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The mutagenicity of airborne particulate pollutants
Cancer Letters, 4 (1977) 5--12 © Elsevier/North-Holland Scientific Publishers Ltd.
5
THE MUTAGENICITY OF AIRBORNE PARTICULATE POLLUTANTS
WALTER DEHNEN, NORBERT PITZ and RENI~ TOMINGAS Medizinisches Institut fi~r Lufthygiene und Silikoseforschung an der Universitdt D~isseldorf, Gurlittstrasse 53, D-4000 Dffsseldorf (G.F.R.)
(Received 19 July 1977) (Revised version received 18 October 1977) (Accepted 19 October 1977)
SUMMARY
The mutagenic effect of extracts derived from airborne particulate matter was investigated with the Ames-test. The whole extract proved to be distinctly mutagenic. To find out whether the content of polycyclic aromatic hydrocarbons may be responsible for the mutagenic effect, the extract was split into several fractions. The fraction containing the polycyclic compounds showed the lowest mutagenic rate that was enzymatically mediated. The other fractions required metabolic activation for some of their components, but some components were active without metabolic activation.
INTRODUCTION
Epidemiological studies suggest a relationship between the particulate matter in the ambient air and the incidence of human bronchus cancer [3,4,6]. The main cancerogenic compounds in airborne particles are thought to be the polycyclic aromatic hydrocarbons (PAH), particularly benzo(a)-pyrene [3,10]. The importance with respect to cancerogenic effects of most of the substances associated with airborne particles is not clear. The bacterial test system introduced by Ames allows rapid detection of mutagens [1]. Initially their test was applied to single compounds [5]. The application was later extended to extracts of particulate matter [7--9]. Talcot and Wei [7] suggest that most of the mutagenic activity of the particulate matter can be attributed to the presence of PAH. Samples of air soot, however, proved to be highly active although no benzo(a)pyrene Could be detected [7]. The correlation between the benzo(a)pyrene content in airborne particulates and mutagenicity was not always positive [8]. These findings, partly contradictory, demand further investigation. The aim of the study presented here was to assess the mutagenic activity in extracts of particulates and that of its fractions with special attention to the PAH-fraction.
MATERIALSAND METHODS 1. Chemicals Methanol, cyclohexene, alumina 90 neutral (Merck, Darmstadt, G.F.R.), were used for extraction and column chromatography. Clophen C, a mixture of polychlorinated biphenyls similar to Aroclor, was a generous gift from Bayer AG, Leverkusen. Histidine was obtained from Merck, glucose 6-phosphate, glucose-6-phosphate
Extract GEX1 j
Liquid--liquidpartition i
Cycl0hexenephase CYCI
\
[Methanol phase MET1
I Column chromatography [Fraction PAHI
]FractionPRO1
Scheme 1. Fractionation of the extract from airborne particulate matter.
were eluted with propanol-2 (fraction PRO). This fraction contains, among a large number of unknown components, aza-heterocyclic-compounds.
5. Analysis PAH were estimated according to the procedure already described [10]. The m e t h o d is based on column chromatography followed b y thin-layer chromatography and direct fluorescence measurement on the plate. 6. Araes test To obtain comparable concentrations of the extracted components in samples to be examined, the volume of all fractions was adjusted to equal the volume of the original whole extract (GEX), usually 180 ml, Thus the concentration of the particular substances remains constant in the subfractions. Aliquots of the fractions prepared in this manner were a d d e d to the tester strain. The mutagenicity test with Salmonella typhimurium was performed as described in detail by Ames et al. [2]. In this study we followed strictly the prescriptions according to Ames. The liver microsomes were obtained from rats pretreated with Clophen C. The tester strains used were TA 1537, TA 1538 and TA 98. Tests were run also with TA 100, b u t no reproducible results were obtained and therefore this strain was discarded. Preliminary experiments showed no mutagenic effect of air pollution extracts in the strain TA 1535, which is susceptible to base pair mutagens. This result is in accordance with the findings of Tokiwa et al. [9]. Dilutions from the extracts and fractions were made with ethanol, and aliquots of 0.1 ml per plate were taken. A volume of 0.1 ml of the original extract (not diluted) refers to 14 m 3 air volume. 7. Plots The values were plotted. In these plots the figures below the abscissa do not represent a certain factor like benzo(a) pyrene content, air volume or residue of the extract, since the question respecting the factor most suited for standardizing is still open. Therefore, the figures stand for a concentration. The original (undiluted) concentration has the value '8' and refers to 0.1 ml extract equal to 14 m 3 air volume and equal to 0.27 pg benzo(a)pyrene in GEX, CYC and PAH fractions. In some cases the original extract concentration was doubled. The curve for the revertants demonstrate the total n u m b e r without deducting the number o f spontaneous revertants. This n u m b e r is plotted for each assay as a line for a better view o f the mutagenic effect. RESULTS
1. The mutagenic activity o f airborne particulate pollutants with metabolic activation In Figs. 1--3 the mutagenic effect o f the whole extract (GEX) and its fractions MET, CYC, PAH and PRO is demonstrated. In all of the three tester
LLI
400
o SPONI REV.TA98 GEX "MET
•
~360 J
'~ CYC • PAH DPRO
~'~ 320 ~m:;280 240 200 160 120 80 40 0 "
" ~"~b'~
c
Fig. i. Mutagenic activity of air pollution extract GEX and its fractions MET, CYC, PAH and PRO in S. typhimurium TA 98 (in the presence of the complete activating system).
ILl 400
o SPONT.REV. TA 1537 • GEX • MET
' .U 360' ' O,_ 320
z~
CYC
PAH
W
r ( 280
D PRO
240 .
2oo4 120 ~
40 0
• i
loo
,
2
" ~1~1
~
" 5ib~
c
Fig. 2. Mutagenie activity of air pollution extract GEX and its fractions MET, CYC, PAH and PRO in S. typhimurium TA 1537 (in the presence of the complete activating system).
LL1400 |
n
o SPONT.REV.TA 1538 • GEX • MET CYC • PAH PRO
"
320. W rr280 240 200 160 120 80 40 0
@
0
0
'
'
O
'
'
c
Fig. 3. Mutagenic activity of air pollution extract GEX and its fractions MET, CYC, PAH and PRO in S. typhimurium TA 1538 (in the presence of the complete activating system). strains, TA 1537, TA 1538 and TA 98, the highest activity is found in the whole extract GEX. After liquid--liquid partition of GEX between cyclohexene and methanol/water about 70% of the activity can be extracted into the cyclohexane phase CYC. The rest is within the methanol phase. Column chromatography on alumina with cyclohexene as eluant transfers the smaller part of the mutagenic effect into the PAH containing fraction PAH. The much larger effect is found in the more polar fraction PRO.
2. The mutagenic activity of airborne particulate pollutants with and without metabolic activation Mutagenic and cancerogenic substances either act directly or require prior metabolism to produce active metabolites. To estimate the relation of these t w o groups present in the particulates, samples were applied to the bacteria w i t h o u t S-9 (liver microsomes and cytoplasma), with S-9 and with S-9 and NADPH together. The results are shown in Table 1 for the tester strains TA 1537 and TA 98, and the corresponding dose-response curves in Figs. 4 and 5. A distinct proportion of the whole extract GEX and the fractions MET, CYC and PRO have a mutagenic effect w i t h o u t metabolic activation. This direct acting mutagenicity is predominant in the fraction MET. In the polycyclic aromatic hydrocarbon containing fraction PAH most of the mutagenic activity requires metabolic activation, which is characteristic for these c o m p o u n d s [ 1]. In some cases the extracts cause toxic effects as judged by the inhibition of the background lawn o f bacterial growth [2] (fractions MET, PAH). The addition
10 TABLE 1 E F F E C T O F E N Z Y M A T I C A C T I V A T I O N ON E X T R A C T I N D U C E D M U T A G E N I C I T Y
Strain
Fraction
S 9 omitted
+S9
+ S 9 + NADPH
T A 98
GEX MET CYC PAH PRO
89 ?* 55 3 22
58 38 38 10 15
205 64 197 93 128
T A 1537
GEX MET CYC PAH PRO
39 ?* 25 ?* 3
70 60 38 8 22
122 80 165 93 80
The figures are the n u m b e r o f revertants after subtraction of s p o n t a n e o u s revertants. Sample Duisburg, 2nd sampling site. C o n c e n t r a t i o n o f CYC, PAH, P R O t w o - f o l d of G E X and MET. S p o n t a n e o u s revertants (s.d.): T A 9 8 : 1 4 ± 4 (S 9 o m i t t e d o r added); 29 -+ 7 (8 9 + N A D P H ) . T A 1 5 3 7 : 1 0 -+ 4 (all conditions). * Toxic effect of extract.
LL1100
o SPONT. REV. TA 98 • GEX • MET
~_..1 9o 13_ "~ 80
•
W n " 70
CYC PAH PRO
60 50 40 30 2O 0
[~
0
,0
'
10 0
• 1
i0 o
,
~
. . . . . .
5
i
101
"
c
Fig. 4. Direct m u t a g e n i c activity o f air p o l l u t i o n e x t r a c t G E X and its subfractions MET, CYC, P A H and P R O in S. typhimurium T A 98 (in the presence o f S-9, b u t w i t h o u t N A D P H )
11 o SPONT.REV. TA 1537 • GEX • MET
LL1100
d 9o n
/
t.U cr 7O 6O
CYC •
P A H
I
PRO
5O 40 3O 20 10 0
• 1
10o
.
.
.
.
.
5
.
.
I
.
½
.
.
.
.
s
.
.
I
10 2
C
Fig. 5. Direct mutagenic activity of air pollution extract GEX and its subfractions MET, CYC, PAH and PRO in S. typhimuriurn TA 1537 (in the presence of S-9, but without NADPH). of S-9 inhibits these toxic effects but also reduces the n u m b e r of revertants, presumably due to nucleophiles present in S-9, which can react with active compounds. However, the changes observed are small and near the limits of experimental variation (about 25% in the range of 20--100 revertants). DISCUSSION The Ames test system is well suited to checking extracts from airborne pollutants for mutagenic effect. In agreement with the results of other authors the whole extract GEX proved to have mutagenic activity [7--9]. But the correlation between the PAH c o n t e n t and the mutagenic effect is n o t unequivocal. It is possible t h a t samples from different collecting sites have different pollutants which account for the different results. But it seems obvious that other substances participate in the mutagenicity. After splitting the whole extract into fractions, the mutagenic effect in these fractions was confirmed. The low activity of the PAH fraction is in accordance with the low concentration of benzo(a)pyrene and other PAH. No mutagenic effect was observed in experiments with pure benzo(a)pyrene in the range of 0.2 pg/plate. Thus the mutagenic activity of our extracts from particulate airborne matter is predominantly based on compounds other than the PAH. A considerable proportion of the mutagenicity occurs w i t h o u t metabolic activation. The low activity of the PAH fraction makes it unsuitable to relate the
12
mutagenicity to the content of PAH. Thus a better standard of the evaluation of the results must be chosen. The dry-weight of the extract [7--9] or the volume o f air from which the used particulate matter was derived [9] are useful standards. The a m o u n t of extract used in our tests refers to the air volume inhaled daily by an adult man. ACKNOWLEDGEMENTS
The assistance of Mrs. Renate Volkening and Mrs. Gisela Voltmer is gratefully acknowledged. REFERENCES 1 Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. USA, 70, 2281--2285. 2 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31,347--364. 3 Carnow, B.W. and Meier, P. (1973) Air pollution and pulmonary cancer. Arch. Environ. Health, 27,207--218. 4 Hendersor~, H.E., Gordon, R.J., Mench, H., Soohoo, J., Martin, S.P. and Pike, M.C. (1975) Lung cancer and air pollution in south-central Los Angeles County. Am. J. Epidemiol., 101,477--488. 5 McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA, 72, 5135--5139. 6 Stocks, P. (1960) On the relations between atmosphere pollution in urban and rural localities and mortality from cancer, bronchitis and pneumonia, with particular reference to 3:4-benzpyrene, beryllium, molybdenum, vanadium, arsenic. Br. J. Cancer, 14, 397--418. 7 Talcot, R. and Wei, E. (1977) Airborne mutagens bioassayed in Salmonella typhimurium. J. Natl. Cancer Inst., 58,449--451. 8 Teranishi, K., Hamada, K., Takeda, N. and Watanabe, H. (1977) Mutagenicity of the tar in air pollutants. Proc. 4th Int. Clean Air Congr., Tokyo, May 1977. 9 Tokiwa, H., Morita, K., Takeyoshi, H., Takahashi, K. and Ohnishi, Y. (1977) Detection of mutagenic activity in particulate air pollutants. Murat. Res., 48,237--248. 10 Tomingas, R., Voltmer, G. and Bednarik, R. (1977) Direct fluorometric analysis of aromatic polycyclic hydrocarbons on thin-layer chromatograms. Sci. Total Environ., 7, 261--267. 11 Wynder, E.L. and Hoffmann, D. (1965) Some laboratory and epidemiological aspects of air pollution carcinogenesis. J. Air Pollut. Control Assoc., 15,155--159.
5
THE MUTAGENICITY OF AIRBORNE PARTICULATE POLLUTANTS
WALTER DEHNEN, NORBERT PITZ and RENI~ TOMINGAS Medizinisches Institut fi~r Lufthygiene und Silikoseforschung an der Universitdt D~isseldorf, Gurlittstrasse 53, D-4000 Dffsseldorf (G.F.R.)
(Received 19 July 1977) (Revised version received 18 October 1977) (Accepted 19 October 1977)
SUMMARY
The mutagenic effect of extracts derived from airborne particulate matter was investigated with the Ames-test. The whole extract proved to be distinctly mutagenic. To find out whether the content of polycyclic aromatic hydrocarbons may be responsible for the mutagenic effect, the extract was split into several fractions. The fraction containing the polycyclic compounds showed the lowest mutagenic rate that was enzymatically mediated. The other fractions required metabolic activation for some of their components, but some components were active without metabolic activation.
INTRODUCTION
Epidemiological studies suggest a relationship between the particulate matter in the ambient air and the incidence of human bronchus cancer [3,4,6]. The main cancerogenic compounds in airborne particles are thought to be the polycyclic aromatic hydrocarbons (PAH), particularly benzo(a)-pyrene [3,10]. The importance with respect to cancerogenic effects of most of the substances associated with airborne particles is not clear. The bacterial test system introduced by Ames allows rapid detection of mutagens [1]. Initially their test was applied to single compounds [5]. The application was later extended to extracts of particulate matter [7--9]. Talcot and Wei [7] suggest that most of the mutagenic activity of the particulate matter can be attributed to the presence of PAH. Samples of air soot, however, proved to be highly active although no benzo(a)pyrene Could be detected [7]. The correlation between the benzo(a)pyrene content in airborne particulates and mutagenicity was not always positive [8]. These findings, partly contradictory, demand further investigation. The aim of the study presented here was to assess the mutagenic activity in extracts of particulates and that of its fractions with special attention to the PAH-fraction.
MATERIALSAND METHODS 1. Chemicals Methanol, cyclohexene, alumina 90 neutral (Merck, Darmstadt, G.F.R.), were used for extraction and column chromatography. Clophen C, a mixture of polychlorinated biphenyls similar to Aroclor, was a generous gift from Bayer AG, Leverkusen. Histidine was obtained from Merck, glucose 6-phosphate, glucose-6-phosphate
Extract GEX1 j
Liquid--liquidpartition i
Cycl0hexenephase CYCI
\
[Methanol phase MET1
I Column chromatography [Fraction PAHI
]FractionPRO1
Scheme 1. Fractionation of the extract from airborne particulate matter.
were eluted with propanol-2 (fraction PRO). This fraction contains, among a large number of unknown components, aza-heterocyclic-compounds.
5. Analysis PAH were estimated according to the procedure already described [10]. The m e t h o d is based on column chromatography followed b y thin-layer chromatography and direct fluorescence measurement on the plate. 6. Araes test To obtain comparable concentrations of the extracted components in samples to be examined, the volume of all fractions was adjusted to equal the volume of the original whole extract (GEX), usually 180 ml, Thus the concentration of the particular substances remains constant in the subfractions. Aliquots of the fractions prepared in this manner were a d d e d to the tester strain. The mutagenicity test with Salmonella typhimurium was performed as described in detail by Ames et al. [2]. In this study we followed strictly the prescriptions according to Ames. The liver microsomes were obtained from rats pretreated with Clophen C. The tester strains used were TA 1537, TA 1538 and TA 98. Tests were run also with TA 100, b u t no reproducible results were obtained and therefore this strain was discarded. Preliminary experiments showed no mutagenic effect of air pollution extracts in the strain TA 1535, which is susceptible to base pair mutagens. This result is in accordance with the findings of Tokiwa et al. [9]. Dilutions from the extracts and fractions were made with ethanol, and aliquots of 0.1 ml per plate were taken. A volume of 0.1 ml of the original extract (not diluted) refers to 14 m 3 air volume. 7. Plots The values were plotted. In these plots the figures below the abscissa do not represent a certain factor like benzo(a) pyrene content, air volume or residue of the extract, since the question respecting the factor most suited for standardizing is still open. Therefore, the figures stand for a concentration. The original (undiluted) concentration has the value '8' and refers to 0.1 ml extract equal to 14 m 3 air volume and equal to 0.27 pg benzo(a)pyrene in GEX, CYC and PAH fractions. In some cases the original extract concentration was doubled. The curve for the revertants demonstrate the total n u m b e r without deducting the number o f spontaneous revertants. This n u m b e r is plotted for each assay as a line for a better view o f the mutagenic effect. RESULTS
1. The mutagenic activity o f airborne particulate pollutants with metabolic activation In Figs. 1--3 the mutagenic effect o f the whole extract (GEX) and its fractions MET, CYC, PAH and PRO is demonstrated. In all of the three tester
LLI
400
o SPONI REV.TA98 GEX "MET
•
~360 J
'~ CYC • PAH DPRO
~'~ 320 ~m:;280 240 200 160 120 80 40 0 "
" ~"~b'~
c
Fig. i. Mutagenic activity of air pollution extract GEX and its fractions MET, CYC, PAH and PRO in S. typhimurium TA 98 (in the presence of the complete activating system).
ILl 400
o SPONT.REV. TA 1537 • GEX • MET
' .U 360' ' O,_ 320
z~
CYC
PAH
W
r ( 280
D PRO
240 .
2oo4 120 ~
40 0
• i
loo
,
2
" ~1~1
~
" 5ib~
c
Fig. 2. Mutagenie activity of air pollution extract GEX and its fractions MET, CYC, PAH and PRO in S. typhimurium TA 1537 (in the presence of the complete activating system).
LL1400 |
n
o SPONT.REV.TA 1538 • GEX • MET CYC • PAH PRO
"
320. W rr280 240 200 160 120 80 40 0
@
0
0
'
'
O
'
'
c
Fig. 3. Mutagenic activity of air pollution extract GEX and its fractions MET, CYC, PAH and PRO in S. typhimurium TA 1538 (in the presence of the complete activating system). strains, TA 1537, TA 1538 and TA 98, the highest activity is found in the whole extract GEX. After liquid--liquid partition of GEX between cyclohexene and methanol/water about 70% of the activity can be extracted into the cyclohexane phase CYC. The rest is within the methanol phase. Column chromatography on alumina with cyclohexene as eluant transfers the smaller part of the mutagenic effect into the PAH containing fraction PAH. The much larger effect is found in the more polar fraction PRO.
2. The mutagenic activity of airborne particulate pollutants with and without metabolic activation Mutagenic and cancerogenic substances either act directly or require prior metabolism to produce active metabolites. To estimate the relation of these t w o groups present in the particulates, samples were applied to the bacteria w i t h o u t S-9 (liver microsomes and cytoplasma), with S-9 and with S-9 and NADPH together. The results are shown in Table 1 for the tester strains TA 1537 and TA 98, and the corresponding dose-response curves in Figs. 4 and 5. A distinct proportion of the whole extract GEX and the fractions MET, CYC and PRO have a mutagenic effect w i t h o u t metabolic activation. This direct acting mutagenicity is predominant in the fraction MET. In the polycyclic aromatic hydrocarbon containing fraction PAH most of the mutagenic activity requires metabolic activation, which is characteristic for these c o m p o u n d s [ 1]. In some cases the extracts cause toxic effects as judged by the inhibition of the background lawn o f bacterial growth [2] (fractions MET, PAH). The addition
10 TABLE 1 E F F E C T O F E N Z Y M A T I C A C T I V A T I O N ON E X T R A C T I N D U C E D M U T A G E N I C I T Y
Strain
Fraction
S 9 omitted
+S9
+ S 9 + NADPH
T A 98
GEX MET CYC PAH PRO
89 ?* 55 3 22
58 38 38 10 15
205 64 197 93 128
T A 1537
GEX MET CYC PAH PRO
39 ?* 25 ?* 3
70 60 38 8 22
122 80 165 93 80
The figures are the n u m b e r o f revertants after subtraction of s p o n t a n e o u s revertants. Sample Duisburg, 2nd sampling site. C o n c e n t r a t i o n o f CYC, PAH, P R O t w o - f o l d of G E X and MET. S p o n t a n e o u s revertants (s.d.): T A 9 8 : 1 4 ± 4 (S 9 o m i t t e d o r added); 29 -+ 7 (8 9 + N A D P H ) . T A 1 5 3 7 : 1 0 -+ 4 (all conditions). * Toxic effect of extract.
LL1100
o SPONT. REV. TA 98 • GEX • MET
~_..1 9o 13_ "~ 80
•
W n " 70
CYC PAH PRO
60 50 40 30 2O 0
[~
0
,0
'
10 0
• 1
i0 o
,
~
. . . . . .
5
i
101
"
c
Fig. 4. Direct m u t a g e n i c activity o f air p o l l u t i o n e x t r a c t G E X and its subfractions MET, CYC, P A H and P R O in S. typhimurium T A 98 (in the presence o f S-9, b u t w i t h o u t N A D P H )
11 o SPONT.REV. TA 1537 • GEX • MET
LL1100
d 9o n
/
t.U cr 7O 6O
CYC •
P A H
I
PRO
5O 40 3O 20 10 0
• 1
10o
.
.
.
.
.
5
.
.
I
.
½
.
.
.
.
s
.
.
I
10 2
C
Fig. 5. Direct mutagenic activity of air pollution extract GEX and its subfractions MET, CYC, PAH and PRO in S. typhimuriurn TA 1537 (in the presence of S-9, but without NADPH). of S-9 inhibits these toxic effects but also reduces the n u m b e r of revertants, presumably due to nucleophiles present in S-9, which can react with active compounds. However, the changes observed are small and near the limits of experimental variation (about 25% in the range of 20--100 revertants). DISCUSSION The Ames test system is well suited to checking extracts from airborne pollutants for mutagenic effect. In agreement with the results of other authors the whole extract GEX proved to have mutagenic activity [7--9]. But the correlation between the PAH c o n t e n t and the mutagenic effect is n o t unequivocal. It is possible t h a t samples from different collecting sites have different pollutants which account for the different results. But it seems obvious that other substances participate in the mutagenicity. After splitting the whole extract into fractions, the mutagenic effect in these fractions was confirmed. The low activity of the PAH fraction is in accordance with the low concentration of benzo(a)pyrene and other PAH. No mutagenic effect was observed in experiments with pure benzo(a)pyrene in the range of 0.2 pg/plate. Thus the mutagenic activity of our extracts from particulate airborne matter is predominantly based on compounds other than the PAH. A considerable proportion of the mutagenicity occurs w i t h o u t metabolic activation. The low activity of the PAH fraction makes it unsuitable to relate the
12
mutagenicity to the content of PAH. Thus a better standard of the evaluation of the results must be chosen. The dry-weight of the extract [7--9] or the volume o f air from which the used particulate matter was derived [9] are useful standards. The a m o u n t of extract used in our tests refers to the air volume inhaled daily by an adult man. ACKNOWLEDGEMENTS
The assistance of Mrs. Renate Volkening and Mrs. Gisela Voltmer is gratefully acknowledged. REFERENCES 1 Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. USA, 70, 2281--2285. 2 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31,347--364. 3 Carnow, B.W. and Meier, P. (1973) Air pollution and pulmonary cancer. Arch. Environ. Health, 27,207--218. 4 Hendersor~, H.E., Gordon, R.J., Mench, H., Soohoo, J., Martin, S.P. and Pike, M.C. (1975) Lung cancer and air pollution in south-central Los Angeles County. Am. J. Epidemiol., 101,477--488. 5 McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA, 72, 5135--5139. 6 Stocks, P. (1960) On the relations between atmosphere pollution in urban and rural localities and mortality from cancer, bronchitis and pneumonia, with particular reference to 3:4-benzpyrene, beryllium, molybdenum, vanadium, arsenic. Br. J. Cancer, 14, 397--418. 7 Talcot, R. and Wei, E. (1977) Airborne mutagens bioassayed in Salmonella typhimurium. J. Natl. Cancer Inst., 58,449--451. 8 Teranishi, K., Hamada, K., Takeda, N. and Watanabe, H. (1977) Mutagenicity of the tar in air pollutants. Proc. 4th Int. Clean Air Congr., Tokyo, May 1977. 9 Tokiwa, H., Morita, K., Takeyoshi, H., Takahashi, K. and Ohnishi, Y. (1977) Detection of mutagenic activity in particulate air pollutants. Murat. Res., 48,237--248. 10 Tomingas, R., Voltmer, G. and Bednarik, R. (1977) Direct fluorometric analysis of aromatic polycyclic hydrocarbons on thin-layer chromatograms. Sci. Total Environ., 7, 261--267. 11 Wynder, E.L. and Hoffmann, D. (1965) Some laboratory and epidemiological aspects of air pollution carcinogenesis. J. Air Pollut. Control Assoc., 15,155--159.