Mutascreen, an automated bacterial mutagenicity assay.

Mutascreen is an automated instrument for bacterial mutagenicity testing. The biological principles of the Mutascreen assay are the same as those of the bacterial reverse-mutation assays, like the Ames test, but several operational principles are different. The Mutascreen assay takes place in wells containing only 400 microliter of liquid medium. Also, the dispensing of the liquid medium, the bacterial tester strains, the metabolic activation system (S9), and the test solutions is all performed by a computer-controlled robot according to the user's preprogrammed instructions. The turbidity in up to 200 wells is monitored intermittently over a 24-h period by a vertical-pathway photometer, thereby avoiding measurement problems caused by sedimentation. The data for the resulting growth curves is stored for analysis. The auxotrophic growth pattern is altered characteristically by test solutions that are toxic or contain endogenous growth factor(s), while prototrophic growth is observed earlier in the 24-h period when revertants have been induced by the test solution. To compare the Mutascreen assay with the conventional plate assay, 36 chemicals including known carcinogens and noncarcinogens were tested. Both assays identified the same chemicals as mutagens and gave quantitatively similar results, thus testifying to the potential usefulness of automated bacterial mutagenicity testing.

Detection of exposure to mutagenic compounds in low-tar and medium-tar cigarette smokers.

A volunteer study was carried out on groups smoking low-tar (5.4 mg/cig.) and medium-tar (16.3 mg/cig.) cigarettes and on non-smokers. The number of cigarettes smoked was recorded daily throughout a 51-day study period, and the effects of smoking were measured in the beginning and after 3-week terms of smoking medium-tar or low-tar cigarettes. The parameters measured were blood carboxyhemoglobin concentration, thioethers excreted into the urine, mutagenic activity in the urine, and the frequency of sister-chromatid exchanges in blood lymphocytes. For all four parameters, the differences between the smokers and the non-smokers were significant. However, within the study design no differences could be detected between low-tar or medium-tar cigarette smoking in the biological-monitoring tests used.

Bacterial urinary assay in monitoring exposure to mutagens and carcinogens.

The bacterial bioassay procedure provides a sensitive test for the presence of mutagenic activity in urine. Its sensitivity for detecting the presence of individual chemicals may not be as high as that of specific analytical methods, but it has the following advantages: (1) many substances and metabolites may be active in a single assay, making it possible to detect mutagenic activity from unanticipated sources; (2) biological activity is demonstrated, rather than simply the presence of substances; or (3) the assay may also reflect the 'integrated' effect of multiple substances, although this capability has not been well characterized and (4) the assay can be easily coupled with a chemical analysis. The chief disadvantages of the test system are lack of sensitivity for certain specific substances, as compared to chemical techniques, and possible interference from substances normally present in urine (such as amino acids). The urinary mutagenicity assay is most useful when exposure to carcinogens and mutagens is suspected but when the specific chemical is unknown, when chemical analytical techniques are not available or when exposure is to undefined complex mixtures.

Correlations of alkylating activity and mutagenicity in bacteria of cytostatic drugs.

Alkylating activity of cytostatic drugs was studied in relation to their mutagenicity and toxicity in E. coli WP2 uvrA. Four classes of directly acting cytostatic drugs were studied: nitrogen mustards (nitrogen mustard, melphalan, chlorambucil and phosphoramide mustard, a metabolite of cyclophosphamide), ethyleneimine derivatives (Thio-TEPA, TEPA and triethylenemelamine), busulfan, and halogenated nitrosoureas. The reference compounds included methyl methanesulfonate, ethyleneimine and methylnitrosourea. Guanosine alkylation was determined by fluorometry. The rate of guanosine and nitrobenzylpyridine alkylation agreed well. Nitrogen mustard derivatives and triethylenemelamine were the most potent alkylating agents among the cytostatic drugs; nitrogen mustard was 5 to 10 times more active than methyl methanesulfonate. Ethyleneimine derivatives, busulfan and the nitrosoureas were relatively weak alkylating agents. Nitrogen mustard and triethylenemelamine were the most potent mutagens to bacteria; they were also among the most toxic drugs studied.

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.

Reactivity, SCE induction and mutagenicity of benzyl chloride derivatives.

Benzyl chloride, benzyl bromide, p-methylbenzyl chloride, and p-nitrobenzyl chloride were used to study chemical reactivity with 4-(p-nitrobenzyl)-pyridine (NBP), and with guanosine in vitro, in relation to mutagenic potency in S. typhimurium and sister chromatid exchange (SCE) induction in CHO cells. Benzyl bromide was found to be the most reactive compound, followed by p-methylbenzyl chloride, benzyl chloride and p-nitrobenzyl chloride. The order of mutagenicity was p-nitrobenzyl chloride much greater than benzyl bromide greater than benzyl chloride approximately equal to p-methylbenzyl chloride. The compounds tested caused base-pair mutations only. The order of SCE-inducing ability decreased as follows: benzyl bromide greater than benzyl chloride approximately equal to p-nitrobenzyl chloride approximately equal to p-methylbenzyl chloride. The particularly high mutagenicity of p-nitrobenzyl chloride in bacteria may be due to reactions other than direct aralkylation, or it may react particularly actively with DNA. Among the other compounds, benzyl bromide was the most active aralkylating compound, mutagen and SCE inducer. The results suggested that reaction of N2 of guanine, as compared with N-7 of guanine, failed to show any remarkable mutagenicity or SCE induction, since p-methylbenzyl chloride, reacting preferentially at N2 of guanosine, failed to show unexceptional potency.

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.

Biological monitoring of occupational exposure to mutagenic chemicals in the rubber industry. Use of the bacterial urinary mutagenicity assay.

The assay of mutagenicity in the urine with the bacterial fluctuation test was used as an approach to monitor the exposure to mutagenic chemicals among 129 rubber workers. Especially those workers exposed to chemical dusts or fumes showed high mutagenicity in their urine. Smoking was also found to increase mutagenicity in the urine, but this effect could be detected only with the frame-shift detector strain Salmonella typhimurium TA 98. Escherichia coli WP 2 uvrA could thus be used to monitor the occupational exposure to base-pair substitution mutagens, without the interfering effect of smoking.

Possibilities for identifying genotoxic risks in the rubber industry: use of the urinary mutagenicity assay and sister chromatid exchange.

Two approaches have been used for biological monitoring of exposure to genotoxins in the rubber industry: (1) assay of mutagenic activity in urine with the bacterial fluctuation test; and (2) analysis of sister chromatid exchange frequencies in peripheral blood lymphocytes. Workers in the rubber industry have significantly more mutagenic activity in their urine than nonexposed controls. Mutagenic activity was tested with Escherichia coli WP2 uvrA as indicator bacteria was significantly higher in urine of workers after a normal work week than in urine samples from the same individuals after a holiday. The mean frequency of sister chromatid exchange in rubber workers who smoked was significantly higher (p less than 0.01) than that in unexposed smokers. The only job categories in which workers who smoked had statistically significantly higher mean frequencies than others were mixing and weighing. People in these jobs generally also had high mutagenic activity in their urine.

Monitoring genotoxicity in the occupational environment.

New methods need to be developed for the biological monitoring and health surveillance of workers occupationally exposed to mutagenic and carcinogenic chemicals. Two potential approaches, the assay of urine for mutagenicity and the analysis of chromosomal changes in lymphocytes of workers, are discussed in this report with respect to experience in two occupational environments, the rubber industry and a hospital oncology unit. The urine of workers in the rubber industry has shown significantly increased mutagenic activity after the workweek in comparison to postvacation values. Handling cancer chemotherapeutic drugs in oncology units may cause exposure to the cytostatics, many of which are known mutagens and carcinogens. This exposure can be detected as an increased sister chromatid exchange frequency in cultured lymphocytes.

Comparison of alkylation rates and mutagenicity of directly acting industrial and laboratory chemicals: epoxides, glycidyl ethers, methylating and ethylating agents, halogenated hydrocarbons, hydrazine derivatives, aldehydes, thiuram and dithiocarbamate derivatives.

Groups of industrial and laboratory chemicals were tested for their alkylation activity using 4-(p-nitrobenzyl)-pyridine and deoxyguanosine as nucleophiles. The alkylation activity was compared with mutagenicity of the chemicals to E. coli WP2 uvrA without metabolic activation. All the epoxide-containing compounds including simple epoxides and glycidyl ethers elicited alkylation activity and mutagenicity. Furthermore there was a reasonable correlation between the rate of alkylation and the mutagenic potency. All the methylating and ethylating compounds tested were active but no correlation was observed between the rate of alkylation and the mutagenic potency, apparently due to the different types of alkylation products formed. The other compounds tested including halogenated hydrocarbons, hydrazine derivatives, aldehydes, thiuram and dithiocarbamate derivatives elicited a slow or no alkylation activity while many of the compounds were mutagenic. There was no evidence among the chemicals tested of an alkylating non-mutagen. Thus evidence of alkylation activity appears to indicate mutagenic risk.

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, nonsmokers 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 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.