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/m³; N-nitrosomorpholine: 0.77–2.40 μg/m³) and PAH (total PAH: 0.34–11.35 μg/m³) 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.     

On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophila

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulates sampled with the inhalable PM-10 high volume sampler in two different streets of Brescia, a heavily industrialized town in northern Italy, using the Tradescantia/micronucleus test and a bacterial mutagenicity test (Kado test, a more sensitive version of the Ames test). In addition, the Tradescantia/micronucleus test was used for in situ monitoring of gaseous pollutants in other urban areas of Brescia and in two car tunnels, one with heavy car traffic in Perugia, a town in central Italy, and one in Brescia with moderate traffic. The Tradescantia-micronucleus test carried out on extracts of airborne particulates gave positive results only for the sample collected in the traffic-congested street where also higher bacterial mutagenicity was found. The in situ monitoring of the urban areas with the Tradescantia/micronucleus test always gave negative results. Monitoring carried out in the two car tunnels showed a significant increase in micronuclei frequency only in flowers exposed in the smaller and more polluted tunnel.     

Mutagens in contaminated soil: a review

The intentional and accidental discharges of toxic pollutants into the lithosphere results in soil contamination. In some cases (e.g., wood preserving wastes, coal-tar, airborne combustion by-products), the contaminated soil constitutes a genotoxic hazard. This work is a comprehensive review of published information on soil mutagenicity. In total, 1312 assessments of genotoxic activity from 118 works were examined. The majority of the assessments (37.6%) employed the Salmonella mutagenicity test with strains TA98 and/or TA100. An additional 37.6% of the assessments employed a variety of plant species (e.g., Tradescantia clone 4430, Vicia faba, Zea mays, Allium cepa) to assess mutagenic activity. The compiled data on Salmonella mutagenicity indicates significant differences (p<0.0001) in mean potency (revertents per gram dry weight) between industrial, urban, and rural/agricultural sites. Additional analyses showed significant empirical relationships between S9-activated TA98 mutagenicity and soil polycyclic aromatic hydrocarbon (PAH) concentration (r2=0.19 to 0.25, p<0.0001), and between direct-acting TA98 mutagenicity and soil dinitropyrene (DNP) concentration (r2=0.87, p<0.0001). The plant assay data revealed excellent response ranges and significant differences between heavily contaminated, industrial, rural/agricultural, and reference sites, for the anaphase aberration in Allium cepa (direct soil contact) and the waxy locus mutation assay in Zea mays (direct soil contact). The Tradescantia assays appeared to be less responsive, particularly for exposures to aqueous soil leachates. Additional data analyses showed empirical relationships between anaphase aberrations in Allium, or mutations in Arabidopsis, and the 137Cs contamination of soils. Induction of micronuclei in Tradescantia is significantly related to the soil concentration of several metals (e.g., Sb, Cu, Cr, As, Pb, Cd, Ni, Zn). Review of published remediation exercises showed effective removal of genotoxic petrochemical wastes within one year. Remediation of more refractory genotoxic material (e.g., explosives, creosote) frequently showed increases in mutagenic hazard that remained for extended periods. Despite substantial contamination and mutagenic hazards, the risk of adverse effect (e.g., mutation, cancer) in humans or terrestrial biota is difficult to quantify.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Mutagenic activity of airborne particulate matter in a petrochemical industrial area

Exposure to airborne particulate matter has adverse effects on human health and ecosystem. Mutagenic activity of airborne particulate organic matter extracts in three time periods from total suspended particles (TSP) and particles less than 10 microm (PM10) was evaluated in an area under the influence of a petrochemical industry located in the town of Triunfo, Brazil. The extracts were investigated using the Salmonella/microsome assay, with the microsuspension method. The extracts were obtained by sonication extracted using dichloromethane (DCM) solvent. The fractions were tested for mutagenicity with the Salmonella typhimurium strains TA98 (with and without metabolic activation), TA98NR and TA98/1,8DNP(6); or YG1021 and YG1024. A positive frameshift mutagenic response was observed for the environmental samples during the different periods. The responses according to percentage of extractable organic matter (EOM%), EOM/m(3), revertants/microg (rev/microg) and revertants/m(3) (rev/m(3)) were lower for TSP than for PM10 extracts. The highest rev/m(3) values were observed in PM10 extract samples collected in winter, July 2005, in the presence (13.79 rev/m(3)) or absence (6.87 rev/m(3)) of S9 fraction. Similarly in the first (1995) or second period (2000) the highest values for TSP were observed in winter, but with lower activity (3.00 and 0.89 rev/m(3) respectively). The responses observed for the nitrosensitive strains suggest the contribution of nitro, amino and/or hydroxylamino derivatives of PAHs to the total mutagenicity of matter extracted from airborne particles. The Salmonella/microsome assay was a sensitive method to define areas contaminated by genotoxic compounds, even in samples with TSP or PM10 values that are acceptable according to legal environmental quality standards, favoring environmental control measures with an effective response seen in the population's improved quality of life.     

Chemical characterization and biological properties of airborne particulate matter

Summary The presence of heavy metals and Polynuclear Aromatic Hydrocarbons (PAH) has been evidenced in airborne particulate matters samples collected in the atmosphere of a non-industrialized middle size Italian town (150.000 inhabitants). The PAH concentrations, higher in winter than in other seasons, was inversely correlated with some meteorological variables such as temperature (p<0.01) and wind speed (p<0.05). The positive correlation found (p<0.01) between lead and total PAH seems to indicate traffic as the most important source of pollution.All samples were mutagenic in the Ames test and the highest mutagenicity levels were obtained in winter samples. The positive correlation found between Benzo(a)Pyrene {B(a)P} concentration and the number of revertants/m³ air, suggests that B(a)P could be an index of mutagenic potential of the matrix considered.The airborne particulate matter extracts obtained by organic solvents were able to inhibit the phagocytosis of mice alveolar macrophages, giving further proof of the possibility of interaction between air-particulate chemical compounds and important biological functions.     

Genotoxic effects of volatile organic compounds in a chemical factory as evaluated by the Tradescantia micronucleus assay and by chemical analysis

The clastogenic effects of volatile organic compounds in the workplace air of a chemical factory were studied by means of the Tradescantia micronucleus (Trad-MCN) assay and chemical analysis. Sampling was performed at a chemical factory producing PVC film in Cheong-ju, South Korea. Inflorescences of Tradescantia BNL 4430 were placed for 2, 6, and 9 h at the height of 1.40 m at two locations in the workplace and one outdoor of the chemical industry. Air samplings were conducted in the same places and the collected tube samples were analyzed by automatic thermal desorption/gas chromatography/mass spectrometry (ATD/GC/MS). The frequencies of micronuclei in specimens exposed for 2 h in sites 1-3 were 6.13 +/- 0.47, 5.40 +/- 1.60, and 2.93 +/- 0.43 MCN per 100 tetrads, respectively. GC/MS analysis proved the presence of various volatile organic compounds such as trichloroethylene, toluene, ethyl benzene, (m, p, o)-xylene, styrene, 1,3,5-trimethyl benzene, and 1,2,4-trimethyl benzene. Mean values of toluene measured by 2 h sampling in sites 1-3 were 1946.6, 1368.3, and 340.1 microg/m3, respectively. The toluene concentrations in sites 1 and 2 were at least four to six times higher than that in site 3. The micronucleus frequencies increased with exposure time. In addition, there was a correlation between the micronucleus frequencies and toluene concentration in the air (R2 = 0.96). The results of this in situ monitoring proved the applicability of the Trad-MCN assay combined with chemical analysis for monitoring genotoxic chemicals in the work environment.     

Genotoxic and mutagenic activity of environmental air samples from different rural, urban and industrial sites in Flanders, Belgium

The present study reports mutagenic and genotoxic activities associated with ambient air collected at 15 sites characteristic for urban, industrial or rural conditions in Flanders. Airborne particulates (PM10) and semi-volatile compounds were collected on quartz filters (QF) and polyurethane foam (PUF) cartridges using a high-volume sampling device. The mutagenic and genotoxic potency of the organic extracts--Soxhlet extraction with acetone--was determined by use of the Salmonella mutagenicity standard plate-incorporation assay and the Vitotox assay, respectively. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). Ambient air samples contained significant PAH levels and mutagenic activities at all 15 sites: direct mutagenicity of up to 47 revertants per cubic meter was found in the QF extracts and more limited activity of up to 11 rev m(-3) in the PUF extracts. Metabolic activation of PUF extracts resulted in an important increase in mutagenic activity, up to 30 rev m(-3), but no such increase was observed for QF extracts. The highest values were observed outside large cities at industrial sites and at a rural site contaminated by pollution from a chemical plant at a distance of 4 km. Also at the background location near the North Sea a significant mutagenic activity was measured in the QF extracts (+S9: 9 rev m(-3); -S9: 7 rev m(-3)). Apparently, there is in Flanders a significant background exposure level to airborne mutagenicity, even in areas with limited or no nearby pollution sources. Based on the concentrations of 10 mutagenic PAHs and supposing additivity of their specific mutagenicities, only a few percent (mean 3%) of the observed indirect mutagenic activity could be explained. This implies that most mutagenic activity originated from other substances that were not identified or measured in our chemical analysis. This underscores the importance of bio-monitoring measurements.     

Genotoxic and mutagenic activity of environmental air samples in Flanders, Belgium

Atmospheric pollution is assumed to play a role in the incidence of respiratory diseases and cancers. Airborne particles are able to penetrate deep into the lung and are composed of complex chemical mixtures, including mutagens and carcinogens such as polycyclic aromatic compounds (PACs). The present study reports mutagenic and genotoxic activities associated with ambient air collected near a busy street in Borgerhout, at an industrial site in Hoboken and in Peer, a rural community 70 km east of Antwerp in Flanders, Belgium. Airborne particulates (PM10) and semi-volatile organic compounds were sampled during winter and summer. Samples were collected with a high-volume sampler using quartz filters (QF) and polyurethane foam (PUF) cartridges. The mutagenic and genotoxic activity of the organic extracts was determined using the Salmonella test/standard plate-incorporation assay and the Vitotox assay. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). The mutagenicity assay, using Salmonella typhimurium strain TA98, demonstrated direct mutagenicity of up to 58 revertants/m3 for the QF extracts and low or no mutagenic activity in the PUF extracts. Metabolic activation of the samples resulted in high indirect mutagenicity for both QF and PUF extracts: up to 96 revertants/m3 were found in QF samples and 62 revertants/m3 in PUF samples. Genotoxic effects of the filter extracts were assessed with the Vitotox assay: some direct genotoxic effects were noted, i.e. without metabolic activation, but almost no effects were observed after metabolic activation. Without activation, most PUF extracts were bacteriotoxic. With metabolic activation this toxicity disappeared, but genotoxic effects were not observed. Statistical analysis showed that the observed biological effects correlated well with the PAH concentrations.     

Mutagenic and clastogenic activity of direct-acting components from air pollutants of the Silesian industrial region

Sequential elution solvent chromatography (SESC) developed by Farcasiu for characterization of coal liquids was used for the fractionation of benzene extracts of airborne particulate pollutants. Mutagenic and clastogenic activity of SESC fractions was determined by the Salmonella/microsome test and the assay for V79 cell chromosomal aberrations (CAs), respectively. Five out of 8 obtained fractions showed differentiated, direct and indirect mutagenic activity. Selected ‘direct’ fractions, examined by the rodent cell chromosome aberration test, also gave a clastogenic response that increased with prolonged treatment time. The SESC system combined with 2 biological assays, the Ames test and the CAs test, seems to be a useful method for examination of genotoxic components of environmental pollutants.     

Analysis and tracing of polycyclic aromatic hydrocarbons and mutagenicity of airborne particulates from the Taipei area

In a 3-year study, we determined the mutagenicity and polycyclic aromatic hydrocarbons (PAHs) of airborne particulates collected during December 1987-September 1988 (216 samples), October 1988-January 1989 (81 samples), and October 1989-April 1990 (52 samples) from 9 locations in the Taipei area. We found that dichloromethane extracts of all the samples were mutagenic to Salmonella typhimurium in the Ames test. Moreover, the mutagenicity was much higher in the presence of rat liver microsomal fraction (S9 mixture) than that observed in its absence, which indicates that airborne particulates contained both direct and indirect mutagens. The average mutagenicity of the samples collected in the 3-year period was 137, 127, and 118 histidine revertants/10 m3 air, respectively. On the other hand, we found that dichloromethane extracts of each airborne particulate sample contained 14 PAHs with wide variations in concentration and relative distribution. The levels of Pha, Flu, Pyr, and Ben were much higher than the PAHs with higher ring numbers such as BaP, BeP, Pr, IP, and DbA. The average PAH content was 8.0, 5.0, and 7.8 ng/m3 air for airborne particulates collected during December 1986-September 1987, October 1988-January 1989, and October 1989-April 1990, respectively. Among the 9 stations, Fu Hsing Elementary School and Chung Hsing University (Taipei campus), which are, respectively, located in the downtown area and a heavy traffic zone, had significantly higher levels of mutagenicity and PAHs than did the other stations. Moreover, comparative analysis of PAH levels of airborne particulates over the 3-year period revealed an interesting season-dependent change of PAH content in airborne particulates from the Taipei area. The concentrations of individual and total PAHs were consistently lower in the summer than those in the winter. A similar pattern of seasonal change was also observed in the mutagenicity of airborne particulate samples examined. It is worth mentioning that neither PAH level nor mutagenicity of airborne particulates showed significant yearly change over the 3-year period of study. As part of an effort to identify pollution sources, we examined the mutagenicity and PAH compounds of air particulates collected from the burning of garbage (14 samples) and motor-vehicle exhaust in the Hsin Hai Tunnel (17 samples), Taipei. The results showed that garbage burning gave rise to air particulates containing several hundred times higher levels of PAHs and about 20 times stronger mutagenicity, while the motor-vehicle exhaust contained about ten times higher PAH content and mutagenicity as compared with those of airborne particulates of the Taipei city.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Anticlastogenic effect of Spirulina maxima extract on the micronuclei induced by maleic hydrazide in Tradescantia

The aim of this investigation was to determine if extracts of Spirulina maxima reduce the genotoxic damage induced by maleic hydrazide (MH) using the Tradescantia biosssay. Two types of extracts from the alga were prepared: an aqueous extract with two different concentrations, 100 and 500 mg/ml, and a second one, the extract of a 1% solution of dimethyl sulfoxide (DMSO) which corresponded to 100 mg/ml of the alga. The capacity of MH to induce micronuclei (MN) was initially established by administering 0.005, 0.01, and 0.015 mg/ml of the chemical to the Tradescantia inflorescences, and observing its effect after 24 h.The results of this experiment showed a significant MN increase with the two high concentrations tested, although no dose-response effect was observed. For the anticlastogenic assay, the extracts of Spirulina were applied to the inflorescences alone or immediately before the application of MH (0.01 mg/ml) and the induced MN were observed 24 h later. We found that none of the extracts increased the MN level with respect to the untreated plants; also, that MH more or less doubled the basal micronuclei frequency, and finally, that all tested extracts reduced the genotoxic damage caused by MH. The inhibitory indices obtained for the aqueous extracts (100 and 500 mg/ml) and for the DMSO extract were respectively 59, 85, and 56.3%. These data indicate that Spirulina is an anticlastogenic agent and suggest that it is advisable to extend studies on this matter using other biological models.     

Comparison of two extraction procedures for the assessment of sediment genotoxicity: implication of polar organic compounds

Four sediment samples (Va?ne Airport VA, Va?ne Center VC, Va?ne North VN and Reference North RN) were collected in the Berre lagoon (France). Sediments were analyzed for polycyclic aromatic hydrocarbons (PAHs) by use of pressurized fluid extraction with a mixture of hexane/dichloromethane followed by HPLC with fluorescence detection analysis. Organic pollutants were also extracted with two solvents for subsequent evaluation of their genotoxicity: a hexane/dichloromethane mixture intended to select non-polar compounds such as PAHs, and 2-propanol intended to select polar contaminants. Sediment extracts were assessed by the Salmonella/microsome mutagenicity test with Salmonella typhimurium TA98+S9 mix and YG1041±S9 mix. Extracts were also assessed for their DNA-damaging activity and their clastogenic/aneugenic properties by the comet assay and the micronucleus test with Chinese Hamster ovary (CHO) cells. The PAH concentrations were 611ngg(-1)dw, 1341ngg(-1) dw, 613ngg(-1)dw and 482ngg(-1)dw for VA, VC, VN and RN, respectively. Two genotoxic profiles were observed, depending on the extraction procedure. All the non-polar extracts were mutagenic for TA98+S9 mix, and VA, VC, VN sediment samples exerted a significant DNA-damaging and clastogenic activity in the presence of S9 mix. All the polar extracts appeared mutagenic for TA98+S9 mix and YG104±S9 mix, and VA, VC, VN were genotoxic and clastogenic both with and without S9 mix. These results indicate that the genotoxic and mutagenic activities mainly originated from PAHs in the non-polar extracts, while these activities came from other genotoxic contaminants, such as aromatic amines and nitroarenes, in the polar extracts. This study focused on the important role of uncharacterized polar contaminants such as nitro-PAHs or aromatic amines in the global mutagenicity of sediments. The necessity to use appropriate extraction solvents to accurately evaluate the genotoxic hazard of aquatic sediments is also highlighted.     

Validation of the SOS/Umu test with mutagenic complex mixtures

The SOS/Umu test, a rapid system for detecting genotoxic agents by monitoring SOS responses, was evaluated with the extracts of 8 mutagenic complex mixtures (airborne particles, coal dust, tobacco snuff, fried beef, fried shredded pork, airborne particles from polyurethane plants). In this system, the SOS function induced by genotoxicants is detected by a colorimetric measurement of beta-galactosidase in tester cells carrying a umuC-lacZ-fused gene on the plasmid. Results from the study show that a higher beta-galactosidase activity was found when the enzyme substrate and treated cells were added simultaneously into the enzyme reaction mixture and post-treatment dilution (10 X dilution with fresh medium) and incubation (for 2 h) were incorporated. The post-treatment dilution is necessary to reduce a possible false positive due to the color of test substances. The extracts of all mutagenic complex mixtures tested were found to induce dose-related SOS responses, indicating that the SOS/Umu test is potentially useful for the detection of mutagenic complex environmental mixtures.     

A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins

In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames+MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames+MNvit--benzyl acetate, toluene, morphine and thiabendazole--and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames+MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit.     

Mutagenicity of processed bidi tobacco: possible relevance to bidi industry workers

The genotoxic potential of bidi tobacco was evaluated by mutagenicity testing of aqueous, aqueous: ethanolic, ethanolic and chloroform extracts of processed tobacco used in the manufacture of 'bidis', indigenous forms of cigarettes smoked in India. The Salmonella/mammalian microsome test (Ames assay) was used to detect mutagenicity in tester strains TA98, TA100 and TA102. The extracts were tested in the absence and presence of metabolic activation using liver S9 from rat and hamster, and following in vitro nitrosation with sodium nitrite at acidic pH. All the extracts were non-mutagenic in the absence of nitrosation. The nitrosated aqueous extract was mutagenic in strains TA98 and TA100. While weak mutagenicity was elicited by the nitrosated aqueous: ethanolic extract in TA100, the nitrosated ethanolic extract induced a 3-fold increase in the number of revertants in the same strain. Moreover both these extracts elicited a strong mutagenic response in TA102, while the chloroform extract was non-mutagenic even after nitrite treatment. The present study indicates that workers employed in the bidi industry are exposed to potentially mutagenic and genotoxic chemicals in the course of their occupation.     

Mutagenic exposure in the rubber manufacturing industry: an industry wide survey

Mutagenic exposure conditions in several rubber manufacturing companies (n=9) in The Netherlands were studied. Mutagenicity of total suspended particulate matter in air (TSPM) and of wipe samples from possible contact surfaces were measured in the Ames mutagenicity assay with Salmonella typhimurium YG1041 in the presence of a metabolic activation system. Large differences in median mutagenicity of TSPM samples were observed between companies (range 49-1056rev/m(3)) and to a lesser extent between production functions (range 129-402rev/m(3)). The production function curing revealed overall the highest TSPM mutagenicity levels. Forty-one percent of the surface wipe samples revealed mutagenic activity ranging from 26 to 665rev/cm(2). Mixing had the largest proportion of positive samples resulting in a median surface mutagenic contamination of 39rev/cm(2). Surface mutagenic contamination, averaged per department/company combination, showed only a weak correlation with TSPM mutagenicity (r=0.28, P=0.05). Company, production function and total soluble matter (e.g. mass collected upon extraction with organic solvents with different polarity) explained 79 and 81% of the variability in mutagenicity of TSPM and surface contamination levels, respectively. "Company" was identified as the most important exposure determinant for mutagenic activity in TSPM and surface wipe samples. This indicates the importance of company specific determinants like production volume and rubber chemicals used for the encountered mutagenic exposure conditions. Detection of substantial mutagenic activity on possible contact surfaces supports furthermore the potential importance of the dermal route in the uptake of genotoxic compounds of workers in the rubber manufacturing industry.     

Genotoxicity studies with pure trans-capsaicin

Both positive and negative effects have been found in classical genetic toxicology assays with capsaicin. However, the capsaicin tested in most studies has been derived from pepper plant extracts, which is likely to display varying degrees of purity and possibly diverse impurity profiles. Therefore, the objective of the series of studies reported here was to test the genotoxic potential of pure, synthetic trans-capsaicin (the only naturally occurring geometric isomer of capsaicin), using four genotoxicity assays widely used to evaluate drug substances. These included the Ames, mouse lymphoma cell mutation, mouse in vivo bone marrow micronucleus and chromosomal aberration in human peripheral blood lymphocytes (HPBL) assays. In the Ames assay, pure trans-capsaicin was not mutagenic to Salmonella typhimurium or Escherichia coli when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. trans-Capsaicin was weakly mutagenic in mouse lymphoma L5178Y cells, in the presence of S9 mix, when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. Limited evidence for very weak activity was also obtained in the absence of S9 mix. trans-Capsaicin did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg per day in male and 200 mg/kg per day in female CD-1 mice using a 0 h plus 24 h oral dosing and 48 h sampling regimen. Finally, trans-capsaicin did not induce structural or numerical chromosomal aberrations when evaluated for its ability to induce clastogenicity in blood lymphocytes. Taken together, these data suggest that the genotoxic potential of pure trans-capsaicin is very low, especially as the clinical significance of weak mutagenicity in the mouse lymphoma assay for catechol-moiety containing compounds is unclear. Moreover, the different genotoxicity profiles of pure trans-capsaicin and purified chili pepper extracts suggest that the purity and source of capsaicin should always be an important consideration for toxicological evaluations.     

Exposure to mutagenic airborne particulate in a rubber manufacturing plant

Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer. Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions. The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes. The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant. Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas. Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding). The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation. Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS). The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples. No mutagenic activity was present in S. typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer. HPLC analysis revealed very low concentrations of PAHs. GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate. We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations. PAHs were not involved in indoor pollution. GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes. These substances are potential carcinogen though their mutagen properties have not been clearly determined.