Dietary factors affecting the urinary mutagenicity assay system. II. The absence of mutagenic activity in human urine following consumption of red wine or grape juice

The mutagenic activity of urine samples from nonsmoking individuals before and after the consumption of either red wine or grape juice was determined. Urine samples collected from individuals on liquid or regular diets were concentrated using XAD-2 resin. No mutagenic activity of urine concentrates was detected with Salmonella tester strains TA98 or TA100 with or without microsomal activation. The addition of 1000 units of beta-glucuronidase into the agar overlay did not show any mutagenic activity. The mutagens in red wine and grape juice, however, were extracted using the XAD-2 column. Concentrates of urine samples spiked with either of the two extracts exhibited mutagenic activity.     

Relationship between mutagenic potency in Salmonella typhimurium and chemical structure of amino- and nitro-substituted biphenyls

All positional isomers of mononitro- and monoaminobiphenyls and those of dinitro-, diamino- and aminonitrobiphenyls, which have one substituent on each benzene ring, were assayed for mutagenicity in Salmonella typhimurium by the Ames method. The results suggest that the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2'-position. The introduction of an amino group to the 3'- or 4'-position of 4-nitrobiphenyl or a nitro group to 3'- or 4'-position of 4-aminobiphenyl enhanced the mutagenicity. Among the mutagenic compounds, 4-nitro analogues were mutagenic in strains TA98 and TA100 in the absence of a microsomal metabolic activation system. Strain TA98NR was not reverted by the direct-acting mutagens, whereas strain TA98/1,8-DNP6 was as revertible as strain TA98; these results suggest that the direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes.     

野生葡萄及葡萄酒抗氧化活性和抗菌性研究

选取广西、湖南等地野生葡萄,与经典酿酒葡萄比较,研究抗氧化活性和活性物质,同时监测葡萄酒发酵过程中各指标的动态变化,并对不同品种葡萄酒的抗菌性进行研究。结果表明:赤霞珠的酚类含量和抗氧化活性高于野生葡萄和玫瑰香葡萄,但野生葡萄酒的抗菌性能显著优于赤霞珠和玫瑰香葡萄酒。葡萄酒在发酵过程中其抗氧化活性和酚类物质含量均随发酵过程的进行而升高;总抗氧化活性与总酚含量、氧自由基清除能力与原花青素含量成显著正相关,相关系数均大于0.989;总花色苷含量在发酵初期上升,后期下降,葡萄酒颜色变浅。     

Mutagenic substances in red and white wine in Chile, a high risk area for gastric cancer

Chilean home-made and commercial wines were analyzed for the presence of mutagenic substances using the Salmonella mutagenicity test with preincubation. Strains TA98 and TA100 were used in the absence and in the presence of S9 mix. 90% of red wines from a total of 30 samples and 54% of white wines from a total of 22 were found to be mutagenic. In all cases, S9 mix did not affect the mutagenicity of the samples. At least in one case, more than one mutagen was present, since the mutagenicity with TA98 could be selectively inactivated without affecting that with TA100. This study supports the hypothesis that wine consumption may be an important risk factor for upper gastrointestinal cancer, particularly for adenocarcinoma of the stomach, which is highly prevalent in Chile.     

Isolation and identification of rutin as the major mutagen of red wine

The polyphenolics of a red wine were concentrated by salt-induced phase separation into acetone-alcohol and fractionated by Sephadex LH-20 and multi-layer counter-current chromatography. The mutagenicity of each fraction was evaluated by the Salmonella mutagenesis assay. The mutagen of red wine required activation by both rat-liver microsomal enzymes and human-fecal enzymes (fecalase). The mutagenic component of red wine was purified to homogeneity by reverse-phase high-performance liquid chromatography (RPHPLC) on Lichrosorb C18 and was identified as rutin by UV spectrometry, co-chromatography with authentic standard on RPHPLC and gas-liquid chromatography/mass spectrometry.     

Chlorophyllin: a potent antimutagen against environmental and dietary complex mixtures

Chlorophyllin, the sodium and copper salt of chlorophyll, was tested for its ability to inhibit the mutagenic activity of a variety of complex mixtures--extracts of fried beef, fried shredded pork, red grape juice, red wine, cigarette smoke, tobacco snuff, chewing tobacco, airborne particles, coal dust and diesel emission particles--in strain TA98 of Salmonella typhimurium. Chlorophyllin was highly effective against the mutagenicity (90-100% inhibition) of 8 of these 10 mixtures. The mutagenicity of the other 2 mixtures was inhibited 75-80% at the highest concentration of chlorophyllin studied. Control and reconstruction experiments showed that chlorophyllin was not toxic to Salmonella at the concentrations used. The antimutagenic activity of chlorophyllin was heat-stable. The mechanism of the antimutagenicity of chlorophyllin in these experiments is not known; however, chlorophyllin is an antioxidant. Scavenging of radicals and/or interaction with the active group of mutagenic compounds may be responsible for its antimutagenic activity. The data reported here indicate that chlorophyllin is potentially useful as an antimutagenic agent.     

The use of the Salmonella/microsomal assay to determine mutagenicity in paired chemical mixtures.

The mutagenicities of two sets of chemicals acting singly and in pairwise combinations were determined by use of the Salmonella/microsomal assay. The first set consisted of the promutagens of benzo(a)pyrene and benzo(rst)pentaphene. The second set contained the direct-acting mutagens methyl-nitro-nitroso-guanidine and ethyl methane sulfonate. In the tests with the promutagens, the quantities of S-9 mix were varied over the range of 0.05 ml to 1.0 ml with increasing quantities of each chemical. The mutagenic responses or production of revertant colonies of the promutagens, acting singly and in pairwise combinations failed to show an additive effect. Excess quantities of S-9 mix appeared to inhibit partially or totally the mutagenic activity of each chemical, although for each particular dose there was an optimal quantity of S-9 mix to induce maximum activity. However, the direct-acting mutagens produced, individually, almost linear dose responses with increasing concentrations. In pairwise combinations, these chemicals also showed linear responses that closely approximated the theoretical additivity indicating that the mutagenicity of the mixtures was the sum of the activities of each component.     

Interaction with microsomal lipid as a major factor responsible for S9-mediated inhibition of 1,8-dinitropyrene mutagenicity

1,8-Dinitropyrene (1,8-DNP), present in polluted air, is a rodent carcinogen and a potent, direct-acting mutagen in salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of mammalian hepatic S9 or microsomes. We demonstrate that at least a substantial part of this effect is attributable to non-enzymatic processes. The microsomal-dependent inhibition was unaffected by omission of an NADPH-generating system or when the cytochrome P-450 inhibitor, SKF-525A, or the cytochrome P-448 inhibitor, ellipticine, was incorporated in the metabolic activation system, suggesting that mixed function oxidases are not involved. Heat inactivation partially decreased the ability of induced S9 to reduce DNP mutagenicity. Substitution of S9 with a similar concentration of bovine serum albumin did not affect DNP activity. Thus non-specific binding to microsomal protein is not involved. However, when lipids, derived from uninduced microsomes, were added to incubations of DNP and S. typhimurium TA98, mutagenicity was decreased. Furthermore, substitution of microsomal lipids with a suspension of phosphatidylcholine (PC), a major lipid constituent of microsomes, affected DNP mutagenicity similarly. An increase in PC concentration resulted in a greater inhibitory effect. The reduction in DNP mutagenicity observed with microsomal lipids or with PC was less than that detected with uninduced S9, whilst the mutagenicity of 2-nitrofluorene was reduced to an approximately equal extent by lipids and S9. This phenomenon may be responsible for the S9-mediated detoxification of other mutagenic nitroaromatic compounds and may have important implications for mutagenicity testing.     

Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.     

Characterisation of yeast microbiota,chemical and sensory properties of organic and biodynamic Sangiovese red wines

Wine quality is closely linked to the fermentation step, which is driven by the microbial ecology of grape and the use of selected microbial strains as well. The microbial species developing during fermentation determines the type and concentration of many substances, which contribute to the sensory properties of wine and its safety. In this view, the present work aims to characterise the yeast microbiota, chemical and sensory properties of Sangiovese red wines obtained from both biodynamic and organic agriculture. The natural yeast populations of grape musts and their evolution during spontaneous were monitored and investigated. In addition, the volatile composition, physico-chemical and safety features (ethyl-carbamate) and sensory properties of wines were evaluated. The results showed that the yeast population was mostly related to the grape management, i.e. organic or biodynamic, while the wine composition was mainly affected by the winemaking process, and then by the grape management.     

Mutagenicity of airborne particles from four cities in Taiwan.

The mutagenicity of airborne particles from 8 urban and suburban locations in each of four cities, Taipei, Hsinchu, Taichung, and Kaohsiung, in Taiwan area were investigated with S. typhimurium strain TA98 by Ames Salmonella/microsomal test. The average mutagenic activity of airborne particulate samples from Taipei and Kaohsiung was higher than that from Hsinchu and Taichung with or without metabolic activation. The major direct-acting mutagenic compounds of airborne particulate samples from Taipei and Kaohsiung was similar to that of standard dinitropyrenes mixture (DNPs) in the retention time of HPLC. Moreover, the contents of DNPs of airborne particulate samples from Taipei and automobile exhaust partially purified through Sephadex LH-20 gel filtration and semipreparative HPLC were determined by HPLC. DNPs was major direct-acting mutagens of the urban air samples from Taipei and their major pollutants might be from automobile exhaust. However, the major mutagenic compounds of airborne particulate samples from Hsinchu and Taichung did not correspond to any of the standard compounds tested. The content of benzo[a]pyrene (B[a]P) of airborne particulate samples was also determined by HPLC. The concentration of B[a] P was 0.05-0.62 ng/m3 air sample. The B[a] P contents of airborne particulate samples from four cities in Taiwan did not show good correlation with their mutagenic activity. Thus, we concluded that B[a] P was not a major indirect-acting mutagenic compound in the tested air samples.     

葡萄酒中白藜芦醇的分析

采用HPLC测定了17种葡萄酒中白藜芦醇含量。干红葡萄酒中白藜芦醇的含量远高于干白葡萄酒。干红葡萄酒中白藜芦醇的含量与葡萄品种有关,同一葡萄品种的干红葡萄酒中白藜芦醇的含量与产地和工艺有关。     

Conversion of IQ, a dietary pyrolysis carcinogen to a direct-acting mutagen by normal intestinal bacteria of humans

The dietary carcinogen, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) is mutagenic in the Salmonella/microsomal mutagenicity assay when activated by microsomal enzymes. IQ is found in many cooked foods, notably fried beef and pork. In laboratory rodents IQ is carcinogenic. We showed that mixed and pure cultures of human intestinal anaerobes, notably Eubacterium spp., metabolized IQ to 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (HOIQ). Unlike IQ, both the synthetic and bacterially produced HOIQ were direct-acting mutagens, i.e. active without microsomal activation. This new direct-acting mutagen, from the bacterial metabolism of a dietary pyrolysis carcinogen, raises new concerns about the possible role of this class of genotoxins in the etiology of human cancer.     

Low levels of ochratocin A in wines from Piedmont

Ochratoxin A (OTA) is a mycotoxin mainly produced by a number of species of Aspergillus, commonly found in warm and tropical climates. OTA poses risks for the human health because of its nephrotoxic, teratogenic, immunotoxic and neurotoxic activity. The mycotoxin, classified as possible human carcinogen (Group 2B) by the IARC, naturally occurs in a wide range of foods, including wine, where the main producer is A. carbonarius. The aim of this work was the validation of a procedure for the analysis of OTA in Piedmontese red and white wines produced after vintage 2003 and 2004, in relationship with the limit of 2.0 microg l(-1) introduced by European Union for wine, must or grape juice (Regulation CE N. 123/2005). An analytical method based on immunoaffinity column (IAC) for clean-up and liquid chromatography with fluorescence detection (LC-FLD) was used to determine the occurrence of OTA in wines. Detection limit (LOD) and quantification Limit (LOQ) were 7.18 pg/ml and 9.31 pg/ml based on statistical method (IUPAC). Average recoveries of OTA from wine samples spiked at levels from 0.1 to 10 ng/ml ranged from 90.8% to 92.4%, with relative standard deviations (RSDs) between 2.64 and 2.71%. Repeatability limit was 8.73 pg/ml for samples spiked with 0.1 ng/ml of OTA. Ninety-one Denomination of Controlled Origin (DOC) wines were analysed, including 41 Barbera (red), 38 Dolcetto (red), and 16 white wines, such as Erbaluce, Cortese and Roero Arneis. The study focused on wines commercialized in Italian supermarkets and wine shops. The white wines resulted, as expected, less contaminated than the red ones. Wines produced after vintage 2003, a season particularly conducive to the growth of A. carbonorius, contained higher levels of OTA than the wines produced in 2004. The samples, resulting positive, contained a concentration of OTA highly inferior to the threshold limits introduced by the European Union. The sample of the highest level of OTA was a Dolcetto produced in 2004, with 1.10 ng/ml of mycotoxin.     

Toxic genetic effects of flunitrazepam

The mutagenic activity of Flunitrazepam, the active ingredient of the drug Rohypnol, has been investigated by using the Salmonella/microsome mutagenicity test. A dose-related mutagenic effect was observed on Salmonella typhimurium strain TA 100 either in the absence or in the presence of a rat liver microsomal fraction (S9) as in vitro metabolic activation system. By adopting a modification of the Salmonella test, the mutagenicity of urines from rats or patients treated with the drug was evaluated. In these cases mutagenic activity was detected toward the Salmonella strains TA 98 and TA 100 both in presence and in absence of the metabolic activation system. The data indicate that Flunitrazepam and/or its urinary metabolites can induce both base-pair substitutions or frame-shift point mutations.     

The determination of total concentration and activity of antioxidants in foodstuffs

We developed a new method for the analysis of active antioxidants that is based on their reactions with the ABTS+. cation radical obtained by oxidation of ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. The feasibility of this method was confirmed by electrochemical and kinetic studies of model antioxidants. ABTS+. was shown to react rapidly with active and slowly with weak antioxidants, which allows it to be used as a model radical for the quantitative determination of the total content of natural antioxidants (antioxidant equivalent) in natural extracts and wines. Another analytical method based on the competitive oxidation of Pyrogallol Red (a detecting molecule) and the examined antioxidants by radicals derived from peroxynitrite was used for measuring the relative activity of antioxidants. A combination of both methods helped measure the total concentration of antioxidants and their average specific activities (per molecule of active compound) in extracts from grape, olive, and tomato and concentrates of various popular beverages (wines, beers, and juices), as well as in the commercial concentrated food product Kréto-A, made from grape, red wine, tomato, and olive. Red wine and red grape juice were shown to be the most rich in antioxidants (up to 20 mM), with their activity being similar to that of polyphenols. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.     

Antimutagenicity studies of chlorophyllin using the Salmonella arabinose-resistant assay system

Studies with the arabinose-resistant Salmonella forward mutation assay system were performed to determine the antimutagenic activity of chlorophyllin against the mutagenic activity of aflatoxin B1 (AFB1), 2-aminoanthracene (2AA), benzo[a]pyrene (BaP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and solvent extracts of coal dust (CD), diesel emission particles (DE), airborne particles (AP), tobacco snuff (TS), black pepper (BP) and red wine (RW). Various concentrations of each chemical and complex mixture extract were assayed for mutagenic activity with and/or without S9 in a preincubation test. One concentration of each chemical and complex mixture extract was then tested with various concentrations of chlorophyllin. Results showed that chlorophyllin, at concentrations of 2.5 mg/plate or less, completely or almost completely inhibited the mutagenicity of 2AA, AFB1, BaP, MNNG and solvent extracts of CD, DE and RW. With concentrations from 1.25 to 5 mg/plate, chlorophyllin inhibited over 50% of the mutagenicity of AP, TS and BP extracts. These results further substantiate the antimutagenic efficacy of chlorophyllin against chemicals and complex mixtures.     

ω-Hydroxymodin, a major hepatic metabolite of emodin in various animals and its mutagenic activity

The hepatic microsomes derived from various animal species transformed emodin (1,3,8-trihydroxy-6-methylanthraquinone), and anthraquinoid pigment present in fungal metabolites and a constituent of plant medicines, into an unidentified anthraquinone h, along with 2-hydroxy-, 4-hydroxy- and 7-hydroxyemodins. TLC, UV, MS and NMR clarified this unidentified major metabolite as ω-hydroxy-emodin (1,3,8-trihydroxy-6-hydroxymethylanthraquinone). Among 7 animal species, the highest activity to produce this ω-hydroxyemodin was observed in the hepatic microsomes of guinea pig and rat, followed by mouse and rabbit. The microsomal activity to convert emodin into ω-hydroxyemodin was accelerated by the pretreatment of animals with phenobarbital, and inhibited by SKF 525A. The microsomal hydroxylation reactions of the methyl residue and the anthraquinoid nucleus of emodin were presumed to be catalyzed regiospecifically by multiple forms of cytochrome P-450.

ω-Hydroxyemodin was not mutagenic to Salmonella typhimurium in the absence of S9, but exhibited mutagenicity in the presence of an activating system. This genotoxic potential was comparable to 2-hydroxyemodin, a direct-acting mutagen.     

Mutagenicity of antiamebic and anthelmintic drugs in the Salmonella typhimurium microsomal test system

4 amebicides (chloroquine diphosphate, diiodohydroxyquin, iodochlorohydroxyquin and dehydroemetine) and 6 anthelmintics (bephenium hydroxynaphthoate, 4-hexylresorcinol, mebendazole, niclosamide, pyrantel pamoate and pyrvinium pamoate) were tested for mutagenicity in the Salmonella typhimurium microsomal test system. Frameshift mutations were induced by dehydroemetine and niclosamide following activation by microsomal enzymes, while pyrvinium pamoate induced both frameshift and base-pair substitution mutations with or without metabolic activation. The urine of mice treated with dehydroemetine or pyrvinium pamoate showed no mutagenic activity. However, urine obtained from mice treated with niclosamide was mutagenic in strains TA98 and TA1538. The fluctuation assay showed chloroquine diphosphate to be mutagenic in TA1537, a strain which detects frameshift mutations.     

A low-cost procedure for production of fresh autochthonous wine yeast

A low-cost procedure was designed for easy and rapid response-on-demand production of fresh wine yeast for local wine-making. The pilot plant produced fresh yeast culture concentrate with good microbial quality and excellent oenological properties from four selected wine yeasts. The best production yields were obtained using 2% sugar beet molasses and a working culture volume of less than 60% of the fermenter capacity. The yeast yield using 2% sugar grape juice was low and had poor cell viability after freeze storage, although the resulting yeast would be directly available for use in the winery. The performance of these yeasts in commercial wineries was excellent; they dominated must fermentation and improved its kinetics, as well as improving the physicochemical parameters and the organoleptic quality of red and white wines.