Bacterial Fluctuation Test Research Articles

Absence of mutagenic action of 5β-cholan-24-oic acid derivatives in the bacterial fluctuation and standard Ames tests

Published data on the mutagenicity of 3 bile acids in the bacterial fluctuation test are conflicting. Eleven 5β-cholanoic acids including 2 of the biie acids were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in the fluctuation tests. In any of these bile acids at the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. Similarly, none of these compounds showed positive mutagenicity in both strains in the standard Ames test either with or without hepatic metabolic activation. Our results support the claim that 3 bile acids are not mutagenic, and indicate that the initiation activity of 5β-cholanoic acids is not demonstrable with a short-term assay using Salmonella strains.

Biological effects in a chemical factory with mutagenic exposure

Eighty-five workers in a chemical plant, in which there was an increased frequency of B-lymphocyte tumours (myelomas and lymphomas) and lung cancer, did not excrete higher amounts of thioethers in urine than 48 control subjects. The chemically exposed workers had, however, a higher excretion of thioethers in urine during shift compared to an exposure-free period. The bacterial fluctuation test showed a statistically significantly increased mutagenic activity for E. coli Wp2 uvrA, but not for Salmonella TA98, in the urine of the exposed workers compared with the controls. However, no difference in excretion was found in the chemical workers between work and an exposure free-period. Further, no association of thioether or mutagen excretion with any specific chemical exposure (e.g. ethylene oxide and formaldehyde) in the plant could firmly be established.

Lack of mutagenic activity of bile acids in bacterial fluctuation tests

3 bile acids (cholic acid, chenodeoxycholic acid and deoxycholic acid) were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in fluctuation tests in the absence of an external source of metabolic activation. At the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. These results do not support the claim (Watabe, J., and H. Bernstein (1985) Mutation Res., 158, 45–51) that these bile acids are mutagenic.

Detection of occupational and environmental exposures by bacterial mutagenesis assays of human body fluids.

Assays of human body fluids provide a means to document human exposure to mutagens in the environment. In contrast to measurements of ambient levels, these assays demonstrate absorption of mutagens and provide estimates of minimal systemic doses. For most studies reviewed here, specimens of urine were concentrated by adsorption to columns of XAD-2 resin or by liquid partition extraction prior to the mutagenesis assays. The resulting extracts most commonly were analyzed for mutagenicity using the Salmonella/mammalian microsomal plate assay. Less frequently used assays included bacterial fluctuation tests instead of the plate assay and assays for the induction of sister chromatid exchanges in cultured mammalian cells. In addition to reviewing literature reports where body fluids were tested, the advantages, disadvantages, and potential role of this approach will be briefly discussed and compared with other approaches to the identification of mutagenic hazards in the workplace.

Excretion of mutagens in sweat and faeces of man, and in serum, gastric juice and urine of rats, after oral dosing of niridazole or metronidazole.

Frameshift mutagens were isolated and concentrated from sweat and faeces of three healthy volunteers (non-smokers) of whom two received 750 mg metronidazole/person and one received 500 mg niridazole. The sweat samples were collected in a sauna a day before and 8 h after oral uptake of the medicaments. The faecal samples were those expelled 12 h before and after drug uptake. All extracts were tested for mutagenicity in the bacterial microtiter fluctuation test employing Salmonella typhimurium TA1538. No mutagenic activity was found with the samples obtained before the drugs were taken, whereas the samples collected after drug treatments were all mutagenic (P less than 0.05). In an animal experiment, female Wistar rats were used to study the time-course of the excretion of mutagens in serum, urine and gastric juice after uptake of 10-20 mg niridazole by gavage. Significant mutagenic activities (P less than 0.001) were found in serum 10 min after and in gastric juice 12h after treatment with niridazole. Non-significant but detectable mutagenicities were found in urine 12 h after treatment, when S. typhimurium G46 was employed as a test organism. These latter mutagenicities were significant (P less than 0.05) 24 h after treatment, they reached a peak of activity (P less than 0.01) 48 h post-administration and disappeared 12 h thereafter.

The metabolic activation of 4,4′‐methylene‐bis‐(2‐chlorobenzeneamine) to a bacterial mutagen by hepatic postmitochondrial supernatant from human and other species

4,4'-Methylene-bis-(2-chlorobenzeneamine) (MbOCA) is a commercially important industrial chemical that is carcinogenic in three animal species and mutagenic in the Ames test. The ability of hepatic postmitochondrial supernatant from humans, dogs, mice, and rats to activate MbOCA to a bacterial mutagen has been investigated using the Ames plate incorporation test and a bacterial fluctuation test. In the Ames plate test, hepatic S9 preparations from mice and Aroclor 1254-induced rats only were sufficiently active to produce a significant mutagenic response. Preincubation of MbOCA with S9 from human liver produced a slight increase in the number of revertants but not a doubling as compared to controls. However, using the more sensitive bacterial fluctuation test, liver S9 from all species activated MbOCA to a bacterial mutagen. The responses produced were dose-related and, for at least part of the dose range, were double the background levels observed in controls. The increases in the mutagenicity of MbOCA produced by liver S9 from humans, dogs, and rats were significant at the 0.1% level of probability. Liver S9 preparations from all species in which MbOCA is carcinogenic have now been demonstrated to be capable of activating this compound to a bacterial mutagen. The finding that S9 from human liver can also activate MbOCA to a mutagen increases the concern that it may be a human carcinogen.

The detection of mutagenic chemicals in the tissues of shellfish exposed to oil pollution

Two species of shellfish (mussels and limpets) were collected from two sites in West Wales (U.K.) extracted in nitric acid and monitored for the presence of mutagenic chemicals using bacterial fluctuation tests over a period of an oil pollution incident. Prior to the observation of oil pollution at the two sites no mutagenic activity could be detected in the tissue extracts. However, within three days of the observation of oil pollution, chemicals capable of inducing both base-substitution and frameshift mutation could be detected in both shellfish species. At one of the sites (St. Brides) mutagenic activity could still be detected in samples collected 12 months after the oil pollution incident, whereas at the other sites (Dale) no activity was detectable after 12 months. The results obtained demonstrate shellfish exposed to crude oil (and dispersants) accumulate mutagenic chemicals that persist for periods longer than pollution could be observed by visual inspection.

Occupational handling of cytostatic drugs.

The bacterial fluctuation test and measurement of the frequency of sister chromatid exchanges were used for evaluation of the exposure of different groups of hospital personnel to cytostatic drugs. Increased mutagenic activity in the urine was detected only in personnel working with inadequate safety precautions, e.g., lack of a ventilated safety cabin for preparation of parenteral solutions. Although such a safety cabin was used within the hospital pharmacy, increased mutagenic activity was detected in the urine of prescriptionists preparing parenteral cytostatic drugs. After a change of glove material and improvement of ventilation in the safety cabin, no work-related increase in urinary mutagenic activity was seen. None of the different groups tested, showed any increase in the frequency of sister chromatid exchanges. It is therefore concluded that handling of cytostatic drugs according to the issued safety recommendations including working in a well ventilated safety cabin, will not result in any enhancement of mutagenic activity in the urine related to work.

Genotoxic hazards in the rubber industry.

The toxicology of chemicals used in the rubber industry is poorly known. In the present investigation an attempt was made to use biological monitoring methods to identify job categories with possible exposure to potentially genotoxic chemicals. Urine samples were collected and analyzed for mutagenic activity by the bacterial fluctuation test. The highest individual mutagenic activities were detected among the workers in the weighing and mixing departments, and some high individual values were found among vulcanizers. Sister chromatid exchanges (SCE) and structural chromosome aberrations were also analyzed from samples of peripheral blood. The effect of smoking was clearly associated with significantly increased numbers of SCEs and chromosome aberrations. Among the workers in different job categories, the highest SCE frequencies were found for mixers who smoked. Also nonsmoking cleaners of the mixing department differed significantly from the nonsmoking referents in this respect. However, only the group of nonsmoking weighers showed an increase in the number of chromosome aberrations (p less than 0.01) when compared with the referents. The results suggest the need for improved occupational hygiene in the weighing and mixing departments of rubber plants so that exposure to potentially hazardous genotoxic chemicals can be decreased.

Urinary mutagenicity assays: a problem arising from the presence of histidine associated growth factors in XAD-2 prepared urine concentrates, with particular relevance to assays carried out using the bacterial fluctuation test.

The appearance of mutagenic activity in urine samples from a group of nurses and from unexposed individuals has been investigated using the bacterial fluctuation test. Apparent mutagenic activity was seen in samples from all subjects and did not appear to be related to any specific occupational or environmental exposure. This activity seems to be related to the presence of histidine or histidine-related auxotrophic growth factors in the urine concentrates, not completely removed by the recommended XAD-2 column procedure. It is suggested that the reliance on XAD-2 columns for the removal of histidine may produce spurious results when using this assay to screen populations for exposure to mutagenic chemicals.

The development of anaerobic methods for bacterial mutation assays: aerobic and anaerobic fluctuation tests of human faecal extracts and reference mutagens

Methods for performing microtitre fluctuation tests in an anaerobic enclosure have been devised in order to study the mutagenic activity of human faecal extracts under conditions of strict anaerobiosis. Under these conditions (monitored by growth of obligate anaerobes), two mutagens (4-chloromethylbiphenyl (4CMB) and potassium dichromate) were reproducibly mutagenic to Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA(pKM101), respectively. We investigated the mutagenicity of aqueous extracts of faeces, prepared aerobically and anaerobically, collected from a single donor. Bioassay of 5 faecal extracts showed that 4 contained substances which promoted auxotrophic growth of his- and tryp- bacteria, and were therefore unsuitable for mutagenicity assay. The remaining extract (half of which was prepared aerobically, the other anaerobically) was assayed for mutagenicity in S. typhimurium TA 100. Reproducible dose-response curves were obtained with the aerobic extract and with the anaerobic extract when both were assayed under aerobic conditions. Negative results were obtained when the anaerobic or aerobic extracts were assayed anaerobically, under conditions where concurrent assays of 4CMB were positive. We conclude that bacterial fluctuation tests using reversion from auxotrophy to prototrophy as the genetic marker can be seriously compromised by the presence of substances (including amino acids) in the test material which promote auxotrophic growth. Moreover, it is clear that the results and interpretation of mutagenicity tests of faecal extracts are very dependent on whether air is present or absent during both extraction and assay.

An assay for mutagenic activity of 4CMB, 4HMB and BC, using the “microtitre” fluctuation test

4CMB, 4HMB and BC were assayed for mutagenic activity using the ‘microtitre’ bacterial fluctuation test without metabolic activation. 4CMB was positive in strains of Salmonella typhimurium detecting both base-substitution and frameshift mutation. BC was weakly positive only in the strain which detects base-substitution mutation. 4HMB was negative in both strains. 4CMB and 4HMB were equally toxic to the strains, whilst BC was comparatively less toxic.

Fluctuation test data on 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and Benzyl Chloride (BC) using Salmonella typhimurium TA98 and TA100

Bacterial fluctuation tests (Green et al., 1976) were performed both with and without metabolic activation using the ‘Ames’ Salmonella typhimurium strains TA98 and TA100 (Ames et al., 1975) to assay the mutagenic potential of 4CMB, 4HMB and BC. 4CMB and 4HMB were tested on the same occasion. However, 4CMB was only compared to BC in one assay. The results also show an independent test of BC.

The mutagenicity of 4CMB in a microwell bacterial fluctuation test

4CMB gave a positive test response in the bacterial fluctuation test. The presence of S9 mix caused attenuation of the dose response indicating that 4CMB is a direct-acting bacterial mutagen that may undergo enzymatic detoxication.

Use of the bacterial fluctuation test to detect mutagenicity in urine of nurses handling cytostatic drugs

Use of the bacterial fluctuation test to detect mutagenicity in urine of nurses handling cytostatic drugs

Mutagenicity of aerosols from the oxidative thermal decomposition of rigid polyurethane foam.

The aerosol fraction of the oxidative thermal decomposition products (700 degrees C) of rigid polyurethane foam was collected on glass fiber filters and fractionated into either-soluble neutral, acidic, and basic fractions and water-soluble compounds. The fractions showed mutagenic activity in a bacterial fluctuation test with Salmonella typhimurium TA98 or Escherichia coli CM891 as the tester strains. All the fractions induced mutations in both strains after metabolic activation with rat liver S-9 mix. The basic and the water-soluble fractions were mutagenic for S. typhimurium TA 98 even without activation. Thin-layer chromatography showed the presence of several primary aromatic amines in the aerosol. Polycyclic aromatic hydrocarbons were not detected by glass capillary gas chromatogaphy.

Mutagenicity in urine of workers in rubber industry

Epidemiological studies have shown that those who work in rubber industry have an increased risk of cancer. In the working environment they are exposed, probably, to several hundred different chemicals some of them being known or suspected carcinogens and mutagens. The bacterial fluctuation test was used to detect the mutagenicity in the urines of exposed workers. A group of unexposed office clerks served as controls. Both groups consisted of smokers and non-smokers, and that was taken into consideration in the results. Rubber workers, either smokers or non-smokers, exhibited significantly higher mutagenic activity in their urine than the occupationally unexposed controls when the base-pair substitution strain E. coli WP2 uvrA was used as indicator organism. Use of the frameshift strain S. typhimurium TA98 revealed increased mutagenicity in the urine of occupationally exposed smokers, non-smokers and unexposed smokers. The extent of mutagenicity in the urine of workers who smoked suggested a synergistic effect of smoking and occupational exposure. The bacterial fluctuation test with urine samples as sources of mutagenicity is able to detect chemical exposure if the excreted compounds are still in active form or can be activated. The method can be used to identify hazardous working conditions long before the manifestation of possible pathological changes in exposed individuals.

Mutagenicity evaluation of the two antimalarial agents chloroquine and mefloquine, using a bacterial fluctuation test

The two antimalarial agents chloroquine and mefloquine have been tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537 and TA1538. Chloroquine was found to revert strain TA1537 at concentrations of 100 and 250 μg/ml, most likely due to intercalation. No mutagenicity was found with mefloquine at concentrations up to 2.5 μg/ml, neither without nor with metabolic activation by Ca 2+-precipitated rat liver microsomes. Higher concentrations of mefloquine and chloroquine inactivated the bacteria.

Detection of mutagenic polycyclic aromatic hydrocarbons in african smoked fish

The mutagenicity of six polycyclic aromatic hydrocarbons found in African smoked fish was tested using several Salmonella typhimurium strains. In the presence of fortified rat-liver post-mitochondrial fractions, mutagenic activity was observed with o-phenylenepyrene, coronene, benzo[ g,h,i]-perylene and triphenylene in the plate incorporation method, and with fluoranthene in the bacterial fluctuation test. No mutagenic effects of benzo[ b]fluoranthene towards any of the tested strains were detected.

Mutagenicity of acrylonitrile

Incubation of Salmonella typhimurium strains in an atmosphere of 0.2% gaseous acrylonitrile increased the numbers of his+ revertants/plate only in the presence of a fortified S9 liver fraction. The mutagenic effect was particularly pronounced with strains TA1530, TA1535 and TA1950 and much weaker with strains TA100, TA98 and TA1978. The results of bacterial fluctuation tests confirmed the necessity of the presence of S9 mix and showed the particular sensitivity of TA1530. The reversion rate varied with the S9 mix composition, the animal species utilized and the type of pretreatments applied to the animals. The mutagenicity of acrylonitrile in S. typhimurium is therefore microsome-mediated and is particularly discernable with strains sensitive to base-substitution mutagens.