[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

in Salmonella typhimurium, using resistance to the purineanalog 8-azaguanine as a genetic marker. A postmitochondrialsupernatant derived from livers of phénobarbital- and/or Aro-clor-pretreated male Sprague-Dawley rats was incorporated

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

tional Institute of Environmental Health Sciences Grants NIH-2-P01-ES00597-08and NIH-5-T32-ES07020-03. Biomédical Research Support Grant NIH-5-S05-RR07047-11 and Grant 1P30-ES-02109-01 from NIH, United States Departmentot Energy Grant EE-77-S-02-4267. Grant EX-76-A-01-2295 from the UnitedStates Department of Energy Institutional Agreement through the MIT EnergyLaboratory, and the MIT Undergraduate Research Opportunity Program.

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).

MATERIALS AND METHODS

Sources of Soot. Nitrogen- and sulfur-containing soots weregenerated from mixtures containing equal parts of pyridine,Decalin, and o-xylene and from thiophene, Decalin, and o-xylene, respectively. Mixtures were burned in an alcoholburner, and soot was collected on the bottom of a water-cooledflask placed directly over the flame. The soot was washed fromthe collection flask with glass wool and méthylènechloride(24). Kerosene soot was obtained by burning a kerosene fuelin a turbulent, continuous-flow combustor, and subsequentcollection was done with a water-cooled probe (23).

Soot samples were extracted in méthylènechloride in aSoxhlet extractor for 18 hr, evaporated by rotary evaporationunder vacuum, and redissolved in dimethyl sulfoxide.

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-

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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 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

Pharmaceuticals, Inc., Plainview, N. Y. Sodium phénobarbitaland méthylènechloride were obtained from Mallinckrodt Chemical Works, St. Louis, Mo. Quinoline and dimethyl sulfoxide,reagent grade, were obtained from Matheson, Coleman & Bell,Norwood, Ohio. 1-Methylisoquinoline was obtained from Pfaltz& Bauer, Stamford, Conn. Anthracene, benzo(a)pyrene, di-benz(a,c)anthracene, dibenz(a,ft)anthracene, 7,12-dimethyl-benz(a)anthracene, 2,3-dimethylquinazoline, m-dinitroben-zene, 3-methylcholanthrene, and 2-methylindole were obtained

from Sigma Chemical Co., St. Louis, Mo.Bacterial Mutation Assay. Mutation assays were carried out

as specified by Skopek ef al. (27, 28). Exponentially growingcultures of Salmonella typhimurium strain TM677 were exposed to several concentrations of the test agent for 2 hr in thepresence of 10% (v/v) of a PMS, prepared as a 25% (w/v)liver homogenate of phénobarbital- or Aroclor-pretreated maleSprague-Dawley rats (Charles River Breeding Laboratories,

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.

Using this criterion, the méthylènechloride extracts of nitrogen-containing, sulfur-containing, furnace black, and kerosene

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

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D.A.Kadenetal.

KEROSENE

SOOT

SULFUR-__— —

CONTAINING SOOT

991 CONFIDENCE LIMIT

J_ _L _L _L J_ _L _L0 10 20 30 40 50 60 70 80 90 100

METHYLENE CHLORIDE EXTRACT (fíg/mfí»2 Hr

Chart 2. Concentration-dependent mutagenicity (open symbols) and toxicity(closed symbols) of the méthylènechloride extracts of kerosene, furnace black,nitrogen-containing, and sulfur-containing soots to S. typhimurium in the presence of Aroclor-induced PMS. Each point represents the average of 2 independent determinations. BaP, benzo(a)pyrene (80 /ÕM);SAG, 8-azaguanine

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

IOO

60

I

sO

•BOP PLUS 8o^g/mJ_NITROGEN -

CONTAININGSOOT EXTRACT

O BaP ALONE

ex

fi I

BENZOfolPYRENE (/iM) x 2 Hr

Chart 3. Concentration-dependent mutagenicity of benzo(a)pyrene (BaP) inthe presence and absence of méthylènechloride nitrogen-containing soot extract(80 jig/ml). Lines are fitted by the method of least-squares linear regression.Slopes obtained by linear regression analysis are: 5.6 X105±1.3X105mutant •survivor ' -fM ' ' for benzo(a)pyrene plus nitrogen-containing soot extract(80 /ig/ml); and 5.4 x 10 5 ± 0.49 x 10~5 mutant-survivor './IM"' for

benzo(a)pyrene alone. SAG, 8-azaguanine.

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Mutagenicity of Soot and Components to Salmonella

Table 1

Mutagenicity ol soot components to S typh/muhum

CompoundBenzenePyridineIndoleNaphthaleneQulnolineIsoquinoline2-Methyl

Indole1-Methylnaphthalene2-Methylquinoline4-Methylquinoline1

-Methylisoquinoline3-MethylisoqulnolineAcenaphthylene1

-Cyanonaphthalene2-CyanonaphthaleneAcenaphthalene2-Phenylpyridlne4-Phenylpyridine2,6-Dimethylnaphthalene2,6-Dimethylquinoline2,7-Dimethylquinoline2,3-DimethylquinazolineFluorene4-Azafluorene1

H-Benz(gjindoleCarbazoleDlbenzo(b,d)thiophenem-Dinitrobenzene2,3,6-TrimethylnaphthaleneAnthracenePhenanthrene3,4-Benzoquinoline5,6-Benzoquinollne7.8-Benzoquinollne4H-Cyclopenta(cteOphenanthrene2-Methylanthracene9-Methylanthracene1

-Methylphenanthrene2-Methylphenanthrene2-PhenylnaphthaleneAnthronePyreneBenzo(o)fluoreneAnthraquinone1

H-Benz(e)indol-2-acid1-MethylpyreneCyclopenta(ct/)pyreneBenz(a)anthraceneChryseneTriphenylene7H-Benzo(de)anthracen-7-oneo-Terphenylm-Terphenyl1

,2-Benzodibenzo(D.cy)thiopheneFluoranthene3,3'.5,5'-TetramethylbenzidineBenzo(a)pyreneBenzo(e)pyrenePerylene1.r-Binaphthyl9-Phenylanthracene7,1

2-Dimethylbenz(a)anthracene3-MethylcholanthreneBenzo(

g/7/)peryleneAnthanthreneDlbenz(a,c)anthraceneDibenz(a

,/7)anthracenePiceneCoroneneDibenz(b,e)fluorantheneM.W.7879117128129129131142143143143143152153153154155155156157157158166167167167168168170178178179179179190192192192192192194202204208210216226228228228230230230234235240252252252254254256268276276278278278300302PMS"A,

PB"A,

PBA,PBA,PBAA,

PBA,PBAA,

PBAA.

PBA,PBA.PBA,PBA.PBA.PBA,PBA.PBA,PBA,PBA.PBA.PBA,PBA,PBA,PBA,PBA,PBAA,

PBA,PBA,PBAA,

PBAA,

PBAA,

PBA.PBA.PBA,PBA.PBAAA.

PBA,PBAAA,

PBA.PBAA,

PBA,PBA,PBA,PBAA,

PBAA.

PBAA.

PBA,PBA,PBA,PBA,PBA.PBA.PBAA,

PBA,PBASignificant

induced mutation" Concentration013

mM+6mM-4mM—2mM-f80/IM—8mM-2mM+

6mM—7mM+

90/IM—7mM-4ITIM'+

1mM-1mM—1mM+490/IM-6mM+

650/IM160/IM'—

2mM-2mM—3mM300

/IM'-

1mM-1mM'—

3mM300/IM'+

2HIM'600/IM'225/IM'300

/IM'+

140/IM+84/IM+280/IM—1mM+

80/IM-1-75/IM+80/IM+40/IM—4mM520

/IM'+

140/IM-H25/IM1

00/IM'—

2mM+

180/IM+7.3/IM+65/IM+45/IM+44/IM+100/IM900/IM'900/IM'500

/IM'+

5/IM420

/IM+4/IM+

90/IM+1.1/IM800UM'400

/IM+25/IM+190/IM+72/IM+40/IM+13/IM+

75/IM36/IM'1

70/IM+26 /IMRelative

mu-tagenic ac- Carclnogenesis in

tivityd animals(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_——-+NANANANANANANANANANA—NANANANANANA-NANANANANANA——NA+NANANANANANANA——NA—NA+NA+±—NANANANA—NA+±—NA-++±±++-—NA

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

concentration tested for negative responses. Mutagenlc activity relative to that of the 80 /IM benzo(a)pyrene-positlve control performedsimultaneously with test compound. " A, Aroclor; PB, phénobarbital; NA, not available. ' Indicates upper limit of solubility.

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D. A. Kaden et al.

e10-

99Z CONFIDENCE LIMIT

_| L

(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.

70 350 700

4-METHYLOUINOLINE (/AM) x 2 HR

700 3500

-METHYLNAPHTHALENE

7000

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Mutagenicity of Soot and Components to Salmonella

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.

Fortunately, a complete chemical analysis of the polycyclicaromatics in our kerosene soot sample has been performed(23) and is summarized in Table 2, along with our calculationsof the expected contribution of each component at total méthylènechloride extract concentrations of 20 and 100 /ig/ml. Wecalculated the expected mutagenic contribution for each compound by calculating the amount of each compound present in

I40 280

CONCENTRATION (¿¿M) 2Hr

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

activity of the kerosene soot méthylènechloride extract. Examinations at 20- and 100-/ig/ml concentrations of kerosene

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

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D. A. Kader) ef al.

Table 2

Mutagenic contribution of individual PAH to kerosene soot extract

CompoundAcenaphthyleneCyclopenta(cd)pyrenePyreneBenzo(gni)perylene

+anthanthreneCoroneneFluorantheneNaphthaleneBenzo(

gnijfluoranthenePhenanthrene+anthraceneBenzacenaphthaleneBenzofluoranthenePeryleneAcenaphthaleneIndenod

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

material0i;

componentcontributionsMéthylènechloride extractWt(%)2315885433222211110.40.318.3100Amount

present(jig/ml)4.63.01.61.61.00.80.60.60.40.40.40.40.20.20.20.20.0080.0063.72020

MI]mlMutation

contribution(induced mutant frac

tion x10s)030026000a0——1.40—0.6—00—34.6201Amountpres

ent(/ig/ml)2315885433222211110.40.318.310000

fig/mlMutation

contribution(induced mutant frac

tion x10s)01651.73.401050__0___340_3.4—00312.5150

" —. 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.

ISO

I60

140

120

80

KEROSENE

SOOT

EXTRACT .

RECONSTITUTED

KEROSENE

SOOT

EXTRACT

60

40

0 10 20 30 40 50 60 70 80 90 100

CONCENTRATION (/ig/ml) x 2 Hr

Chart 9. Concentration-dependent mutagenicity (open symbols) and toxicity(closed symbols') of a méthylènechloride kerosene soot extract (O. •)and a

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.

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OCTOBER 1979 4159

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1979;39:4152-4159. Cancer Res   Debra A. Kaden, Ronald A. Hites and William G. Thilly 

Salmonella typhimuriumHydrocarbons to Mutagenicity of Soot and Associated Polycyclic Aromatic

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