Mutagenicity of Soot and Associated Polycyclic Aromatic Hydrocarbons … · Mutagenicity of Soot and Associated Polycyclic Aromatic Hydrocarbons to Salmonella typhimuriumi Debra A.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
[CANCER RESEARCH 39. 4152-4159, October 1979]0008-5472/79/0039-OOOOS02.00
Mutagenicity of Soot and Associated Polycyclic Aromatic Hydrocarbonsto Salmonella typhimuriumi
Debra A. Kaden,2 Ronald A. Hites, and William G. Thilly
Department of Nutrition and Food Science ¡D.A. K., W. G. T.], and Department of Chemical Engineering ¡R.A. H ¡,Massachusetts Institute of Technology,Cambridge. Massachusetts 02139
ABSTRACT
The mutagenic activity of the polycyclic aromatic hydrocarbon-containing fraction of several soot samples was measured
into all assays to allow metabolism of promutagens to theiractive forms.
The mutagenic activity of the soot extracts ranged from 10to 20 times higher than could be accounted for by the amountof benzo(a)pyrene present. The possibility that synergism occurs between benzo(a)pyrene and some component in the sootextracts was discounted by the finding of a simple additiverelationship of mutagenicity of a soot extract and addedbenzo(a)pyrene.
To examine the alternative explanation that other components of soot may have undiscovered mutagenic activity, 70polycyclic aromatic hydrocarbons were quantitatively assayedfor their mutagenic potential; 34 of these compounds induceda significant increase in the mutant fraction resistant to 8-azaguanine. Of particular interest are the extreme muta-genicities of perylene, cyclopenta(ccOpyrene, and fluoran-
thene, all of which exhibit greater mutagenicity than doesbenzo(a)pyrene at equimolar concentrations.
Using the measured activities of each polycyclic aromatichydrocarbon constituent in a kerosene soot, we are able toaccount for the mutagenic activity of the whole polycyclicaromatic hydrocarbon fraction in terms of the additive mutagenicity of its individual components.
INTRODUCTION
PAH3 are found throughout the environment (2, 12, 32).
They are formed by the incomplete combustion of organicmaterial. Sources of PAH include the decomposition of organicmatter in soil and sediments (1), heat and power generation,refuse burning, coke production, and motor vehicles (19).
PAH from fuel combustion found in the atmosphere are
' Supported by National Cancer Institute Grant NIH-2-R01-CA15010-04, Na
2 Partially supported by Sigma Xi. To whom requests for reprints should be
addressed, at Department of Nutrition and Food Science, Room E18-666.Massachusetts Institute of Technology, Cambridge, Mass. 02139.
3 The abbreviations used are: PAH. polycyclic aromatic hydrocarbons; PMS,
postmitochondrial supernatant.Received July 10. 1978; accepted May 16. 1979.
generally bound to particulate matter such as soot or fly ash.Soot comprises 2 to 15% of the fine particle mass in a typicalurban atmosphere (16).
Numerous experiments have demonstrated that soot is carcinogenic to experimental animals (3, 8, 15, 18, 20, 21, 25,26), and epidemiological observations suggest similar activityin humans (11). Extracts of particulate matter induce transformation in rat and hamster embryo cells in culture (10), as wellas mutation in bacterial cultures (4, 6, 18, 22, 29, 30).
Benzo(a)pyrene, a known mutagen and carcinogen, hasbeen identified as one of the active constituents of soot, flyash, and particulate samples (9, 12, 32). Several other mutagenic and carcinogenic constituents have also been identified(7,9, 12-14, 23, 24). However, in soot or its total PAH fraction,
the mutagenic and carcinogenic potency seems greater thancould be accounted for on the basis of the amounts of constituents with known activity (8, 22).
We have begun analysis of this problem with knowledge ofthe compound distributions in soots (13, 14, 23) and a newquantitative bacterial assay for forward mutation which is particularly useful in the analysis of complex mixtures (27, 28).
Sources of Chemicals. Chemicals were obtained from thefollowing sources. Cyclopenta(cd)pyrene was a generous giftof Dr. Lawrence Wallcave, University of Nebraska MedicalCenter, Omaha, Nebr. 1,2-Benzodibenzo(b, cOthiophene was
generously supplied by Dr. LeRoy H. Klemm, University ofOregon, Eugene, Oreg. 1H-Benz(g)indole and 1H-benz(e)-indol-2-acid were donated by Dr. Stewart W. Schneller, University of South Florida, Tampa, Fla. Dibenzo(a,e)fluoranthenewas supplied by Dr. R. C. Lao, Environmental Health Centre,Ottawa, Ontario, Canada. Acenaphthylene, 4-azafluorene,benzene, 7/-/-benz(d,e)anthracen-7-one, benzo(b)fluorene,benzo(gft/)perylene, benzo(e)pyrene, 5,6-benzoquinoline, 7,8-benzoquinoline, 4H-cyclopenta(deOphenanthrene, 2,6-di-methylquinoline, 2,6-dimethylnaphthalene, isoquinoline, 3-
ronene, and fluoranthene were purchased from Aldrich Chemical Co., Milwaukee, Wis. Anthanthrene, Aroclor 1254, 1,1'-
binaphthyl, 9-phenylanthracene, picene, o-terphenyl, and m-
terphenyl were purchased from Analabs, Inc., North Haven,Conn. 2,3,6-Trimethylnaphthalene was obtained from Chemical Samples Co., Columbus, Ohio. 3,4-Benzoquinoline was
purchased from Eastern Chemicals, Hauppauge, N. Y.Benz(a)anthracene, chrysene, fluorene, 1-methylnaphthalene,
naphthalene, and phenanthracene were obtained from Eastman Chemical Co., Rochester, N. Y. Anthraquinone, anthrone,and indole were purchased from Fisher Scientific Co., Medford,Mass. Dibenzo(o,d)thiophene was obtained from Fluka AGChemische Fabrik, Buchs, Switzerland. Acenaphthalene, 1-cyanonaphthalene, 2-cyanonaphthalene, 1-methylpyrene, 1-methylphenanthrene, and 2-methylphenanthrene were obtained from ICN Life Sciences Group, Plainview, N. Y. 2-Meth-ylanthracene and 9-methylanthracene were obtained from ICN
Wilmington, Mass.). Details of PMS preparation are reportedelsewhere (28). Glucose 6-phosphate (1 mg/ml), NADP* (1
mg/ml), MgCI (670/¿g/ml), and glucose-6-phosphate dehydro-genase (0.4 unit/ml) were included as cofactors for the drug-metabolizing system. Following the 2-hr incubation at 37°,
bacteria were centrifuged (2000 rpm for 15 min), resuspendedin phosphate-buffered saline [NaCI (8 mg/ml), KCI (0.2 mg/
ml), Na2HPO4 (1.15 mg/ml), and KH2PO4 (0.2 mg/ml)], andplated under selective conditions [8-azaguanine (50 fig/ml)]
and nonselective conditions. Colonies were counted aftergrowth for 2 days at 37°.
Mutant fraction was calculated by dividing the number ofcolonies observed under selective conditions by the number ofcolonies observed under permissive conditions and multiplyingby appropriate dilution factors.
RESULTS AND DISCUSSION
All experiments were performed using one of 2 frozenbatches of bacterial strain TM677 (28). Analysis of the variationamong assays shows an approximately normal distribution ofthe background mutant fraction (Chart 1). The mean background mutant fraction for experiments performed from thefirst frozen batch (all experiments between June 26, 1977, and
24 I • 10 II 14 MBACKGROUND MUTANT FRACTION «IO5
246 8 IO 12 H 16BACKGROUND MUTANT FRACTION »IO5
Chart 1. Distribution of background mutant fraction Each event represents asingle determination of the background mutant fraction. A, all experiments usingbacterial batch frozen June 24. 1977. B, all experiments using bacterial batchfrozen September 26. 1977. ñ.total number of determinations; x. mean. S,, S.D.
September 28, 1977) was 7.1 x 10~6. The mean background
mutant fraction for all experiments performed from the secondfrozen batch (all experiments between October 1, 1977, andDecember 1, 1978) was 5.6 x 10~5. Standard deviations were4.0 x 10~5 (n = 157) and 2.2 x 10~6 (n = 146), respectively.
The 99% confidence limit on the mean background fraction(mean + 3 S.D.) was our criterion of minimum significance;i.e., an observed mutant fraction higher than this level for atreated culture was considered statistically significant.
soots were all found to be mutagenic at concentrations of 20to 50 /ig per ml culture medium in a 2-hr exposure (Aroclor-
preinduced rat liver PMS). Initial slopes of the concentrationdependence of induced mutation (Chart 2) show the followingpercentages of the activity of pure benzo(a)pyrene: sulfur-containing soot, 10%; nitrogen-containing soot, 10%; furnaceblack, 13%; and kerosene soot, 17%. When phenobarbital-preinduced rat liver PMS was substituted for Aroclor-prein-
duced rat liver PMS, significantly higher concentrations ofbenzo(a)pyrene or soot extract were required to induce significant amounts of mutation (data not presented).
Benzo(a)pyrene, often considered the highest contributor tothe mutagenicity of soot, constitutes less than 1% of thekerosene soot extract (23) and accounts for less than 3% ofthe observed mutagenicity of that soot extract. Thus, the observed mutagenicity could not be explained by the mutagenicityof benzo(a)pyrene.
Two possibilities could logically account for this phenomenon: nonmutagenic components could act synergistically withbenzo(a)pyrene; alternatively, other components of the sootextract could have yet undiscovered significant mutagenic
activity which could cumulatively account for the mutagenicactivity of the soot extract.
To test the hypothesis of synergism, the mutagenic activityof benzo(a)pyrene was measured in the presence of nitrogen-
containing soot extract (80 jug/ml). We observed strictly additive mutagenicity when benzo(a)pyrene was added to nitrogen-
containing soot extract (Chart 3). This observation is, of course,inconsistent with a significant contribution of synergism of sootcomponents with benzo(a)pyrene, which would increase theobserved mutation for the combination.
To test the second hypothesis, 70 PAH components ofvarious soots were quantitatively assayed for mutagenic activityin the presence of PMS from Aroclor-pretreated rats. (Whenmutagenicity was not observed with PMS from Aroclor-pre
treated rats, the compounds were retested in the presence ofPMS from phenobarbital-pretreated rats. This testing with phe-nobarbital-induced PMS was performed to increase our general
knowledge about the importance of different metabolizing conditions, rather than as a part of our overall analysis of themutagenicity of soot and its components.)
Of the tested components, 34 induced a significant increasein mutant fraction as measured by 8-azaguanine resistance
(Table 1). Data for 3 of the 36 remaining components[4-azafluorene, anthracene, and 1,2-benzodibenzo(o,d)thio-phene] suggest possible low-level mutagenicity. Solubility
problems encountered with several of the compounds prevented testing at higher concentrations (Table 1). The lowest
concentration yielding significant mutation for each compound(Table 1) was calculated by interpolation from the concentration response curve [see Skopek ef al. (27) for the method ofcalculation]. In addition, the mutagenic potency relative tobenzo(a)pyrene (Table 1, Column 6) was calculated by dividingthe initial slope of the concentration response curve (mutantfraction in 2 hr/concentration) by the "slope" of the simulta
neously performed (80 UM) benzo(a)pyrene standard, whichlies on the linear portion of the benzo(a)pyrene concentrationresponse curve.
For those interested in comparing the mutagenicity to thereported carcinogenicities of these compounds, available animal carcinogenicity data are given in Table 1 (31).
Although mutagenicity of some of the compounds [quinoline,acepyrylene, benz(a)anthracene, chrysene, benzo(a)pyrene,benzo(e)pyrene, 7,12-dimethylbenz(a)anthracene, 3-methyl-
cholanthrene, dibenz(a,c)anthracene, benzo( g/7/)perylene, anddibenz(a,ft)anthracene] has been recognized previously (e.g.,Refs. 5 and 17), this is the first report of mutagenic activity inbacteria of 23 of the PAH associated with soot. Of particularinterest is the extreme mutagenic activity of perylene, cyclo-
penta(cd)pyrene, and fluoranthene, all of which exhibit greatermutagenicity than benzo(a)pyrene at equimolar concentrations(Chart 4). Although the mutagenic response of perylenereaches a stable maximum at concentrations greater than 12juM, it induces significant mutation at concentrations as low as
Where there is more than one pretreatment listed, calculations refer to italicized pretreatment. —,no significant Induced mutation;+ , significant induced mutation. c Number listed Is lowest concentration of significant Induced mutation for positive responses, highest
(Charts 4 to 6) illustrate the absolute necessity of simultaneously measuring the toxicity incurred as a result of treatmentwhen examining chemicals of unknown biological activity. Allare mutagenic only at concentrations that are also toxic to thebacteria. Failure to account for this toxicity in calculating mutagenic potency leads to the erroneous conclusion that none
0 20 40 60 80
CONCENTRATION (U.M) x 2 Hr
Chart 4. Concentration-dependent mutagenicity (open symbols) and toxicity(closed symbols) of cyclopenta(cd)pyrene (D, •), fluoranthene (A, A),benzcKa)pyrene (O, •).and perylene (C, 0) to S. typhimurium. All points wereassayed in the presence of Aroclor-induced PMS. Each point represents theaverage of 2 independent determinations. SAG, 8-azaguanine.
1.1 fiM. as compared to 4.0 JUMfor benzo(a)pyrene when a10% (v/v) PMS from Aroclor-pretreated rats is used.
The compounds acenaphthalene, acenaphthylene, 4-phen-ylpyridine, 5,6-benzoquinoline, and 1-methylnaphthalene
ACENAPHTHYLENE
I I _L0 125 525 650 800 1300 1600 3200
CONCENTRATION (p.M) «2 HR
Chart 5. Concentration-dependent mutagenicity (open symbols) and toxicity(closed symbols) of acenaphthylene (D. •),acenaphthalene (O. •),and 4-phenylpyridine (A, A) to S. typhimurium. Acenaphthalene was assayed in thepresence of Aroclor-induced PMS. 4-Phenylpyridine and acenaphthylene wereassayed in the presence of phenobarbital-induced PMS Each point representsthe average of 2 independent determinations BAG, 8-azaguanine.
Chart 6. Concentration-dependent mutagenicity (open symbols) and toxicity (closed symbols) of 4-methylquinoline and 1-methylnaphtha-lene to S. typhimurium in the presence of Aroclor-induced PMS. Each point represents theaverage of 2 independent determinations. BAG,8-azaguanine.
of these compounds is mutagenic to S. typhimurium, since theactual number of mutants is not increased by treatment.
It is necessary to emphasize further that the potency calculations of Table 1 are all based on 10% PMS (v/v) determination. The dependence of the apparent mutagenicity of manypolycyclics on PMS concentration is not simple monotonieincreasing or saturation relationship but often demonstrates amaximum of 1 to 20% (v/v). Thus, the activity relative tobenzo(a)pyrene will vary for many compounds if differentamounts of PMS are used or if PMS concentration is optimizedfor each compound.
Bearing these facts in mind, however, we note some interesting relationships between structure and activity in the dataof Table 1. For instance, the mutagenic activity of a given PAHoften seems lower than that of the corresponding aza compound (Charts 6 to 8), which may be important in analyzing thesoots of fuels, such as coal, that contain significant amounts ofnitrogen. Present limitations in chemical analytical techniqueprevent a quantitative analysis of all the aza aromatics innitrogen-containing soot.
Chart 7. Concentration-dependent mutagenicity (open symbols) and toxicity(closed symbols) of 5,6-benzoquinoline (A, A). 7,8-benzoquinoline (O. •).3,4-benzoquinoline (O, •).and phenanthrene O, Ö)to S. typhimurium. 3,4-Benzo-quinoline, 5,6-benzoquinoline. and 7,8-benzoquinoline were assayed in the presence of Aroclor-induced PMS. Phenanthrene was assayed separately in thepresence of either Aroclor- or phenobarbital-induced PMS. Each point representsthe average of 2 independent determinations. SAG, 8-azaguanine.
99Z CONFIDENCELIMIT
I L
CONCENTRATION (mMI i 2 Hr
Chart 8. Concentration-dependent mutagenicity (open symbols) and toxicity(closed symbols) of quinoline (O. •).isoquinoline (D. •).and naphthalene (A,A) to S. typhimurium. Quinoline and isoquinoline were assayed in the presenceof Aroclor-induced PMS. Naphthalene was assayed separately in the presenceof Aroclor- or phenobarbital-induced PMS. Each pomi represents the average of2 independent determinations BAG, 8-azaguanine
an experiment with kerosene soot extract (20 or 100 fig/ml)and determining the mutant fraction that this amount would beexpected to induce from the individual concentration responsecurve.
As can be seen in Table 2, the sum of the contributions ofindividual PAH constituents is about 2-fold greater than the
soot extract show that compounds may contribute to the mutagenicity of the extract to different extents, depending on theconcentration present, due to nonlinearity of dose response forindividual compounds. However, at all levels examined, thereis sufficient activity in the individual components to account forthe high activity of the extract. The fact that the sum of themutagenicity of the components was greater than that of thekerosene soot extract could be caused by several factors, suchas the imprecision of our estimates or a partial competitiveinhibition of metabolizing reactions.
To test directly this hypothesis of additivity, a mixture thataccurately mimics the chemical composition of the kerosenesoot extract (Table 2) was constructed and assayed simultaneously with the crude kerosene soot extract. Results of theseassays indicate that the mutagenicity of the kerosene sootextract was wholly reproduced by this reconstituted mixture ofknown components (Chart 9).
Thus, the mutagenic activity of the PAH fraction of the
" —. component not available for testing.6 Material lost in the characterization process, plus those compounds which could not be identified by gas chromatography-mass
reconstituted kerosene soot extract (A, A) to S typhimurium in the presence ofAroclor-induced PMS. The reconstitution mixture to which the cells were exposedcontained acenaphthylene (2.3 mg/ml), acepyrylene (1.5 mg/ml). pyrene (800jig/ml), coronene (500 /ig/ml), benzo(g/iOperylene (400 /ig/ml), anthanthrene(400 /ig/ml). fluoranthene (400 ng/ml), naphthalene (300 /ig/ml), perylene (200
kerosene soot extract seems to be due to simple additivecontributions of its mutagenic components.
ACKNOWLEDGMENTS
We acknowledge C. Crespi. R. DiPietro. D. Fleiscnaker. J. Herland, G. Kurz-ban, J. Maupin. G. McKillop, J. McSpedon, J. Ng, B. Penman, R. Roy, J. Seixas,and C. Wang for their technical assistance: R. Glover for administrative aid; andJ. Larsen for typing and technical drawings.
REFERENCES
1. Blumer. M. Polycyclic aromatic compounds in nature. Sei. Am., 234 35-45, 1976.
2. Braunstein, H. M., Copenhaver. E. D , and Pfuder, H. A. (eds.). Environmental Health and Control Aspects of Coal Conversion: An Informative Overview.Oak Ridge, Tenn.: Oak Ridge National Laboratories, 1977.
3. Campbell, J. A. Carcinogenic agents present in the atmosphere and incidence of primary lung tumors in mice. Br. J. Exp. Pathol., 20 122-132,1939.
4. Chrisp, C. E.. Fisher, G. L, and Lammert, J. E. Mutagenicity of filtrates fromrespiratilo coat fly ash. Science, 799. 73-75. 1978.
5. Coombs, M. M., Dixon. C., and Kissonerghis, A-M. Evaluations of themutagenicity of compounds of known carcinogenicity, belonging to thebenz[a]anthracene, chrysene, and cyclopenta[a)phenanthrene series, usingAmes's test. Cancer Res., 36. 4525-4529, 1976.
6. Daisey, J. M., Hawryluk, I., Kneip, T. J., and Mukai, F. H. Mutagenic activityin organic fractions of airborne paniculate matter. In: Proceedings of theConference on Carbonaceous Particles in the Atmosphere. Berkeley, Calif :National Science Foundation and Lawrence Berkeley Laboratory, 1978.
7. Dong, M. W.. and Locke, D. C. Characterization of aza-arenes in basicorganic portion of suspended particulate matter Environ. Sci. Technol., 77.612-618, 1977.
8. Epstein, S. S., Joshi, S.. Andrea, J., Mantel, N.. Sawicki, E., Stanley, T..and Tabor, E. C. Carcinogenicity of organic particulate pollutants in urbanair after administration of trace quantities to neonatal mice. Nature (Lond.),272. 1305-1307. 1966.
9 Falk, H. L., and Steiner, P. E. The identification of aromatic polycyclichydrocarbons in carbon'blacks. Cancer Res., 12: 30-39, 1952.
/ig/ml), phenanthrene (100 fig/ml), anthracene (100 /ig/ml), acenaphthalene(100 /ig/ml) benzo(a)pyrene (50 /ig/ml), and benzo(e)pyrene (50 /ig/ml). Eachpoint represents the average of 2 independent determinations. BaP,benzo(a)pyrene (80 /IM); SAG, 8-azaguanine.
10. Freeman. A. E., Price. P. J.. Bryan, R. J.. Gordon. R. J.. Gilden. R. V..Kellotf, G. J., and Huebner, R. J. Transformation of rat and hamster embryocells by extracts of city smog. Proc. Nati. Acad. Sei. U. S. A. 68 445-449.
1971.11. International Agency for Research on Cancer. IARC Evaluation of Carcino
genic Risk of Chemicals to Man, Vol. 3. Lyon. France: International Agencyfor Research on Cancer, 1973.
12. Kotin, P.. Falk. H. L, Mader. P., and Thomas, M. Aromatic hydrocarbons. I.Presence in Los Angeles atmosphere and the carcinogenicity of atmosphericextracts. A. M. A. Arch. Indus!. Hyg., 9. 153-163, 1954.
13. Lee, M. L., and Hites. R. A. Characterization of sulfur-containing polycyclicaromatic compounds in carbon blacks. Anal. Chem.. 48: 1890-1893. 1976.
14. Lee. M. L.. Prado, G. P., Howard, J. B., and Hites, R. A. Source identificationof urban airborne polycyclic aromatic hydrocarbons by gas Chromatographiemass spectrometry and high resolution mass spectrometry. Biomed. MassSpectrom. 4: 182-186. 1977.
15. Leiter, J.. Shimkin, M. B.. and Shear. M. J. Production of subcutaneoussarcomas in mice with tars extracted from atmospheric dusts. J. Nati. CancerInst.. 3. 155-165. 1942.
16. Macias, E. S. The determination, speciation. and behavior of particulatecarbon. In: Proceedings of the Conference on Carbonaceous Particles inthe Atmosphere. Berkeley. Calif.: National Science Foundation and Lawrence Berkeley Laboratory, 1978.
17. McCann, J., Choi. E.. Yamasaki, E.. and Ames, B. N. Detection of carcinogens as mutagens in the Sa/mone//a/microsome test: assay of 300 chemicals. Proc. Nati. Acad. Sei. U. S. A.. 72. 5135-5139, 1975.
18. McDonald. J. C., Drinker, P., and Gordon, J. The epidemiology and socialsignificance of atmospheric smoke pollution. Am. J Med. Sci.. 221: 325-342. 1951.
19. National Academy of Sciences. Biological Effects of Atmospheric Pollutants:Particulate Polycyclic Organic Matter. Washington, D. C.: National Academyof Sciences. 1972.
20. Passey, R. D. Experimental soot cancer. Br. J. Med., 2. 1112-1113. 1922.21. Passey. R. D., and Carter-Braine, J. Experimental soot cancer. J. Pathol.
Bacteriol., 28: 133-144, 1925.22. Pitts. J. M., Jr.. Grosjean. D.. and Mischke. T. M Mutagenic activity of
Mutagenicity of Soot and Components to Salmonella
airborne particulate organic pollutants. Toxicol. Lett.. / 65-70, 1977.23. Prado, G. P., Lee, M. L.. Hites. R. A.. Houli. D. P.. and Howard. J. B. Soot
and hydrocarbon formation in a turbulent diffusion flame. In: 16th International Symposium on Combustion, pp. 649-661. Cambridge. Mass.: The
Combustion Institute. 1973.24. Schultz. J. A. Analysis of polycyclic aromatic compounds in the combustion
products of thiophene and pyridine. S.B. Thesis. Massachusetts Institute ofTechnology, Cambridge, Mass.. 1977.
25. Seelig. M. G., and Beningnus. A. B. Coal smoke soot and tumors of the lungin mice. Am. J. Cancer. 28. 96. 1936.
26. Shinkin, M. B., and Leiter, J. Induced pulmonary tumors in mice. III. The roleof chronic irritation in the production of pulmonary tumors in strain A mice.J. Nati. Cancer Inst.. 1: 241-254. 1948.
27. Skopek. T. R.. Liber, H. L., Kaden, D. A., and Thilly. W. G. Relative sensitivityof forward and reverse mutation assays in Salmonella typhimurium. Proc.Nati. Acad. Sei. U. S. A., 75. 4470-4473, 1978.
28. Skopek, T. R.. Liber. H. L.. Krolewski. J. J.. and Thilly. W. G. Quantitativeforward mutation assay in Salmonella typhimurium using 8-azaguanine resistance as a genetic marker. Proc. Nati. Acad. Sei. U. S. A.. 75: 410-414.1978.
29. Teranishi, K.. Hamada. K., and Watanabe. H. Mutagenicity in Salmonellatyphimurium mutants of the benzene-soluble organic matter derived fromair-borne particulate matter and its five fractions. Mutât.Res.. 56 373-380.1978.
30. Tokiwa, H., Morita, K., Takeyoshi, H., Takahashi. K., and Ohnishi, Y.Detection of mutagenic activity in particulate air pollutants. Mutât.Res., 48:237-248. 1977.
31. United States Department of Health. Education, and Welfare. Survey ofcompounds which have been tested for carcinogenic activity United StatesPublic Health Service Publication 149, 1968-1969 Vol.. 1972: 1961-1967Vol., Sect. I and Sect. II. 1973: 1970-1971 Vol.. 1974; 1972-1973 Vol..1975: Suppl. 1. 1967: Suppl. 2. 1969. Washington. D. C : United StatesGovernment Printing Office.
32. Waller. R. E. The benzpyrene content of town air. Br. J. Cancer. 6 8-21.1952.