Biomonitoring of urine mutagenicity with the Ames test: improvement of the extraction/concentration method

Comparative extraction efficiency of the pre-packed Bakerbond^®-spe-SDB-1 resin and of Amberlite^®-AD2 (XAD-2) resin, for the preparation of urine extracts in biomonitoring studies. Urine extracts were prepared in parallel with the Bakerbond^® column and with the classical XAD-2 resin from urines (1) spiked with mutagenic chemicals, (2) collected from patients after chemotherapy, and (3) from smokers. Mutagenic activities were evaluated on Salmonella typhimurium tester strains TA97a, TA98, TA100 and TA102 with and without S9 mix. Mutagenic activities obtained with Bakerbond^® extracts were almost always higher or at least equivalent to those prepared on XAD-2 resin. Similar results were observed for the three urine sample groups. When fully validated, the use of the pre-packed columns will be more convenient and time-saving for large population studies.     

Detection of mutagenic activity in urine samples using a new concentration procedure

A study performed with cyclophosphamide (CP) and nor-nitrogen mustard (NNM), one of its main urinary metabolites, has shown that separation on Polygosil C-18 resin is preferable to one on XAD-2 resin as a means of concentrating the mutagenic activity present in urine of rats given cytostatics such as CP. Mutagenic activity was detected, using Salmonella typhimurium tester strain TA1535. While NNM is irreversibly bound to XAD-2 resin, it can be recovered after elution with methanol on Polygosil C-18. The better efficacity of Polygosil C-18 in concentrating CP and its metabolite(s) was confirmed with experiments with urine of rats treated with increasing doses of CP.     

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.     

Mutagens in rat urine after dermal application of 1,3-diaminobenzene

The mutagenicity of urine from rats treated topically on the skin with 1,3-diaminobenzene was studied by the Salmonella/mammalian-microsome assay. Urine samples were either passed directly through micropore filters or extracts were prepared using XAD-2 resin before testing in the frameshift strain TA98. Significant mutagenic activity was found only after metabolic activation with rat-liver microsomes. The activity was higher in extracts from rats treated with a mixture of hydrogen peroxide and 1,3-diaminobenzene than from rats which were exposed to 1,3-diaminobenzene only. After fractionation of the urine by HPLC it could be demonstrated that the mutagenic activity was not due to the parent amine but related to metabolites in two of the fractions. To a lesser extent these two partially purified fractions were also mutagenic without S9 activation even though it was not possible to demonstrate this effect in unfractionated urine extracts. A third fraction containing two metabolites did not exert demonstrable mutagenic activity. The implications for the assessment of hazard to man are discussed.     

Genetic toxicology evaluation of commercial beers. II. Mutagenic activity of commercial beer products in Salmonella typhimurium strains TA98, TA100 and TA102

5 concentrated extracts of commercial beers were prepared using XAD-2 resin. The residues were subjected to evaluation for mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA102. The tests were conducted using preincubation protocols including provisions for S9 metabolic activation. Although the extracts did produce moderate toxicity to the Salmonella organisms used in the assays, none of the residues were found to induce mutation up to their maximum testable concentrations.     

Sensitivity of different bacterial assays in detecting mutagens in urine of humans exposed to polycyclic aromatic hydrocarbons.

The urine mutagenicity and excretion of 1-hydroxypyrene (1-OH PYR) in non-smoking psoriatic patients treated topically with coal-tar-based ointments were analysed in order to find the most appropriate procedure for monitoring occupational PAH exposure. The bacterial mutagenicity assays used were the plate incorporation, macro-scale fluctuation and microsuspension tests, all on Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. The sensitivities of the three assays in detecting mutagenic urinary PAH metabolites were compared. The efficiencies of XAD-2 and C18 resins for concentrating PAH urinary mutagens were evaluated in the microsuspension assay. The plate and fluctuation tests on XAD-2 urine extracts were shown to be insufficiently sensitive to detect low urinary levels of mutagens, being positive on urine samples with very high PAH metabolite content, estimated as more than 30 micrograms/g of creatinine of 1-OH PYR. The microsuspension assay on XAD-2 or, even better, on C18 urine extracts was very sensitive in detecting up to 5 micrograms/g of creatinine of 1-OH PYR. It therefore seems to be applicable to the biological monitoring of most occupational low exposures to coal tar.     

Mutagenic activity in wastewater concentrates from dye plants.

Wastewater concentrates from the wastewater treatment systems of three dye plants were tested for mutagenic activity in Salmonella typhimurium TA98 and Escherichia coli WP2uvrA using a fluctuation assay. Concentrates were prepared by passing samples of wastewater (5-6 or 30 litres) through two porous resins (XAD-2 and XAD-7) in series. S. typhimurium in the presence of microsomal activation proved to be the more sensitive marker of mutagenicity. Mutagenic responses were observed in concentrates from all three plants tested. The results show that mutagenic activity was particularly high in the incoming waters and increased after active, biological treatment. Physico-chemical treatment may be effective in decreasing mutagenic activity, but only if appropriately used.     

Genetic toxicology evaluation of commercial beers. I. Development of procedures for the preparation of extracts from commercial beer products

Commercial beer was subjected to an investigation in order to establish standard conditions for preparing organic solvent extracts to be used in short-term genetic screening assays. Test samples for use in the evaluation were prepared by mixing several brands of commercially available beer into a composite pool which was then spiked with the mutagen, 2-nitrofluorene. The composite sample was then concentrated using varying ratios of beer to XAD-2 resin in a 1.5 cm X 30 cm column. Dry-weight analyses indicated that significant amounts of residue could be trapped by XAD-2 resin. Columns were sequentially eluted by methylene chloride, acetone and methanol followed by evaporation of the solvents under nitrogen gas. Residues from commercial products were not mutagenic, but mutagenic activity could be detected in residues from spiked beer, yielding nearly 90% of the expected biological activity in S. typhimurium TA98. A standard method amenable to processing large volumes of beer products was devised for application to other projects.     

Mutagenicity in Salmonella typhimurium tester strains of XAD-2-ether extract, recovered from Katsura River water in Kyoto City, and its fractions

The mutagenic activity of XAD-2-ether extracts recovered from Katsura River water at monthly intervals during September to December 1980 was tested on S. typhimurium TA1538, TA1535, TA98 and TA100. The extracts showed strong mutagenic activity towards TA1538 nd TA98, especially in the presence of S9 mix. They were more active to TA1538 than to TA98. Some of each of the XAD-2-ether extracts were pooled and separated into neutral, basic and acidic fractions, and their mutagenic activities were tested on TA1538 and TA98 to determine their contribution to the total mutagenic activity of the parent extract. The neutral fraction was responsible for most of the total mutagenic activity of the parent extract. Although the basic fraction was only 5.4% by weight of the parent extract, it was much more mutagenic than any other fraction. Its contribution to the total mutagenic activity was higher than the acidic fraction which was 30.5% by weight of the parent extract.     

Mutagenicity of azo dyes in the Salmonella/microsome assay using in vitro and in vivo activation

The mutagenicity of 6 azo dyes, including direct black 38 (DB38), direct black 19 (DB19), direct brown 95 (DB95), solvent yellow 3 (SY3), trypan blue (TPB), and food black 2 (FB2), was examined in the Salmonella/microsome assay. The effect of chemical azo reduction (dithionite) and in vivo metabolism on the mutagenicity of the dyes was also studied. In vivo azo-dye metabolites were isolated from the urine of rats intubated with dyes by XAD-2 column chromatography. Urinary metabolites from all the treated animals, except animals treated with FB2, induced frame-shift mutations in strains TA1538 and TA98 in the presence of liver S9 activation. The control urine did not increase the incidence of revertants in strains TA1538 and TA98. Thus, XAD-2 chromatography can be used to isolate genotoxic metabolites from the urine of animals intubated with azo dyes.     

昆明水源水和自来水水质致突变性及化学背景值1．Ames试验和水源水及自来水微量有机、无机污染物化学背景值

本文报道：用ＸＡＤ－２离子树脂吸附富集法制备自来水和滇池及松华坝水库源水提取物,以Ａｍｅｓ鼠伤寒沙门氏菌回复突变系统诱发致突变性。Ａｍｅｓ试验结果如下：来自滇池源水经氯化的自来水提取物,用ＴＡ９８菌株加Ｓ９或不加Ｓ９混合物,突变率＞２,结果为阳性。滇池源水提取物致突变性为可疑。使用化学分析法结合生物分析法评价昆明自来水和滇池及松华坝水库水中的化学微量污染物,估价在水中的微量污染物对人体健康的潜在致癌效应是有意义的。     

The use of rat urine extracted on XAD-2 resin and assayed in a microsuspension-modified Ames test as an in vivo indicator of genotoxic exposure

The measurement of urinary mutagenicity is a non-invasive monitoring tool which often reflects an animal's recent exposure to genotoxic agents. Although studies in man are indispensable for monitoring industrial and/or environmental exposure to genotoxins, a sensitive laboratory animal model is necessary for mechanistic studies on the role of specific chemical exposure in altering urinary mutagenicity. The objective of this study was to enhance the sensitivity of the methodology used for detecting urinary mutagenicity in rats by using XAD-2 resin to extract and concentrate the urine and a microsuspension-modified Ames test to quantify mutagenicity. The polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) and the aromatic amine 2-acetylaminofluorene (AAF) were used as test compounds. Under the conditions of our study, AAF administered to rats by gavage at doses of 1 mg/kg or higher induced a dose-dependent increase in urine mutagenicity. The greatest mutagenic response was seen when S9 was present during the microsuspension-modified Ames test and beta-glucuronidase (BG) was not included. Similarly, BP administered to rats by gavage at doses of 10 mg/kg or higher induced a dose-dependent increase in urinary mutagenicity. The relative importance of BG and S9 were quite different with BP than with AAF. With BP, mutagenicity was greatest when both S9 and BG were present during the microsuspension-modified Ames test, and least with S9 and without BG. In both AAF- and BP-treated animals, extraction of the urine on XAD-2 resin markedly enhanced the mutagenic response compared to neat urine, but partitioning of the XAD-2 eluate into methylene chloride always diminished the mutagenicity of the urine extract. The results demonstrate the sensitivity and reproducibility of rodent urinary mutagenicity assays when XAD-2 resin is used to extract and concentrate the urine and a microsuspension-modified Ames test is used to quantify mutagenicity. This sensitive method should facilitate mechanistic studies on the roles of specific environmental agents in affecting urinary mutagenicity and, in addition, may be used during acute, subchronic and chronic rodent bioassays as a non-invasive in vivo indicator of genotoxic exposure.     

Genotoxicity of drinking water from three Korean cities

Organic content of drinking tap water from Seoul, Taejon, and Suwon was extracted with an XAD-2 resin column and organic solvents. Four doses of the extract equivalent to 4, 2, 1, and 0.5 l water were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 mix. The organic extracts of the water from all three cities were mutagenic in TA 98 without S9 mix and in TA 100 with and without S9 mix. The highest number of revertants per plate was found in the absence of S9 mix. Three doses of the extract (equivalent to 22, 11, and 3.7 l water) were also tested in the bone marrow micronucleus test using BDF1 mice. At the highest dose, a significant increase of the micronucleus frequency was observed. The time required to be on the effect, however, varied with the source of the water. Our results indicate that the drinking tap waters from the three cities were genotoxic clearly in the bacterial test and also in the in vivo assay with mice. As we found no genotoxicity of the source water as seen in a previous study, it is likely that the chlorination process leads to the genotoxicity of the tap water.     

Mutagenicity in Salmonella typhimurium mutants of serum extracts from airborne particulates

Airborne particulates collected from urban and non-urban air were extracted with calf serum or benzene, and their mutagenic potencies were evaluated in the Salmonella reversion assay. The serum extracts were mutagenic to strains TA98 and TA100 and contained both direct- and indirect-acting mutagens. Mutagenic activities for TA98 recovered from the particulates by serum or benzene extraction were much less in the serum extracts than in the benzene extracts. There was no significant difference in mutagenic potencies of the extracts between the urban and non-urban particulates, irrespective of the presence of S9 mix. The calculated mutagenic activities per m3 of air, however, were greater for urban air than for non-urban air, because of higher concentration of particulates in urban air than in non-urban air. Serum effectively reduced both direct and indirect mutagenic activities of the benzene extracts except for an insufficient reduction in direct mutagenicity at a high dose of benzene extracts. These findings suggest that serum could contribute greatly to decrease the mutagenicity of airborne particulates by mechanisms such as less efficient solubilization of mutagenic components and inactivation by protein binding. Biological availability of mutagens, therefore, should be considered for evaluation of actual mutagenic hazard by airborne particulates.     

Use of Salmonella typhimurium TA 98, YG 1024 and YG 1021 and deconjugating enzymes for evaluating the mutagenicity from smokers'' urine

Four smokers were chosen for their different smoking habits, and their declared cigarette consumption confirmed by urinary measurement of nicotine and its metabolites. The promutagenicity of their urine was evaluated by the Ames test, modified according to Kado et al. (Mutation Res., 31 (1983) 25–32) after extraction on XAD2 Amberlite resin. The different Salmonella typhimurium strains TA 98, YG 1021 and YG 1024 were compared to determine the presence of amino aromatic compounds in the urine of smokers of blond and black tobacco. The strain YG 1024 shows higher mutagenicity than TA 98 for extracts from the smoker's urine and more particularly from black tobacco smokers. In addition, the pretreatment of urine by external enzymatic systems (β-glucuronidase or arylsulfatase) reveals the presence in the urine of glucurono- and sulfoconjugated forms of promutagens, including amino aromatic compounds.     

Analysis of urinary mutagens produced by cigarette-smoking baboons

Urine concentrates from 17 cigarette-smoking baboons and 12 sham puffers were analyzed for mutagenic activity in S. typhimurium tester strain TA1538. Both the proportion of animals exhibiting measurable mutagenic activity in urine and the mean level of mutagenic activity present were significantly greater in cigarette smoking baboons (P less than 0.05). Mutagenic activity in the urine of male and female cigarette-smoking baboons was not significantly different. Age and smoking history did not, but mean blood carboxyhemoglobin did, correlate with mutagenic activity of the urine concentrate from individual animals. Fractionation of the urine concentrates on silicic acid separated the concentrate into fractions that were more active in TA100 and others that were more active in TA1538. Further fractionation was accomplished by HPLC.     

The kinetics of mutagen excretion in the urine of cigarette smokers

The excretion of mutagens in the urine of cigarette smokers was studied as a model for absorption and elimination of complex carcinogenic and mutagenic mixtures in humans. Urine was collected from an occasional smoker who smoked 1 cigarette (17 mg tar/cigarette) and from a heavy smoker (smokes approximately 20 cigarettes/day) who quit for 2 days and then resumed smoking. Urine samples were collected for 6 days, including a 2-day pre-smoking period for the occasional smoker and pre-abstention period for the heavy smoker, respectively. Mutagen excretion patterns were determined by extracting the mutagens in each urine sample with XAD-2 resin and testing the extract in a microsuspension modification of the Salmonella/microsome liquid-incubation assay using bacterial strain TA98 with metabolic activation. Peak mutagenic activity of the urine collected from the two smokers appeared 4-5 h after the beginning of smoking. Activity decreased to pre-smoking "baseline' levels in approximately 12 h for the occasional smoker, and the activity for the heavy smoker approached the occasional smoker's 'baseline' in approximately 18 h after the cessation of smoking. The mutagen excretion patterns of the occasional smoker after smoking a single cigarette suggests that, the mutagens, as detected by the Salmonella assay, are absorbed rapidly (3-5 h) and are eliminated from the body following first order kinetics. The excretion rate constant for the occasional smoker was approximately 0.1 h-1 and the half-life (T1/2) was approximately 7 h.     

Mutagenicity testing of protein-containing and biological samples using the Ames/Salmonella plate incorporation test and the fluctuation test.

Mutagenicity testing of biological samples and proteins is complicated by the presence of histidine and histidine-related growth factors which may produce a false positive result in the Ames/Salmonella plate incorporation test. A bioassay method, utilizing an automated dispenser-photometer and Salmonella typhimurium strain TA1535 as the indicator bacteria, was used to estimate the presence of histidine-related growth factors in three enzyme solutions submitted for mutagenicity testing. One of the solutions was clearly positive in the Ames/Salmonella test and also contained the highest amount of L-histidine-HCl-equivalents. The two other solutions, with low or undetectable amounts of L-histidine-HCl-equivalents, gave equivocal and negative results, respectively, in the Ames/Salmonella test. Studies were also performed with strains TA98, TA100 and TA1535 to determine the amount of added L-histidine-HCl that would result in a 'positive' result in the Ames/Salmonella test. Because the minimum amount of L-histidine-HCl required to double the number of revertant colonies was 150 nmol/plate, and the maximum amount of L-histidine-HCl-equivalents supplied by the enzyme preparations was 40 nmol/plate at the highest tested dose, the mutagenicity test results of the enzyme solutions cannot be explained solely by histidine or related compounds. Smokers' and non-smokers' urines, concentrated with liquid extraction (CHCl3) and adsorbent (XAD-2 and XAD-2/Sep-Pak C18) techniques, were studied to reveal differences in efficiencies to extract histidine and histidine-related compounds in the urines. Amounts of 'histidine' in concentrates of urine were measured using the bioassay method and a chemical method employing derivatization with fluorescamine. The fluorescamine method also efficiently detected 3-methyl-L-histidine, a product of muscle metabolism excreted in urine, which was found to be unable to support auxotrophic growth in TA1535, leading to exaggerated estimations of the auxotrophic growth enhancing properties of urine extracts. The urine extracts, and pure L-histidine-HCl, were tested using a two-step fluctuation test to estimate auxotrophic growth factor effects in this type of test. Because of a strong dilution effect when adding the histidine-free selection medium, the fluctuation test employed in this study was not found to be particularly sensitive to growth factors. The results of this study indicate that use of a bioassay, employing the same indicator bacteria as the mutagenicity test themselves, is a reliable way to measure histidine-related growth factors in biological samples.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-pressure liquid chromatography for isolation of clastogenic agents from urine

Small columns of XAD-2 resin have been widely used to extract and concentrate mutagenic materials from urine. Using analytical HPLC and assays for clastogenicity with Chinese hamster ovary cells, we found that small columns of XAD-2 resin (1.5 ml bed volume) retain only a small percentage of organic material and undetectable amounts of genotoxic activity in urine samples. Increasing the size of the XAD resin bed resulted in better recoveries, but much organic material was still lost by overloading of the column. In contrast, when urine was acidified and chromatographed by preparative reversed-phase HPLC using large-bed-volume (500 ml) commercial columns, retention of hydrophobic organic material from urine was excellent. Subsequent stepwise elution of the column with increasing concentrations of acetone produce 3 fractions of organic material of increasing hydrophobicity. When urine from smokers was analysed, all 3 fractions contained material which was clastogenic to Chinese hamster ovary cells. The procedures developed are suggested as a new general purpose approach to the isolation of genotoxic materials from urine.     

Mutagenic/carcinogenic agents in indoor pollutants; the dinitropyrenes generated by kerosene heaters and fuel gas and liquefied petroleum gas burners

Incomplete combustion of kerosene heater, and fuel gas and liquefied petroleum gas-burner emissions produces indoor pollutants that may be carcinogenic. The incomplete-combustion products from each type of appliance were therefore collected by adsorption on about 3 g of XAD-2 resin, and were extracted with benzene-methanol as a solvent for determination and identification of mutagens in the Salmonella-microsome test system. Benzene-methanol extracts of the particulates generated by a heater and two burners showed extreme mutagenicity for strains TA97 and TA98 without S9 mix. Based on the results of analysis, a combination of high performance liquid chromatography (h.p.l.c.) and gas chromatography (GC), about 40-80% of the direct-acting mutagenicity in each crude extract showed the same h.p.l.c. and GC retention times as dinitropyrenes (1,3-, 1,6- and 1,8-isomers), and 1-nitropyrene. Moreover, other nitroarenes, 2-nitrofluorene, 1,5- and 1,8-dinitronaphthalene, and 4,4'-dinitrobiphenyl, were detectable in almost all samples, but their contribution to the mutagenicity of each extract was very low. Kerosene heaters were found to generate small amounts (0.2 ng/h) of dinitropyrenes, which are potential mutagens/carcinogens, only after 1 h of operation.