Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Methods for analysis of the mutagenicity of indirect mutagens/carcinogens in eukaryotic cells

Summary The review discusses the variety of methods for activation of indirect mutagens/carcinogens and testing them in cell cultures, especially in mammalian cell cultures.After the necessity for including metabolizing components in mutagenicity tests has been pointed out, the enzymes that transform foreign compounds metabolically, and the factors influencing them, are described. In the main section the various methods of activating indirect mutagens/carcinogens are presented. The methods of including in vivo metabolism in mutagenicity tests are: Analysis of cells from organisms contaminated with a chemical (III.1.a); body fluid-mediated mutagenesis (III.1.b); host-mediated assay (III.1.c).The following activation systems are suitable for including in vitro metabolism of test compounds in mutagenicity tests: Liver and lung perfusion (III.2.a.); organ slices and homogenates (III.2.a.); subcellular fractions (III.2.a.); cultivated cells (cell-mediated mutagenesis) (III.2.b); nonenzymatic activation systems (III.2.c).Finally the main factors that influence the metabolism of test substances are summarized. Two figures illustrate the mutagenicity tests with regard to the metabolism of mammalian livers and the methods of performing mutagenicity tests in man.     

Biotransformation and recovery of the isoflavones genistein and daidzein from industrial antibiotic fermentations

The objective of this study was to follow the metabolic fate of isoflavone glucosides from the soybean meal in a model industrial fermentation to determine if commercially useful isoflavones could be harvested as coproducts from the spent broth at the end of the fermentation. The isoflavone aglycones, genistein, and daidzein together make up 0.1–0.2 % of the soybean meal by weight but serve no known function in the manufacturing process. After feeding genistein to washed cells of the erythromycin-producing organism, Saccharopolyspora erythraea, the first biotransformation product (Gbp1) was determined by X-ray crystallography to be genistein-7-O-α-rhamnoside (rhamnosylgenistein). Subsequent feeding of rhamnosylgenistein to growing cells of Saccharopolyspora erythraea led to the production of a second biotransformation product, Gbp2. Chromatographic evidence suggested that Gbp2 accumulated in the spent broth of the erythromycin fermentation. When the spent broth was hydrolyzed with acid or industrial enzyme preparations, the isoflavone biotransformation products were returned back to their parental forms, genistein and daidzein, which were then recovered as coproducts. Desirable features of this method are that it does not require modification of the erythromycin manufacturing process or genetic engineering of the producing organism to be put into practice. A preliminary investigation of five additional antibiotic fermentations of industrial importance also found isoflavone coproduct potential.     

Metabolism of the soy isoflavones daidzein and genistein by fungi used in the preparation of various fermented soybean foods

The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein was investigated. A total of 21 fungal strains from dou-chi, miso, sake, soy sauce, and sufu were screened. The genera of the tested fungi included Actinomucor, Aspergillus, Candida, Debaryomyces, Monascus, Mucor, Rhizopus, Saccharomyces, and Zygosaccharomyces. The results were that all tested Aspergillus strains from these soybean foods, including five A. oryzae strains, one A. sojae strain, and one A. tamarii strain, metabolized both daidzein and genistein. In contrast, no other tested fungi from the fermented soybean foods metabolized either daidzein or genistein. The metabolites of daidzein and genistein by Aspergillus strains were identified as 8-hydroxydaidzein and 8-hydroxygenistein, respectively, based on their mass, (1)H-, and (13)C-NMR spectra.     

Effect of cigarette-smoke condensate and norharman on the induction of SCEs by direct and indirect mutagens in CHO cells

Cigarette-smoke condensate and norharman were investigated either alone or in combination with a number of direct or indirect mutagens for the induction of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Cigarette-smoke condensate and norharman induced SCEs in these cells, only in the presence of a metabolic activation system. The number of SCEs induced by the direct-acting mutagens mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine was decreased in the presence of cigarette-smoke condensate or norharman. However, cigarette-smoke condensate and norharman showed synergistic effects in combination with the indirect mutagens 2-acetylaminofluorene, 2-aminofluorene, N-hydroxy-acetylaminofluorene, 2-aminoanthracene and benzo[a]pyrene. No synergism was observed when CHO cells were treated simultaneously with cigarette-smoke condensate or norharman and the indirect mutagen cyclophosphamide.     

Effects of antimutagenic flavourings on SCEs induced by chemical mutagens in cultured Chinese hamster cells

Effects of antimutagenic flavourings such as vanillin, ethylvanillin, anisaldehyde, cinnamaldehyde, coumarin and umbelliferone on the induction of SCEs by MMC were investigated in cultured Chinese hamster ovary cells. None of these 6 flavourings showed any SCE-inducing activity by themselves. However, an obvious increase in the frequencies of SCEs was observed when MMC-pretreated cells were cultured in the presence of each flavouring. All these compounds have either an alpha, beta-unsaturated carbonyl group or a carbonyl functionality neighbouring the phenyl group which may react with an enzyme SH-group and cause higher-order structure changes. SCE-enhancing effects of vanillin were further investigated on 6 other kinds of mutagens. Vanillin was also effective on SCEs induced by EMS, ENNG, ENU or MNU. On the other hand, MMS- or MNNG-induced SCEs were not influenced at all by vanillin. SCE-enhancing effects of vanillin seemed to be dependent on the quality of lesions in DNA.     

Sister-chromatid exchange induction by indirect mutagens/carcinogens, aryl hydrocarbon hydroxylase activity and benzo[alpha]pyrene metabolism in cultured human hepatoma cells

2 human hepatoma cell lines (C-HC-4 and C-HC-20), in which aryl hydrocarbon hydroxylase activity was induced with benz[alpha]anthracene in vitro to about 140- and 64-fold of the respective basal levels, yielded an increased frequency of sister-chromatid exchanges (SCEs) when exposed to benzo[alpha]pyrene (BP), 7,12-dimethylbenz[alpha]anthracene and 3-methylcholanthrene in vitro. Analysis of the metabolism of BP by these cells by high-pressure liquid chromatography revealed that both cell lines produced various BP metabolites including the proximate form BP-7,8-dihydrodiol which has been reported to be the most potent inducer of SCEs among the metabolites of BP. In addition, aflatoxin B1 and cyclophosphamide also induced SCEs in these cell lines. The above findings suggest that these cells may be capable of metabolizing a range of indirect mutagens/carcinogens into DNA-active forms. These cells may therefore serve as a useful test system in vitro for the detection of genotoxic agents, without the use of an exogenous activating system.     

DNA damage induced by indirect and direct acting mutagens in catalase-deficient transgenic tobacco. Cellular and acellular Comet assays

We have measured the level of DNA damage induced by treating roots (cellular Comet assay) and isolated root nuclei (acellular Comet assay) of catalase-deficient (CAT1AS) and wild-type (SR1) tobacco with the promutagen o-phenylenediamine (o-PDA) and the direct acting genotoxic agents hydrogen peroxide and ethyl methanesulphonate (EMS). The roots of CAT1AS have about 60% less catalase activity compared to the roots of SR1. The promutagen o-PDA applied on tobacco roots induced significantly higher levels of DNA damage in the CAT1AS transgenic line than in SR1, while after application of o-PDA on isolated root nuclei, no DNA damage could be detected. In the catalase-deficient line CAT1AS about six-fold lower concentrations of H(2)O(2) are sufficient to induce the same levels of DNA damage as in SR1. By contrast, after treatment of isolated root nuclei with H(2)O(2) no difference in the induced levels of DNA damage was observed between CAT1AS and SR1. The DNA damaging effect of EMS was not affected by the presence of catalase in the tobacco roots and the levels of DNA damage measured by the cellular and acellular assay were similar.Comparing the effects of genotoxic agents in both the cellular and acellular Comet assays may help to elucidate their mechanism of action. Differences in both systems may reveal the participation of scavengers and of repair and metabolic enzymes on the activity of the genotoxic agent and the role of the cell wall in preventing the agent from reacting with nuclear DNA.     

Chemoprotective action of resveratrol and genistein from apoptosis induced in human peripheral blood lymphocytes

Extensive research is being carried out to analyse the importance of plant products such as resveratrol and genistein, which are known to exert a variety of pharmacological effects. This study aims at evaluating the protective role of these compounds against the apoptosis induced in normal cells by cytotoxic anticancer agents such as cisplatin and mytomycin C during therapy. Despite the broad antineoplastic action of cisplatin and mitomycin C, their genotoxicity in normal cell might lead to the induction of secondary malignancies. Therefore, the problem of identifying plant compounds, which might exert protective action in normal cells, gains lot of significance. We have analyzed the chemoprotective effect of plant compounds on peripheral blood human lymphocytes when exposed to cisplatin and mitomycin C by pre-treating and post-treating them with resveratrol and genistein at 100 microM concentration Biochemical alterations occurring in many cells during apoptosis include loss of plasma membrane phospholipid asymmetry, DNA fragmentation, and activation of caspase-3, et cetera, and have been assessed. Fluorescence microscopy, flow cytometric techniques have clearly demonstrated that resveratrol and genistein are efficient in protecting lymphocytes undergoing DNA damage when exposed to cisplatin and mitomycin C and exerted their activity by reducing the caspase 3 expression. An interesting observation is that, these compounds offered their protective effect by reducing their apoptotic potential on membrane and nucleic acids against cytotoxic agents, cisplatin, and mitomycin C. These results suggest that resveratrol and genistein might be useful for risk assessments in advance of clinical trials and could be considered as a strong candidate in pharmacogenomics or nutriprotective arena.     

Pharmacokinetics of isoflavones,daidzein and genistein,after ingestion of soy beverage compared with soy extract capsules in postmenopausal Thai women

Background  

Isoflavones from soybeans may provide some beneficial impacts on postmenopausal health. The purpose of this study was to compare the pharmacokinetics and bioavailability of plasma isoflavones (daidzein and genistein) after a single dose of orally administered soy beverage and soy extract capsules in postmenopausal Thai women.     

Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography–mass spectrometry (GC–MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.     

Degradation activity of adhered and suspended Pseudomonas cells cultured on 2,4,6-trichlorophenol, measured by indirect conductimetry

The degradation activity (expressed as specific CO₂ production rates) of adhered and suspended Pseudomonas cells, strains SP1 and SP2, during the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), was compared using indirect conductimetry technique. This technique is defined as the measurement of CO₂ ionization in an alkaline solution and expressed as the negative conductance change values of such solution. The attachment surfaces were porous glass and silicone rubber. The 2,4,6-TCP concentrations ranged from 10 to 500 mg 1⁻¹. Specific respiration rates were determined from CO₂ evolution rates and biomass yields of both suspended and adhered cell cultures. CO₂ evolution rates were determined after conversion of conductance change values into CO₂ produced values. Results indicate that glass-adhered cells reached a higher maximum specific CO₂ evolution rate ( Q _CO2max) than both suspended and silicone rubber-adhered cells. However, suspended cells showed a lower saturation constant ( K_s ) than the adhered cells. These results suggest that depending on support nature the respiration activity of adhered cells could be higher than of suspended cells. Moreover, the indirect conductimetry technique could efficiently be used by measurements of respiration activities of both attached or suspended xenobiotic-degrading micro-organisms.     

Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe

Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions. All strains converted glycitein and daidzein to 6,7,4′-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4′-trihydroxyisoflavone. Three strains transformed daidzein to 7,8,3′,4′-tetrahydroxyisoflavone and 6,7,3′,4′-tetrahydroxyisoflavone. In addition, two strains formed 6,7,8,4′-tetrahydroxyisoflavone from daidzein. Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3′,4′-tetrahydroxyisoflavone. The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation. Revision received: 9 September 1995 / Accepted: 21 September 1995     

Induction of sister-chromatid exchanges by indirect mutagens/carcinogens in cultured rat hepatoma and esophageal tumor cells and in Chinese hamster Don cells co-cultivated with rat cells

2 rat cell lines originated from ascites hepatoma AH66-B and esophageal tumor R1 were examined for their inducibility of sister-chromatid exchanges (SCEs) after treatment with 14 kinds of indirect mutagens/carcinogens, including 6 amine derivatives, 4 azo compounds, 3 aromatic hydrocarbons and 1 steroid. Of the 14 chemicals tested, 2-acetylaminofluorene (AAF), butylbutanolnitrosamine (BBN), dimethylnitrosamine (DMN), cyclophosphamide (CP), urethane, 2-methyl-4-dimethylaminoazobenzene (2-MeDAB), 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB), 4-o-tolylazo-o-toluidine (4-TT), benzo[a]pyrene (BP), 7,12-dimethyl-benz[a]anthracene (DMBA) and diethylstilbestrol (DES) were estimated to be effective inducers of SCEs in AH66-B and/or R1 cells, without the use of exogenous activating systems. Cell-mediated SCE tests with 6 selected chemicals, CP, 2-MeDAB, 4-TT, BP, DMBA and DES, showed a significant increase of SCEs in Chinese hamster Don-6 cells co-cultivated with AH66-B or R1 cells, depending on the number and sensitivity of AH66-B or R1 cells, as well as on the dose of chemicals tested, whereas singly cultured Don-6 cells were much less sensitive or almost insensitive to these chemicals. The above findings suggest that AH66-B and R1 cells may retain metabolic activities to convert a wide range of indirect mutagens/carcinogens into their active forms to induce SCEs, and that these cell lines provide simple and reliable screening systems in vitro, including the cell-mediated SCE assay, for detection of genotoxic agents, without the use of exogenous activation systems.     

Chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The protective effects of carnosine as a natural dipeptide were investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were injected with solutions of carnosine at three different doses (10, 50 and 100?mg kg(-1) bw) for five consecutive days. On the fifth day of treatment, mice were injected cyclophosphamide and killed after 24?h. The frequency of micronuclei in polychromatic erythrocytes and the ratio of polychromatic erythrocyte/polychromatic erythrocyte?+?normochromatic erythrocyte [PCE/(PCE?+?NCE)] were evaluated by May-Grunwald/Giemsa staining. Histopathology of bone marrow was examined in mice treated with cyclophosphamide and carnosine. Carnosine significantly reduced micronucleated polychromatic erythrocytes (MnPCEs) induced by cyclophosphamide at all three doses. Carnosine at dose of 100?mg kg(-1) bw reduced MnPCEs 3.76-fold and completely normalized the PCE/(PCE?+?NCE) ratio. Administration of carnosine inhibited bone marrow toxicity induced by cyclophosphamide. It appeared that carnosine with protective activity reduced the oxidative stress and genotoxicity induced by cyclophosphamide in bone marrow cells of mice. Copyright ? 2012 John Wiley & Sons, Ltd.     

Inhibition of repair activity induced by gamma-, UV-irradiation and radiomimetics in cultured cells from schizophrenia patients

A study was made of the processes of repair, virus reactivation, and formation of sister chromatid exchanges (SCE) in blood cells of patients with schizophrenia after the effect of gamma-radiation and 4-nitroquinoline-1-oxide. These processes were estimated by 12 criteria. The mutagen-induced disturbances in the processes of repair and SCE formation were found in cells of patients with schizophrenia and were absent in the control cells of healthy donors.     

Effects of genistein and daidzein on the cell growth,cell cycle,and differentiation of human and murine melanoma cells(1)

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G(2)/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G(1) phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.