Immunoassay procedures to detect exposure to aflatoxin B1 and benzo(a)pyrene in animals and man at the DNA level

Summary Immunological methods were used to examine human liver for the presence of aflatoxin-DNA adducts and human lung for benzo(a)pyrene diol-epoxide DNA (BPDE-DNA) adducts. Eight liver samples obtained from Czechoslovakian patients with primary hepatocellular carcinoma were studied, seven of which had detectable anti-aflatoxin inhibitory material. Values ranged between 0.63 and 3.51 picomoles aflatoxin per mg DNA. In a separate, independent study performed in another laboratory the one sample with no aflatoxin bound to DNA also had no free aflatoxin present in the liver. In the case of the human lung DNA samples, 12 samples were examined, the samples having been removed during thoracic surgery, and five had detectable anti-BPDE-DNA antibody activity. The positive samples were all from smokers and had inhibitory values ranging from 4 to 12 femtomoles per mg DNA. Samples were prepared by immunoconcentration prior to analysis. These preliminary results support the view that immunological methods can be used to examine human tissue DNA for carcinogen adducts.     

绿茶抑制大鼠体内杂环胺-DNA加合物的形成

食物中的致突变物２－氨基－１－甲基－６－苯基咪唑并「４，５－ｂ」吡啶可诱发大鼠结肠和乳腺肿瘤，而且与人类癌症特别是结肠和直肠癌的密切关系。本研究以致癌物－ＤＮＡ加合物为生物标记物，观察绿茶对ＰｈＩＰ致癌作用的预防效果及机理。     

Prevention of coronary heart disease and cancer by tea,a review

Biomedical research has uncovered the mechanisms whereby tea promotes good health and lowers the risk of major chronic diseases, such as heart disease and many types of cancer. The active components in tea are polyphenols, epigallocatechin gallate in green tea, theaflavins and thearubigins in black tea. Green and black tea and the polyphenols have similar beneficial effects. The mechanisms are categorized into 5 groups. 1) Tea polyphenols are powerful antioxidants. They decrease the oxidation of LDL cholesterol and lower the risk of heart disease, and also inhibit action of reactive oxygen species mediating the oxidation of DNA associated with carcinogenesis 2) Tea polyphenols induce detoxifying enzymes, glucuronosyl transferases, eliminating active forms of carcinogens and other toxicants, accounting for the lower cancer risk. 3) Tea polyphenols lower duplication rates of cancer cells and inhibit the growth of cancer, increase apoptosis and lower angiogenesis. 4) Tea polyphenols alter the intestinal bacterial flora, suppressing undesirable bacteria and favoring growth of beneficial bacteria. 5) Aging phenomena, and diseases associated with the formation of reactive oxygen species (ROS) are inhibited. Presented at the 2001 International Conference on O-CHA (tea) Culture and Science, Shizuoka, Japan, October 7, 2001.     

Green Tea and Cancer Prevention

Extracts of green tea and green tea polyphenols have exhibited inhibitory effects against the formation and development of tumors at different organ sites in animals. These include animal models for skin, lung, oral cavity, esophagus, stomach, intestine, colon, liver, pancreas, bladder, mammary gland, and prostate cancers. In addition to suppressing cell proliferation, promoting apoptosis, and modulating signaling transduction, green tea polyphenols, especially (-)-epigallocatechin-3-gallate, also inhibit cell invasion, angiogenesis, and metastasis. This article reviews data on the cancer preventive activities of green tea polyphenols, possible mechanisms involved, and the relationship between green tea consumption and human cancer risk.     

Green tea and cancer prevention

Extracts of green tea and green tea polyphenols have exhibited inhibitory effects against the formation and development of tumors at different organ sites in animals. These include animal models for skin, lung, oral cavity, esophagus, stomach, intestine, colon, liver, pancreas, bladder, mammary gland, and prostate cancers. In addition to suppressing cell proliferation, promoting apoptosis, and modulating signaling transduction, green tea polyphenols, especially (-)-epigallocatechin-3-gallate, also inhibit cell invasion, angiogenesis, and metastasis. This article reviews data on the cancer preventive activities of green tea polyphenols, possible mechanisms involved, and the relationship between green tea consumption and human cancer risk.     

Bioavailability and antioxidant activity of tea flavanols after consumption of green tea, black tea, or a green tea extract supplement

BACKGROUND: Green and black tea polyphenols have been extensively studied as cancer chemopreventive agents. Many in vitro experiments have supported their strong antioxidant activity. Additional in vivo studies are needed to examine the pharmacokinetic relation of absorption and antioxidant activity of tea polyphenols administered in the form of green or black tea or tea extract supplements. OBJECTIVE: The purpose of this study was to compare the pharmacokinetic disposition of tea polyphenols and their effect on the antioxidant capacity in plasma 8 h after a bolus consumption of either green tea, black tea, or a green tea extract supplement. DESIGN: Thirty healthy subjects were randomly assigned to 3 different sequences of green tea, black tea, or a green tea extract supplement in a 3 x 3 crossover design with a 1-wk washout period in between treatments. RESULTS: Flavanol absorption was enhanced when tea polyphenols were administered as a green tea supplement in capsule form and led to a small but significant increase in plasma antioxidant activity compared with when tea polyphenols were consumed as black tea or green tea. All 3 interventions provided similar amounts of (-)-epigallocatechin-3-gallate. CONCLUSIONS: Our observations suggest that green tea extract supplements retain the beneficial effects of green and black tea and may be used in future chemoprevention studies to provide a large dose of tea polyphenols without the side effects of caffeine associated with green and black tea beverages.     

EGCG和红茶多酚对HepG_2细胞脂质代谢相关基因表达的影响

目的:探讨表没食子儿茶素没食子酸酯(EGCG)和红茶多酚对人肝细胞癌HepG2细胞脂质代谢相关基因表达的影响。方法:HepG2细胞经EGCG(5μmol/L),红茶多酚(5μg/ml)或0.1%二甲亚枫(对照组)处理8h后,抽提RNA,并杂交至人14kcDNA芯片进行基因表达谱分析。应用实时RT-PCR技术对芯片结果进行验证。结果:HepG2细胞经EGCG和红茶多酚处理后表达差异的脂质代谢相关基因数分别为13条和29条,其中表达差异一致的基因有6条。实时RT-PCR结果与芯片结果一致。结论:EGCG和红茶多酚影响脂质代谢的机制是复杂的,此次实验发现的新靶标为今后茶多酚降脂作用研究提供了新的依据。     

茶多酚、茶色素对人肝癌细胞株HepG2端粒酶活性的影响

目的　利用人肝癌细胞株HepG2观察茶多酚和茶色素对端粒酶活性的影响。方法采用TRAP PCR ELISA方法对HepG2细胞端粒酶活性进行定性和定量检测。结果　TRAP PCR定性分析表明空白对照组、茶多酚组和茶色素组均端粒酶阳性 ;ELISA定量结果显示 ,5 0mg/L和 10 0mg/L茶多酚组端粒酶活性 (A450 690 值 )分别为 1 5 6和 1 4 6 ;5 0mg/L和 10 0mg/L茶色素处理组为 1 5 5和1 4 9,与空白对照组 (A450 690 值为 2 11)相比 ,茶多酚和茶色素处理后HepG2细胞端粒酶活性有统计学意义。结论　茶多酚和茶色素显著抑制HepG2细胞端粒酶活性 ,端粒酶活性可能是癌症化学预防研究的一个有用的生物标志物     

Green Tea Polyphenols Inhibit Colorectal Tumorigenesis in Azoxymethane-Treated F344 Rats

In studying the cancer-preventive activities of green tea polyphenols, we previously demonstrated that dietary administration of polyphenon E (PPE) inhibited the formation of aberrant crypt foci (ACF) in the colon of azoxymethane (AOM)-treated F344 rats. Herein, we reported cancer-preventive activity of PPE using colorectal cancer as an end point. F344 rats were given two weekly injections of AOM, and then maintained on a 20% high-fat diet with or without 0.24% PPE for 34 wk. In the control group, 83% of rats developed colorectal tumors. Dietary PPE treatment significantly increased the plasma and colonic levels of tea polyphenols, and decreased tumor multiplicity and tumor size. Histological analysis indicated that PPE significantly decreased the incidence of adenocarcinoma, and the multiplicity of adenocarcinoma as well as the multiplicity of adenoma. PPE treatment significantly decreased plasma levels of proinflammatory eicosanoids, prostaglandin E2, and leukotriene B4. It also decreased β-catenin nuclear expression, induced apoptosis, and increased expression levels of RXRα, β, and γ in adenocarcinomas. In conclusion, our results convincingly demonstrated the inhibitory effects of orally administered PPE on colon carcinogenesis in AOM-treated rats and suggested possible biomarkers for the biological effects of green tea polyphenols.     

Effects of chemoprotective agents on the metabolic activation of the carcinogenic arylamines PhIP and 4-aminobiphenyl in human and rat liver microsomes

Carcinogenic aromatic amines, including the heterocyclic amines, may pose a significant health risk to humans. To determine the potential for chemoprotective intervention against the carcinogenicity of these arylamines and to better understand their mechanism of action, a range of agents, most of them natural dietary constituents, was examined in vitro for their ability to modulate the N-hydroxylation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (ABP), an initial step in their bioactivation. Experiments were conducted with rat and human liver microsomes. The agents (diallyl sulfide, indole-3-carbinol, alpha-angelicalactone, cafestol/kahweol palmitates, cafestol, kahweol, benzylisothiocyanate, genistin, formononetin, daidzin, equol, biochanin A, Oltipraz, tannic acid, quercetin, ethoxyquin, green tea, and black tea) comprised a variety of chemical classes that included sulfur-containing compounds, antioxidants, flavonoids, phytoestrogens, diterpenes, and polyphenols. Several of these agents, quercetin, ethoxyquin, and black tea, were found to strongly inhibit PhIP N-hydroxylation in rat liver microsomes, resulting in a nearly 85-90% decrease in activity at 100 microM or 0.2%. Tannic acid and green tea, in addition to these agents, were also strong inhibitors of ABP N-hydroxylation. In human liver microsomes, each of these agents was strongly inhibitory (approx 85-95% at 100 microM or 0.02%) of PhIP and ABP N-hydroxylation. Theaflavins and polyphenols were judged to be the primary inhibiting components in the teas, the theaflavins showing the most potent effect. These results demonstrate that chemoprotective agents can inhibit the bioactivation of carcinogenic arylamines, and this is likely to be one of the mechanisms of protection.     

Reduction of Hexavalent Chromium by Green Tea Polyphenols and Green Tea Nano Zero-Valent Iron (GT-nZVI)

This study reports on the direct reduction of hexavalent chromium [Cr(VI)] by green tea polyphenols, including a green tea solution and pure epigallocatechin gallate (EGCG) solution. A linear trend was observed between the amount of reduced Cr(VI) and the amount of added polyphenols. The green tea solution showed a continued decrease in the observed stoichiometry with increasing pH, from a maximum of 1.4 mol per gallic acid equivalent (GAE) of green tea at pH 2.5, to 0.2 mol/GAE at pH 8.8. The EGCG solution exhibited different behavior, with a maximum stoichiometry of 2 at pH 7 and minimum of 1.6 at pH 4.4 and 8.9. When green tea was used to first react with Fe³⁺ and form GT-nZVI, the amount of Cr(VI) reduced by a certain volume of GT-nZVI was double compared to green tea, and 6 times as high considering that GT-nZVI only contains 33 % green tea.     

Tea as a potential chemopreventive agent in PhIP carcinogenesis: effects of green tea and black tea on PhIP-DNA adduct formation in female F-344 rats

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of proteinaceous animal foods (meat, chicken, and fish). PhIP is a carcinogen in the Fischer 344 (F-344) rat; it induces mammary tumors in female rats and lymphomas and colon and prostate tumors in male rats. In F-344 rats, PhIP forms DNA adducts in various organs, including the target organs. Inhibition of PhIP-DNA adduct formation is likely to lead to inhibition of PhIP tumorigenicity. We have examined the chemopreventive properties of green tea and black tea in PhIP carcinogenesis by evaluating their effects on PhIP-DNA adduct formation in the female F-344 rat. Young adult animals were maintained on powdered AIN-76A diet while receiving regular drinking water or 2% (wt/vol) infusions of green tea or black tea for a total of six weeks. During Weeks 3, 4, and 5, all animals received PhIP by gavage (1 mg/kg/day). Three rats per group were euthanized on Days 1 and 8 after termination of PhIP exposure. DNA was isolated from a number of organs and analyzed for PhIP-DNA adducts by 32P-postlabeling assays. Compared with animals on regular drinking water, PhIP-DNA adduct formation was inhibited in small intestine, colon, liver, and mammary epithelial cells (MECs) of animals receiving green tea or black tea as the sole source of drinking fluid. Green tea inhibited adduct formation in colon, liver, and MECs (33.3-80.0%) on both days, but only on Day 8 (54.4%) in small intestine. Black tea inhibited adduct formation on both days in liver (71.4-80.0%), on Day 1 in colon (40.0%), and on Day 8 in small intestine (81.8%); it had no effect on MEC adducts. Neither green tea nor black tea had an effect on adduct levels in pancreas, lungs, white blood cells, heart, kidneys, spleen, cecum, or stomach. Similarly, these teas did not affect the rate of adduct removal (percent change from Day 1 to Day 8) in any organ. It is concluded that green tea and black tea are potential chemopreventive agents in PhIP-induced tumorigenesis in the F-344 rat.     

Tea polyphenols and theaflavins are present in prostate tissue of humans and mice after green and black tea consumption

Green and black tea have shown promise in the chemoprevention of prostate cancer. The objective of this study was to determine the bioavailability and bioactivity of tea polyphenols (PP) and theaflavins in human serum and human and mouse tissues. A decaffeinated black tea diet was administered to C57BL/6 mice. PPs and theaflavins were found in the small and large intestine, liver, and prostate in conjugated and free forms. The relative prostate bioavailability of theaflavin was 70% higher than that of epigallocatechin gallate (EGCG). In the second mouse study, a green tea (GT) diet was administered followed by the control diet for 1-5 d. Epicatechin (EC), EGCG, and epicatechin gallate (ECG) concentrations in prostate tissue were significantly decreased after 1 d of consuming the control diet. Epigallocatechin gallate (EGC), however, did not decrease significantly. For the human study, 20 men scheduled for surgical prostatectomy were randomly assigned to consume 1.42 L daily of GT, BT, or a caffeine-matched soda control (SC) for 5 d before radical prostatectomy. Tea PPs were greater in prostate samples from men consuming BT and GT than in men consuming SC (P = 0.0025). Although tea PP were not detectable in serum, ex vivo LNCaP prostate cancer cell proliferation was less when cells were grown in media containing patient serum collected after BT (P < 0.001) and GT (P = 0.025) consumption relative to baseline serum This is the first human study to show that tea polyphenols and theaflavins are bioavailable in the prostate where they may be active in the prevention of prostate cancer.     

Modulatory Effects of Curcumin in Conjunction with Piperine on Benzo(A)Pyrene-Mediated DNA Adducts and Biotransformation Enzymes

The antigenotoxic effects of curcumin alone and with piperine on benzo(a)pyrene-diol (BaP) epoxide DNA adducts (BaPDE-DNA adducts), and carcinogen biotransformation enzymes was investigated in liver and lung of mice. Male Swiss albino mice received curcumin (100 mgkg^?1 body weight) and piperine (20 mgkg^?1 body weight) separately as well as in combination orally in corn oil for 7 days as pretreatments and thereafter 2 h, BaP (125 mgkg^?1 body weight) was administered orally in corn oil. A single dose of BaP to normal mice increased the activities of ethoxyresorufin o-deethylase (EROD), pentoxyresorufin o-depentylase (PROD) and levels of BaPDE-DNA adducts in both the tissues. Quinone reductase (QR) activity was also elevated in the BaP-treated group in both liver and lung when compared with normal control group. Pretreatment of curcumin and curcumin plus piperine before administration of BaP significantly decreased the activities of EROD, PROD, and the level of BaPDE-DNA adducts with consequent increase in QR activities. The study clearly indicates that curcumin, when given in combination with piperine, is more effective in modulating BaPDE-DNA adducts (liver and lung), and activity of EROD (liver).     

Mechanisms of cancer prevention by tea constituents

Consumption of tea (Camellia sinensis) has been suggested to prevent cancer, heart disease and other diseases. Animal studies have shown that tea and tea constituents inhibit carcinogenesis of the skin, lung, oral cavity, esophagus, stomach, liver, prostate and other organs. In some studies, the inhibition correlated with an increase in tumor cell apoptosis and a decrease in cell proliferation. Studies with human cancer cell lines have demonstrated that epigallocatechin-3-gallate (EGCG), a major tea polyphenol, inhibits mitogen-activated protein kinases, cyclin-dependent kinases, growth factor-related cell signaling, activation of activator protein 1 (AP-1) and nuclear factor kappaB (NFkappaB), topoisomerase I and matrix metalloproteinases as well as other potential targets. Although some studies report effects of EGCG at submicromolar levels, most experiments require concentrations of >10 or 20 micromol/L to demonstrate the effect. In humans, tea polyphenols undergo glucuronidation, sulfation, methylation, and ring fission. The peak plasma concentration of EGCG is approximately 1 micromol/L. The possible relevance of each of the proposed mechanisms to human cancer prevention is discussed in light of current bioavailability data for tea polyphenols and the potential limitations of animal models of carcinogenesis. Such discussion, it is hoped, will clarify some misunderstandings of cancer prevention by tea and stimulate new research efforts.     

Interaction of soy food and tea consumption with CYP19A1 genetic polymorphisms in the development of endometrial cancer

Certain polyphenols inhibit the activity of aromatase, a critical enzyme in estrogen synthesis that is coded by the CYP19A1 gene. Consumption of polyphenol-rich foods and beverages, thus, may interact with CYP19A1 genetic polymorphisms in the development of endometrial cancer. The authors tested this hypothesis in the Shanghai Endometrial Cancer Study (1997-2003), a population-based case-control study of 1,204 endometrial cancer cases and 1,212 controls. Dietary information was obtained by use of a validated food frequency questionnaire. Genotypes of CYP19A1 at rs28566535, rs1065779, rs752760, rs700519, and rs1870050 were available for 1,042 cases and 1,035 controls. Unconditional logistic regression models were used to calculate odds ratios and their 95% confidence intervals after adjustment for potential confounding factors. Higher intake of soy foods and tea consumption were both inversely associated with the risk of endometrial cancer, with odds ratios of 0.8 (95% confidence interval: 0.6, 1.0) for the highest versus the lowest tertiles of intake of soy and 0.8 (95% confidence interval: 06, 0.9) for ever tea consumption. The association of single nucleotide polymorphisms rs1065779, rs752760, and rs1870050 with endometrial cancer was modified by tea consumption (p(interaction) < 0.05) but not by soy isoflavone intake. The authors' findings suggest that tea polyphenols may modify the effect of CYP19A1 genetic polymorphisms on the development of endometrial cancer.     

Phosphorylation of hepatic AMP-activated protein kinase and liver kinase B1 is increased after a single oral dose of green tea extract to mice

We have previously shown that green and black tea extracts increase the phosphorylation of AMP-activated protein kinase (AMPK) and HMG-CoA reductase in rat hepatoma cells in culture, concomitant with a decrease in cholesterol synthesis. In the present study, we evaluated the ability of a single oral dose of green or black tea extract to promote the phosphorylation of AMPK, liver kinase B1 (LKB1, an AMPK-kinase), and HMG-CoA reductase in mouse liver. Green tea extract administered by gavage at 50 and 100 mg/kg caused a 2- to 3-fold increase in hepatic AMPK phosphorylation at 3 and 6 hours after dosing and a 1.5- to 2-fold increase in LKB1 phosphorylation at these same time points. The phosphorylation of HMG-CoA reductase at these and later time points was not significantly increased. Black tea administered by gavage at up to 250 mg/kg was ineffective in increasing hepatic AMPK phosphorylation. Both green and black tea extracts increased LKB1 phosphorylation in hepatoma cells in culture at 15 μg/mL, and black tea also increased the phosphorylation of protein kinase A in hepatoma cells. These results suggest that compounds in both tea extracts activate AMPK by activating its upstream kinase, LKB1, and that black tea may do so by first activating protein kinase A, a known kinase for LKB1. Only green tea, at 50 and 100 mg/kg, was able to activate AMPK and LKB1 in mouse liver after oral dosing, suggesting that the polymerized catechins present in black tea do not reach the liver in sufficient concentration to affect AMPK activity.     

茶多酚、茶色素对大鼠肝癌癌前病变抑制作用及机理研究

用腹腔注射二乙基亚硝胺及四氯化碳和部分肝切除建立的改良Solt Farber大鼠肝癌癌前病变模型以观察茶多酚和茶色素对其抑制作用 ,并对其可能机制进行探讨。结果表明 ,与阳性对照组相比 ,饮0 1%茶多酚和茶色素动物的谷胱甘肽硫转移酶P型 (GST P)阳性灶的面积和数目显著减少。Westernblot分析表明茶多酚和茶色素诱导了p2 1WAF1 表达 ,抑制了Bcl 2蛋白的表达 ,诱导了Bax蛋白的表达。本研究表明茶多酚和茶色素对大鼠肝癌癌前病变具有明显的抑制作用 ,而抑制细胞增殖、诱导细胞凋亡可能是其重要机制。     

茶多酚,茶色素对大鼠肝癌癌前病变抑制作用的研究

采用腹腔注射二乙基亚硝胺（ Ｄ Ｅ Ｎ）启动、并用 Ｃ Ｃｌ４ 加于三分之二肝切除促进形成的大鼠肝癌癌前病变模型,以研究茶多酚、茶色素对肝癌癌前病变的抑制作用。试验组分别饮用含０１％ 茶多酚或０１％ 茶色素的水,用谷胱甘肽硫转移酶（ Ｇ Ｓ Ｔ） Ｐｉ阳性灶的密度和面积作为判断肝癌癌前病变发生和进展程度的指标,同时用免疫印迹法测定肝脏组织 Ｇ Ｓ Ｔ Ｐｉ蛋白水平,并用 Ｎｏｒｔｈｅｒｎ Ｂｌｏｔ方法检测 Ｇ Ｓ Ｔ Ｐｉｍ Ｒ Ｎ Ａ 的转录活性。结果显示：茶多酚和茶色素组对 Ｇ Ｓ Ｔ Ｐｉ阳性灶的面积抑制率分别为７２％ 和６０％ ,对阳性灶数目的抑制率分别为４４％ 和５０％ 。用 Ｇ Ｓ Ｔ Ｐｉ多克隆抗体进行的免疫印迹分析显示：肝癌癌前病变模型中 Ｇ Ｓ Ｔ Ｐｉ蛋白表达明显升高,茶多酚和茶色素有不同程度的抑制作用。 Ｇ Ｓ Ｔ Ｐｉｍ Ｒ Ｎ Ａ 的改变基本与 Ｇ Ｓ Ｔ Ｐｉ蛋白的变化相一致。试验表明：茶叶中抗氧化成分茶多酚和茶色素能够在不同水平上拮抗 Ｇ Ｓ Ｔ Ｐｉ的过度表达,这可能是茶多酚、茶色素抑制肿瘤发生和发展的机制之一。