Mutagenicity of Soot and Associated Polycyclic Aromatic Hydrocarbons to Salmonella Typhimuriumi

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[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 Â¡D.A. K., W. G. T.], and Department of Chemical Engineering Â¡R.A. H Â¡,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 phÃ©nobarbital- 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 mÃ©thylÃ¨nechloride (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 mÃ©thylÃ¨nechloride 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. BiomÃ©dical 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-

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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, perylene, 2-phenylnaphthalene, 2-phenylpyridine, 4-phenylpyridine, pyrene, pyridine, triphenylene, 2,7-dimethylquinoline, coronene, 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 â€¢ 10 II 14 M naphthalene, and phenanthracene were obtained from East BACKGROUND MUTANT FRACTION Â«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 phÃ©nobarbital and mÃ©thylÃ¨nechloride 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 Â»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. Ã±.total number of determinations; x. mean. S,, S.D. benz(a)anthracene, 2,3-dimethylquinazoline, m-dinitrobenzene, 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 phÃ©nobarbital- 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 mÃ©thylÃ¨nechloride extracts of nitro mg/ml), MgCI (670/Â¿g/ml), and glucose-6-phosphate dehydro- gen-containing, sulfur-containing, furnace black, and kerosene genase (0.4 unit/ml) were included as cofactors for the drug- soots were all found to be mutagenic at concentrations of 20 metabolizing system. Following the 2-hr incubation at 37Â°, to 50 /ig per ml culture medium in a 2-hr exposure (Aroclor- bacteria were centrifuged (2000 rpm for 15 min), resuspended preinduced rat liver PMS). Initial slopes of the concentration in phosphate-buffered saline [NaCI (8 mg/ml), KCI (0.2 mg/ dependence of induced mutation (Chart 2) show the following percentages of the activity of pure benzo(a)pyrene: sulfur- ml), Na2HPO4 (1.15 mg/ml), and KH2PO4 (0.2 mg/ml)], and plated under selective conditions [8-azaguanine (50 fig/ml)] containing soot, 10%; nitrogen-containing soot, 10%; furnace and nonselective conditions. Colonies were counted after black, 13%; and kerosene soot, 17%. When phenobarbital- growth for 2 days at 37Â°. preinduced rat liver PMS was substituted for Aroclor-prein- Mutant fraction was calculated by dividing the number of duced rat liver PMS, significantly higher concentrations of colonies observed under selective conditions by the number of benzo(a)pyrene or soot extract were required to induce signif colonies observed under permissive conditions and multiplying icant amounts of mutation (data not presented). by appropriate dilution factors. Benzo(a)pyrene, often considered the highest contributor to the mutagenicity of soot, constitutes less than 1% of the RESULTS AND DISCUSSION kerosene soot extract (23) and accounts for less than 3% of the observed mutagenicity of that soot extract. Thus, the ob All experiments were performed using one of 2 frozen served mutagenicity could not be explained by the mutagenicity batches of bacterial strain TM677 (28). Analysis of the variation of benzo(a)pyrene. among assays shows an approximately normal distribution of Two possibilities could logically account for this phenome the background mutant fraction (Chart 1). The mean back non: nonmutagenic components could act synergistically with ground mutant fraction for experiments performed from the benzo(a)pyrene; alternatively, other components of the soot first frozen batch (all experiments between June 26, 1977, and extract could have yet undiscovered significant mutagenic

OCTOBER 1979 4153

concentration yielding significant mutation for each compound (Table 1) was calculated by interpolation from the concentra tion response curve [see Skopek ef al. (27) for the method of calculation]. In addition, the mutagenic potency relative to benzo(a)pyrene (Table 1, Column 6) was calculated by dividing the initial slope of the concentration response curve (mutant fraction in 2 hr/concentration) by the "slope" of the simulta neously performed (80 UM) benzo(a)pyrene standard, which lies on the linear portion of the benzo(a)pyrene concentration response curve. For those interested in comparing the mutagenicity to the KEROSENE reported carcinogenicities of these compounds, available ani SOOT mal 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-methylcholanthrene, dibenz(a,c)anthracene, benzo( g/7/)perylene, and dibenz(a,ft)anthracene] has been recognized previously (e.g., Refs. 5 and 17), this is the first report of mutagenic activity in bacteria of 23 of the PAH associated with soot. Of particular interest is the extreme mutagenic activity of perylene, cyclopenta(cd)pyrene, and fluoranthene, all of which exhibit greater SULFUR- __â€” â€” mutagenicity than benzo(a)pyrene at equimolar concentrations CONTAINING SOOT (Chart 4). Although the mutagenic response of perylene 991 CONFIDENCE LIMIT reaches a stable maximum at concentrations greater than 12 juM, it induces significant mutation at concentrations as low as J_ _L _L _L J_ _L _L 0 10 20 30 40 50 60 70 80 90 100

METHYLENE CHLORIDE EXTRACT (fÃg/mfÃÂ»2 Hr I Chart 2. Concentration-dependent mutagenicity (open symbols) and toxicity

(closed symbols) of the mÃ©thylÃ¨nechloride extracts of kerosene, furnace black, IOO nitrogen-containing, and sulfur-containing soots to S. typhimurium in the pres ence of Aroclor-induced PMS. Each point represents the average of 2 independ ent determinations. BaP, benzo(a)pyrene (80 /Ã•M);SAG, 8-azaguanine

activity which could cumulatively account for the mutagenic activity of the soot extract. To test the hypothesis of synergism, the mutagenic activity of benzo(a)pyrene was measured in the presence of nitrogen- containing soot extract (80 jug/ml). We observed strictly addi tive mutagenicity when benzo(a)pyrene was added to nitrogen- 60 containing soot extract (Chart 3). This observation is, of course, inconsistent with a significant contribution of synergism of soot components with benzo(a)pyrene, which would increase the s observed mutation for the combination. O To test the second hypothesis, 70 PAH components of

various soots were quantitatively assayed for mutagenic activity â€¢BOPPLUS 8o^g/mJ_ in the presence of PMS from Aroclor-pretreated rats. (When NITROGEN - CONTAINING mutagenicity was not observed with PMS from Aroclor-pre SOOT EXTRACT

treated rats, the compounds were retested in the presence of O BaP ALONE PMS from phenobarbital-pretreated rats. This testing with phenobarbital-induced PMS was performed to increase our general knowledge about the importance of different metabolizing con ex ditions, rather than as a part of our overall analysis of the mutagenicity of soot and its components.) fi I Of the tested components, 34 induced a significant increase in mutant fraction as measured by 8-azaguanine resistance BENZOfolPYRENE (/iM) x 2 Hr Chart 3. Concentration-dependent mutagenicity of benzo(a)pyrene (BaP) in (Table 1). Data for 3 of the 36 remaining components the presence and absence of mÃ©thylÃ¨nechloride nitrogen-containing soot extract [4-azafluorene, anthracene, and 1,2-benzodibenzo(o,d)thio- (80 jig/ml). Lines are fitted by the method of least-squares linear regression. Slopes obtained by linear regression analysis are: 5.6 X105Â±1.3X105 phene] suggest possible low-level mutagenicity. Solubility mutant â€¢survivor' -fM ' ' for benzo(a)pyrene plus nitrogen-containing soot extract problems encountered with several of the compounds pre (80 /ig/ml); and 5.4 x 10 5 Â± 0.49 x 10~5 mutant-survivor './IM"' for vented testing at higher concentrations (Table 1). The lowest benzo(a)pyrene alone. SAG, 8-azaguanine.

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Table 1 Mutagenicity ol soot components to S typh/muhum mu- induced mu tagenic ac- Carclnogenesis in tation" Concentration013 (32)<0.010.100.010.050.070.010.10<0.010.050.110.030.150.080.500.300.070.080.051.510.140.200.070.201.001.000.116.000.440.060.080.080.770.080.88_â€”â€”-+NANANANANANANANANANAâ€”NANANANANANA-NANANANANANAâ€”â€”NA+NANANANANANANAâ€”â€”NAâ€”NA+NA+Â±â€”NANANANAâ€”NA+Â±â€”NA-++Â±Â±++-â€”NAtivityd animals CompoundBenzenePyridineIndoleNaphthaleneQulnolineIsoquinoline2-Methyl PB"A, mM+ PBA, mM- 6 PBA, mMâ€” 4 PBAA, mM-f 2 /IMâ€” 80 PBA, mM- 8 Indole1 PBAA, mM+ 2 -Methylnaphthalene2-Methylquinoline4-Methylquinoline1 mMâ€” 6 PBAA. mM+ 7 /IMâ€” 90 -Methylisoquinoline3-MethylisoqulnolineAcenaphthylene1 PBA, mM- 7 ITIM'+ 4 PBA. PBA, mM- 1 -Cyanonaphthalene2-CyanonaphthaleneAcenaphthalene2-Phenylpyridlne4-Phenylpyridine2,6-Dimethylnaphthalene2,6-Dimethylquinoline2,7-Dimethylquinoline2,3-DimethylquinazolineFluorene4-Azafluorene1PBA. mMâ€” 1 PBA. mM+ 1 PBA, /IM- 490 PBA. mM+ 6 PBA, 650/IM160 /IM'â€” PBA, PBA. mM- 2 PBA. mMâ€” 2 PBA, 3mM300 /IM'- PBA, PBA, mM- 1 mM'â€” 1 H-Benz(gjindoleCarbazoleDlbenzo(b,d)thiophenem-Dinitrobenzene2,3,6-TrimethylnaphthaleneAnthracenePhenanthrene3,4-Benzoquinoline5,6-Benzoquinollne7.8-Benzoquinollne4H-Cyclopenta(cteOphenanthrene2-Methylanthracene9-Methylanthracene1PBA, PBA, 3mM300 /IM'+ PBAA, 2HIM'600 /IM'225 PBA, /IM'300 PBA, /IM'+ PBAA, /IM+ 140 PBAA, /IM+ 84 /IMâ€” 280 PBAA, mM+ 1 /IM-1- 80 PBA. /IM+ 75 -Methylphenanthrene2-Methylphenanthrene2-PhenylnaphthaleneAnthronePyreneBenzo(o)fluoreneAnthraquinone1PBA. /IM+ 80 PBA, /IMâ€” 40 PBA. 4mM520 /IM'+ PBAAA. /IM-H 140 25/IM1 /IM'â€” 00 PBA, H-Benz(e)indol-2-acid1-MethylpyreneCyclopenta(ct/)pyreneBenz(a)anthraceneChryseneTriphenylene7H-Benzo(de)anthracen-7-oneo-Terphenylm-Terphenyl1PBAAA, mM+ 2 /IM+ 180 /IM+ 7.3 PBA. /IM+ 65 PBAA, /IM+ 45 /IM+ 44 PBA, 100/IM900 /IM'900 PBA, /IM'500 PBA, /IM'+ ,2-Benzodibenzo(D.cy)thiopheneFluoranthene3,3'.5,5'-TetramethylbenzidineBenzo(a)pyreneBenzo(e)pyrenePerylene1.r-Binaphthyl9-Phenylanthracene7,1PBAA, 5/IM420 PBAA. /IM+ /IM+ 4 PBAA. /IM+ 90 UM'4001.1/IM800 PBA, PBA, /IM+ 2-Dimethylbenz(a)anthracene3-MethylcholanthreneBenzo( PBA, /IM+ 25 PBA, /IM+ 190 g/7/)peryleneAnthanthreneDlbenz(a,c)anthraceneDibenz(a PBA. /IM+ 72 PBA. /IM+ 40 PBAA, /IM+ 13 ,/7)anthracenePiceneCoroneneDibenz(b,e)fluorantheneM.W.7879117128129129131142143143143143152153153154155155156157157158166167167167168168170178178179179179190192192192192192194202204208210216226228228228230230230234235240252252252254254256268276276278278278300302PMS"A,75/IM36 /IM'1 PBA, PBASignificant /IM+ 70 26 /IMRelative Where there is more than one pretreatment listed, calculations refer to italicized pretreatment. â€”,no significant Induced mutation; + , significant induced mutation. c Number listed Is lowest concentration of significant Induced mutation for positive responses, highest concentration tested for negative responses. Mutagenlc activity relative to that of the 80 /IM benzo(a)pyrene-positlve control performed simultaneously with test compound. " A, Aroclor; PB, phÃ©nobarbital; NA, not available. ' Indicates upper limit of solubility.

OCTOBER 1979 4155

(Charts 4 to 6) illustrate the absolute necessity of simultane e10- ously measuring the toxicity incurred as a result of treatment when examining chemicals of unknown biological activity. All are mutagenic only at concentrations that are also toxic to the bacteria. Failure to account for this toxicity in calculating mu tagenic potency leads to the erroneous conclusion that none

ACENAPHTHYLENE

99Z CONFIDENCE LIMIT _| L 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, â€¢), fluoranthene (A, A), benzcKa)pyrene (O, â€¢).and perylene (C, 0) to S. typhimurium. All points were assayed in the presence of Aroclor-induced PMS. Each point represents the II _L 0 125 525 650 800 1300 1600 3200 average of 2 independent determinations. SAG, 8-azaguanine. CONCENTRATION (p.M) Â«2 HR Chart 5. Concentration-dependent mutagenicity (open symbols) and toxicity 1.1 fiM. as compared to 4.0 JUMfor benzo(a)pyrene when a (closed symbols) of acenaphthylene (D. â€¢),acenaphthalene (O. â€¢),and 4- 10% (v/v) PMS from Aroclor-pretreated rats is used. phenylpyridine (A, A) to S. typhimurium. Acenaphthalene was assayed in the presence of Aroclor-induced PMS. 4-Phenylpyridine and acenaphthylene were The compounds acenaphthalene, acenaphthylene, 4-phen- assayed in the presence of phenobarbital-induced PMS Each point represents ylpyridine, 5,6-benzoquinoline, and 1-methylnaphthalene the average of 2 independent determinations BAG, 8-azaguanine.

Chart 6. Concentration-dependent mutagen icity (open symbols) and toxicity (closed sym bols) of 4-methylquinoline and 1-methylnaphthalene to S. typhimurium in the presence of Aro clor-induced PMS. Each point represents the average of 2 independent determinations. BAG, 8-azaguanine.

70 350 700 700 3500 7000 4-METHYLOUINOLINE (/AM) x 2 HR -METHYLNAPHTHALENE

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Mutagenicity of Soot and Components to Salmonella of these compounds is mutagenic to S. typhimurium, since the actual number of mutants is not increased by treatment. It is necessary to emphasize further that the potency calcu lations of Table 1 are all based on 10% PMS (v/v) determina tion. The dependence of the apparent mutagenicity of many polycyclics on PMS concentration is not simple monotonie increasing or saturation relationship but often demonstrates a maximum of 1 to 20% (v/v). Thus, the activity relative to benzo(a)pyrene will vary for many compounds if different amounts of PMS are used or if PMS concentration is optimized for each compound. Bearing these facts in mind, however, we note some inter esting relationships between structure and activity in the data of Table 1. For instance, the mutagenic activity of a given PAH often seems lower than that of the corresponding aza com pound (Charts 6 to 8), which may be important in analyzing the soots of fuels, such as coal, that contain significant amounts of nitrogen. Present limitations in chemical analytical technique prevent a quantitative analysis of all the aza aromatics in nitrogen-containing soot. Fortunately, a complete chemical analysis of the polycyclic aromatics in our kerosene soot sample has been performed (23) and is summarized in Table 2, along with our calculations of the expected contribution of each component at total mÃ©th ylÃ¨nechloride extract concentrations of 20 and 100 /ig/ml. We calculated the expected mutagenic contribution for each com 99Z CONFIDENCELIMIT pound by calculating the amount of each compound present in I L

CONCENTRATION (mMI i 2 Hr Chart 8. Concentration-dependent mutagenicity (open symbols) and toxicity (closed symbols) of quinoline (O. â€¢).isoquinoline (D. â€¢).and naphthalene (A, A) to S. typhimurium. Quinoline and isoquinoline were assayed in the presence of Aroclor-induced PMS. Naphthalene was assayed separately in the presence of Aroclor- or phenobarbital-induced PMS. Each pomi represents the average of 2 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 be expected to induce from the individual concentration response curve. As can be seen in Table 2, the sum of the contributions of individual PAH constituents is about 2-fold greater than the activity of the kerosene soot mÃ©thylÃ¨nechloride extract. Ex aminations at 20- and 100-/ig/ml concentrations of kerosene soot extract show that compounds may contribute to the mu tagenicity of the extract to different extents, depending on the concentration present, due to nonlinearity of dose response for individual compounds. However, at all levels examined, there is sufficient activity in the individual components to account for the high activity of the extract. The fact that the sum of the mutagenicity of the components was greater than that of the kerosene soot extract could be caused by several factors, such as the imprecision of our estimates or a partial competitive inhibition of metabolizing reactions. To test directly this hypothesis of additivity, a mixture that I40 280 accurately mimics the chemical composition of the kerosene CONCENTRATION (Â¿Â¿M) 2Hr soot extract (Table 2) was constructed and assayed simulta Chart 7. Concentration-dependent mutagenicity (open symbols) and toxicity (closed symbols) of 5,6-benzoquinoline (A, A). 7,8-benzoquinoline (O. â€¢).3,4- neously with the crude kerosene soot extract. Results of these benzoquinoline (O, â€¢).and phenanthrene O, Ã–)to S. typhimurium. 3,4-Benzo- assays indicate that the mutagenicity of the kerosene soot quinoline, 5,6-benzoquinoline. and 7,8-benzoquinoline were assayed in the pres extract was wholly reproduced by this reconstituted mixture of ence of Aroclor-induced PMS. Phenanthrene was assayed separately in the presence of either Aroclor- or phenobarbital-induced PMS. Each point represents known components (Chart 9). the average of 2 independent determinations. SAG, 8-azaguanine. Thus, the mutagenic activity of the PAH fraction of the

OCTOBER 1979 4157

Table 2 Mutagenic contribution of individual PAH to kerosene soot extract MI]mlMutation fig/mlMutation

contribution contribution pres (induced mutant frac pres (induced mutant frac CompoundAcenaphthyleneCyclopenta(cd)pyrenePyreneBenzo(gni)peryleneent(jig/ml)4.63.01.61.61.00.80.60.60.40.40.40.40.20.20.20.20.0080.0063.72020tion x10s)030026000a0â€”â€”1.40â€”0.6â€”00â€”34.6201Amountent(/ig/ml)2315885433222211110.40.318.310000tion x10s)01651.73.401050__0___340_3.4â€”00312.5150

anthanthreneCoroneneFluorantheneNaphthaleneBenzo(+

gnijfluoranthenePhenanthrene anthraceneBenzacenaphthaleneBenzofluoranthenePeryleneAcenaphthaleneIndenod+

.2.3-cd)pyreneBenzo(a)pyrene benzo(e)pyrene4H-Cyclopenta(deOphenanthreneBenzofluoreneFluoreneUncharacterized+

material0i;

contributionsMÃ©thylÃ¨necomponent chloride extractWt(%)2315885433222211110.40.318.3100Amount " â€”. component not available for testing. 6 Material lost in the characterization process, plus those compounds which could not be identified by gas chromatography-mass spectrometry.

kerosene soot extract seems to be due to simple additive contributions of its mutagenic components.

ACKNOWLEDGMENTS

We acknowledge C. Crespi. R. DiPietro. D. Fleiscnaker. J. Herland, G. Kurz- ISO 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; and J. Larsen for typing and technical drawings. I60

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Debra A. Kaden, Ronald A. Hites and William G. Thilly

Cancer Res 1979;39:4152-4159.

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