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Chemical characterization of direct-acting airborne mutagens: The functional group
Mutation Research, 91 (1981)433-436 Elsevier/North-Holland BiomedicalPress
433
C H E M I C A L C H A R A C T E R I Z A T I O N OF D I R E C T - A C T I N G A I R B O R N E MUTAGENS: THE FUNCTIONAL GROUP
RONALD E. TALCOTT and WILLIAM HARGER Division of Toxicology and Physiology, Department of Entomology, Universityof California, Riverside, CA (U.S.A.)
(Received 25 March 1981) (Accepted 7 April 1981) SUMMARY Durham NC air was sampled, extracted, and bioassayed for mutagenic activity in Salmonella typhimurium TA98. Portions o f the extracts were treated with sodium borohydride over copper(II) acetylacetonate to reduce any nitroaromatic substances to their corresponding amines. All o f the reduced extracts were not directly mutagenic, but the 2 that were derived from cold-weather air samplings did contain substances that could be activated oxidatively. These "indirect-acting" mutagens were present in the same 2 reduced extracts that contained detectable concentrations o f aromatic amines. These results suggest that a major portion o f the total mutagenic activity in air-pollution particles is contributed by nitro-substituted compounds that are detectable as their corresponding amines. They also suggest that the atmospheric concentrations o f these substances may be high in the colder months of Durham's year.
Direct-acting frameshift mutagens have been detected in city air [1,2,6,7,10] on pollutant particles small enough to penetrate into the parenchyma of human lungs [8,9]. Hence it is possible that air pollution and lung cancer are meaningfully associated, as suggested by the epidemiological interpretations of Pike et al. [5], but a satisfactory assessment cannot be accomplished prior to an understanding of the chemical nature of the airborne mutagenic substances. The nature o f the functional group involved in the mutagenic mechanism is the subject o f this report. The organic matter extracted from city air is preferentially mutagenic to the Salmonella typhimurium tester strain TA98 [1,2,6,7,10], as are many nitroaromatic compounds [4]. The aromatic amines derived from nitroaromatics are nonmutagenic unless the assay mixture is supplemented with liver oxidases, so, if nitroaromatics are responsible for the liver-independent (direct-acting) mutagenic activity of 0165-7992/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
434 city air, this a c t i v i t y s h o u l d d i s a p p e a r w h e n t h e n i t r o g r o u p s a r e fully r e d u c e d . T o d e t e r m i n e w h e t h e r o r n o t this w o u l d o c c u r , w e t r e a t e d 5 m u t a g e n i c a i r - p o l l u t i o n e x t r a c t s w i t h a r e d u c i n g a g e n t [3], a n d a s s a y e d t h e r e d u c e d e x t r a c t s f o r r e s i d u a l mutagenic activity. P a r t i c u l a t e air p o l l u t i o n s a m p l e s f o r this s t u d y w e r e c o l l e c t e d at t h e U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y ' s s a m p l i n g site in D u r h a m , N C , e x t r a c t e d w i t h a c e t o n e , a n d a n a l y z e d a c c o r d i n g to t h e p r o t o c o l p r e s e n t e d in F i g . 1. I n s u m m a r y , a p o r t i o n of each acetone extract was tested for primary aromatic amines and bioassayed for m u t a g e n i c i t y , t h e n a s e c o n d p o r t i o n was t r e a t e d w i t h s o d i u m b o r o h y d r i d e o v e r a c o p p e r ( I I ) c a t a l y s t to r e d u c e t h e n i t r o a r o m a t i c s u b s t a n c e s to t h e i r c o r r e s p o n d i n g a m i n e s [3]. F o l l o w i n g r e d u c t i o n , e a c h e x t r a c t was t e s t e d f o r a m i n e s a n d b i o a s s a y e d
Collectiona
Extraction
and
I
Concentration b
I
I
Mutagenicity bioassay d
Amine spot test c
Reduction
I
Amine spot test c
I
Mutagenicity bioassay d
Fig. 1. Collection, extraction and assay of air pollution extracts. a Respirable-sized particles were collected from Durham NC air by drawing it through Andersen 2000 samplers continuously for 1 week. Large particles, deposited on the upper stages, were not analyzed; the respirable-sized particles, deposited on the backup filters, were extracted with acetone. bThe filters containing the deposited particles were extracted 6 h with acetone in a Soxhlet apparatus [7-9]. The acetone was removed in vacuo, and the residue was redissolved in either acetone (amine spot test) or dimethyl sulfoxide (mutagenicity bioassay) to a final concentration of l0 mg/ml. c 25 #l (250 #g) of the acetone-stock solutions were applied to silicic acid thin-layer chromatography plates and sprayed with nitrous acid followed by l-naphthol. The presence of aromatic primary amines was indicated by the sandy orange to burgundy color of the diazo products. The limit of sensitivity was estimated to be 2 moles of amine/spot. d Serial 1 : 1 dilutions of the DMSO stock solutions were tested for mutagenic activity toward S. typhimurium TA98 [7-9]. The presence of mutagenic substances was indicated by an increase in the rate of reversion to histidine prototroph. If this occurred in the absence of the liver 9000 x g supernatant enzymes, the mutagenic substances were termed, "direct-acting". e Extracts (10-50 mg) were reduced under nitrogen with copper acetylacetonate-sodium borohydride catalyst at 30°C for 4 h. The components of the mixture were added in the order described by Hanaya et al. [3].
435 TABLE 1 THE EFFECT OF C H E M I C A L REDUCTION ON THE MUTAGENICITY OF AIR POLLUTION EXTRACTS Sampling week, extract treatment
Amine content (+ or - )
Mutagenic activity (TA98 revertants/mg ext.)
$9 absent
$9 present a
August 9, 1979, untreated August 9, 1979, reduced
37 0
11 4
August 21, 1979, untreated August 21, 1979, reduced
74 0
0 9
October 16, 1979, untreated October 16, 1979, reduced
283 0
186 13
December 6, 1978, untreated December 6, 1978, reduced
+
322 8
205 175
January 15, 1980, untreated January 15, 1980, reduced
+
130 0
321 224
a $9 from Aroclor 1254 pretreated rats was purchased from Litton Bionetics, Inc.
for mutagenicity. Amines were detected with a spot test [11], which was scored positive or negative depending on whether diazotized products could be detected in 250/zg of extract. All mutagenicity bioassays were done with Salmonella typhimurium TA98 as the target organism, with or without supplementation with ratliver 9000 x g supernatant fraction. Chemical reduction of the 5 air-pollution extracts eliminated the liver-independent mutagenic activities and clearly produced amines in the 2 extracts derived from coldweather sampling (Table 1). Reduction may have produced amines in the other samples as well, but they were not detected by our spot test. The 2 "amine-negative" reduced extracts were not metabolically activated. The loss of mutagenic activity after reduction, and its retrieval by oxidative activation of amine-rich extracts, are observations that support the conclusion that nitroaromatic substances comprise a major portion of the total quantity of the direct-acting mutagenic substances extracted from Durham NC air-pollutant particles. The seasonal variability of the results, though too preliminary to justify a conclusion, does suggest the following explanation for the seasonal pattern of atmospheric mutagenicity that seems to prevail in Durham: The mutagenic burden increases in the winter months [9] as the air becomes enriched with nitroaromatic substances, possibly through increased fuel consumption. This hypothesis could be tested in Durham, and in other cities, by collecting air-particulate samples at various
436
times o f year a n d a n a l y z i n g t h e m according to the p r o t o c o l presented in Fig. 1. T h e results o f such a survey, w h e n correlated with energy-use data, c o u l d be i n t r i g u i n g . W e wish to t h a n k Lester Spiller a n d his associates at the A e r o s o l Section, U.S. E . P . A . D u r h a m , N C , for p r o v i d i n g us with the a i r - p o l l u t i o n samples, Dr. Bruce A m e s , U n i v e r s i t y o f C a l i f o r n i a , Berkeley, for p r o v i d i n g us with the Salmonella
typhimurium T A 9 8 , a n d the C a l i f o r n i a L u n g A s s o c i a t i o n for their s u p p o r t o f this project. REFERENCES 1 Commoner, B., P. Madyastha, A. Bronsdon and A.J. Vithayathil, Environmental mutagens in urban air particulates, J. Toxicol. Environ. Hlth., 4 (1978) 59-77. 2 Dehnen, W., N. Pitz and R. Tomingas, The mutagenicity of airborne particulate pollutants, Cancer Lett., 4 (1977) 5-12. 3 Hanaya, K., T. Muramatsu, H. Kudo and Y.L. Chow, Reduction of aromatic nitro compounds to amines with sodium borohydride-copper(II) acetylacetonate, J. Chem. Soc. Parkin Trans., (1979) 2409-2410. 4 McCan, J., E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the Salmonella microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 5 Pike, M.C., R.J. Gordon, B.E. Henderson, H.R. Mench and J. Sootloo, Air Pollution, in: J. Fraumeni (Ed.), Persons at High Risk of Cancer, Academic Press, New York, 1975, pp. 225-240. 6 Pitts, J.N., D. Grosjean, T.M. Mischke, V.F. Simmon and D. Poole, Mutagenic activity of airborne particulate organic pollutants, Toxicol. Lett., 1 (1977) 65-70. 7 Talcott, R.E., and E.T. Wei, Airborne mutagens bioassayed in Salmonella typhimurium, J. Natl. Cancer Inst., 58 (1977) 449-451. 8 Talcott, R.E., and W. Harger, Mutagenic activity of aerosol size fractions, E.P.A. Report No. 600/3-79-032, (1979). 9 Talcott, R.E., and W. Harger, Airborne mutagens extracted from particles of respirable size, Mutation Res., 79 (1980) 177-180. 10 Tokiwa, H., K. Morita, H. Takeyoshi, T. Kasumi and Y. Ohnishi, Detection of mutagenic activity of particulate air pollutants, Mutation Res., 48 (1977) 237-248. 11 Waldi, D., Spray reagents for thin layer chromatography, in: E. Stahl (Ed.), Thin Layer Chromatography, Academic Press, New York, 1965, p. 489.
433
C H E M I C A L C H A R A C T E R I Z A T I O N OF D I R E C T - A C T I N G A I R B O R N E MUTAGENS: THE FUNCTIONAL GROUP
RONALD E. TALCOTT and WILLIAM HARGER Division of Toxicology and Physiology, Department of Entomology, Universityof California, Riverside, CA (U.S.A.)
(Received 25 March 1981) (Accepted 7 April 1981) SUMMARY Durham NC air was sampled, extracted, and bioassayed for mutagenic activity in Salmonella typhimurium TA98. Portions o f the extracts were treated with sodium borohydride over copper(II) acetylacetonate to reduce any nitroaromatic substances to their corresponding amines. All o f the reduced extracts were not directly mutagenic, but the 2 that were derived from cold-weather air samplings did contain substances that could be activated oxidatively. These "indirect-acting" mutagens were present in the same 2 reduced extracts that contained detectable concentrations o f aromatic amines. These results suggest that a major portion o f the total mutagenic activity in air-pollution particles is contributed by nitro-substituted compounds that are detectable as their corresponding amines. They also suggest that the atmospheric concentrations o f these substances may be high in the colder months of Durham's year.
Direct-acting frameshift mutagens have been detected in city air [1,2,6,7,10] on pollutant particles small enough to penetrate into the parenchyma of human lungs [8,9]. Hence it is possible that air pollution and lung cancer are meaningfully associated, as suggested by the epidemiological interpretations of Pike et al. [5], but a satisfactory assessment cannot be accomplished prior to an understanding of the chemical nature of the airborne mutagenic substances. The nature o f the functional group involved in the mutagenic mechanism is the subject o f this report. The organic matter extracted from city air is preferentially mutagenic to the Salmonella typhimurium tester strain TA98 [1,2,6,7,10], as are many nitroaromatic compounds [4]. The aromatic amines derived from nitroaromatics are nonmutagenic unless the assay mixture is supplemented with liver oxidases, so, if nitroaromatics are responsible for the liver-independent (direct-acting) mutagenic activity of 0165-7992/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
434 city air, this a c t i v i t y s h o u l d d i s a p p e a r w h e n t h e n i t r o g r o u p s a r e fully r e d u c e d . T o d e t e r m i n e w h e t h e r o r n o t this w o u l d o c c u r , w e t r e a t e d 5 m u t a g e n i c a i r - p o l l u t i o n e x t r a c t s w i t h a r e d u c i n g a g e n t [3], a n d a s s a y e d t h e r e d u c e d e x t r a c t s f o r r e s i d u a l mutagenic activity. P a r t i c u l a t e air p o l l u t i o n s a m p l e s f o r this s t u d y w e r e c o l l e c t e d at t h e U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y ' s s a m p l i n g site in D u r h a m , N C , e x t r a c t e d w i t h a c e t o n e , a n d a n a l y z e d a c c o r d i n g to t h e p r o t o c o l p r e s e n t e d in F i g . 1. I n s u m m a r y , a p o r t i o n of each acetone extract was tested for primary aromatic amines and bioassayed for m u t a g e n i c i t y , t h e n a s e c o n d p o r t i o n was t r e a t e d w i t h s o d i u m b o r o h y d r i d e o v e r a c o p p e r ( I I ) c a t a l y s t to r e d u c e t h e n i t r o a r o m a t i c s u b s t a n c e s to t h e i r c o r r e s p o n d i n g a m i n e s [3]. F o l l o w i n g r e d u c t i o n , e a c h e x t r a c t was t e s t e d f o r a m i n e s a n d b i o a s s a y e d
Collectiona
Extraction
and
I
Concentration b
I
I
Mutagenicity bioassay d
Amine spot test c
Reduction
I
Amine spot test c
I
Mutagenicity bioassay d
Fig. 1. Collection, extraction and assay of air pollution extracts. a Respirable-sized particles were collected from Durham NC air by drawing it through Andersen 2000 samplers continuously for 1 week. Large particles, deposited on the upper stages, were not analyzed; the respirable-sized particles, deposited on the backup filters, were extracted with acetone. bThe filters containing the deposited particles were extracted 6 h with acetone in a Soxhlet apparatus [7-9]. The acetone was removed in vacuo, and the residue was redissolved in either acetone (amine spot test) or dimethyl sulfoxide (mutagenicity bioassay) to a final concentration of l0 mg/ml. c 25 #l (250 #g) of the acetone-stock solutions were applied to silicic acid thin-layer chromatography plates and sprayed with nitrous acid followed by l-naphthol. The presence of aromatic primary amines was indicated by the sandy orange to burgundy color of the diazo products. The limit of sensitivity was estimated to be 2 moles of amine/spot. d Serial 1 : 1 dilutions of the DMSO stock solutions were tested for mutagenic activity toward S. typhimurium TA98 [7-9]. The presence of mutagenic substances was indicated by an increase in the rate of reversion to histidine prototroph. If this occurred in the absence of the liver 9000 x g supernatant enzymes, the mutagenic substances were termed, "direct-acting". e Extracts (10-50 mg) were reduced under nitrogen with copper acetylacetonate-sodium borohydride catalyst at 30°C for 4 h. The components of the mixture were added in the order described by Hanaya et al. [3].
435 TABLE 1 THE EFFECT OF C H E M I C A L REDUCTION ON THE MUTAGENICITY OF AIR POLLUTION EXTRACTS Sampling week, extract treatment
Amine content (+ or - )
Mutagenic activity (TA98 revertants/mg ext.)
$9 absent
$9 present a
August 9, 1979, untreated August 9, 1979, reduced
37 0
11 4
August 21, 1979, untreated August 21, 1979, reduced
74 0
0 9
October 16, 1979, untreated October 16, 1979, reduced
283 0
186 13
December 6, 1978, untreated December 6, 1978, reduced
+
322 8
205 175
January 15, 1980, untreated January 15, 1980, reduced
+
130 0
321 224
a $9 from Aroclor 1254 pretreated rats was purchased from Litton Bionetics, Inc.
for mutagenicity. Amines were detected with a spot test [11], which was scored positive or negative depending on whether diazotized products could be detected in 250/zg of extract. All mutagenicity bioassays were done with Salmonella typhimurium TA98 as the target organism, with or without supplementation with ratliver 9000 x g supernatant fraction. Chemical reduction of the 5 air-pollution extracts eliminated the liver-independent mutagenic activities and clearly produced amines in the 2 extracts derived from coldweather sampling (Table 1). Reduction may have produced amines in the other samples as well, but they were not detected by our spot test. The 2 "amine-negative" reduced extracts were not metabolically activated. The loss of mutagenic activity after reduction, and its retrieval by oxidative activation of amine-rich extracts, are observations that support the conclusion that nitroaromatic substances comprise a major portion of the total quantity of the direct-acting mutagenic substances extracted from Durham NC air-pollutant particles. The seasonal variability of the results, though too preliminary to justify a conclusion, does suggest the following explanation for the seasonal pattern of atmospheric mutagenicity that seems to prevail in Durham: The mutagenic burden increases in the winter months [9] as the air becomes enriched with nitroaromatic substances, possibly through increased fuel consumption. This hypothesis could be tested in Durham, and in other cities, by collecting air-particulate samples at various
436
times o f year a n d a n a l y z i n g t h e m according to the p r o t o c o l presented in Fig. 1. T h e results o f such a survey, w h e n correlated with energy-use data, c o u l d be i n t r i g u i n g . W e wish to t h a n k Lester Spiller a n d his associates at the A e r o s o l Section, U.S. E . P . A . D u r h a m , N C , for p r o v i d i n g us with the a i r - p o l l u t i o n samples, Dr. Bruce A m e s , U n i v e r s i t y o f C a l i f o r n i a , Berkeley, for p r o v i d i n g us with the Salmonella
typhimurium T A 9 8 , a n d the C a l i f o r n i a L u n g A s s o c i a t i o n for their s u p p o r t o f this project. REFERENCES 1 Commoner, B., P. Madyastha, A. Bronsdon and A.J. Vithayathil, Environmental mutagens in urban air particulates, J. Toxicol. Environ. Hlth., 4 (1978) 59-77. 2 Dehnen, W., N. Pitz and R. Tomingas, The mutagenicity of airborne particulate pollutants, Cancer Lett., 4 (1977) 5-12. 3 Hanaya, K., T. Muramatsu, H. Kudo and Y.L. Chow, Reduction of aromatic nitro compounds to amines with sodium borohydride-copper(II) acetylacetonate, J. Chem. Soc. Parkin Trans., (1979) 2409-2410. 4 McCan, J., E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the Salmonella microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 5 Pike, M.C., R.J. Gordon, B.E. Henderson, H.R. Mench and J. Sootloo, Air Pollution, in: J. Fraumeni (Ed.), Persons at High Risk of Cancer, Academic Press, New York, 1975, pp. 225-240. 6 Pitts, J.N., D. Grosjean, T.M. Mischke, V.F. Simmon and D. Poole, Mutagenic activity of airborne particulate organic pollutants, Toxicol. Lett., 1 (1977) 65-70. 7 Talcott, R.E., and E.T. Wei, Airborne mutagens bioassayed in Salmonella typhimurium, J. Natl. Cancer Inst., 58 (1977) 449-451. 8 Talcott, R.E., and W. Harger, Mutagenic activity of aerosol size fractions, E.P.A. Report No. 600/3-79-032, (1979). 9 Talcott, R.E., and W. Harger, Airborne mutagens extracted from particles of respirable size, Mutation Res., 79 (1980) 177-180. 10 Tokiwa, H., K. Morita, H. Takeyoshi, T. Kasumi and Y. Ohnishi, Detection of mutagenic activity of particulate air pollutants, Mutation Res., 48 (1977) 237-248. 11 Waldi, D., Spray reagents for thin layer chromatography, in: E. Stahl (Ed.), Thin Layer Chromatography, Academic Press, New York, 1965, p. 489.