Biological and chemical studies on overheated brewed coffee

Vapour formed from overheated decaffeinated coffee was condensed and tested for mutagenicity using the Ames assay in Salmonella typhimurium strains TA98 and TA100. Vapour produced at 73 and 100 degrees C exhibited no mutagenicity. The basic fraction of vapour produced at 350 degrees C showed weak mutagenicity towards strains TA98 with metabolic activation. The chemical analysis of this fraction identified pyridines and pyrazines as the major constituents. None of the compounds identified in this fraction has been reported as mutagenic when tested in the Ames assay.     

Ames mutagenicity tests of repeatedly heated edible oils

Six cooking oils with different levels of unsaturation were each heated at 180 +/- 3 degrees C for 6 hr on each of four consecutive days and cut potatoes were fried in the oils at hourly intervals. Samples of the heated and unheated oils were tested in the Ames mutagenicity assay. None of the oil samples (taken before or after 24 hr heating) showed any mutagenicity in the Ames test with or without metabolic activation.     

Ames mutagenicity tests on purified 3-nitropropionic acid

3- Nitropropionic acid is a toxic compound produced by several moulds involved in food fermentation or spoilage. An impure commercial sample of this compound was previously reported as being mutagenic to Salmonella typhimurium strains TA1535 and TA100. In the present study, a sample from the same lot of 3- nitropropionic acid was mutagenic in strain TA100 without metabolic activation, but this activity was diminished after recrystallization. This sample was not mutagenic in strain TA98, before or after recrystallization. A new, purer commercial sample was non-mutagenic in strains TA98, TA100 and TA1538, with or without metabolic activation. Therefore the mutagenicity reported to be due to 3- nitropropionic acid was considered to be due to the impurity(ies).     

Ames mutagenicity tests of products from a heated potato-starch system

A charred sample was prepared from potato starch heated with ammonium carbonate at 600°C in a flask under a nitrogen stream. The water produced was collected and extracted with methylene chloride. The basic fraction obtained from the extract exhibited strong mutagenicity in Ames assays using Salmonella typhimurium strains TA98 or TA100 with metabolic activation (rat-liver S-9 mix). The basic fraction was further fractionated by silica gel column chromatography and subsequently by Scphadex column chromatography. Some of the resulting fractions exhibited strong mutagenic activities in S. typhimurium strain TA98 with S-9 mix.     

Contribution of coffee aroma constituents to the mutagenicity of coffee

About 40 coffee aroma constituents belonging to the classes of dicarbonyls, sulphur-containing compounds, furfuryls, N-heterocyclics and others were systematically evaluated in three Ames tester strains. Only aliphatic dicarbonyl compounds showed notable direct mutagenic activity, which mainly affected 'base-pair substitution' in Ames tester strains TA100 and TA102. Very weak effects were also seen with some N-heterocyclics, mainly affecting frameshift tester strain TA98 upon metabolic activation. However, it was shown that these N-heterocyclics do not contribute substantially to the mutagenicity in coffee. The hydrogen peroxide and methylglyoxal contents of coffee were determined up to 26 hr after preparation. Their concentrations tended to decrease whereas mutagenic activity decreased significantly with time in tester strains TA100 and TA102. It is concluded that several highly labile coffee constituents contribute to the bacterial mutagenicity and also that the synergism between hydrogen peroxide and methylglyoxal is not the main factor. The absence of coffee mutagenicity/carcinogenicity in rodents with these highly reactive coffee aroma compounds can be explained in part by detoxification of microsomal enzyme systems.     

Instant and brewed coffees in the in vitro human lymphocyte mutagenicity test

Incubation of instant and 'home brew' coffees (caffeinated and decaffeinated) and of coffee aroma with cultured human lymphocytes in the presence and absence of S-9 increased the number of total aberrations. However, the increase was smaller in the presence of S-9 than in its absence. Pure caffeine tested with or without S-9 at doses equivalent to levels in caffeine-containing coffee did not give statistically significant increases of any type of aberration when compared with controls. In all in vitro test systems used to date, coffee and coffee aroma or their reactive compounds were metabolically deactivated in the presence of S-9. This could explain the negative results obtained in mutagenicity assays in vivo.     

Detection of genotoxicity and mutagenicity of chlorthiophos using micronucleus,chromosome aberration,sister chromatid exchange,and Ames tests

Potential mutagenic and genotoxic effects of Chlorthiophos, an organophosphate pesticide, were evaluated using four standard assays. Five different concentrations of the pesticide were tested by an Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without S9 metabolic activation. No concentrations of Chlorthiophos showed mutagenic activity on the TA97, TA100, and TA102 strains, with and without S9 fraction, but were all mutagenic to the TA98 strain without S9. Sister chromatid exchange (SCE), chromosome aberration (CA), and micronucleus (MN) tests were used to investigate the genotoxic effects of Chlorthiophos in human peripheral lymphocytes treated with 25, 50, 100, and 200 µg/mL concentrations of Chlorthiophos for 24 and 48 h. The nuclear division index (NDI), replication index (RI), and mitotic index (MI) were also calculated to determine the cytotoxicity of Chlorthiophos. No increase in SCE frequency was seen for any treatment period or concentration, but Chlorthiophos at 200 µg/mL increased the frequency of CAs. Increases in MN formation were only observed at Chlorthiophos concentrations of 200 µg/mL following 24 and 48 h treatments. Chlorthiophos treatment reduced the MI and NDI significantly, but had no effect on the RI. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 937–945, 2015.     

Small numbers in mutagenicity tests

Experimental control material for statistical analysis of the results of the micronuclei test in the mouse (NMRI strain) and the Chinese hamster and for the host-mediated assay in the mouse (NMRI strain) using auxotrophic bacterial strains are presented. The binomial distribution of the micronuclei makes it possible to analyse the sample size according to the formula of Cochran and Cox (1957).For the host-mediated assay, the experimental principles are given which make it possible to evaluate the results obtained even with a weakly mutagenic, unknown substance.Critical points in comparative tests are not only the methodological questions, but also pharmacokinetic problems of the substance being tested which can only be clarified in the species used for the mutagenicity test. If this is ignored then even experimentally based findings can only be recorded as speculations.Presented at the 3rd Meeting of the Gesellschaft für Umwelt-Mutationsforschung e. V., Neuherberg, July 1–2, 1976     

Methemoglobinogenic potential of primaquine and its mutagenicity in the Ames test

Single doses of primaquine did not produce methemoglobinemia in beagle bitches. Repeated daily administration for 12 days produced a gradually rising level of methemoglobin over that time period, unaccompanied by depletion of erythrocytic reduced glutathione. Primaquine was mutagenic in the Ames test in Salmonella typhimurium strain TA 1537, with or without S9, using a liquid preincubation assay. Primaquine was non-mutagenic in this assay to strains TA 1535, TA 1538, TA 98 and TA 100, regardless of the presence or absence of S9. In the standard overpour Ames test, the drug was non-mutagenic in all 5 Salmonella strains, both with and without S9 metabolic activation.     

The effect of brewed and instant coffee on reproduction and teratogenesis in the rat

A blend of roasted and ground coffees was brewed and an instant coffee was prepared daily and given to rats in place of their drinking water, at either full-strength (100%) or as 50 or 25% dilutions. A control group was given water. The administration of the coffee solutions began shortly after weaning and continued for about 30 weeks, through two pregnancies. During pregnancy and lactation the caffeine intakes were approximately 80, 40, or 20 mg/kg/day, being slightly higher in the groups given brewed coffee and slightly lower in the groups give instant coffee. The parent rats given either brewed or instant coffee grew as well as or better than controls and ate more feed, although feed efficiencies were not different. The rats given full-strength coffees drank significantly less than controls, but the ones given 50 or 25% solutions drank more. Both male and female rats given 100% brewed or instant coffee to replace their drinking water had enlarged kidneys at 13 and 30 weeks, while only the females given 50% solutions of either coffee had enlarged kidneys. The females given full-strength coffees had enlarged livers at both times, while the females given 50% brewed coffee had enlarged livers only at 30 weeks. No effects on the organ weights were seen in the groups given 25% coffee solutions. No effects were seen on reproduction or lactation, except that the pups in the groups given 100% brewed or instant coffee to replace their drinking water weighed significantly less than the controls at weaning. Neither embryotoxicity nor frank teratogenicity was seen as a result of coffee ingestion. However, the fetuses in the groups given 100 or 50% solutions of the brewed coffee and 100% instant coffee in place of drinking water had a significantly higher incidence of unossified sternebrae, indicating a delay in calcification.     

Comparative metabolism and mutagenicity of azo and hydrazone dyes in the Ames test

Enteric bacterial and hepatic azoreductase enzymes are capable of reducing azo dyes to yield the constituent aromatic amines. Azo dyes based on benzidine and benzidine congeners have received particular attention because of their widespread use and the known carcinogenicity of benzidine to humans. Azo dyes based on beta-diketone coupling components exist preferentially as the tautomeric hydrazones. A series of hydrazone dyes based on benzidine and benzidine congeners was prepared and characterized by NMR and UV-visible spectroscopy. These dyes were tested for mutagenicity using a modified Ames assay and, unlike the true azo dyes, showed no significant mutagenic activity. The hydrazone dyes were resistant to enzymatic reduction by FMN-supplemented hamster-liver post-mitochondrial supernatant (S-9); under identical conditions, azo dyes such as trypan blue were rapidly reduced.     

Investigation of coffee in Drosophila genotoxicity tests

Two preparations of coffee (instant coffee and freeze-dried home-brew coffee) were tested in different mutagenicity assays in germ cells as well as in somatic cells of Drosophila melanogaster. The three end-points assayed in germ cells were sex-linked recessive lethals (mainly gene mutations and small chromosome aberrations), dominant lethals (cytotoxic effects as well as genotoxic effects) and sex-chromosome losses (chromosome breakage and non-disjunction). The aqueous coffee solutions were fed either to adult male flies for 3 days or to growing larvae during the whole larval development. Treated males were crossed with appropriately marked females, and the different genetic end-points were analysed in the F1 or F2 generation. The test concentrations (instant coffee 4% (w/v), home-brew coffee 3%) were acutely toxic in adult males (killing approximately 75 and 90% of the exposed flies, respectively). No increase in deaths was caused in larvae by the same concentrations. Only cytotoxic effects were observed in the test for dominant lethals. No conclusive genotoxic effects could be detected in any of the three germ cell assays. The coffee preparations were also tested for induction of mutation and mitotic recombination in somatic cells of the wing imaginal disc. Larvae trans-heterozygous for two recessive wing hair markers were fed high concentrations of the coffees for varying periods of time. Wings of surviving adult flies were analysed for mosaic spots. Twin spots exhibiting both mutant phenotypes are produced by mitotic recombination; single spots showing one or the other phenotype are the result of somatic mutation, such as gene mutation or deletion, or of mitotic recombination. Both coffees had weak effects on normal (repair-proficient) cells as well as on excision repair-defective cells in this assay. Additional experiments with pure caffeine and decaffeinated coffee show that these weak effects in somatic cells were most probably caused by the caffeine present in the two coffees.     

An evaluation of instant and regular coffee in the ames mutagenicity test

High concentrations of “home brew” and instant coffee induced. revertants 2–3-fold the spontaneous level with the Ames Salmonella tester strain TA 100 but not with the strains TA 98, TA 1535, TA 1537 and TA 1538. This borderline effect, which may also have been due to non-mutagenic interactions (false positives) occurred only at bactericidal levels of coffees and was completely abolished in the presence of the microsomal “metabolic activation system”. Negative results were obtained in host-mediated assays when mice received up to 6 g instant coffee/kg body weight. An extrapolation in respect of possible carcinogenic risks is dubious.     

Metabolic activation of drugs in mutagenicity tests in vitro

The metabolic pathways of chemical compounds of pharmaceutical interest are reviewed in relation to the role of activation and detoxification in the process of mutagenicity. The properties and subcellular localization of the enzymes involved are given together with the main reactions they catalyze.The role of metabolism in mutagenicity testing in vitro is discussed with special emphasis on the choice of the enzymatic system. Parameters such as species, strain, age, sex, diet, and induction are considered. The effect of various enzymatic effectors added in vitro is also discussed. It is concluded that the metabolism of drugs is very complex, involving both activation and detoxification processes catalyzed by a large variety of enzymes. Production of mutations in vitro in prokaryotic or eukaryotic cells is the results of a balance between all those pathways. Metabolic activation thus merits special attention.     

Salmonella/microsome mutagenicity tests of heat-processed milk samples

Samples of whole milk were heat treated by commercial heat-sterilization, by commercial heat-pasteurization or by a laboratory heat-pasteurization (65 degrees C for 30 min). Each sample was tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 with and without S-9 mix. Dichloromethane extracts of milk heated at 100, 135 and 150 degrees C for 5 hr were also tested for mutagenicity using the same assay. None of these samples exhibited mutagenicity in the Ames assay used.     

应用遗传毒理短期测试研究氰戊菊酯致突变性

报道的氰戊菊酯致突变性结果不一,为此用多种方法研究了氰戊菊酯标准品和工业品的致突变性。结果发现:小鼠骨髓多染红细胞微核试验阴性,但见雄性较大剂量时有随剂量增高趋势。小鼠睾丸初级精母细胞染色体数目畸变阴性,染色体结构畸变阳性。人体外周血淋巴细胞SCE频率虽与阴性对照比差别有显著意义,但无剂量反应关系,为阴性。人体外周血淋巴细胞UDS 试验,氰戊菊酯工业品油剂及日产粉剂呈较强阳性;标准品阳性较弱。综合上述材料,认为氰戊菊酯标准品是一弱阳性致突变剂,其工业品致突变性增强,可见杂质起了重要作用。因此,减少我国工业品中之杂质、提高纯度,可望降低其致突变性能。     

基于Ames实验的玉屏风胶囊致突变研究

目的:对玉屏风胶囊进行细菌回复突变试验,以评价其遗传毒性。方法:采用直接平皿掺入法,将胶囊以每皿40、200、1000、5000μg玉屏风胶囊药粉为终浓度在活化与非活化条件下,与鼠伤寒沙门氏菌组氨酸缺陷型突变菌株,37℃接触48h,计数回变菌落数。结果:玉屏风胶囊药粉每皿40～5000μg各组在+S9与-S92种测试系统条件下,各菌株回变菌落数与阴性对照相比差异无统计学意义,回变菌落背景正常,未显示其有抑菌作用和沉淀,且无剂量-反应关系。结论:玉屏风胶囊药粉在该实验条件下对测试菌株无致突变性。