cells (HEK293), and the levels of lipid peroxides (LP) in PBMCs were respectively investigated.Materials and methods: Cancer, kidney, and PBMCs from several donors were each exposed to each of the flavonoids at concentrations of 0, 5, 10, 15, 20, and 25 µM. Our hypotheses were that exposure of cancer and kidney cells to genistein, kaempferol, and quercetin can increase PTEN and decrease lipid peroxides in PBMCs levels respectively to better cope with oxidative stress.Results: The results indicate that the flavonoids increased total PTEN levels in a dose-dependent manner. The effect of quercetin was more pronounced followed by genistein and kaempferol. Furthermore, decreases in lipid peroxides were observed in the PBMCs for the flavonoid-treated samples compared to those exposed to flavonoids and with oxidative stress as described by Fenton's chemistry. Levels of LP in quercetin-treated samples were lower compared to kaempferol and genistein.Conclusions: The findings suggest that the flavonoids play an important role in controlling oxidative stress in several human cells.

Effect of quercetin, genistein and kaempferol on glutathione and glutathione-redox cycle enzymes in 3T3-L1 preadipocytes.

CONTEXT AND OBJECTIVE: Many studies have shown that cellular redox potential is largely determined by glutathione (GSH), which accounts for more than 90% of cellular nonprotein thiols. The aim of this study was to delineate the effect of three flavonoids - namely, quercetin, kaempferol and genistein - and exogenous GSH on oxidative damage by the Fenton's pathway through the GSH and GSH-redox cycle enzymes in 3T3-L1 cells. MATERIALS AND METHODS: 3T3-L1 preadipocytes were exposed to each flavonoid and GSH at concentrations of 0, 5, 10, 15, 20 and 25 µM and then GSH levels and activities of glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rx) and superoxide dismutase (SOD) were measured. RESULTS: Exogenous GSH did not have significant effect on intracellular GSH although slight decrease was observed at 15-25 µM doses. However, each of the three flavonoids sustained intracellular GSH levels in the cells as compared to the respective controls. Quercetin had the most profound effect, followed by kaempferol and genistein in that order. GSH-Px, GSH-Rx and SOD activities increased for all the doses tested compared to their respective controls. Again, quercetin had the maximum increase in enzyme activities followed by kaempferol and genistein for the enzymes tested. DISCUSSION AND CONCLUSION: These findings suggest that the flavonoids play an important role in diminishing oxidation-induced biochemical damages. The enhancement of these enzymes may increase the resistance of the organism against oxidative damage by the Fenton's pathway.

Lipid peroxides and glutathione status in human progenitor mononuclear (U937) cells following exposure to low doses of nickel and copper.

Effects of Cu(2+), Ni(2+) or Cu(2+) + Ni(2+) on lipid peroxide and glutathione (GSH) levels in U937 cells were investigated. Cells were treated with 0, 5, 10, and 20 µM of Cu(2+) and/or Ni(2+) and H(2)O(2) (0.01 mM) and incubated for 24 hours at 37°C. Lipid peroxides were measured by the thiobarbituric acid assay (TBA). GSH intracellular levels were assayed by the GSH assay kit from EMD/Calbiochem (San Diego, California, USA). Cu(2+) or Ni(2+) significantly (P < 0.01) increased lipid peroxides in a dose-dependent manner, compared to controls. The effect was more pronounced for Cu(2+), compared to the Ni(2+)-treated samples. Cu(2+) + Ni(2+) increased lipid peroxides in a significant (P < 0.001), dose-dependent manner, compared to Cu(2+) or Ni(2+) alone (i.e., ratio of 2.5:1-fold for combined versus single treatments, respectively). Cu(2+) or Ni(2+) significantly decreased GSH levels in U937 cells, with the effect being pronounced for Cu(2+). Cu(2+) + Ni(2+) metal ions significantly (P < 0.001) depleted cells of GSH in a dose-dependent manner. Ethylene diamine tetraacetic acid (EDTA) at 50 or 100 µM moderately reduced the Cu(2+)- or Ni(2+)-induced effects on GSH levels. Interestingly, GSH levels generally decreased to half (except for the combined metal dose of 20 µM at 100 µM EDTA) of its level at the highest metal concentration tested for both the single or combined treatments. In conclusion, multiple exposures of cells to metal ions may be lethal to cells, compared to their single treatments.

Effects of Quercetin, Kaempferol, and Exogenous Glutathione on Phospho- and Total-AKT in 3T3-L1 Preadipocytes.

Obesity has been reported to be a risk factor for some types of cancer, such as prostate and lung. The AKT or PI3K-AKT is a signal transduction pathway that promotes survival and growth in response to extracellular signals. The aim of this study was to investigate the effects of two flavonoids, quercetin and kaempferol, and exogenous glutathione (GSH) on the expressions of phospho- and total-AKT levels in 3T3-L1 preadipocytes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were measured in the treated samples and used as the internal standard. 3T3-L1 preadipocytes were exposed to each flavonoid and GSH at concentrations of 0, 5, 10, 15, 20, and 25 µM, and the levels of phospho- and total-Akt were measured by the MILLIPLEX MAP mates protocol, based on the Luminex xMAP technology (Millipore Corp., St. Charles, MI, USA). GAPDH levels in the preadipocytes were not significantly different at the doses tested for the flavonoids and exogenous GSH. However, significant (p <.05) decreases in phospho-AKT levels in cells treated with quercetin, kaempferol, and GSH at certain doses were observed compared to their respective controls. Total-AKT levels showed the same profile for all the tested compounds. Significant (p <.01) differences were observed for kaempferol (15-25 µM), quercetin at 10 and 20 µM, and GSH at 10 µM compared to their respective controls. Findings suggest that exposure of 3T3-L1 preadipocytes to quercetin, kaempferol, and GSH may block the activation of AKT, suggesting the role such compounds play in cell differentiation in 3T3-L1 cells.

Effects of low doses of quercetin and genistein on oxidation and carbonylation in hemoglobin and myoglobin.

Protein-bound carbonyls have been shown to increase with age as well as in numerous diseases including rheumatoid arthritis, adult respiratory syndrome pulmonary fibrosis, diabetes, Parkinson's disease, and Alzheimer's just to mention a few. The effects of the flavonoids quercetin and genistein were investigated according to their ability to inhibit the oxidation of hemoglobin and myoglobin via the Fenton's pathway. Antioxidative activity of the flavonoids were determined by oxidizing hemoglobin and myoglobin in separate experiments with 50 µM Fe(2+) and 0.01 mM hydrogen peroxide (H2O2) with and without quercetin and/or genistein. The samples were treated singly with either quercetin, genistein, or in combination at concentrations of 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 µM, respectively, dissolved in dimethyl sulfoxide (DMSO). Samples were then incubated in a water bath at 37°C for 8, 12, and 24 hr, respectively. Levels of carbonylation were assayed by the protein carbonyl assay and the carbonyl levels quantified and expressed per mg of protein. The results indicate that protein carbonyls for samples treated with quercetin or genistein decreased in a dose-dependent manner compared to the controls. That of quercetin compared to genistein was more efficient in reducing the levels of protein carbonylation in hemoglobin and myoglobin, respectively. The combination of both flavonoids did show a gradual decrease in carbonyl compounds for only hemoglobin for all the doses and times tested. The results indicate that both flavonoids at low doses inhibited carbonylation in both hemoglobin and myoglobin and the inhibition may be attributed to the prevention of protein oxidation.

In vitro exposure to quercetin and genistein alters lipid peroxides and prevents the loss of glutathione in human progenitor mononuclear (U937) cells.

The effects of flavonoids quercetin and genistein were investigated according to their potency to inhibit the oxidation of U937 cells via Fenton's pathway through the analysis of lipid peroxides and glutathione (GSH) levels. Human leukemia (U937) cells from the American Type Culture Collection were maintained at 37 degrees C for 24 h under 5% CO2 tension in RPMI-1640 medium containing 10% fetal bovine serum and 50 units ml(-1) each of penicillin and streptomycin. Cells were oxidized with iron 50 microM) or copper (50 microM) in H2O2 (0.01 mM) without or with a flavonoid sample (10 or 20 microM) for the lipid peroxidation studies. The GSH levels were measured (GSH Kit) before and after oxidation as above with different concentrations of flavonoids (0-40 microM). Lipid peroxide was measured by the thiobarbituric acid assay. Both quercetin and genistein at either the 10 or 20 microM level decreased lipid peroxidation significantly compared with their respective controls (P < 0.01). Lipid peroxides by Fe compared to the Cu-treated samples did not differ significantly from each other. However, the combination of flavonoids at the doses tested significantly (P < 0.001) decreased lipid peroxides, the effect being the same for both metal ions. The GSH levels increased significantly before exposure to the metal ions (for the different doses for the differences between the flavonoid samples and their respective untreated levels). For quercetin and genistein the increases in GSH above their untreated levels were 4.5, 8.3, 11.7 and 15 and 3.8, 7.9, 12.5 and 14.6 nmol 10(-6) cells, respectively, for the 5-40 microM levels tested for each flavonoid. Following the exposure to the metal ions, GSH levels remained almost the same for the different concentrations for each of the flavonoids tested but significantly above all of the controls and same for those of the untreated samples. The results indicate that both flavonoids inhibited lipid peroxides and the inhibition may be attributed to the prevention of loss of intracellular GSH levels in U937 cells.

Effect of quercetin and genistein on copper- and iron-induced lipid peroxidation in methyl linolenate.

The single and combined effects of two abundant flavonoids, namely quercetin and genistein, were investigated according to their ability to inhibit the oxidation of methyl linolenate via Fenton's pathway. Antioxidative activity was determined by oxidizing methyl linolenate suspended in a buffer solution with either Fe2+ (50 microM) or Cu2+ (50 microM) and hydrogen peroxide (0.01 mM) without or with a flavonoid sample (10 or 20 microM). Lipid peroxidation products were measured by the thiobarbituric acid (TBA) assay and the amounts of thiobarbituric acid-reactive substances (TBARS) were calculated from a calibration curve using 1,1,3,3-tetraethoxypropane as the standard. Both quercetin and genistein at the 10 or 20 microM level decreased lipid peroxidation significantly compared with their respective controls. Of the two flavonoids tested, quercetin had a more marked effect on inhibiting lipid peroxides. Peroxidation products for the control samples were higher for the Fe2+-treated samples compared with the Cu2+ samples. Combination of both flavonoids at the same dose levels continued to decrease lipid peroxidation, the effect being the same for both metal ions. The data suggest that the combined flavonoids offered better protection than the single treatments and this may be attributed to the better radical scavenging or increased chelating capabilities of the combined over the single treatments. The differences in peroxide levels for the single treatment of quercetin compared with the genistein-treated samples may reflect the structural differences between these compounds in combating oxidative stress.

Supramolecular aggregation in 4,4'-bipyridin-1,1'-ium dichloride, 4,4'-bipyridin-1,1'-ium dinitrate and 4,4'-bipyridin-1-ium bromide.

4,4'-Bipyridin-1,1'-ium dichloride [C(10)H(10)Cl(2)N(2) (I)] and 4,4'-bipyridin-1,1'-ium dinitrate [C(10)H(10)N(4)O(6) (II)] have been prepared and the crystal structures determined at 90.0 (2) K. Molecules of (I) are linked by two chlorine-bridged, three-centered N-H...Cl hydrogen bonds into chains along the b axis. The chains are coupled by weak C-H...Cl interactions into a molecular ladder along the c direction. In (II) each nitrate is coordinated to four bipyridinium ions through the interplay of the N-H...O and C-H...O contacts, resulting in a three-dimensional zigzag sheet on the ab plane. The sheets stack along the c axis. In 4,4'-bipyridin-1-ium bromide monohydrate [C(10)H(9)N(2)(+).Br(-).H(2)O (III)] the bipyridinium ions are linked by three-center N-H.N' hydrogen bonds in a head-to-tail fashion to form chains along the b axis. The chains are linked by C-H...Br and C-H...OH2 into a three-dimensional framework.