Volatile Mutagens Research Articles

4-Oxo-2-hexenal, a Mutagen Formed by .OMEGA.-3 Fat Peroxidation, Causes DNA Adduct Formation in Mouse Organs

To identify mutagens formed in a model reaction of lipid peroxidation, linolenic acid methyl ester and hemin were reacted with dG. As a result, a 4-oxo-2-hexenal-dG adduct (dG*) was identified in the model reaction mixture. The 4-oxo-2-hexenal (4-OHE) showed mutagenic activity in the Salmonella typhimurium strains TA100 and TA104. After 4-OHE was orally administered to mice, dG, 4-OHE-dC- and 4-OHE-5-methyl-dC adducts were detected in esophageal, stomach and intestinal DNA. In the vapor phase released from the methyl linolenate-hemin model system, and in the smoke released during the broiling of fish, 4-OHE was detected by GCMS. The 4-OHE seems to be produced by the auto-oxidation of omega-3 polyunsaturated fatty acids. These results provide a warning to workers dealing with omega-3 fats, who may be exposed to this volatile mutagen.

Monitoring airborne genotoxicants in the rubber industry using genotoxicity tests and chemical analyses

This research was designed to examine the presence of mutagenic/carcinogenic compounds in airborne pollutants in the rubber industry using an integrated chemical/biological approach. Inhalable airborne particulate matter (PM-10: <10 μm) was collected in four rubber factories using a high-volume sampler equipped with a cascade impactor for particle fractionation. The organic extracts of two different fractions (0.5–10 μm and <0.5 μm) were examined for mutagenicity with the Ames test and for in vitro DNA-damaging activity in human leukocytes by single-cell microgel electrophoresis (Comet assay). The extracts were also studied by gas chromatography/mass spectrometry (GC/MS) for polycyclic aromatic hydrocarbon (PAH) content. Nitrosamines in ambient air were sampled on cartridges and analysed by GC with a thermal energy analyser (TEA) detector. Airborne volatile genotoxins were monitored in situ using a clastogenicity plant test ( Tradescantia/micronuclei test). The results showed that airborne particulates were mainly very fine (<0.5 μm) and that trace amounts of genotoxic nitrosamines ( N-nitrosodimethylamine: 0.10–0.98 μg/m 3; N-nitrosomorpholine: 0.77–2.40 μg/m 3) and PAH (total PAH: 0.34–11.35 μg/m 3) were present in air samples. Some extracts, particularly those obtained from the finest fractions, were mutagenic with the Ames test and genotoxic with the Comet assay. In situ monitoring of volatile mutagens using the Tradescantia/micronuclei test gave positive results in two working environments. The results showed the applicability of this integrated chemical–biological approach for detecting volatile and non-volatile genotoxins and for monitoring genotoxic hazards in the rubber industry.

Mutagenicity of bi-, tri- and tetra-cyclic aromatic hydrocarbons in the “taped-plate assay” and in the conventional Salmonella mutagenicity assay

Aromatic hydrocarbons in the range of 1–4 nuclear rings were examined for mutagenicity in the so-called “taped-plate assay”. This modification of the Ames assay is particularly equipped for the detection of volatile mutagens. Of the many compounds tested only phenanthrene, pyrene, benzo[ c]phenanthrene and benzoacenaphthylene were positive in this assay. The present data underline the exceptional behaviour of fluoranthene by being a rather potent bacterial mutagen with a volatile nature (as found in a previous study).

Application of polyurethane foam for sampling volatile mutagens from ambient air

This paper reports the use of polyurethane foam (PUF) in the sampling of airborne mutagens too volatile to be retained by glass fibre filters. Air was sampled simultaneously with a glass fibre filter and PUF plugs, and the two extracts of the samples were tested for mutagenicity and analysed for polycyclic aromatic hydrocarbons (PAH). As PUF itself was found to contain mutagens, it was thoroughly rinsed, and a blank extract of the same PUF used for sampling was tested as a control. The resulting blank extracts were not mutagenic. In the first series of experiments, in which methanol was used as the extraction solvent, sampling induced clear-cut mutagenic effects in the PUF extracts. As the mutagenicity of the PUF itself appeared only with the use of methanol or other alcohols as extraction solvents, a second series of experiments was carried out in which acetone was used as the extraction solvent. In these experiments sampling induced no mutagenicity in the extracts. The discrepancy between the two series of experiments may be due to the absence of volatile mutagens in the second series, but another explanation may be an artefact induced by the solvent in the first series. Additional experiments suggest that sampling induces the formation, from PUF, of compounds mutagenic upon extraction with methanol. The study leads to the provisional conclusion that no volatile mutagens could be demonstrated in ambient air sampled by the use of PUF as an absorbent, notwithstanding the clear-cut effects of the filter extracts and the presence of volatile PAH in the PUF extracts.

Fluoranthene, a volatile mutagenic compound, present in creosote and coal tar

Creosote, a coal-tar distillation product, contains mutagens which are volatile at 37°C. After distillation of creosote we found that these volatile mutagens were present in the distillation fraction with the highest boiling range (> 360°C). The “volatile mutagenic activity” was connected with the presence of fluoranthene, a polycyclic aromatic hydrocarbon. Commercially available fluoranthene was positive in the so-called “taped-plate assay” (the test system used for the detection of volatile mutagens) towards the strains TA98 and TA100 in the presence of S9 mix. The tested creosote and coal tar contained fluoranthene in concentrations of 5.2 and 2.2%, respectively.

Vaporization technique to measure mutagenic activity of volatile organic chemicals in the ames/Salmonella assay

The purpose of this research was to develop and characterize a sensitive test method to detect mutagenic activity of volatile liquid organic chemicals (i.e, volatiles) in the Ames/Salmonella assay. A Tedlar bag vaporization technique was developed, which increased contact time between the volatiles and bacterial test system, circumvented volatilization limitations in the standard plate incorporation and preincubation methods, allowed chemical analysis during incubation, and was flexible in design. The vaporization technique was evaluated concurrently against the plate incorporation and preincubation techniques with eight liquid volatile mutagens in the Ames/Salmonella mutagenicity assay with Salmonella typhimurium strains TA100 and TA102. Results suggested that when volatile organic chemicals with boiling points below 63 degrees C were tested for mutagenic activity, the most sensitive test conditions were the vaporization technique with TA100. GC analysis of epichlorohydrin and butylene oxide concentrations within Tedlar bags suggested that these two chemicals volatilized and were contained in the media after 1 hr of incubation at 37 degrees C. The mutagenic activity of six volatile liquid mutagens was similar in single and triple plate Tedlar bags. Three general test groups of volatile organic chemicals were identified by test data: chemicals which had boiling points below 63 degrees C, for which the vaporization technique was the most sensitive test procedure (ethylene, propylene, and butylene oxides and methylene chloride); chemicals which had boiling points from 107 degrees to 132 degrees C, for which the vaporization technique was still useful, but where sensitivity was only slightly increased over the preincubation technique (1-bromo-2-chloroethane, epichlorohydrin, and ethylene dibromide); and 3) a chemical which had a boiling point at 194 degrees C, where the preincubation technique was the most appropriate test method (styrene oxide).

Detection of volatile mutagens in creosote and coal tar

Creosote and coal tar were examined for the presence of volatile mutagens by use of the so-called “taped plate assay”. Application of this method, recently published by Distlerath et al., reveals that vapour escaping from creosote and coal tar at 37°C was able to revert the Salmonella typhimurium strains TA98 and TA100 in the presence of S9 mix. The simplicity of this method makes it useful for routine screening of industrial fluids or solid products for the presence of volatile mutagens.

Model systems demonstrating the volatile mutagenicity and carcinogenicity of sodium nitrite in rats.

Volatile mutagens derived from sodium nitrite buffered at various pH values or in the presence of human feces were detected using Ames Salmonella tester strain TA 1535 on petrie plates inverted over samples. Volatile mutagenicity increased as the pH decreased and was primarily a function of the nitrous acid produced from sodium nitrite and hydrogen ions. Sodium nitrite administered intracecally to 3 Wistar rats through surgically implanted cannula caused tumors (fibrosarcoma: 1/3 and squamous cell, 2/3). The possible role of nitrite-derived mutagens in GI cancer is discussed.

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37°C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50–80% of the activity of these compounds observed in the standard assay. Moreover, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.

Development of an in situ microbial mutagenicity test system for airborne workplace mutagens: laboratory evaluation

A simple on-site Salmonella mutagenicity test system for the detection of airborne mutagens in the workplace is being developed. The system permits entrapment of mutagenic airborne particles and vapors by impinging unfiltered ambient air into trapping medium containing bacterial tester cells. The trapping device consists mainly of a pump, an impinger and a cyclone. The impinging air flow generated by the pump is approximately 3 1/min. New Salmonella typhimurium testers which are resistant to streptomycin (Str) and 8-azaguanine (AG) were derived from the Ames testers TA98 and TA100 and the arabinose-resistant tester SV50, and were used as mutation indicators. Microbial contamination was sufficiently controlled by addition of ampicillin, Str, AG, and cycloheximide to the trapping and plating media. New tester strains retained a high mutagenic sensitivity from their parent strains. Laboratory studies with volatile mutagens (methyl methanesulfonate, ethyl methanesulfonate, and dimethylnitrosamine) showed that the vapor trapping of this system is promising. The study with suspended silica particles coated with a known mutagen (2,4,7-trinitro-9-fluorenone) indicated that the particle trapping of the system is satisfactory. Incorporation of metabolic activation into the trapping medium by confining S9 mix and tester cells in dialysis tubing enabled this system to detect promutagens. This in situ system may be useful for mutagenic monitoring in the workplace.

Evaluation of an exposure system using cells grown on collagen gels for detecting highly volatile mutagens in the CHO/HGPRT mutation assay.

Chinese hamster ovary (CHO) cells were grown on hydrated collagen gels, the overlaying medium removed leaving the cells at an air/collagen interface, and the cells exposed to a dynamic flow of ethylene oxide. Increases in CHO cell mutant frequency and decreases in cell viability were observed. To establish if the exposure system could be simplified, cells were exposed in sealed bottles (static system) to ethylene oxide. No substantial changes in cytotoxicity, mutant frequency, or effective concentration were noted when comparing static versus dynamic exposure systems. The general usefulness of the exposure system using cells grown on collagen gels was evaluated in a static system using propylene oxide and 1,2-dichloroethane, both of which were found to be mutagenic and cytotoxic. Comparatively, the exposure of cells by the collagen gel method was as effective in detecting genotoxic damage as were conventional methods (cells covered with medium) using cells grown on glass substrates. The exposure of CHO cells on collagen gels to highly volatile mutagens was simple and inexpensive, and may be generally useful in the detection of gaseous or volatile mutagens.

Nitrite-induced volatile fecal mutagens: some preliminary studies.

Further studies are reported on the formation of volatile mutagens from normal human feces incubated with sodium nitrite at 37 degrees C. Volatile mutagens were detected using Ames' Salmonella typhimurium tester strain TA1535 on petrie plates inverted over samples in sealed containers. An optimal exposure period was shown to be approximately 24 hours. When plates inverted over samples were replaced periodically, mutagenicity during serial 24 hour or 48 hour periods could be measured. Mutagenicity was optimal during the second 24 hour period and the first 48 hour period with 0.6 M sodium nitrite, and in the first 24- and 48-hour period with 0.2 M sodium nitrite. Subsequent exposure periods showed decreasing mutagenicity with time. Because of the great differences in reactivity with nitrite in stools of healthy volunteers, the effect of mixing "high reacting" samples with "low reacting" samples were investigated. When "low reacting" samples were mixed with "high reacting" samples, a lower mutagenicity of the mixture was observed compared to a predicted "average" value. Autoclaving stool samples made very little difference in nitrite-induced mutagenicity and behavior in mixing experiments. Thus, fecal mutagens arise as a spontaneous chemical process involving nitrite ions and both dietary and flora factors may be important in their production or absence.

Nitrite-induced volatile mutagens from normal human feces.

Volatile mutagens (putative carcinogens) were produced from normal human and animal feces upon incubation with sodium nitrite in saline at 37 C for 48 hours. The mutagens were detected by using Ames' Salmonella typhimurium tester strain TA1535 without microsomes, on plates inverted over samples in sealed containers. Mutagenicity was maximal at 0.2 to 0.6 M NaNO2 and at pH 6.2 to 6.8. Reversions per plate varied from approximately 30 to 450 (1.5 to 25 x background) within the normal human population. Sodium ascorbate and alpha-Tocopherol (at one-half [NaNO2]) each reduced the mutagenicity by approximately 30%. Two standard N-nitroso-compounds were mutagenic in the system. We propose that the mutagenicity in our system is probably caused by the formation of volatile N-nitroso-compounds and that addition of nitrite to human feces in vitro enhances a process that occurs in vivo.

Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/mammalian-microsome assay

About 300 compounds have been reported in the literature as constituents of pulp-mill effluent. Previously, in our screening program, 10 resin acids identified in effluent were examined for potential mutagenicity in the Salmonella/mammalian-microsome assay. Neoabietic acid was the only resin acid which was found to be mutagenic. Now, a program to screen for mutagenicity of 48 additional compounds, belonging to chemical classes of chlorinated aliphatic and aromatic hydrocarbons, phenols, aldehydes, quinos, and carboxylitic acids, has been completed. Only 2 of these compounds, tetrachloropropene and pentachloropropene, were found to be mutagenic, showing dose-related increases in His + reversion mutations, in the standard Salmonella test. Metabolic activation with a preparation of Aroclor 1254-induced liver homogenate (S9) greatly reduced the mutagenic responses of these 2 compounds. Modification of the Salmonella test for volatile mutagens enabled the detection of the mutagenicity of 3 additional chlorinated aliphatic hydrocarbons, dichloromethane, dichloroethane and trichloroethane.

Induction of forward mutations to colicin E2 resistance in repair deficient strains of Escherichia coli: Experiments with ultraviolet light and captan

The quantitative induction of Escherichia coli of mutants resistant to colicin E2 is described. The kinetics of induction by both ultraviolet light and the fungicide captan were approximately dose-squared, and the induction of mutations after UV was photoreversible to the same extent as for mutations to Trp + known to be predominantly base-pair substitutions. As with mutations to Trp +, there was no detectable induction of colicin E2-resistant mutations in strains deficient at the exrA gene. Because of the high sensitivity of this mutation system, extensive dose-response curves for mutation induction could be obtained in the absence of appreciable lethality. The UV sensitivity for induction of mutations resistant to colicin E2 was considerably greater than that reported for mutations to colicin B resistance, most of which are known to be deletions. It is suggested that the presumed failure of the colicin B sensitivity genes to be rendered non-functional by base-pair substitution mutations may reflect a fundamental difference in the function of the cell surface receptors between the two colicins. Captan must be considered as a potent mutagen; in the excision-deficient strain WP2 uvrA induced mutations were detected following exposure to less than 0.1 μg/ml captan. Premutagenic lesions produced by captan were excisable as judged by the comparative mutabilities of Uvr + and Uvr − bacteria, and did not lead to mutations in the presence of the exrA allele. Exr − bacteria were also more sensitive to the toxic action of captan. In all these properties (except photoreversibility) captan was similar to ultraviolet light. No evidence was found for mutagenesis of bacteria suspended in phosphate buffer by the exrA-independent volatile mutagen previously detected using a spot-test plate method and believed to be a hydrolysis product of captan. It is assumed therefore to be highly unstable in aqueous solution.

Repair-deficient bacterial strains suitable for mutagenicity screening: Tests with the fungicide captan

A spot test with selected repair-deficient strains of Escherichia coli is described which not only provides a sensitive assay for the mutagenic activity of chemicals, but also gives useful information about the characteristics of the mutagenic process. It has been shown that a substantial part of the mutagenic activity of the fungicide captan is due to excisable DNA damage mediated by a volatile breakdown product. The production of this volatile mutagen is greater at alkaline pH and is probably a result of hydrolysis. It may be very short-lived. Unlike other excisable DNA damage, that produced by the volatile mutagen does not depend on the ExrA + and RecA + repair functions for its mutagenicity. There is also a diffusible mutagen producing excisable damage the mutagenic action of which is Exr +-dependent in contrast to that of the volatile mutagen. It is suggested that a possible carcinogenic hazard might exist if captan is inhaled.