Yukiaki Kuroda

Antimutagenic and anticarcinogenic activity of tea polyphenols

Tea is the most popular beverage, consumed by over two thirds of the worlds population. Tea is processed differently in different parts of the world to give green (20%), black (78%) or oolong tea (2%). Green tea is consumed mostly in Japan and China. The antimutagenic and anticarcinogenic activities of green tea are extensively examined. The chemical components of green and black tea are polyphenols, which include EC, ECG, EGC, EGCG and TFs. This article reviews the epidemiological and experimental studies on the antimutagenicity and anticarcinogenicity of tea extracts and tea polyphenols. In Japan, an epidemiological study showed an inverse relationship between habitual green tea drinking and the standardized mortality rates for cancer. Some cohort studies on Chanoyu (Japanese tea ceremony) women teachers also showed that their mortality ratio including deaths caused by malignant neoplasms were surprisingly low. The antimutagenic activity against various mutagens of tea extracts and polyphenols including ECG and EGCG has been demonstrated in microbial systems (Salmonella typhimurium and Escherichia coli), mammalian cell systems and in vivo animal tests. The anticarcinogenic activity of tea phenols has been shown in experimental animals such as rats and mice, in transplantable tumors, carcinogen-induced tumors in digestive organs, mammary glands, hepatocarcinomas, lung cancers, skin tumors, leukemia, tumor promotion and metastasis. The mechanisms of antimutagenesis and anticarcinogenesis of tea polyphenols suggest that the inhibition of tumors may be due to both extracellular and intracellular mechanisms including the modulation of metabolism, blocking or suppression, modulation of DNA replication and repair effects, promotion, inhibition of invasion and metastasis, and induction of novel mechanisms.

Antimutagenesis Studies in Japan

Studies on antimutagenic factors were initially carried out in the 1950s in the field of microbial genetics [for review, see Clarke and Shankel (3)]. During the past ten years, a wide variety of chemical mutagens and carcinogens has been detected in foods, medicines, cosmetics, insecticides, and even in the atmosphere and water which we utilize daily. Some mutagens act directly on cells to produce mutations, and others act following their modification by other factors. Some of these indirect mutagens are metabolically activated by enzymes in organs or tissues, and others may be inactivated and inhibited by some dietary foods and by components of our cells.

Antimutagenic Activity of Vitamins in Cultured Mammalian Cells

Cultured mammalian cell systems are useful for examining the quantitative effects of mutagens and antimutagens on cell survival and gene mutations and the mechanisms of the interaction of two chemicals in the process of mutation induction. In the present article, the antimutagenic effects of vitamins C, E, and A, and derivatives of vitamin C on EMS-induced 6TG-resistant mutations in Chinese hamster V79 cells were examined. Vitamin C was most effective in inhibiting EMS-induced cytotoxicity and 6TG-resistant mutations. In the presence of vitamin C at a concentration of 100 micrograms/ml, EMS-induced mutations were reduced to about one-third or one-fourth of those in control cultures treated with EMS alone. Dehydro-vitamin C and iso-vitamin C also inhibited EMS-induced mutations to about one-half or one-third of the control level. The fact that vitamin C was effective in reducing EMS-induced mutations when EMS was previously incubated together with vitamin C for 3 hr suggests that vitamin C may react directly with EMS as a desmutagen and thus inactivate its mutation-inducing activity in Chinese hamster V79 cells. Vitamin E had an additive cytotoxic effect on EMS-induced cytotoxicity. This vitamin enhanced the frequencies of 6TG-resistant mutations induced by EMS. Pretreatment with vitamin E before treatment with EMS resulted in no detectable effect in modifying the EMS-induced mutations. On the contrary, vitamin A markedly enhanced EMS-induced mutation frequencies.

Mutagenesis in cultured human diploid cells III. Induction of 8-azaguanine-resistant mutations by furylfuramide

Trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide: FF or AF2) was tested for ability to induce 8-azaguanine (8AG) resistant mutations in cultured human diploid cells. FF had a relatively severe cytotoxic effect on the cells. From the concentration-survival curve, the D0 value for 2-h treatment with FF was estimated to be 11 mug/ml. When cells were treated with FF at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected in medium containing 8AG at 30 mug/ml, the induced mutation frequency increased gradually with increase in concentration of FF. When cells were treated with FF at 10 mug/ml for 2 h, cultured in normal medium for various periods of mutation expression time, and selected with 8AG at 30 mug/ml, the highest induced mutation frequency was obtained with 48 h of mutation expression time. Microscopic examination of the numbers of cells in colonies indicated that the total number of cells increased by half during this mutation expression time of 48 h.

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.

Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells

The effects of cobaltous chloride on 8-azaguanine (8AG)-resistant mutations induced by gamma-rays or ultraviolet (UV) light in cultured Chinese hamster V79 cells were examined. Cobaltous chloride alone had no significant effects on survival and mutations of V79 cells at concentrations less than 1 x 10(-5) M. Cobaltous chloride at a concentration of 3 x 10(-6) M had a marked effect in reducing 8AG-resistant mutations induced by gamma-rays of 2-6 Gy, when cells were incubated for 6-7 days in the presence of cobaltous chloride after gamma-ray irradiation (posttreatment). The pretreatment of cells with cobaltous chloride for 6 days before gamma-ray irradiation reduced 8AG-resistant mutations induced by gamma-rays. Pre- or post-treatment with cobaltous chloride had no such effect on UV-induced mutations, however. The difference in responsiveness to cobaltous chloride between bacterial and mammalian cell systems is discussed.

Effect of lectins on chondrogenesis of cultured quail limb bud cells

Abstract Mesenchyme cells from the limb buds of quail embryos at stages 20–21 were treated with various lectins to study the role of cell aggregation on cartilage differentiation. When cells were cultured in a succinyl Con A medium (50 μg/ml) for 24 hr, they formed several times more cartilage nodules than control cultures. This study shows that the lectin enhances the process of cartilage nodule formation of mesenchyme cells.

Dose-rate effects of ethyl methanesulfonate on survival and mutation induction in cultured Chinese hamster cells

The dose-rate effects of ethyl methanesulfonate (EMS) on the survival and induction of mutations in Chinese hamster Don cells were investigated. The most effective time of exposure to EMS for reducing the surviving fraction of cells was 4 h, shorter and longer exposure times being less effective. The threshold or minimal concentration of EMS giving a surviving fraction of 0.5 was 0.05 mg/ml. The minimal effective time of exposure to EMS for cell death was 1 h. Corrected survival curves showed that longer exposure times at lower dose rates of EMS had less cytotoxic effect than shorter exposure times at higher dose rates. After exposure of Don cells to various doses of EMS for various times, the frequencies of mutations resistant to 6--thioguanine (6TG) were measured. An exposure time of 4 h produced a lower mutation frequency than shorter or longer exposure times that resulted in the same surviving fraction of cells. An exposure time of 20 h produced the highest induced mutation frequency. This system using cultured Chinese hamster cells should be useful as a sensitive procedure for detecting the mutagenic actions of chemicals.

Genetic and Chemical Factors Affecting Chemical Mutagenesis in Cultured Mammalian Cells

The genetic and chemical factors affecting chemical mutagenesis in cultured Chinese hamster V79 cells are described. The frequency of mutations induced by the same chemicals at the same concentrations was markedly affected by the genetic markers used. Some chemicals that were positive in the Ames tests induced 8AGr and 6TGr mutations at higher frequencies than OUAr mutations. Carcinogens that were negative in the Ames tests induced only 8AGr mutations, but did not induce OUAr mutations. As a compound affecting mutations induced by EMS, vitamin C showed marked effects in reducing the cytotoxicity and frequency of 6TGr mutations induced by EMS when cells were treated simultaneously with vitamin C at a concentration of 100 micrograms/ml.

Radiosensitization of cultured mammalian cells by 5-iodouridine.

Radiosensitization of cultured mammalian cells was studied with halogenated pyrimidines, such as 5-iodouridine or 6-chloropurine, which have been shown to promote bacterial cell lethality when combined with gamma-irradiation. When Chinese hamster cells were exposed to gamma-rays to acidic pH values and the number of colonies was scored after 6 to 11 days of incubation, many more cells were inactivated in the presence of the drug than in its absence. This may be due to radiation-induced cytotoxic iodine radicals from the reagent in the case of 5-iodouridine, because the cells were inactivated efficiently only be contact with the previously-irradiated drug solution. The toxicity of the irradiated drug solution increased remarkably when the pH shifted to acidic side. The radiosensitization and the cytotoxic effects of gamma-irradiated drug solution were not found with 6-chloropurine. This may be the first observation on the lethal effect of chemical radicals on mammalian cells, and it is concluded that radiosensitization with 5-iodouridine does not require the drug incorporation into cellular DNA, at least under the conditions adopted in the present studies.