[CANCER RESEARCH 39. 4152-4159, October 1979] 0008-5472/79/0039-OOOOS02.00 Mutagenicity of Soot and Associated Polycyclic Aromatic Hydrocarbons to 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 generally bound to particulate matter such as soot or fly ash. Soot comprises 2 to 15% of the fine particle mass in a typical The mutagenic activity of the polycyclic aromatic hydrocar urban atmosphere (16). bon-containing fraction of several soot samples was measured Numerous experiments have demonstrated that soot is car in Salmonella typhimurium, using resistance to the purine cinogenic to experimental animals (3, 8, 15, 18, 20, 21, 25, analog 8-azaguanine as a genetic marker. A postmitochondrial 26), and epidemiological observations suggest similar activity supernatant derived from livers of phénobarbital- and/or Aro- in humans (11). Extracts of particulate matter induce transfor clor-pretreated male Sprague-Dawley rats was incorporated mation in rat and hamster embryo cells in culture (10), as well into all assays to allow metabolism of promutagens to their as mutation in bacterial cultures (4, 6, 18, 22, 29, 30). active forms. Benzo(a)pyrene, a known mutagen and carcinogen, has The mutagenic activity of the soot extracts ranged from 10 been identified as one of the active constituents of soot, fly to 20 times higher than could be accounted for by the amount ash, and particulate samples (9, 12, 32). Several other muta of benzo(a)pyrene present. The possibility that synergism oc genic and carcinogenic constituents have also been identified curs between benzo(a)pyrene and some component in the soot (7,9, 12-14, 23, 24). However, in soot or its total PAH fraction, extracts was discounted by the finding of a simple additive the mutagenic and carcinogenic potency seems greater than relationship of mutagenicity of a soot extract and added could be accounted for on the basis of the amounts of constit benzo(a)pyrene. uents with known activity (8, 22). To examine the alternative explanation that other compo We have begun analysis of this problem with knowledge of nents of soot may have undiscovered mutagenic activity, 70 the compound distributions in soots (13, 14, 23) and a new polycyclic aromatic hydrocarbons were quantitatively assayed quantitative bacterial assay for forward mutation which is par for their mutagenic potential; 34 of these compounds induced ticularly useful in the analysis of complex mixtures (27, 28). a significant increase in the mutant fraction resistant to 8- azaguanine. Of particular interest are the extreme muta- genicities of perylene, cyclopenta(ccOpyrene, and fluoran- MATERIALS AND METHODS thene, all of which exhibit greater mutagenicity than does Sources of Soot. Nitrogen- and sulfur-containing soots were benzo(a)pyrene at equimolar concentrations. generated from mixtures containing equal parts of pyridine, Using the measured activities of each polycyclic aromatic Decalin, and o-xylene and from thiophene, Decalin, and o- hydrocarbon constituent in a kerosene soot, we are able to xylene, respectively. Mixtures were burned in an alcohol account for the mutagenic activity of the whole polycyclic burner, and soot was collected on the bottom of a water-cooled aromatic hydrocarbon fraction in terms of the additive muta flask placed directly over the flame. The soot was washed from genicity of its individual components. the collection flask with glass wool and méthylènechloride (24). Kerosene soot was obtained by burning a kerosene fuel INTRODUCTION in a turbulent, continuous-flow combustor, and subsequent collection was done with a water-cooled probe (23). PAH3 are found throughout the environment (2, 12, 32). Soot samples were extracted in méthylènechloride in a They are formed by the incomplete combustion of organic Soxhlet extractor for 18 hr, evaporated by rotary evaporation material. Sources of PAH include the decomposition of organic under vacuum, and redissolved in dimethyl sulfoxide. matter in soil and sediments (1), heat and power generation, Sources of Chemicals. Chemicals were obtained from the refuse burning, coke production, and motor vehicles (19). following sources. Cyclopenta(cd)pyrene was a generous gift PAH from fuel combustion found in the atmosphere are of Dr. Lawrence Wallcave, University of Nebraska Medical Center, Omaha, Nebr. 1,2-Benzodibenzo(b, cOthiophene was ' Supported by National Cancer Institute Grant NIH-2-R01-CA15010-04, Na generously supplied by Dr. LeRoy H. Klemm, University of tional Institute of Environmental Health Sciences Grants NIH-2-P01-ES00597-08 Oregon, Eugene, Oreg. 1H-Benz(g)indole and 1H-benz(e)- and NIH-5-T32-ES07020-03. Biomédical Research Support Grant NIH-5-S05- indol-2-acid were donated by Dr. Stewart W. Schneller, Uni RR07047-11 and Grant 1P30-ES-02109-01 from NIH, United States Department ot Energy Grant EE-77-S-02-4267. Grant EX-76-A-01-2295 from the United versity of South Florida, Tampa, Fla. Dibenzo(a,e)fluoranthene States Department of Energy Institutional Agreement through the MIT Energy was supplied by Dr. R. C. Lao, Environmental Health Centre, Laboratory, and the MIT Undergraduate Research Opportunity Program. 2 Partially supported by Sigma Xi. To whom requests for reprints should be Ottawa, Ontario, Canada. Acenaphthylene, 4-azafluorene, addressed, at Department of Nutrition and Food Science, Room E18-666. benzene, 7/-/-benz(d,e)anthracen-7-one, benzo(b)fluorene, Massachusetts Institute of Technology, Cambridge, Mass. 02139. benzo(gft/)perylene, benzo(e)pyrene, 5,6-benzoquinoline, 7,8- 3 The abbreviations used are: PAH. polycyclic aromatic hydrocarbons; PMS, benzoquinoline, 4H-cyclopenta(deOphenanthrene, 2,6-di- postmitochondrial supernatant. Received July 10. 1978; accepted May 16. 1979. methylquinoline, 2,6-dimethylnaphthalene, isoquinoline, 3- 4152 CANCER RESEARCH VOL. 39 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Mutagenicity of Soot and Components to Salmonella methylisoquinoline, 2-methylquinoline, 4-methylquinoline, per- ylene, 2-phenylnaphthalene, 2-phenylpyridine, 4-phenylpyri- dine, pyrene, pyridine, triphenylene, 2,7-dimethylquinoline, co- ronene, and fluoranthene were purchased from Aldrich Chem ical 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 Chemi cal Samples Co., Columbus, Ohio. 3,4-Benzoquinoline was purchased from Eastern Chemicals, Hauppauge, N. Y. Benz(a)anthracene, chrysene, fluorene, 1-methylnaphthalene, 24 I • 10 II 14 M naphthalene, and phenanthracene were obtained from East BACKGROUND MUTANT FRACTION «IO5 man 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 AG Chemische Fabrik, Buchs, Switzerland. Acenaphthalene, 1- cyanonaphthalene, 2-cyanonaphthalene, 1-methylpyrene, 1- methylphenanthrene, and 2-methylphenanthrene were ob tained from ICN Life Sciences Group, Plainview, N. Y. 2-Meth- ylanthracene and 9-methylanthracene were obtained from ICN Pharmaceuticals, Inc., Plainview, N. Y. Sodium phénobarbital and méthylènechloride were obtained from Mallinckrodt Chem ical Works, St. Louis, Mo. Quinoline and dimethyl sulfoxide, 246 8 IO 12 H 16 reagent grade, were obtained from Matheson, Coleman & Bell, BACKGROUND MUTANT FRACTION »IO5 Norwood, Ohio. 1-Methylisoquinoline was obtained from Pfaltz Chart 1. Distribution of background mutant fraction Each event represents a & Bauer, Stamford, Conn. Anthracene, benzo(a)pyrene, di- single determination of the background mutant fraction. A, all experiments using bacterial batch frozen June 24. 1977. B, all experiments using bacterial batch benz(a,c)anthracene, dibenz(a,ft)anthracene, 7,12-dimethyl- frozen September 26. 1977. ñ.total number of determinations; x. mean. S,, S.D. benz(a)anthracene, 2,3-dimethylquinazoline, m-dinitroben- zene, 3-methylcholanthrene, and 2-methylindole were obtained September 28, 1977) was 7.1 x 10~6. The mean background from Sigma Chemical Co., St. Louis, Mo. Bacterial Mutation Assay. Mutation assays were carried out mutant fraction for all experiments performed from the second frozen batch (all experiments between October 1, 1977, and as specified by Skopek ef al. (27, 28). Exponentially growing December 1, 1978) was 5.6 x 10~5. Standard deviations were cultures of Salmonella typhimurium strain TM677 were ex 4.0 x 10~5 (n = 157) and 2.2 x 10~6 (n = 146), respectively. posed to several concentrations of the test agent for 2 hr in the presence of 10% (v/v) of a PMS, prepared as a 25% (w/v) The 99% confidence limit on the mean background fraction liver homogenate of phénobarbital- or Aroclor-pretreated male (mean + 3 S.D.) was our criterion of minimum significance; Sprague-Dawley rats (Charles River Breeding Laboratories, i.e., an observed mutant fraction higher than this level for a Wilmington, Mass.). Details of PMS preparation are reported treated culture was considered statistically significant. elsewhere (28). Glucose 6-phosphate (1 mg/ml), NADP* (1 Using this criterion, the méthylènechloride extracts of nitro mg/ml), MgCI (670/¿g/ml), and glucose-6-phosphate