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Bioassay-directed chemical analysis and detection of mutagenicity in ambient air of the coke oven
Mutation Research 445 Ž1999. 285–293 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres
Bioassay-directed chemical analysis and detection of mutagenicity in ambient air of the coke oven a,)
L. Dobias ´ˇ
, J. Kusova ˚ ´ a, O. Gajdosˇ b, P. Vidova´ b, D. Gajdosova ˇ ´ b, J. Havrankova ´ ´ a, M. Fried c , B. Binkova´ d , J. Topinka d
a
b
Department of Genetic Toxicology, Regional Institute of Hygiene, 728 92 OstraÕa, Czech Republic Department of Genetic Toxicology, Specialized National Health Institute, 040 11 Kosice, ˇ SloÕak Republic c Hygienic Institute, UniÕersity of Heidelberg, 69 120 Heidelberg, Germany d LGE, c r o LGE-RIH of Central Bohemia and IEM AS CR, Prague, Czech Republic Received 17 August 1998; accepted 15 December 1998
Abstract In the present study, we summarize the results of studies on the mutagenic potential of the main fractions and subfractions of extractable organic material ŽEOM. in the ambient air at the workplaces of the coke oven. The objective of our experiments was to apply the Bioassay-Directed Chemical Analysis Žwith the use of the Ames test. for the identification of the differences in the mutagenicity of these fractions, in relationship to the complex mixture of EOM in occupational air. From the evaluation of results, it is possible to deduce the following conclusions: Ž1. The comparison of the mutagenicity in the main fractions Žbasic, acidic, neutral. demonstrates the existence of differences in mutagenic potential. Of the total mutagenicity, 20.4% is in the basic fraction, 25.4% in the acidic fraction and 54.2% in the neutral fraction. Ž2. In general, 90.1% of the mutagenicity found in the basic, acidic and neutral fractions together was associated with the requirement of metabolic activation in vitro ŽqS9.. In the case of the neutral fraction, it was 51.8%. Ž3. These results also suggest that frameshift mutations are the major component Ž53.8%. of the total mutagenicity of the main fractions. Ž4. With regards to the mutagenicity of organic compounds in the neutral fraction it appeared that genotoxicants of its subfractions Žslightly and moderately polar and aromatic. play the main role. Carcinogenic polycyclic aromatic hydrocarbons ŽPAH. and genotoxic nitrocompounds play an important role as determinants of the mutagenic potential of complex mixtures of harmful compounds in ambient air. This is confirmed first by the results of short-term bacterial tests. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Mutagenicity; Bioassay-directed chemical analysis; Carcinogenic PAH; Coke-oven emissions
1. Introduction The increasing occurrence of genotoxic pollutants in the environment has become a matter of interest )
Corresponding author. Fax: q42-69-611-86-61
as a complex public health problem. Questions arose as to the emission, distribution and fate of the genotoxicants in the environment and the exposure levels of the human population w1,2x. At the same time, the identification and control of dangerous genotoxic substances in the work environment play an extraor-
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 3 3 - 3
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dinary role in the prevention of occupational diseases w3x. To answer these questions, investigators had to rely initially on chemical analysis of individual genotoxic compounds and subsequently on biological tests for biomonitoring exposure to complex mixtures. The environment contains a wide variety of manmade genotoxic agents including mutagens and carcinogens. The development of short-term genetic bioassays in the mid-1970s rapidly led to the use of these assays in environmental monitoring w4x. The most widely used bacterial mutagenicity bioassay has been the Salmonella typhimurium plate-incorporation assay described by Ames et al. in 1975 and validated as an initial bioassay to screen for potential carcinogens w5,21,22x. Ames’ bacterial bioassay was used as a short-term test to detect and quantify the mutagenicity associated with complex mixtures of harmful substances in air, water, industrial effluents and commercial products. Generally, short-term microbial tests are being considered an integral part of environmental monitoring studies aimed at cancer or genetic risk assessment. Many studies have clearly demonstrated the mutagenicity of airborne particles from areas with anthropogenic air pollution w6–8x, combustion sources and industrial manufacturing emissions w9,10x. The present study was aimed at the application of bioassaydirected chemical analysis to identify the mutagenic potential of a fractionated complex mixture of extractable organic material ŽEOM. emitted into the air surrounding the coke-oven battery. Attention was especially drawn to the evaluation of the biological significance of PAH and nitro-compounds. The genotoxic effect of exposure to complex mixtures of carcinogenic PAH in the workplaces is a general risk factor for the health status of workers w11–14x. However, the complex mixture of EOM in coke-oven batteries is represented by a large quantity of heterogeneous chemical substances among which the PAH dominate w11x. The complexity of these mixtures limits the possibilities of chemical detection during identification of harmful substances with genotoxic effects, and at the same time hampers their biological detection. Therefore, it was felt necessary to use fractionation of the complex mixture in connection with biological detection of genotoxicity of the main frac-
tions and subfractions Žbioassay-directed chemical analysis.. In currently published studies in which the above-mentioned procedure was applied it has been shown that, as a rule, specific groups of chemical substances play a major role in genotoxicity of complex mixtures w15–19x. The aim of this work is to show that the methods used in our study provide suitable indicators which could help control exposure to carcinogenic PAH in ambient air of coke ovens.
2. Material and methods 2.1. Air sampling and sampling sites During November 1996, air samples were taken on the topside of a coke-oven battery ŽIron Works, Kosice, Slovak Republic. using a high-volume ˇ ŽHiVOL. sampler ŽSierra Misco, USA.. The sampler was located on a lorry car on the topside of the oven. Air was sampled continuously during 5 h of each shift during 5 days; the mean air flow rate during sampling was 88 m3rh. The HiVOL sampler was equipped with teflon-impregnated glass fibre filters PALLFLEX T60 A20 for collection of total particulate matter ŽTPM.. 2.2. Storage of filters The exposed filters were weighed to five decimals after drying for 24 h in a desiccator, at room temperature in the dark. Prior to extraction and separation, the filters were kept wrapped in Al-foil at y708C in a freezing box. 2.3. Extraction of exposed filters Individual filters were extracted as described w20x. Extraction was performed by sonication in an ultrasound bath Ž140 W at 258C, 3 = 10 min.. Dichloromethane p.a. ŽMerck, FRG. ŽDCM. was used as an extraction medium. The extraction was carried out with three sequential volumes of solvent, which were finally pooled into a total crude extract. After filtration of the crude extract Žvolume 140 . ml , aliquots were removed for the determination of
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extractable organic matter ŽEOM.. The concentration of total EOM was 21.790 mg per ml of crude extract. 2.4. Fractionation of crude extract Crude EOM extract ŽCR. was fractionated by acid–base partitioning into three main fractions, viz. a basic ŽKOS2., an acidic ŽKOS3. and a neutral ŽKOS4. fraction. The latter was fractionated further by use of silica-gel column chromatography into aliphatic ŽKOS5., aromatic ŽKOS6., slightly polar ŽKOS7., moderately polar ŽKOS8. and highly polar ŽKOS9. subfractions, on the basis of polarity of the mobile phase w17,19x. 2.5. Analysis of PAH and nitro-PAH analysis PAH in the crude EOM and the aromatic subfraction ŽKOS6. and nitro-PAH in all subfractions ŽKOS5, KOS6, KOS7, KOS8, KOS9. of the neutral fraction were analyzed by electron-impact GC-MS with selected ion monitoring ŽSIM mode. w28x. A total of 0.25 mg of EOM was dissolved in acetone Ž10 mgrul. and precleaned on an SPE cartridge ŽBaker. filled with 1 g of SiOH. The column was conditioned and prewashed with n-hexane and PAH were eluted with n-hexane:DCM Ž5:1 vrv.. Samples were concentrated under a stream of nitrogen, diluted by toluene and transferred to vials for GCrMS analysis. For the gas-chromatographic analysis a HewlettPackard GC Ž589011.rMSD Ž5971. was used. GC parameters were: injector temperature 2908C; transferline temperature 2908C; oven programme: start at 1008C for 2 min, ramp 108Crmin from 1008C to 2908C, then 2908C for 9 min. A total of 16 analysed PAH was divided into 5 groups according to their boiling points and for each of these groups one internal standard was used Žacenaphtalene-d10, phenanthrene-d10, fluoranthene-d10, benzŽ a . anthracene-d12 and perylene-d12.. 2.6. Bioassay sample preparation For the plate-incorporation mutagenicity assay, the EOM Žin DCM. of each sample was transferred to dimethylsulphoxide ŽDMSO. ŽMerck, FRG.. Aliquots of the crude extract and all fractions and
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subfractions Žin DCM. were evaporated to dryness under a stream of nitrogen and the residual EOM were dissolved in a standard volume of DMSO. After their transfer to DMSO, the samples were tested in the mutagenicity bioassay at four doses of the stock extracts corresponding to 10, 20, 40 and 80 mg EOM per plate. 2.7. Mutagenicity bioassay The Ames S. typhimurium assay Ža variant of the plate- incorporation test. was used to determine the mutagenicity. The following indicator strains were used: TA100 Žbase-pair substitutions. and TA98 Žframe-shift mutations., kindly provided by Prof. B.N. Ames, Berkeley, CA, USA, and YG1041 Ža derivative of the TA98 parent strain., kindly donated by Drs. T. Nohmi and M. Watanabe, National Institute of Hygienic Science, Japan. The genetic properties of the tester strains were checked according to recommended procedures w21,22x. The sensitivity of the tester strains to mutagens was determined by use of the following standard compounds: 2-aminofluorene ŽSigma, FRG., p-nitro-o-phenylenediamine ŽAldrich Chemie, FRG. for TA98 and YG1041, natrium azide ŽSigma. and 2-anthramine ŽAldrich. for TA100. Cultures of the tester strains were grown overnight in Oxoid Nutrient Broth No. 2 in a shaking water bath at 378C. Bioassay experiments were conducted as previously described w22x, on individual sample extracts using duplicate plates with and without metabolic activation in vitro ŽqS9 and yS9; DELOR 103-induced S9 fraction from liver of rats. for each dose w22x. After a 72-h incubation at 378C, colony counts on Petri plates were determined using a ZEBRA System’s ŽCzech Republic. automatic colony counter. 2.8. Statistical eÕaluation of the bioassay The results of mutagenicity tests were evaluated with the GeneTox Manager programme ŽU.S. EPA, HERL, RTP, NC 27711. w23x, the software was acquired by courtesy of Dr. L. Claxton. The maximum mutagenic potency Žnumber of revertantsrmg EOM. and the dose-response of each sample were determined with the GeneTox Manager.
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The linear slope values Ž B1 . for both test variants were calculated from numbers of revertantsrmg EOM from the Bernstein model w24x. The statistical significance for differences of positive responses Ždefined as B1 GeneTox values with P - 0.05. was evaluated. The mutagenicity of individual fractions and subfractions Žwith the indicator strains, q and yS9. was expressed as percentage of the total mutagenicity of the complex mixtures. The Student’s t-test was used for statistical evaluation of the results.
3. Results and discussion The study was aimed towards the objectivization of the mutagenic potential of complex mixtures of EOM in HiVOL air samples at the coke oven by use of the Ames mutagenicity assay. In the experimental part we used the bioassay-directed chemical analysis vs. identification of the mutagenicity in major fractions and subfractions of organic chemical substances in emissions from the coke-oven batteries. To assess hazardous pollutants in the occupational environment and to evaluate the exposure to genotoxic substances, relevant exposure data are necessary. The profile of HiVOL air samples corresponds to standard working conditions at the coke-oven batteries in the winter season Žwith deteriorated dispersion of the pollutants in air.. The important role of carcinogenic PAH is visible from a survey of the PAH content in the crude extract ŽTable 1.. The mutagenic potential of the main fractions and subfractions of the EOM mixture contained in occupational air samples from the coke oven was evaluated by the Ames test Žplate-incorporation assay. with the use of the S. typhimurium TA100 and TA98 indicator strains Žin the absence and presence of S9.. Strain YG1041 was used to determine the mutagenicity of the neutral fraction. This strain is a derivative of the TA98 parent strain, and has elevated levels of both nitroreductase and O-acetyltransferase activities w25x. This strain was used because in the slightly polar subfraction Ža component of the neutral fraction. the presence of 3nitrophenanthrene Ž66 ngrmg EOM. and 1-nitropyrene Ž27 ngrmg EOM. was detected.
Table 1 PAH analysis in crude EOM PAH
Concentrations Žmgr mg EOM. crude EOM
BenzoŽ a.pyrenea BenzoŽ a.anthracenea Chrysenea BenzoŽ b .fluoranthenea BenzoŽ k .fluoranthenea IndenoŽ1,2,3-c,d .pyrenea DibenzoŽ a,h.anthracenea BenzoŽ g,h,i .perylenea Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Naphthalene Acenaphthylene Acenaphthalene Sum of carcinogenic PAH Sum of PAH
6.4 8.8 8.9 7.0 6.9 4.9 0.8 4.2 n.d. 0.9 0.3 10.9 8.2 n.d. n.d. n.d. 47.9 Ž4.8%. 68.2 Ž6.8%.
n.d.s Not detectable Ždetection limits 0.1.. a Carcinogenic PAH Žsee IARC Monographs, 1983..
Comparison of the overall results of the mutagenicity assays of the main fractions Žbasic, acidic, neutral. of EOM from occupational airborne TPM sampled in the coke oven shows differences in mutagenic activity ŽFig. 1.. The differences in mutagenic potential Žin % of the mutagenicity of the total extract. between basic, acidic and neutral fractions are statistically significant Ž P - 0.01.. Of the total mutagenicity of EOM in airborne TPM, 20.4% is in the basic fraction ŽKOS2., 25.4% in the acidic fraction ŽKOS3. and 54.2% in the neutral fraction ŽKOS4.. The levels of mutagenicity were determined from the statistical evaluation of results of each mutagenicity assay Žas percentage of the total mutagenic potential. using results from GeneTox Manager software. Simultaneously obtained data demonstrate that, in general, 90% of the mutagenic potential found in the KOS2, KOS3 and KOS4 fractions together is revealed after metabolic activation in vitro ŽFig. 1.. The effect of metabolic activation on the mutagenicity of the sample was manifested mainly in the case of the neutral fraction ŽKOS4.. The influence of metabolic activation Žwith S9. on the mutagenic
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Fig. 1. Distribution of total mutagenic potential over the major fractions of EOM, analysed with indicator strains TA100 and TA98 in response to metabolic activation in vitro ŽyS9 and qS9..
potential of organic chemical compounds in this fraction is very high, a ; 20-fold difference being observed between tests with and without S9. The mutagenicity in fractions KOS2, KOS3 and KOS4 was dependent on the biological effect of genotoxicants having indirect mechanisms of action, and on their various reactive intermediates. The analysis of the carcinogenic PAH in the crude extract ŽCR. ŽTable 1. showed their different concentrations in complex mixtures. In view of their overall mutagenicity, carcinogenic PAH play an undeniable role in the total mutagenic potential of EOM from airborne TPM in coke-oven emissions. The character
of the mutagenic effect of the main fractions of CR confirms above-mentioned results Žsee Fig. 1.. The summary data ŽFigs. 2 and 3. demonstrate that the effect of frameshift mutagens among the genotoxicants in EOM is more important for the total mutagenicity of the main fractions ŽKOS2, 3 and 4 together. than the impact of genotoxicants acting on the basis of substitution mutations. About 54% of the total mutagenicity of the major fractions is due to frameshift mutagens in the complex mixtures, as revealed with strain TA98. From the total mutagenicity of the fractions ŽFig. 2., the mutagenic potential Žin %. observed with strain TA98 Žwith and without
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Fig. 2. Frameshift mutagenicity in the major fractions of EOM, analysed with indicator strain TA98 in response to metabolic activation in vitro ŽyS9 and qS9..
S9. prevails. Base-pair substitution mutagens Žwith mutagenic activity on indicator strain TA100. con-
tribute up to about 60% to the total mutagenic potential ŽFig. 3..
Fig. 3. Base-substitution mutagenicity in the major fractions of EOM, analysed with indicator strain TA98 in response to metabolic activation in vitro ŽyS9 and qS9..
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These results can be considered as the biological evidence of the importance of frameshift mutagens Žincluding carcinogenic PAH. for the overall genotoxic potential of EOM bound on TPM in the ambient air of coke ovens. As is shown from data given in the literature, PAH-like substances and their derivatives dominate in the neutral fraction of total EOM after fractionation of complex mixtures w15,17x. In connection with this finding, the results of mutagenicity tests of the main EOM fractions Žsee Fig. 1. demonstrate that compounds contained in the neutral fraction are the most important for total genotoxic
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potential of the complex mixture of genotoxic organic substances in the air of coke ovens. The results in Fig. 1 also suggest that the importance of the chemical compounds in the acidic and basic fractions is not negligible for the total mutagenicity of EOM. This is confirmed by the data on the distribution of the percentage of frameshift and base-substitution mutations for each major fraction ŽFigs. 2 and 3.. No reasonable set of chemical analyses can fully characterize all the potentially genotoxic agents in complex mixtures w4x. Short-term genetic bioassays Žespecially bacterial assays. are
Fig. 4. Mutagenicity in the subfractions of the neutral main fraction of EOM, analysed with indicator strains TA100 and TA98 in response to metabolic activation in vitro ŽyS9 and qS9.. NS s not significant; UU P - 0.01.
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being considered as an integral part of environmental monitoring studies aimed at cancer or genetic risk evaluation. In the case of EOM from airborne TPM at coke ovens, short-term genetic bioassays complement chemical analysis to provide direct data on the genotoxic activity of complex mixtures. Fig. 4 shows results obtained in the mutagenicity tests of the aliphatic ŽKOS5., aromatic ŽKOS6., slightly polar ŽKOS7., moderately polar ŽKOS8. and highly polar ŽKOS9. subfractions of the main neutral fraction. The tester strains TA100 and TA98 were used, with and without metabolic activation in vitro. Most of individual mutagenicity results were significant according to the B1 GeneTox linear slope value ŽRev.rmg EOM. but the values show large differences in the mutagenic potential of each subfraction. Based on the analyses of the results obtained, the following variances were found: Ø The aliphatic subfraction ŽKOS5. does not contribute significantly to the total genotoxic effect of the complex mixture of EOM in the main neutral fraction. Ø The highly polar ŽKOS9., moderately polar ŽKOS8. and aromatic ŽKOS6. subfractions play a minor role, but the slightly polar ŽKOS6. fraction contributes most to the mutagenicity of organic chemical substances in the main neutral fraction. The mutagenicity values of each of the subfractions KOS7, KOS9, KOS8, and KOS6 were statistically significant Ž P - 0.01. according to the linear slope B1 of each subfraction. The main objective of this study was to ascertain whether there are any differences in genotoxic potential of main fractions and subfractions of complex mixtures of chemical pollutants in the ambient air of the coke oven. The results show that the highest mutagenic potential was bound on EOM contained in the neutral fraction ŽFig. 1.. Carcinogenic PAH and nitrated polycyclic aromatic hydrocarbons ŽnitroPAH. were also present in this fraction Žsee abovementioned results.. In addition to PAH in the occupational environment, nitro-PAH are also released as a result of incomplete combustion processes w26x. This fact is confirmed by the finding of the direct mutagenicity of the EOM fractions and subfractions in our study w27x. Nitro-PAH are known to be potent bacterial mutagens. They require metabolic activation by both nitroreductase and acetyltransferase in
Fig. 5. Total mutagenicity in the neutral main fraction of EOM, analysed with indicator strains TA98 and YG1041 in response to metabolic activation in vitro ŽyS9 and qS9.. NSs not significant; UU P - 0.01.
order to exert their mutagenicity. These enzymes are present in bacterial indicator strains as well as in mammalian cells. The overproduction of nitroreductase and O-acetyltransferase in the YG1041 strain results in its stronger ability to transform nitro-PAH into mutagenic metabolites. For that reason this strain was used to obtain biological evidence for the effect of nitroarenes on the overall mutagenicity of genotoxicants contained in the neutral fraction ŽFig. 5.. The present results show that the mutagenicity of the neutral fraction was significantly increased Ž P 0.01. on strain YG1041 in comparison with the standard strain TA98. Indeed, the increase of direct and indirect mutagenicity with strain YG1041, as observed in our experiments, may be indicative of the presence of nitro-PAH.
Acknowledgements The authors would like to thank J. Lenıcek, ´ˇ PhD ´ ´ n.Labem, Czech Republic. for the fracŽRIH, Ustı tionation of the EOM from HiVOL air samples. We are grateful to Mrs. J. Tvrda´ and Mrs. M. Skopalova´ for their excellent technical assistance in experimental part of the work. This work was supported by Grant No. 3159-3 of the Czech Ministry of Health and by EU Grant No. CIPA-CT-94-0113.
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Bioassay-directed chemical analysis and detection of mutagenicity in ambient air of the coke oven a,)
L. Dobias ´ˇ
, J. Kusova ˚ ´ a, O. Gajdosˇ b, P. Vidova´ b, D. Gajdosova ˇ ´ b, J. Havrankova ´ ´ a, M. Fried c , B. Binkova´ d , J. Topinka d
a
b
Department of Genetic Toxicology, Regional Institute of Hygiene, 728 92 OstraÕa, Czech Republic Department of Genetic Toxicology, Specialized National Health Institute, 040 11 Kosice, ˇ SloÕak Republic c Hygienic Institute, UniÕersity of Heidelberg, 69 120 Heidelberg, Germany d LGE, c r o LGE-RIH of Central Bohemia and IEM AS CR, Prague, Czech Republic Received 17 August 1998; accepted 15 December 1998
Abstract In the present study, we summarize the results of studies on the mutagenic potential of the main fractions and subfractions of extractable organic material ŽEOM. in the ambient air at the workplaces of the coke oven. The objective of our experiments was to apply the Bioassay-Directed Chemical Analysis Žwith the use of the Ames test. for the identification of the differences in the mutagenicity of these fractions, in relationship to the complex mixture of EOM in occupational air. From the evaluation of results, it is possible to deduce the following conclusions: Ž1. The comparison of the mutagenicity in the main fractions Žbasic, acidic, neutral. demonstrates the existence of differences in mutagenic potential. Of the total mutagenicity, 20.4% is in the basic fraction, 25.4% in the acidic fraction and 54.2% in the neutral fraction. Ž2. In general, 90.1% of the mutagenicity found in the basic, acidic and neutral fractions together was associated with the requirement of metabolic activation in vitro ŽqS9.. In the case of the neutral fraction, it was 51.8%. Ž3. These results also suggest that frameshift mutations are the major component Ž53.8%. of the total mutagenicity of the main fractions. Ž4. With regards to the mutagenicity of organic compounds in the neutral fraction it appeared that genotoxicants of its subfractions Žslightly and moderately polar and aromatic. play the main role. Carcinogenic polycyclic aromatic hydrocarbons ŽPAH. and genotoxic nitrocompounds play an important role as determinants of the mutagenic potential of complex mixtures of harmful compounds in ambient air. This is confirmed first by the results of short-term bacterial tests. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Mutagenicity; Bioassay-directed chemical analysis; Carcinogenic PAH; Coke-oven emissions
1. Introduction The increasing occurrence of genotoxic pollutants in the environment has become a matter of interest )
Corresponding author. Fax: q42-69-611-86-61
as a complex public health problem. Questions arose as to the emission, distribution and fate of the genotoxicants in the environment and the exposure levels of the human population w1,2x. At the same time, the identification and control of dangerous genotoxic substances in the work environment play an extraor-
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 3 3 - 3
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dinary role in the prevention of occupational diseases w3x. To answer these questions, investigators had to rely initially on chemical analysis of individual genotoxic compounds and subsequently on biological tests for biomonitoring exposure to complex mixtures. The environment contains a wide variety of manmade genotoxic agents including mutagens and carcinogens. The development of short-term genetic bioassays in the mid-1970s rapidly led to the use of these assays in environmental monitoring w4x. The most widely used bacterial mutagenicity bioassay has been the Salmonella typhimurium plate-incorporation assay described by Ames et al. in 1975 and validated as an initial bioassay to screen for potential carcinogens w5,21,22x. Ames’ bacterial bioassay was used as a short-term test to detect and quantify the mutagenicity associated with complex mixtures of harmful substances in air, water, industrial effluents and commercial products. Generally, short-term microbial tests are being considered an integral part of environmental monitoring studies aimed at cancer or genetic risk assessment. Many studies have clearly demonstrated the mutagenicity of airborne particles from areas with anthropogenic air pollution w6–8x, combustion sources and industrial manufacturing emissions w9,10x. The present study was aimed at the application of bioassaydirected chemical analysis to identify the mutagenic potential of a fractionated complex mixture of extractable organic material ŽEOM. emitted into the air surrounding the coke-oven battery. Attention was especially drawn to the evaluation of the biological significance of PAH and nitro-compounds. The genotoxic effect of exposure to complex mixtures of carcinogenic PAH in the workplaces is a general risk factor for the health status of workers w11–14x. However, the complex mixture of EOM in coke-oven batteries is represented by a large quantity of heterogeneous chemical substances among which the PAH dominate w11x. The complexity of these mixtures limits the possibilities of chemical detection during identification of harmful substances with genotoxic effects, and at the same time hampers their biological detection. Therefore, it was felt necessary to use fractionation of the complex mixture in connection with biological detection of genotoxicity of the main frac-
tions and subfractions Žbioassay-directed chemical analysis.. In currently published studies in which the above-mentioned procedure was applied it has been shown that, as a rule, specific groups of chemical substances play a major role in genotoxicity of complex mixtures w15–19x. The aim of this work is to show that the methods used in our study provide suitable indicators which could help control exposure to carcinogenic PAH in ambient air of coke ovens.
2. Material and methods 2.1. Air sampling and sampling sites During November 1996, air samples were taken on the topside of a coke-oven battery ŽIron Works, Kosice, Slovak Republic. using a high-volume ˇ ŽHiVOL. sampler ŽSierra Misco, USA.. The sampler was located on a lorry car on the topside of the oven. Air was sampled continuously during 5 h of each shift during 5 days; the mean air flow rate during sampling was 88 m3rh. The HiVOL sampler was equipped with teflon-impregnated glass fibre filters PALLFLEX T60 A20 for collection of total particulate matter ŽTPM.. 2.2. Storage of filters The exposed filters were weighed to five decimals after drying for 24 h in a desiccator, at room temperature in the dark. Prior to extraction and separation, the filters were kept wrapped in Al-foil at y708C in a freezing box. 2.3. Extraction of exposed filters Individual filters were extracted as described w20x. Extraction was performed by sonication in an ultrasound bath Ž140 W at 258C, 3 = 10 min.. Dichloromethane p.a. ŽMerck, FRG. ŽDCM. was used as an extraction medium. The extraction was carried out with three sequential volumes of solvent, which were finally pooled into a total crude extract. After filtration of the crude extract Žvolume 140 . ml , aliquots were removed for the determination of
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extractable organic matter ŽEOM.. The concentration of total EOM was 21.790 mg per ml of crude extract. 2.4. Fractionation of crude extract Crude EOM extract ŽCR. was fractionated by acid–base partitioning into three main fractions, viz. a basic ŽKOS2., an acidic ŽKOS3. and a neutral ŽKOS4. fraction. The latter was fractionated further by use of silica-gel column chromatography into aliphatic ŽKOS5., aromatic ŽKOS6., slightly polar ŽKOS7., moderately polar ŽKOS8. and highly polar ŽKOS9. subfractions, on the basis of polarity of the mobile phase w17,19x. 2.5. Analysis of PAH and nitro-PAH analysis PAH in the crude EOM and the aromatic subfraction ŽKOS6. and nitro-PAH in all subfractions ŽKOS5, KOS6, KOS7, KOS8, KOS9. of the neutral fraction were analyzed by electron-impact GC-MS with selected ion monitoring ŽSIM mode. w28x. A total of 0.25 mg of EOM was dissolved in acetone Ž10 mgrul. and precleaned on an SPE cartridge ŽBaker. filled with 1 g of SiOH. The column was conditioned and prewashed with n-hexane and PAH were eluted with n-hexane:DCM Ž5:1 vrv.. Samples were concentrated under a stream of nitrogen, diluted by toluene and transferred to vials for GCrMS analysis. For the gas-chromatographic analysis a HewlettPackard GC Ž589011.rMSD Ž5971. was used. GC parameters were: injector temperature 2908C; transferline temperature 2908C; oven programme: start at 1008C for 2 min, ramp 108Crmin from 1008C to 2908C, then 2908C for 9 min. A total of 16 analysed PAH was divided into 5 groups according to their boiling points and for each of these groups one internal standard was used Žacenaphtalene-d10, phenanthrene-d10, fluoranthene-d10, benzŽ a . anthracene-d12 and perylene-d12.. 2.6. Bioassay sample preparation For the plate-incorporation mutagenicity assay, the EOM Žin DCM. of each sample was transferred to dimethylsulphoxide ŽDMSO. ŽMerck, FRG.. Aliquots of the crude extract and all fractions and
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subfractions Žin DCM. were evaporated to dryness under a stream of nitrogen and the residual EOM were dissolved in a standard volume of DMSO. After their transfer to DMSO, the samples were tested in the mutagenicity bioassay at four doses of the stock extracts corresponding to 10, 20, 40 and 80 mg EOM per plate. 2.7. Mutagenicity bioassay The Ames S. typhimurium assay Ža variant of the plate- incorporation test. was used to determine the mutagenicity. The following indicator strains were used: TA100 Žbase-pair substitutions. and TA98 Žframe-shift mutations., kindly provided by Prof. B.N. Ames, Berkeley, CA, USA, and YG1041 Ža derivative of the TA98 parent strain., kindly donated by Drs. T. Nohmi and M. Watanabe, National Institute of Hygienic Science, Japan. The genetic properties of the tester strains were checked according to recommended procedures w21,22x. The sensitivity of the tester strains to mutagens was determined by use of the following standard compounds: 2-aminofluorene ŽSigma, FRG., p-nitro-o-phenylenediamine ŽAldrich Chemie, FRG. for TA98 and YG1041, natrium azide ŽSigma. and 2-anthramine ŽAldrich. for TA100. Cultures of the tester strains were grown overnight in Oxoid Nutrient Broth No. 2 in a shaking water bath at 378C. Bioassay experiments were conducted as previously described w22x, on individual sample extracts using duplicate plates with and without metabolic activation in vitro ŽqS9 and yS9; DELOR 103-induced S9 fraction from liver of rats. for each dose w22x. After a 72-h incubation at 378C, colony counts on Petri plates were determined using a ZEBRA System’s ŽCzech Republic. automatic colony counter. 2.8. Statistical eÕaluation of the bioassay The results of mutagenicity tests were evaluated with the GeneTox Manager programme ŽU.S. EPA, HERL, RTP, NC 27711. w23x, the software was acquired by courtesy of Dr. L. Claxton. The maximum mutagenic potency Žnumber of revertantsrmg EOM. and the dose-response of each sample were determined with the GeneTox Manager.
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The linear slope values Ž B1 . for both test variants were calculated from numbers of revertantsrmg EOM from the Bernstein model w24x. The statistical significance for differences of positive responses Ždefined as B1 GeneTox values with P - 0.05. was evaluated. The mutagenicity of individual fractions and subfractions Žwith the indicator strains, q and yS9. was expressed as percentage of the total mutagenicity of the complex mixtures. The Student’s t-test was used for statistical evaluation of the results.
3. Results and discussion The study was aimed towards the objectivization of the mutagenic potential of complex mixtures of EOM in HiVOL air samples at the coke oven by use of the Ames mutagenicity assay. In the experimental part we used the bioassay-directed chemical analysis vs. identification of the mutagenicity in major fractions and subfractions of organic chemical substances in emissions from the coke-oven batteries. To assess hazardous pollutants in the occupational environment and to evaluate the exposure to genotoxic substances, relevant exposure data are necessary. The profile of HiVOL air samples corresponds to standard working conditions at the coke-oven batteries in the winter season Žwith deteriorated dispersion of the pollutants in air.. The important role of carcinogenic PAH is visible from a survey of the PAH content in the crude extract ŽTable 1.. The mutagenic potential of the main fractions and subfractions of the EOM mixture contained in occupational air samples from the coke oven was evaluated by the Ames test Žplate-incorporation assay. with the use of the S. typhimurium TA100 and TA98 indicator strains Žin the absence and presence of S9.. Strain YG1041 was used to determine the mutagenicity of the neutral fraction. This strain is a derivative of the TA98 parent strain, and has elevated levels of both nitroreductase and O-acetyltransferase activities w25x. This strain was used because in the slightly polar subfraction Ža component of the neutral fraction. the presence of 3nitrophenanthrene Ž66 ngrmg EOM. and 1-nitropyrene Ž27 ngrmg EOM. was detected.
Table 1 PAH analysis in crude EOM PAH
Concentrations Žmgr mg EOM. crude EOM
BenzoŽ a.pyrenea BenzoŽ a.anthracenea Chrysenea BenzoŽ b .fluoranthenea BenzoŽ k .fluoranthenea IndenoŽ1,2,3-c,d .pyrenea DibenzoŽ a,h.anthracenea BenzoŽ g,h,i .perylenea Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Naphthalene Acenaphthylene Acenaphthalene Sum of carcinogenic PAH Sum of PAH
6.4 8.8 8.9 7.0 6.9 4.9 0.8 4.2 n.d. 0.9 0.3 10.9 8.2 n.d. n.d. n.d. 47.9 Ž4.8%. 68.2 Ž6.8%.
n.d.s Not detectable Ždetection limits 0.1.. a Carcinogenic PAH Žsee IARC Monographs, 1983..
Comparison of the overall results of the mutagenicity assays of the main fractions Žbasic, acidic, neutral. of EOM from occupational airborne TPM sampled in the coke oven shows differences in mutagenic activity ŽFig. 1.. The differences in mutagenic potential Žin % of the mutagenicity of the total extract. between basic, acidic and neutral fractions are statistically significant Ž P - 0.01.. Of the total mutagenicity of EOM in airborne TPM, 20.4% is in the basic fraction ŽKOS2., 25.4% in the acidic fraction ŽKOS3. and 54.2% in the neutral fraction ŽKOS4.. The levels of mutagenicity were determined from the statistical evaluation of results of each mutagenicity assay Žas percentage of the total mutagenic potential. using results from GeneTox Manager software. Simultaneously obtained data demonstrate that, in general, 90% of the mutagenic potential found in the KOS2, KOS3 and KOS4 fractions together is revealed after metabolic activation in vitro ŽFig. 1.. The effect of metabolic activation on the mutagenicity of the sample was manifested mainly in the case of the neutral fraction ŽKOS4.. The influence of metabolic activation Žwith S9. on the mutagenic
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Fig. 1. Distribution of total mutagenic potential over the major fractions of EOM, analysed with indicator strains TA100 and TA98 in response to metabolic activation in vitro ŽyS9 and qS9..
potential of organic chemical compounds in this fraction is very high, a ; 20-fold difference being observed between tests with and without S9. The mutagenicity in fractions KOS2, KOS3 and KOS4 was dependent on the biological effect of genotoxicants having indirect mechanisms of action, and on their various reactive intermediates. The analysis of the carcinogenic PAH in the crude extract ŽCR. ŽTable 1. showed their different concentrations in complex mixtures. In view of their overall mutagenicity, carcinogenic PAH play an undeniable role in the total mutagenic potential of EOM from airborne TPM in coke-oven emissions. The character
of the mutagenic effect of the main fractions of CR confirms above-mentioned results Žsee Fig. 1.. The summary data ŽFigs. 2 and 3. demonstrate that the effect of frameshift mutagens among the genotoxicants in EOM is more important for the total mutagenicity of the main fractions ŽKOS2, 3 and 4 together. than the impact of genotoxicants acting on the basis of substitution mutations. About 54% of the total mutagenicity of the major fractions is due to frameshift mutagens in the complex mixtures, as revealed with strain TA98. From the total mutagenicity of the fractions ŽFig. 2., the mutagenic potential Žin %. observed with strain TA98 Žwith and without
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Fig. 2. Frameshift mutagenicity in the major fractions of EOM, analysed with indicator strain TA98 in response to metabolic activation in vitro ŽyS9 and qS9..
S9. prevails. Base-pair substitution mutagens Žwith mutagenic activity on indicator strain TA100. con-
tribute up to about 60% to the total mutagenic potential ŽFig. 3..
Fig. 3. Base-substitution mutagenicity in the major fractions of EOM, analysed with indicator strain TA98 in response to metabolic activation in vitro ŽyS9 and qS9..
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These results can be considered as the biological evidence of the importance of frameshift mutagens Žincluding carcinogenic PAH. for the overall genotoxic potential of EOM bound on TPM in the ambient air of coke ovens. As is shown from data given in the literature, PAH-like substances and their derivatives dominate in the neutral fraction of total EOM after fractionation of complex mixtures w15,17x. In connection with this finding, the results of mutagenicity tests of the main EOM fractions Žsee Fig. 1. demonstrate that compounds contained in the neutral fraction are the most important for total genotoxic
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potential of the complex mixture of genotoxic organic substances in the air of coke ovens. The results in Fig. 1 also suggest that the importance of the chemical compounds in the acidic and basic fractions is not negligible for the total mutagenicity of EOM. This is confirmed by the data on the distribution of the percentage of frameshift and base-substitution mutations for each major fraction ŽFigs. 2 and 3.. No reasonable set of chemical analyses can fully characterize all the potentially genotoxic agents in complex mixtures w4x. Short-term genetic bioassays Žespecially bacterial assays. are
Fig. 4. Mutagenicity in the subfractions of the neutral main fraction of EOM, analysed with indicator strains TA100 and TA98 in response to metabolic activation in vitro ŽyS9 and qS9.. NS s not significant; UU P - 0.01.
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being considered as an integral part of environmental monitoring studies aimed at cancer or genetic risk evaluation. In the case of EOM from airborne TPM at coke ovens, short-term genetic bioassays complement chemical analysis to provide direct data on the genotoxic activity of complex mixtures. Fig. 4 shows results obtained in the mutagenicity tests of the aliphatic ŽKOS5., aromatic ŽKOS6., slightly polar ŽKOS7., moderately polar ŽKOS8. and highly polar ŽKOS9. subfractions of the main neutral fraction. The tester strains TA100 and TA98 were used, with and without metabolic activation in vitro. Most of individual mutagenicity results were significant according to the B1 GeneTox linear slope value ŽRev.rmg EOM. but the values show large differences in the mutagenic potential of each subfraction. Based on the analyses of the results obtained, the following variances were found: Ø The aliphatic subfraction ŽKOS5. does not contribute significantly to the total genotoxic effect of the complex mixture of EOM in the main neutral fraction. Ø The highly polar ŽKOS9., moderately polar ŽKOS8. and aromatic ŽKOS6. subfractions play a minor role, but the slightly polar ŽKOS6. fraction contributes most to the mutagenicity of organic chemical substances in the main neutral fraction. The mutagenicity values of each of the subfractions KOS7, KOS9, KOS8, and KOS6 were statistically significant Ž P - 0.01. according to the linear slope B1 of each subfraction. The main objective of this study was to ascertain whether there are any differences in genotoxic potential of main fractions and subfractions of complex mixtures of chemical pollutants in the ambient air of the coke oven. The results show that the highest mutagenic potential was bound on EOM contained in the neutral fraction ŽFig. 1.. Carcinogenic PAH and nitrated polycyclic aromatic hydrocarbons ŽnitroPAH. were also present in this fraction Žsee abovementioned results.. In addition to PAH in the occupational environment, nitro-PAH are also released as a result of incomplete combustion processes w26x. This fact is confirmed by the finding of the direct mutagenicity of the EOM fractions and subfractions in our study w27x. Nitro-PAH are known to be potent bacterial mutagens. They require metabolic activation by both nitroreductase and acetyltransferase in
Fig. 5. Total mutagenicity in the neutral main fraction of EOM, analysed with indicator strains TA98 and YG1041 in response to metabolic activation in vitro ŽyS9 and qS9.. NSs not significant; UU P - 0.01.
order to exert their mutagenicity. These enzymes are present in bacterial indicator strains as well as in mammalian cells. The overproduction of nitroreductase and O-acetyltransferase in the YG1041 strain results in its stronger ability to transform nitro-PAH into mutagenic metabolites. For that reason this strain was used to obtain biological evidence for the effect of nitroarenes on the overall mutagenicity of genotoxicants contained in the neutral fraction ŽFig. 5.. The present results show that the mutagenicity of the neutral fraction was significantly increased Ž P 0.01. on strain YG1041 in comparison with the standard strain TA98. Indeed, the increase of direct and indirect mutagenicity with strain YG1041, as observed in our experiments, may be indicative of the presence of nitro-PAH.
Acknowledgements The authors would like to thank J. Lenıcek, ´ˇ PhD ´ ´ n.Labem, Czech Republic. for the fracŽRIH, Ustı tionation of the EOM from HiVOL air samples. We are grateful to Mrs. J. Tvrda´ and Mrs. M. Skopalova´ for their excellent technical assistance in experimental part of the work. This work was supported by Grant No. 3159-3 of the Czech Ministry of Health and by EU Grant No. CIPA-CT-94-0113.
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