Benjamin H.S. Lau

S-Allyl Cysteine Inhibits Activation of Nuclear Factor Kappa B in Human T Cells

Reactive oxygen species are involved in signal transduction pathways leading to nuclear factor kappa B (NF-kappa B) activation which has been implicated in the regulation of gene transcription. We recently reported that a garlic compound, S-allyl cysteine (SAC), protects bovine pulmonary artery endothelial cells from oxidant injury induced by hydrogen peroxide (H2O2). In this study we determined the effects of SAC on NF-kappa B activation in human T lymphocytes (Jurkat cells) induced by tumor necrosis factor alpha (TNF- alpha) and H2O2. Activated NF-kappa B in nuclear extracts was measured by an electrophoretic mobility shift assay using 32P-labeled probe. SAC consistently exhibited a dose-dependent inhibition of NF-kappa B activation induced by both TNF-alpha and H2O2. Supershift with specific antibodies to NF-kappa B subunits confirmed that the inducible retarded bands observed in the EMSA and p65-p50 heterodimer of the NF-kappa B/Rel protein. Our data suggest that SAC may act via antioxidant mechanisms to block NF-kappa B activation in Jurkat cells.

GARLIC INHIBITS FREE RADICAL GENERATION AND AUGMENTS ANTIOXIDANT ENZYME ACTIVITY IN VASCULAR ENDOTHELIAL CELLS

Abstract Oxygen free radicals have been implicated in mediating various pathological processes such as ischemia, inflammatory diseases, diabetes, and atherosclerosis. The antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)—play an important role in scavenging oxidants and preventing cell injury. Aged Garlic Extract (AGE) has been shown to prevent oxidant-induced injury of endothelial cells. The present study determined the effects of AGE on the generation of hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 − ) and the activity of three antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with AGE, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H 2 O 2 . AGE exhibited both concentration- and time-dependent suppression of H 2 O 2 and O 2 − generation, and it also significantly increased the activities of SOD, CAT and GPX. The results suggest that AGE may be an effective antioxidant in preventing or treating disorders related to endothelial cell injury associated with free radicals.

Ginkgo biloba attenuates oxidative stress in macrophages and endothelial cells.

The action of Ginkgo biloba extract (GBE) as an antioxidant was studied using various models of oxidative stress in macrophages and vascular endothelial cells. GBE was incubated with murine macrophages (J774) at 37 degrees C and 5% CO2 for 1 h; oxidative burst was triggered by zymosan. The intensity of fluorescence was measured directly in 96-well plates using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. GBE exhibited both time- and concentration-dependent suppression of oxidative burst. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were preincubated with different concentrations of GBE for 16 h, washed, and then exposed to an organic oxidant tert-butyl hydroperoxide (tBHP) for 2 h. Lipid peroxidation products of PAEC were determined by measuring thiobarbituric acid-reactive substances (TBARS). Cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH), and cell viability was determined by the methylthiazol tetrazolium (MTT) assay. tBHP increased production of TBARS in PAEC. Preincubation with GBE inhibited the increase of TBARS induced by tBHP. GBE protected biomembranes from oxidative injury by decreasing intracellular LDH leakage from PAEC. MTT assay showed that GBE minimized loss of cell viability induced by oxidative injury. The extensive antioxidant effect of GBE may be valuable to the prevention and treatment of various disorders related to free radical-induced pathology.

Aged garlic extract modulates glutathione redox cycle and superoxide dismutase activity in vascular endothelial cells

In this study the effect of an aged garlic extract (AGE) on glutathione (GSH) redox cycle and activity of antioxidant enzymes was investigated. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were incubated with varying concentrations of AGE for 8–48 h, washed, and then lysed with Triton X‐100. Biochemical assays were performed with the lysate. AGE caused both dose‐ and time‐dependent increases in intracellular GSH level, glutathione disulphide (GSSG) reductase and superoxide dismutase (SOD) activity while GSSG level was decreased. These results suggest that the antioxidant effect of AGE may be due to its modulation of the GSH redox cycle and SOD activity in vascular endothelial cells.

Pycnogenol inhibits generation of inflammatory mediators in macrophages

Abstract Pycnogenol (procyanidins extracted from the bark of Pinus maritima Aiton) is a potent free radical scavenger. In this study, we used macrophages (a major cell type in inflammation) to study the antiinflammatory effect of pycnogenol. We determined the effects of pycnogenol on peroxide generation and on glutathione (GSH)-dependent and GSH-independent antioxidant systems. J774 cells were incubated with pycnogenol at 37°C and 5% CO 2 for 2 h. Generation of peroxides (one of the major mediators of inflammation) was triggered by zymosan and measured with a fluorometric assay. Pycnogenol exhibited a concentration-dependent suppression of peroxide release. To investigate possible mechanisms involved in pycnogenols suppression of peroxide release, confluent monolayers of J774 cells were incubated with pycnogenol for 18 h, washed, resuspended in phosphate buffered saline, and sonicated to obtain a cell lysate. Biochemical assays were performed with the lysate. Pycnogenol increased the intracellular GSH content and activities of GSH peroxidase and GSH disulfide reductase, indicating its ability to modulate the GSH redox cycle. The activity of antioxidant enzymes catalase and superoxide dismutase also increased with pycnogenol treatment. These results suggest that the antiinflammatory effect of pycnogenol may at least in part be due to its ability to modulate the GSH redox cycle and activities of catalase and superoxide dismutase.

Effect of allixin, a phytoalexin produced by garlic, on mutagenesis, DNA-binding and metabolism of aflatoxin B1

Allixin, a phytoalexin isolated from garlic, was examined for its effects on aflatoxin B1(AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver S9 fraction as the metabolic activation system. The effects of allixin on the binding of [3H]AFB1 to calf thymus DNA and on the formation of metabolites of [3H]AFB1 were also determined. Allixin showed a dose-related inhibition of Histidine+ revertants induced by AFB1. Allixin at 75 micrograms/ml inhibited [3H]AFB1 binding to calf thymus DNA and reduced formation of AFB1-DNA adducts. In addition, allixin exhibited a concentration-dependent inhibition of the formation of organosoluble metabolites and the glutathione conjugates of [3H]AFB1. The data indicate that the effect of allixin on AFB1-induced mutagenesis and binding of metabolites to DNA may be mediated through an inhibition of microsomal P-450 enzymes. Allixin may thus be useful in the chemoprevention of cancer.

Selective Inhibition of Cell Proliferation by Lycopene in MCF-7 Breast Cancer Cells In vitro: A Proteomic Analysis

Lycopene, a red pigmented carotenoid present in many fruits and vegetables such as tomatoes, has been associated with the reduced risk of breast cancer. This study sought to identify proteins modulated by lycopene during cell proliferation of the breast cancer cell line MCF‐7 to gain an understanding into its mechanism of action. MCF‐7 breast cancer cells and MCF‐10 normal breast cells were treated with 0, 2, 4, 6, 8, and 10 μM of lycopene for 72 h. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) tetrazolium reduction assay was used to measure cell proliferation and two‐dimensional fluorescence difference gel electrophoresis to assess the changes in protein expression, which were identified using MALDI‐ToF/ToF (matrix‐assisted laser desorption ionization tandem time‐of‐flight) and Mascot database search. MTT and cell proliferation assays showed that lycopene selectively inhibited the growth of MCF‐7 but not MCF‐10 cells. Difference gel electrophoresis analysis revealed that proteins in the MCF‐7 cells respond differently to lycopene compared with the MCF‐10 cells. Lycopene altered the expression levels of proteins such as Cytokeratin 8/18 (CK8/18), CK19 and their post translational status. We have shown that lycopene inhibits cell proliferation in MCF‐7 human breast cancer cells but not in the MCF‐10 mammary epithelial cells. Lycopene was shown to modulate cell cycle proteins such as beta tubulin, CK8/18, CK19 and heat shock proteins. Copyright

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of aflatoxin B1

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors while Astragalus membranaceus (AM) and Ligustrum lucidum (LL) are often used as an adjunct in cancer therapy. In this study, we determined the effects of aqueous extracts of these four herbs on aflatoxin B1 (AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant as the activation system. The effects of these herbs on [3H]AFB1 binding to calf-thymus DNA were assessed. Organosoluble and water-soluble metabolites of AFB1 were extracted and analyzed by high-performance liquid chromatography (HPLC). Mutagenesis assays revealed that all of these herbs produced a concentration-dependent inhibition of histidine-independent revertant (His+) colonies induced by AFB1. At a concentration of 1.5 mg/plate, SB and OD in combination exhibited an additive effect. The trend of inhibition of these four herbs on AFB1-induced mutagenesis was: SB greater than LL greater than AM. LL, OD and SB significantly inhibited AFB1 binding to DNA, reduced AFB1-DNA adduct formation, and also significantly decreased the formation of organosoluble metabolites of AFB1. Our data suggest that these Chinese medicinal herbs possess cancer chemopreventive properties.

Modulation of cytochrome P-450IA1-mediated mutagenicity, DNA binding and metabolism of benzo[a]pyrene by Chinese medicinal herbs

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. We previously showed that they inhibited mutagenesis, DNA binding and metabolism of benzo[a]pyrene (BaP) and aflatoxin B1 (AFB1) bioactivated by Aroclor 1254-induced rat hepatic S9. The purpose of this study was to investigate the effects of OD and SB on the cytochrome P-450IA1-mediated mutagenicity of BaP in Salmonella typhimurium TA100 using beta-naphthoflavone (beta NF)-induced rat hepatic S9. We also determined the effects of OD and SB on cytochrome P-450IA1-linked ethoxyresorufin O-deethylase (EROD) activity in beta NF-induced hepatic microsomes. In addition, we studied the effects of these two herbs on BaP metabolite binding to calf thymus DNA and using high performance liquid chromatography (HPLC) we investigated the effects of OD and SB on the metabolism of BaP by beta NF-induced S9. Our experimental results showed that OD and SB inhibited the mutagenicity of BaP in the presence of either non-induced or beta NF-induced S9. SB significantly inhibited BaP binding to DNA. These effects correlated with the inhibition of cytochrome P-450IA1-linked EROD activity in beta NF-induced microsomes and with an inhibition of beta NF-induced S9 mediated metabolism of [3H]BaP as determined by HPLC. These results suggest that OD and SB may possess antimutagenic activity by inhibiting P-450IA-mediated metabolism of BaP.

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of benzo[a]pyrene 7,8-dihydrodiol and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide

Oldenlandia diffusa(OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. In this study, the effects of aqueous extracts of these two herbs on benzo[a]pyrene 7,8-dihydrodiol. (BaP 7,8-DHD) and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system were assessed. We also determined the effects of these two herbs on BaP 7,8-DHD and BPDE binding to calf thymus DNA. Organosoluble metabolites of BaP 7,8-DHD and water-soluble conjugates of BaP 7,8-DHD and BPDE were analyzed by high-performance liquid chromatography (HPLC) and alumina column liquid chromatography. Mutagenesis assays revealed that these two herbs produced a significant concentration-dependent inhibition of histidine-independent (His+) revertants induced by BaP 7,8-DHD and BPDE. OD and SB also inhibited BPDE-induced mutagenesis in a concentration-dependent manner in the absence of S9. SB had a greater inhibitory effect than OD. SB significantly inhibited BaP 7,8-DHD and BPDE binding to DNA while OD significantly enhanced DNA binding of both compounds. OD and SB inhibited the formation of organosoluble metabolites of BaP 7,8-DHD and decreased the formation of water-soluble conjugates of BaP 7,8-DHD and BPDE. However, the fraction of the total radioactivity in the water-soluble conjugates present as sulfate and glutathione was increased by OD and SB. Glucuronide fraction was decreased. The results of this study affirm our previous work suggesting that these two Chinese medicinal herbs possess antimutagenic properties and further suggest that they act as blocking agents through a scavenging mechanism.