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Mutagenic and clastogenic activity of direct-acting components from air pollutants of the Silesian industrial region
Mutation Research, 204 (1988) 289-296 Elsevier
289
MTR 01240
Mutagenic and clastogenic activity of direct-acting components from air pollutants of the Silesian industrial region G. Motykiewicz 1, j. Michalska 1, j. Szeliga i and B. Cimander 2 I Department of Tumor Biology, Institute of Oncology, 44-100 Gliwice (Poland) and 2 District Sanitary-Epidemiologic Station, 44-075 Katowice (Poland)
(Received 13 January 1987) (Revision received 6 July 1987) (Accepted 20 July 1987)
Keywords: Pollutant, environmental; Mutagenic activity; Clastogenic activity.
Summary 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.
Epidemiological data have suggested an association of lung cancer mortality with the mutagenicity of airborne particulate pollutants, assayed by the Ames test (Walker et al., 1982). Urban air, particularly in industrial regions, contains many known mutagens and carcinogens (IARC, 1983). Only a small part of the observed mutagenic activity can be explained by 'classical' polycyclic aromatic hydrocarbons (PAHs) a n d / o r their known direct-acting derivatives, e.g., nitropyrenes (Rosenkranz, 1982; Gibson, 1983). Most of the mutagens present in the total extractable organic matter, and acting directly, have not been identified. Several studies have shown that mutagenic Correspondence: Dr. G. Motykiewicz, Department of Tumor Biology, Institute of Oncology, Wybrze~:e Armii Czerwonej 15, 44-100 Gliwice (Poland).
activity was present in the polar fractions not containing conventional PAHs (Dehnen et al., 1977; Tokiwa et al., 1980; De Wiest et al., 1982; Alfheim et al., 1983). Great methodological variations exist between studies since no standardized fractionation protocol has been established. However, Meller and Lrfroth (1982), in a review of genotoxic air pollutants, have recommended high-performance liquid chromatographic (HPLC) methods. The Ames Salmonella assay is a useful tool for identification of the mutagenic potency of air pollutants (Tokiwa et al., 1977; Moller and Alfheim, 1980; Fukino et al., 1982; Madsen et al., 1982; Tokiwa et al., 1983; Garner et al., 1986), although as a prokaryotic test it is not quite adequate to risk estimation for humans. Up to now, only a few studies with mammalian cells have been carried
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
290
out. The organics associated with air particulate matter have mainly been examined by sister-chromatid exchange (SCE) tests (Lockard et al., 1981; Alink et al., 1983; De Raat, 1983), whereas induction of chromosomal aberrations (CAs) in human lymphocytes was presented only by Krishna et al. (1984) and Hadnagy et al. (1986). In our earlier studies (Motykiewicz et al., 1985) benzene extracts of airborne particulate material, collected at 23 locations in the industrial Silesian region during a 6-month period were evaluated in the Salmonella/microsome system with TA100 and TA98 strains. We observed seasonal variations in mutagenic activity of samples collected in summer and winter. Crude extracts derived from winter samples showed high direct mutagenic activity. In the present paper we have further used the Ames Salmonella assay with strain TA100, together with a chromosome aberration test in V79 cells, for examination of the direct-acting fractions obtained by sequential elution solvent chromatography (SESC) of benzene extracts of airborne particulates collected in highly polluted locations in Silesia (December 1984 and 1985). Materials and methods
Airborne samples Particulate matter was collected on fiber-glass filters (Staplex Co.) by Staplex high-volume samplers at a flow rate of about 90 m3/h. Samples were taken at 8 'high-pollution' locations (Motykiewicz et al., 1985), 6 × 24 h / m o n t h in December
1984 and 1985. After sampling, filters were combined and then organic matter was extracted in a Soxhlet apparatus with benzene for 7 h. The solvent was removed to dryness by rotary evaporator and the extraction yield was determined. Samples were stored under gaseous nitrogen at - 20 o C.
Sequential elution solvent chromatography (SESC) The method developed by Farcasiu (1977) and modified by Jacobs and Filby (1983) for the separation of coal liquefaction products was applied to the fractionation of benzene extracts. This scheme used silica gel and a solvent sequence that provided a systematic variation in the Hildebrand solubility parameters and the specific solubility parameters (Table 1). The separation was performed on a silica gel column (80 x 0 . 8 cm ID) containing 25 g of Koch-Light 5000 h silica gel 'Davison', 100-200 mesh. The silica gel was dried and the water content was adjusted to 4% by weight. The sample (0.2 g) was first loaded onto glass beads, 60 mesh. The coated glass beads were then poured over the silica gel column. For elution of each fraction, 200 ml of each solvent were used. The collected fractions were evaporated to dryness by rotary evaporator at 50°C, weighed and stored under gaseous nitrogen at - 20 o C.
Mutagenicity testing The test was carried out according to the procedure of Ames et al. (1975). The bacteria used were
TABLE 1 SEQUENTIAL ELUTION SOLVENT CHROMATOGRAPHY (SESC) SCHEME The method was developed and the compound types were identified by Farcasiu (1977). Fraction Number
Eluent
Compound types eluted
I II III IV V VI VII VIII
Hexane 15% Toluene in hexane " Chloroform 10% Diethylether in chloroform 3% Ethanol in diethylether Methanol 3% Ethanol in chloroform 3% Ethanol in tetrahydofuran
Saturated hydrocarbons Aromatic hydrocarbons Polar aromatics, non-basic N, S, O heterocyclics Monophenols Basic nitrogen heterocyclics Highly functional molecules containing heteroatoms at 10% by weight Polyphenols Molecules with high O and N content
a Modification by Jacobs and Filby (1983).
291
Salmonella typhimurium strain TA100, kindly supplied by Prof. B.N. Ames. Because our earlier studies, performed with strains TA100 and TA98 indicated that strain TA100 was more sensitive (Motykiewicz et al., 1985), this strain was used in all experiments. Studies were performed in the presence and absence of an Aroclor 1254-induced rat liver microsomal fraction ($9). The protein content of the activation mixture, the so-called $9 mix, varied from 6.6 to 6.8 m g / m l (Miiller et al., 1980) and 0.5 ml of $9 mix was applied per plate. Samples dissolved in DMSO were tested in duplicate for each concentration in 2 independent experiments. Benzo[a]pyrene and 1-nitro-9-Y-[NN-dimethyl-aminopropyl]aminoacridine were used as positive control mutagens. Determination of mutagenic response was based on criteria recommended by Ames et al. (1975).
wt%
40 35 30 25 20
,,\\
10 5
I
II
III
I~
V
'f-i ~/II "4111
Fraction No. Fig. 1. Relative weight distribution of SESC fractions of samp l e s collected in December 1984 and 1985. Mean v a l u e s + standard deviation.
TA100) for crude extract and SESC fractions of samples collected in December 1984. Mutagenic activity was observed in fraction Nos. II-VI. TABLE 2 M U T A G E N I C I T Y O F SEPARATE SESC F R A C T I O N S The number of revertants per /~g of crude extract or SESC fractions, and per m 3 of air was calculated from a dose of 125 /~g/plate (linear parts of the curves, Fig. 2). Fraction N umbe r
Results
Benzene extracts of airborne particulates were separated into 8 fractions by the SESC system. Fig. 1 shows the relative weight distribution of the obtained fractions. Samples from December 1984 and December 1985 gave very similar fractionation profiles. Fraction Nos. I I - V comprised 84% of the extracts by weight, and the major types of chemical compounds expected in these fractions are given in Table 1. Fig. 2 presents results of the Ames test (strain
\
\
/
15
Chromosome aberration analysis The CA test was performed according to the procedure described previously (Michalska, 1986). Briefly, the appropriate amounts of test fractions dissolved in 50 #1 DMSO were added per plate to exponentially growing V79 cells in 10 ml of Dulbecco's MEM medium supplemented with 10% fetal calf serum, for 5, 14, and 24 h. Fraction No. III was appfied in 100 /~1 due to low solubility. DMSO was used as control. Colchicine at a final concentration of 0.25 /~g/ml was added for 2 h before harvesting the cells. Preparations were made by the air-drying method and were stained by Giemsa. 200 or 300 metaphases were examined for each experimental point, and the chi-square test was used to analyze the chromosome aberration frequency.
/// / .// /// /// ../, /// /// ///
Calculated number of revertants for TA100 per ~tg - $9
per m 3 + $9
- $9
+ $9
2.56
3.32
0.10
0.00
4.41 3.61 3.28 1.68 1.40 0.10 0.13
186.5 0.5 17.9 47.0 51.2 14.1 5.6 0.2 0.1
241.5 0.2 38.3 91.1 53.5 15.4 9.5 0.1 0.1
-
136.6
208.0
Crude extract I
II III IV V VI VII VIII Sum of fractions
2.06 1.86 3.14 1.54 0.83 0.22 0.23 -
292
Fraction
Nos. II and III showed both a direct and an indirect mutagenic activity. In these fractions, however, the higher doses (250 and 500/~g/plate) I&l cI c~
gave a nonlinear response especially with S9 mix. This effect is most probably due to the specific toxicity of material sampled in winter. Under the
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400
300
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100
Ft. No.IV
800
ll
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12o
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300 20O
i I
100 100 200 300 400 500
100 200 300 400 500
mo 2oo 30o 4o0 5oo j.tglpiote
Fig. 2. Mutagenic activities of SESC fractions of air-pollutant benzene extracts, collected in December 1984; S. typhimurium TA100, O) and without $9 ( O O); subtracted spontaneous revertant backgrounds were: 103-151 ( - $ 9 ) and 113-156 ( + $9). Hatched columns represent the contribution of each fraction, in / ~ g / m 3 of air. with $9 (o . . . . . .
293 same
experimental
centration summer,
conditions
(the
same
con-
Nos. II and III (Motykiewicz
of $9 mix used) for samples collected in we
dose-related
observed
a linear
response
for
or
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of
aration).
linear
The
(Nos. IV-VI)
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other
and Szeliga, in prep-
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active fractions
only direct mutagenic
activ-
ity.
TABLE3 C H R O M O S O M A L A B E R R A T I O N S P R O D U C E D IN V79 CELLS BY SESC F R A C T I O N S Fraction and dose ( / l g / m l ) III
IV
V
0 5 0 ( 2 . 3 m 3) 1~ 2~
Time (h)
Mitotic index (%)
Aberrant metaphases + G (%)
Aberrant metaphases - G (%)
Chromosomal aberrations/100 cells End CdG CdB Ex C1G C1B
Dic
R
P (Ab - G)
5
6.2 7.4 5.6 3.0
1.0 0.5 2.5 3.5
0.5 0 1.0 2.0
0 0 0 0
0.5 0.5 1.5 1.5
0 0 1.0 1.5
0 0 0 0
0 0 0 0
0 0 0 0
0.5 0 0 0.5
0 0 0 0
N N N N
0 50 100 200
14
5.2 4.9 2.5 1.1
2.5 1.5 3.0 7.0
1.5 0.5 3.0 4.5
0 0.5 0.5 1.0
0.5 1.0 1.5 2.5
1.0 0 1.0 3.0
0.5 0 0.5 0.5
0 0 0 0
0 0 0 0
0 0 0 0
0 0 1.0 0
N N N N
0 50 100 200
24
7.3 4.0 3.5 0.9
2.0 3.0 7.5 16.5
1.0 1.5 4.0 12.5
0 0 0 4.0
1.0 1.5 3.5 4.0
1.0 1.5 3.0 4.5
0 0 0.5 1.5
0 0 0.5 0
0 0 0 0
0 0 0.5 3.0
0 0 0.5 0
N N N <0.001
5
4.0 2.5 1.2
2.0 2.0 2.3 ~w ~toses
1.0 1.3 1.0
0 0 0
0.3 0.7 1.0
1.0 1.3 1.0
0 0 0
0.7 0 0.3
0 0 0
0 0 0
0 0 0
N N N
0 50 1~ 200
14
4.7 2.1 0.8
1.3 1.7 5.3 no ~ t o s e s
1.0 1.7 4.3
0 0 0.7
0.3 0 1.0
1.0 1.3 4.3
0 0 0
0 0 0
0 0 0.3
0 0.3 0
0 0 0.7
N N <0.025
0 50 1~ 200
24
4.1 2.5 1.5
2.3 8.7 8.3 no ~ t o s e s
1.7 6.0 6.7
0 2.3 1.0
0.3 2.7 1.7
0.7 2.7 4.3
0 2.0 3.0
0.3 0 0
0.3 0.3 0
0.7 0.7 0.3
0 0.3 0.7
N <0.01 <0.01
5
7.3 8.0 5.6 1.9
1.5 1.5 2.5 11.0
0.5 0.5 1.0 6.5
0 0 0 1.5
1.0 1.0 1.0 4.5
0.5 0.5 1.0 3.0
0 0 0 1.0
0 0 0 0
0 0 0 0
0 0 0.5 0
0 0 0 1.0
N N N <0.001
0 50 100 200
14
7.0 5.8 3.8
1.5 2.5 3.5 few ~ t o s e s
1.0 1.5 2.0
0.5 1.0 0
0.5 1.5 1.5
0.5 1.0 1.0
0 0 0.5
0 0 0
0 0 0
0 0 0.5
0 0 0.5
N N N
0 50 100 200
24
7.9 6.6 4.6
2.0 6.0 7.3 no ~ t o s e s
1.7 5.0 5.7
0 3.0 1.0
0.3 1.0 2.0
1.3 1.7 5.7
0 0 0.7
0.3 0 0
0 0 0
0 0 0
0.3 0.3 0.3
N <0.025 <0.01
0 5 0 ( 2 . 2 5 m 3) 1~ 2~
0 50(7.6 ~ ) 100 2~
G, gaps; End, Endoreduplication; CdG, chromatid gaps; CdB, chromatid breaks; Ex, chromatid interchanges; C1G, chromosomal gaps; C1B, chromosomal breaks; Dic, dicentrics; R, Rings. N, not significant statistically, P was calculated from the n u m b e r of aberrant metaphases without gaps.
294
The recovery of mutagenic activity (Table 2) was slightly decreased for the sum of the mutagenic activity of the separate fractions when compared to the activity of the unfractionated sample (crude extract). The number of revertants per/~g and m 3 of air was calculated from the linear part of the curves (Fig. 2). The results of the Ames test led to the selection of 'direct' fractions for examination for clastogenic potential in the CA test. A pilot experiment was carried out only with fraction No. IV from the December 1984 sample because the amount of material was limited. In this experiment the number of aberrant metaphases for a 24-h treatment with 100 ~tg/ml was statistically significant ( P < 0.01). The main experiment for CA (sample from December 1985) was performed with 3 selected fractions and the results are presented in Table 3. All of the tested fractions showed clastogenic activity that increased with prolonged treatment time. However, for 24-h exposures, toxic effects were observed at a dose of 200 # g / m l , which was comparable to the toxicity obtained with 500 /~g/plate in the Ames test. Only fraction No. V exhibited statistically significant clastogenic effects for the shortest treatment time (5 h). In each of the tested samples a high number of endoreduplicated cells was present; this result was especially characteristic of fraction No. III (24-h exposure). Only 1 endoreduplicated metaphase was noticed in all of the analyzed control cells. Discussion
Extracts from airborne particulate pollutants present a complex mixture and their preliminary separation provides more information about the mixture. Pollutants collected from the Silesian region mostly originate from coal combustion, encompassing industrial operations, coke production, power plants using fossil fuels and private domestic heating by coal. Therefore, benzene extracts of organic matter of air pollutants in this region may have a chemical resemblance to coal liquefaction products. It was assumed that a simple SESC method would be useful for separation of the air pollutant samples without simultaneous degradation of their genotoxic activity. Five out of 8 obtained fractions showed a dif-
ferential potency between direct and indirect mutagenic activity in the Salmonella test with strain TA100 (Fig. 2), expressed as the number of revertants per t~g and m 3 of air (Table 2). It should be noted that the recovery of mutagenic activity from the silica column was satisfactory; however, some material was irreversibly adsorbed on the silica as found by Farcasiu (1977) and Jacobs and Filby (1983). The highest direct mutagenic activity detected in the Ames test was shown by fraction No. IV, which probably contained monophenols (Table 1). The observed reduction in the mutagenicity by $9 addition was noted earlier, and unspecific reactions between active mutagens and nucleophiles presented in the S9 mix were suggested (Dehnen et al., 1977; Talkott and Wei, 1977; Takeda et al., 1984). The results from the CA test demonstrated the highest number of aberrant metaphases at a dose of 100 ~ g / m l for fraction No. IV (Table 3), which correlated well with the Ames test. For the other direct mutagenic fractions (Nos. V and VI), clastogenic effects could only be determined for fraction No. V because of the low amount of material available in fraction No. VI. Fraction No. III was examined for CA induction even though the Ames test for this material showed both direct and indirect activity. This fraction was chosen for CA testing due to its high weight percentage content in the total extract and therefore its quantitatively significant contribution to air pollution. Organic extracts of airborne particulate matter induced increases in SCEs in human lymphocytes and rodent cells (Lockard et al., 1981; Alink et al., 1983; Hadnagy et al., 1986). While the CA test compared with SCE induction appears to be less sensitive (Perry and Evans, 1975), the complementation of bacterial mutagenicity assays by CA tests revealing structural chromosomal damage in mammalian cells seems to be justified. It should be mentioned that SCEs would be comparable to gene mutation tests; therefore, chromosomal aberration assays were recommended as a useful addition to the Ames test for the detection of a genotoxic hazard (Sobels, 1985; Natarajan and Obe, 1986). Little information exists on the clastogenic effects of air pollutants measured by the CA test
295 a n d these studies were p e r f o r m e d with h u m a n lymphocytes ( K r i s h n a et al., 1984; H a d n a g y et al., 1986). I n experiments carried out b y K r i s h n a et al. (1984), h u m a n lymphocytes were exposed to very high doses of extracted material, b u t only for the S a n d G 2 phases of the cell cycle. Obviously, most k n o w n chemical clastogens are S-phase d e p e n d e n t (Hollstein a n d M c C a n n , 1979), b u t the presence of other clastogen types in such a complex mixture as a i r b o r n e p o l l u t a n t s c a n n o t be excluded. F o r each tested fraction at a given dose, we observed that the highest frequency of chrom o s o m a l a b e r r a t i o n was o b t a i n e d after 24-h treatment, although this t r e a t m e n t time theoretically exceeds 1 cell cycle for the V79 cells (average 1 0 - 1 2 h). This effect m a y be observed in an a s y n c h r o n o u s cell p o p u l a t i o n due to the mitotic cycle delay. This was noticed i n h u m a n l y m p h o cytes exposed to city-smog extracts b y H a d n a g y et al. (1986). It is possible that metabolic changes in the indicator V79 cells occurred d u r i n g the long exposure period leading to the f o r m a t i o n of m o r e active degradation products from the tested complex mixtures. Considering the h u m a n exposure to environm e n t a l genotoxic factors, the use of the Salmonella assay in c o m b i n a t i o n with the c h r o m o s o m a l aberration analysis is a m e t h o d of choice in a comparative e v a l u a t i o n of h u m a n health hazards related to a m b i e n t air pollution.
Acknowledgments W e t h a n k Prof. M. Sorsa a n d Prof. M. C h o r ~ y for constructive criticisms of the manuscript. W e are also i n d e b t e d to Mrs A. Kostowska for excellent technical assistance a n d Mrs G. Mafika for help in sampling. This work was s u p p o r t e d in part b y the Polish N a t i o n a l Cancer P r o g r a m PR-6, G r a n t No. 2101.
References Alfheim, I., G. L~Sfroth and M. Moller (1983) Bioassay of extracts of ambient particulate matter, Environ. Health Perspect., 47, 227-238. Alink, G.M., H.A. Smit, J.J. van Houdt, J.R. Kolkman and J.S.M. Boleij (1983) Mutagenic activity of airborne particulates at nonindustrial locations, Mutation Res., 116, 21-34.
Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella mammalian microsome mutagenicity test, Mutation Res., 31, 347-363. Dehnen, W., N. Pitz and R. Tomingas (1977) The mutagenicity of airborne particulate pollutants, Cancer Lett., 4, 5-12. De Ratt, W.K. (1983) Genotoxicity of aerosol extracts. Some methodological aspects and the contribution of urban and industrial locations, Mutation Res., 116, 47-63. De Wiest, F., D. Rondia, R. Gol-Winkler and J. Gielen (1982) Mutagenic activity of non-volatile organic matter associated with suspended matter in urban air, Mutation Res., 104, 201-207. Farcasiu, M. (1977) Fractionation and structural characterization of coal liquids, Fuel, 56, 9-14. Fukino, H., S. Mimura, K. Inone and Y. Yamane (1982) Mutagenicity of airborne particles, Mutation Res., 102, 237-247. Garner, R.C., C.A. Stanton, C.N. Martin, F.L. Chow, W. Thomas, D. Hubner and R. Herrmann (1986) Bacterial mutagenicity and chemical analysis of polycyclic aromatic hydrocarbons and some nitro derivatives in environmental samples collected in West Germany, Environ. Mutagen., 8, 109-117. Gibson, T.L. (1983) Sources of direct-acting nitroarene mutagens in airborne particulate matter, Mutation Res., 122, 115-121. Hadnagy, W., N.H. Seemayer and R. Tomingas (1986) Cytogenetic effects of airborne particulate matter in human lymphocytes in vitro, Mutation Res., 175, 97-101. Hollstein, M., and J. McCann (1979) Short-term tests for carcinogens and mutagens, Mutation Res., 65, 133-226. IARC (1983) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 32, Polynuclear Aromatic Compounds Part 1, Chemical, Environmental and Experimental Data, IARC, Lyon. Jacobs, F.S., and R.H. Filby (1983) Liquid chromatographic fraction of oil-sand and crude oil asphaltenes, Fuel, 62, 1186-1192. Krishna, G., J. Nath and T. Ong (1984) Correlative genotoxicity studies of airborne particles in Salmonella typhimurium and cultured human lymphocytes, Environ. Mutagen., 6, 585-592. Lockard, J.M., C.J. Viau, C. Lee-Stephens, J.C. Caldwell, J.P. Wojciechowski, H.G. Enoch and P.S. Sabharwal (1981) Induction of sister chromatid exchanges and bacterial revertants by organic extracts of airborne particles, Environ. Mutagen., 3, 671-681. Madsen, E.S, P.A. Nielsen and J.C. Pederson (1982) The distribution and origin of mutagens in airborne particulate, detected by the Salmonella/microsome assay in relation to level of lead, vanadium and PAH, Sci. Total Environ., 24, 13-25. Michalska, J. (1986) Chromosome aberrations induced by 9aminoacridine derivatives, Mutation Res., 170, 119-123. Moiler, M., and I. Alfheim (1980) Mutagenicity and PAHanalysis of airborne particulate matter, Atmosph. Environ., 14, 117-119.
296 Mailer, M., and G. Lt~froth (1982) Genotoxic components in polluted air, in: Mutagens in our Environment, Alan R. Liss, New York, pp. 221-238. Motykiewicz, G.~ G. Mahka, B. Cimander and M. Chor~£y (1985) Mutagenic activity in airborne particulate pollutants at industrial district of Silesia, Bull. Pol. Acad. Sci., 33, 1-6. MUller, D., J. Nelles, E. Deparade and D. Arni (1980) The activity of $9 liver fraction from seven species in the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 70, 279-300. Natarajan, A.T., and G. Obe (1986) How do in vivo mammalian assays compare to in vitro assays in their abihty to detect mutagens?, Mutation Res., 167, 189-201. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Rosenkranz, H.S. (1982) Direct-acting mutagens in diesel exhaust: magnitude of the problem, Mutation Res., 101, 1-10. Sobels, F.H. (1985) Studies in comparative chemical mutagenesis, Environ. Mutagen., 7, 759-773.
Takeda, N., K. Teranishi and K. Hamada (1984) Mutagenicity of air pollutants collected at industrial, urban-residential and rural areas, Bull. Environ. Contam. Toxicol., 32, 688-692. Talkott, R., and E. Wei (1977) Airborne mutagens bioassayed in Salmonella typhimurium, J. Natl. Cancer Inst., 58, 449-451. Tokiwa, H., K. Morita, H. Takeyoshi, K. Takahashi and Y. Ohnishi (1977) Detection of mutagenic activity in particulate air pollutants, Mutation Res., 48, 237-248. Tokiwa, H., S. Kitamori, K. Takahashi and Y. Ohnishi (1980) Mutagenic and chemical assay of extracts of airborne particulates, Mutation Res., 77, 99-108. Tokiwa, H., S. Kitamori, K. Horikawa and R. Nakagawa (1983) Some findings on mutagenicity in airborne particulate pollutants, Environ. Mutagen., 5, 87-100. Walker, R.D., T.H. Connor, E.J. MacDonald, N.M. Trieff, M.S. Legator, K.W. MacKenzie, Jr. and J.G. Dobbins (1982) Correlation of mutagenic assessment of Houston air particulate extracts in relation to lung cancer mortality rates, Environ Res., 28, 303-312.
289
MTR 01240
Mutagenic and clastogenic activity of direct-acting components from air pollutants of the Silesian industrial region G. Motykiewicz 1, j. Michalska 1, j. Szeliga i and B. Cimander 2 I Department of Tumor Biology, Institute of Oncology, 44-100 Gliwice (Poland) and 2 District Sanitary-Epidemiologic Station, 44-075 Katowice (Poland)
(Received 13 January 1987) (Revision received 6 July 1987) (Accepted 20 July 1987)
Keywords: Pollutant, environmental; Mutagenic activity; Clastogenic activity.
Summary 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.
Epidemiological data have suggested an association of lung cancer mortality with the mutagenicity of airborne particulate pollutants, assayed by the Ames test (Walker et al., 1982). Urban air, particularly in industrial regions, contains many known mutagens and carcinogens (IARC, 1983). Only a small part of the observed mutagenic activity can be explained by 'classical' polycyclic aromatic hydrocarbons (PAHs) a n d / o r their known direct-acting derivatives, e.g., nitropyrenes (Rosenkranz, 1982; Gibson, 1983). Most of the mutagens present in the total extractable organic matter, and acting directly, have not been identified. Several studies have shown that mutagenic Correspondence: Dr. G. Motykiewicz, Department of Tumor Biology, Institute of Oncology, Wybrze~:e Armii Czerwonej 15, 44-100 Gliwice (Poland).
activity was present in the polar fractions not containing conventional PAHs (Dehnen et al., 1977; Tokiwa et al., 1980; De Wiest et al., 1982; Alfheim et al., 1983). Great methodological variations exist between studies since no standardized fractionation protocol has been established. However, Meller and Lrfroth (1982), in a review of genotoxic air pollutants, have recommended high-performance liquid chromatographic (HPLC) methods. The Ames Salmonella assay is a useful tool for identification of the mutagenic potency of air pollutants (Tokiwa et al., 1977; Moller and Alfheim, 1980; Fukino et al., 1982; Madsen et al., 1982; Tokiwa et al., 1983; Garner et al., 1986), although as a prokaryotic test it is not quite adequate to risk estimation for humans. Up to now, only a few studies with mammalian cells have been carried
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
290
out. The organics associated with air particulate matter have mainly been examined by sister-chromatid exchange (SCE) tests (Lockard et al., 1981; Alink et al., 1983; De Raat, 1983), whereas induction of chromosomal aberrations (CAs) in human lymphocytes was presented only by Krishna et al. (1984) and Hadnagy et al. (1986). In our earlier studies (Motykiewicz et al., 1985) benzene extracts of airborne particulate material, collected at 23 locations in the industrial Silesian region during a 6-month period were evaluated in the Salmonella/microsome system with TA100 and TA98 strains. We observed seasonal variations in mutagenic activity of samples collected in summer and winter. Crude extracts derived from winter samples showed high direct mutagenic activity. In the present paper we have further used the Ames Salmonella assay with strain TA100, together with a chromosome aberration test in V79 cells, for examination of the direct-acting fractions obtained by sequential elution solvent chromatography (SESC) of benzene extracts of airborne particulates collected in highly polluted locations in Silesia (December 1984 and 1985). Materials and methods
Airborne samples Particulate matter was collected on fiber-glass filters (Staplex Co.) by Staplex high-volume samplers at a flow rate of about 90 m3/h. Samples were taken at 8 'high-pollution' locations (Motykiewicz et al., 1985), 6 × 24 h / m o n t h in December
1984 and 1985. After sampling, filters were combined and then organic matter was extracted in a Soxhlet apparatus with benzene for 7 h. The solvent was removed to dryness by rotary evaporator and the extraction yield was determined. Samples were stored under gaseous nitrogen at - 20 o C.
Sequential elution solvent chromatography (SESC) The method developed by Farcasiu (1977) and modified by Jacobs and Filby (1983) for the separation of coal liquefaction products was applied to the fractionation of benzene extracts. This scheme used silica gel and a solvent sequence that provided a systematic variation in the Hildebrand solubility parameters and the specific solubility parameters (Table 1). The separation was performed on a silica gel column (80 x 0 . 8 cm ID) containing 25 g of Koch-Light 5000 h silica gel 'Davison', 100-200 mesh. The silica gel was dried and the water content was adjusted to 4% by weight. The sample (0.2 g) was first loaded onto glass beads, 60 mesh. The coated glass beads were then poured over the silica gel column. For elution of each fraction, 200 ml of each solvent were used. The collected fractions were evaporated to dryness by rotary evaporator at 50°C, weighed and stored under gaseous nitrogen at - 20 o C.
Mutagenicity testing The test was carried out according to the procedure of Ames et al. (1975). The bacteria used were
TABLE 1 SEQUENTIAL ELUTION SOLVENT CHROMATOGRAPHY (SESC) SCHEME The method was developed and the compound types were identified by Farcasiu (1977). Fraction Number
Eluent
Compound types eluted
I II III IV V VI VII VIII
Hexane 15% Toluene in hexane " Chloroform 10% Diethylether in chloroform 3% Ethanol in diethylether Methanol 3% Ethanol in chloroform 3% Ethanol in tetrahydofuran
Saturated hydrocarbons Aromatic hydrocarbons Polar aromatics, non-basic N, S, O heterocyclics Monophenols Basic nitrogen heterocyclics Highly functional molecules containing heteroatoms at 10% by weight Polyphenols Molecules with high O and N content
a Modification by Jacobs and Filby (1983).
291
Salmonella typhimurium strain TA100, kindly supplied by Prof. B.N. Ames. Because our earlier studies, performed with strains TA100 and TA98 indicated that strain TA100 was more sensitive (Motykiewicz et al., 1985), this strain was used in all experiments. Studies were performed in the presence and absence of an Aroclor 1254-induced rat liver microsomal fraction ($9). The protein content of the activation mixture, the so-called $9 mix, varied from 6.6 to 6.8 m g / m l (Miiller et al., 1980) and 0.5 ml of $9 mix was applied per plate. Samples dissolved in DMSO were tested in duplicate for each concentration in 2 independent experiments. Benzo[a]pyrene and 1-nitro-9-Y-[NN-dimethyl-aminopropyl]aminoacridine were used as positive control mutagens. Determination of mutagenic response was based on criteria recommended by Ames et al. (1975).
wt%
40 35 30 25 20
,,\\
10 5
I
II
III
I~
V
'f-i ~/II "4111
Fraction No. Fig. 1. Relative weight distribution of SESC fractions of samp l e s collected in December 1984 and 1985. Mean v a l u e s + standard deviation.
TA100) for crude extract and SESC fractions of samples collected in December 1984. Mutagenic activity was observed in fraction Nos. II-VI. TABLE 2 M U T A G E N I C I T Y O F SEPARATE SESC F R A C T I O N S The number of revertants per /~g of crude extract or SESC fractions, and per m 3 of air was calculated from a dose of 125 /~g/plate (linear parts of the curves, Fig. 2). Fraction N umbe r
Results
Benzene extracts of airborne particulates were separated into 8 fractions by the SESC system. Fig. 1 shows the relative weight distribution of the obtained fractions. Samples from December 1984 and December 1985 gave very similar fractionation profiles. Fraction Nos. I I - V comprised 84% of the extracts by weight, and the major types of chemical compounds expected in these fractions are given in Table 1. Fig. 2 presents results of the Ames test (strain
\
\
/
15
Chromosome aberration analysis The CA test was performed according to the procedure described previously (Michalska, 1986). Briefly, the appropriate amounts of test fractions dissolved in 50 #1 DMSO were added per plate to exponentially growing V79 cells in 10 ml of Dulbecco's MEM medium supplemented with 10% fetal calf serum, for 5, 14, and 24 h. Fraction No. III was appfied in 100 /~1 due to low solubility. DMSO was used as control. Colchicine at a final concentration of 0.25 /~g/ml was added for 2 h before harvesting the cells. Preparations were made by the air-drying method and were stained by Giemsa. 200 or 300 metaphases were examined for each experimental point, and the chi-square test was used to analyze the chromosome aberration frequency.
/// / .// /// /// ../, /// /// ///
Calculated number of revertants for TA100 per ~tg - $9
per m 3 + $9
- $9
+ $9
2.56
3.32
0.10
0.00
4.41 3.61 3.28 1.68 1.40 0.10 0.13
186.5 0.5 17.9 47.0 51.2 14.1 5.6 0.2 0.1
241.5 0.2 38.3 91.1 53.5 15.4 9.5 0.1 0.1
-
136.6
208.0
Crude extract I
II III IV V VI VII VIII Sum of fractions
2.06 1.86 3.14 1.54 0.83 0.22 0.23 -
292
Fraction
Nos. II and III showed both a direct and an indirect mutagenic activity. In these fractions, however, the higher doses (250 and 500/~g/plate) I&l cI c~
gave a nonlinear response especially with S9 mix. This effect is most probably due to the specific toxicity of material sampled in winter. Under the
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800 700 600 500
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400
300
n:y
200
100
Ft. No.IV
800
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Ft. No, V
12o
Fr. No.VI
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600 500 400
: 10
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300 200 100 i
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i
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i
Ft. No.Vii
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300 20O
i I
100 100 200 300 400 500
100 200 300 400 500
mo 2oo 30o 4o0 5oo j.tglpiote
Fig. 2. Mutagenic activities of SESC fractions of air-pollutant benzene extracts, collected in December 1984; S. typhimurium TA100, O) and without $9 ( O O); subtracted spontaneous revertant backgrounds were: 103-151 ( - $ 9 ) and 113-156 ( + $9). Hatched columns represent the contribution of each fraction, in / ~ g / m 3 of air. with $9 (o . . . . . .
293 same
experimental
centration summer,
conditions
(the
same
con-
Nos. II and III (Motykiewicz
of $9 mix used) for samples collected in we
dose-related
observed
a linear
response
for
or
almost
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of
aration).
linear
The
(Nos. IV-VI)
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other
and Szeliga, in prep-
genotoxically
showed
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only direct mutagenic
activ-
ity.
TABLE3 C H R O M O S O M A L A B E R R A T I O N S P R O D U C E D IN V79 CELLS BY SESC F R A C T I O N S Fraction and dose ( / l g / m l ) III
IV
V
0 5 0 ( 2 . 3 m 3) 1~ 2~
Time (h)
Mitotic index (%)
Aberrant metaphases + G (%)
Aberrant metaphases - G (%)
Chromosomal aberrations/100 cells End CdG CdB Ex C1G C1B
Dic
R
P (Ab - G)
5
6.2 7.4 5.6 3.0
1.0 0.5 2.5 3.5
0.5 0 1.0 2.0
0 0 0 0
0.5 0.5 1.5 1.5
0 0 1.0 1.5
0 0 0 0
0 0 0 0
0 0 0 0
0.5 0 0 0.5
0 0 0 0
N N N N
0 50 100 200
14
5.2 4.9 2.5 1.1
2.5 1.5 3.0 7.0
1.5 0.5 3.0 4.5
0 0.5 0.5 1.0
0.5 1.0 1.5 2.5
1.0 0 1.0 3.0
0.5 0 0.5 0.5
0 0 0 0
0 0 0 0
0 0 0 0
0 0 1.0 0
N N N N
0 50 100 200
24
7.3 4.0 3.5 0.9
2.0 3.0 7.5 16.5
1.0 1.5 4.0 12.5
0 0 0 4.0
1.0 1.5 3.5 4.0
1.0 1.5 3.0 4.5
0 0 0.5 1.5
0 0 0.5 0
0 0 0 0
0 0 0.5 3.0
0 0 0.5 0
N N N <0.001
5
4.0 2.5 1.2
2.0 2.0 2.3 ~w ~toses
1.0 1.3 1.0
0 0 0
0.3 0.7 1.0
1.0 1.3 1.0
0 0 0
0.7 0 0.3
0 0 0
0 0 0
0 0 0
N N N
0 50 1~ 200
14
4.7 2.1 0.8
1.3 1.7 5.3 no ~ t o s e s
1.0 1.7 4.3
0 0 0.7
0.3 0 1.0
1.0 1.3 4.3
0 0 0
0 0 0
0 0 0.3
0 0.3 0
0 0 0.7
N N <0.025
0 50 1~ 200
24
4.1 2.5 1.5
2.3 8.7 8.3 no ~ t o s e s
1.7 6.0 6.7
0 2.3 1.0
0.3 2.7 1.7
0.7 2.7 4.3
0 2.0 3.0
0.3 0 0
0.3 0.3 0
0.7 0.7 0.3
0 0.3 0.7
N <0.01 <0.01
5
7.3 8.0 5.6 1.9
1.5 1.5 2.5 11.0
0.5 0.5 1.0 6.5
0 0 0 1.5
1.0 1.0 1.0 4.5
0.5 0.5 1.0 3.0
0 0 0 1.0
0 0 0 0
0 0 0 0
0 0 0.5 0
0 0 0 1.0
N N N <0.001
0 50 100 200
14
7.0 5.8 3.8
1.5 2.5 3.5 few ~ t o s e s
1.0 1.5 2.0
0.5 1.0 0
0.5 1.5 1.5
0.5 1.0 1.0
0 0 0.5
0 0 0
0 0 0
0 0 0.5
0 0 0.5
N N N
0 50 100 200
24
7.9 6.6 4.6
2.0 6.0 7.3 no ~ t o s e s
1.7 5.0 5.7
0 3.0 1.0
0.3 1.0 2.0
1.3 1.7 5.7
0 0 0.7
0.3 0 0
0 0 0
0 0 0
0.3 0.3 0.3
N <0.025 <0.01
0 5 0 ( 2 . 2 5 m 3) 1~ 2~
0 50(7.6 ~ ) 100 2~
G, gaps; End, Endoreduplication; CdG, chromatid gaps; CdB, chromatid breaks; Ex, chromatid interchanges; C1G, chromosomal gaps; C1B, chromosomal breaks; Dic, dicentrics; R, Rings. N, not significant statistically, P was calculated from the n u m b e r of aberrant metaphases without gaps.
294
The recovery of mutagenic activity (Table 2) was slightly decreased for the sum of the mutagenic activity of the separate fractions when compared to the activity of the unfractionated sample (crude extract). The number of revertants per/~g and m 3 of air was calculated from the linear part of the curves (Fig. 2). The results of the Ames test led to the selection of 'direct' fractions for examination for clastogenic potential in the CA test. A pilot experiment was carried out only with fraction No. IV from the December 1984 sample because the amount of material was limited. In this experiment the number of aberrant metaphases for a 24-h treatment with 100 ~tg/ml was statistically significant ( P < 0.01). The main experiment for CA (sample from December 1985) was performed with 3 selected fractions and the results are presented in Table 3. All of the tested fractions showed clastogenic activity that increased with prolonged treatment time. However, for 24-h exposures, toxic effects were observed at a dose of 200 # g / m l , which was comparable to the toxicity obtained with 500 /~g/plate in the Ames test. Only fraction No. V exhibited statistically significant clastogenic effects for the shortest treatment time (5 h). In each of the tested samples a high number of endoreduplicated cells was present; this result was especially characteristic of fraction No. III (24-h exposure). Only 1 endoreduplicated metaphase was noticed in all of the analyzed control cells. Discussion
Extracts from airborne particulate pollutants present a complex mixture and their preliminary separation provides more information about the mixture. Pollutants collected from the Silesian region mostly originate from coal combustion, encompassing industrial operations, coke production, power plants using fossil fuels and private domestic heating by coal. Therefore, benzene extracts of organic matter of air pollutants in this region may have a chemical resemblance to coal liquefaction products. It was assumed that a simple SESC method would be useful for separation of the air pollutant samples without simultaneous degradation of their genotoxic activity. Five out of 8 obtained fractions showed a dif-
ferential potency between direct and indirect mutagenic activity in the Salmonella test with strain TA100 (Fig. 2), expressed as the number of revertants per t~g and m 3 of air (Table 2). It should be noted that the recovery of mutagenic activity from the silica column was satisfactory; however, some material was irreversibly adsorbed on the silica as found by Farcasiu (1977) and Jacobs and Filby (1983). The highest direct mutagenic activity detected in the Ames test was shown by fraction No. IV, which probably contained monophenols (Table 1). The observed reduction in the mutagenicity by $9 addition was noted earlier, and unspecific reactions between active mutagens and nucleophiles presented in the S9 mix were suggested (Dehnen et al., 1977; Talkott and Wei, 1977; Takeda et al., 1984). The results from the CA test demonstrated the highest number of aberrant metaphases at a dose of 100 ~ g / m l for fraction No. IV (Table 3), which correlated well with the Ames test. For the other direct mutagenic fractions (Nos. V and VI), clastogenic effects could only be determined for fraction No. V because of the low amount of material available in fraction No. VI. Fraction No. III was examined for CA induction even though the Ames test for this material showed both direct and indirect activity. This fraction was chosen for CA testing due to its high weight percentage content in the total extract and therefore its quantitatively significant contribution to air pollution. Organic extracts of airborne particulate matter induced increases in SCEs in human lymphocytes and rodent cells (Lockard et al., 1981; Alink et al., 1983; Hadnagy et al., 1986). While the CA test compared with SCE induction appears to be less sensitive (Perry and Evans, 1975), the complementation of bacterial mutagenicity assays by CA tests revealing structural chromosomal damage in mammalian cells seems to be justified. It should be mentioned that SCEs would be comparable to gene mutation tests; therefore, chromosomal aberration assays were recommended as a useful addition to the Ames test for the detection of a genotoxic hazard (Sobels, 1985; Natarajan and Obe, 1986). Little information exists on the clastogenic effects of air pollutants measured by the CA test
295 a n d these studies were p e r f o r m e d with h u m a n lymphocytes ( K r i s h n a et al., 1984; H a d n a g y et al., 1986). I n experiments carried out b y K r i s h n a et al. (1984), h u m a n lymphocytes were exposed to very high doses of extracted material, b u t only for the S a n d G 2 phases of the cell cycle. Obviously, most k n o w n chemical clastogens are S-phase d e p e n d e n t (Hollstein a n d M c C a n n , 1979), b u t the presence of other clastogen types in such a complex mixture as a i r b o r n e p o l l u t a n t s c a n n o t be excluded. F o r each tested fraction at a given dose, we observed that the highest frequency of chrom o s o m a l a b e r r a t i o n was o b t a i n e d after 24-h treatment, although this t r e a t m e n t time theoretically exceeds 1 cell cycle for the V79 cells (average 1 0 - 1 2 h). This effect m a y be observed in an a s y n c h r o n o u s cell p o p u l a t i o n due to the mitotic cycle delay. This was noticed i n h u m a n l y m p h o cytes exposed to city-smog extracts b y H a d n a g y et al. (1986). It is possible that metabolic changes in the indicator V79 cells occurred d u r i n g the long exposure period leading to the f o r m a t i o n of m o r e active degradation products from the tested complex mixtures. Considering the h u m a n exposure to environm e n t a l genotoxic factors, the use of the Salmonella assay in c o m b i n a t i o n with the c h r o m o s o m a l aberration analysis is a m e t h o d of choice in a comparative e v a l u a t i o n of h u m a n health hazards related to a m b i e n t air pollution.
Acknowledgments W e t h a n k Prof. M. Sorsa a n d Prof. M. C h o r ~ y for constructive criticisms of the manuscript. W e are also i n d e b t e d to Mrs A. Kostowska for excellent technical assistance a n d Mrs G. Mafika for help in sampling. This work was s u p p o r t e d in part b y the Polish N a t i o n a l Cancer P r o g r a m PR-6, G r a n t No. 2101.
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Takeda, N., K. Teranishi and K. Hamada (1984) Mutagenicity of air pollutants collected at industrial, urban-residential and rural areas, Bull. Environ. Contam. Toxicol., 32, 688-692. Talkott, R., and E. Wei (1977) Airborne mutagens bioassayed in Salmonella typhimurium, J. Natl. Cancer Inst., 58, 449-451. Tokiwa, H., K. Morita, H. Takeyoshi, K. Takahashi and Y. Ohnishi (1977) Detection of mutagenic activity in particulate air pollutants, Mutation Res., 48, 237-248. Tokiwa, H., S. Kitamori, K. Takahashi and Y. Ohnishi (1980) Mutagenic and chemical assay of extracts of airborne particulates, Mutation Res., 77, 99-108. Tokiwa, H., S. Kitamori, K. Horikawa and R. Nakagawa (1983) Some findings on mutagenicity in airborne particulate pollutants, Environ. Mutagen., 5, 87-100. Walker, R.D., T.H. Connor, E.J. MacDonald, N.M. Trieff, M.S. Legator, K.W. MacKenzie, Jr. and J.G. Dobbins (1982) Correlation of mutagenic assessment of Houston air particulate extracts in relation to lung cancer mortality rates, Environ Res., 28, 303-312.