mutagenicity of diesel exhaust particle extracts inhalation toxicology ...

LMF-96 UC 48 APRIL 1982

MUTAGENICITY DIESEL

EXHAUST PARTICLE

CHARLES

R.

OF

EXTRACTS

CLARK,

Ph.D.

INHALATION LOVELACE P.O.

Prepared for Department

TOXICOLOGY RESEARCH INSTITUTE BIOMEDICAL & ENVIRONMENTAL RESEARCH INSTITUTE Box 5890 Albuquerque, NM 87185

the Office of Energy

of Health and Environmental under Contract

Research of the U.S.

Number DE-ACO4-76EVOI013

NOTICE

This report was prepared as an account of work sponsored partially by the United States Government. Neither the United States nor the United States Department of Energy, nor any of their employees, nor any of their contractors, subcontractors, or their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness or usefulness of any information, apparatus, product or process disclosed, or represents that its use would not infringe privately owned rights.

The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Printed

in the United States of America Available

from

National Technical Information Service U. S. Department of Commerce 5285 Port Royal Road Springfield, VA 22161

LMF-96 Category:UC-48

MUTAGENICITYOF

DIESEL EXHAUST PARTICLE EXTRACTS

CharlesR. Clark

InhalationToxicologyResearchInstitute LovelaceBiomedicaland EnvironmentalResearchInstitute P. O. Box 5890 Albuquerque,New Mexico 87185

April 1982

Questionsor Commentsconcerningthis report should be addressedto Dr. Roger O. McClellan at the above addressor by callingFTS 844-6835or Commercial505-844-6835.

Preparedfor the Office of the Health and EnvironmentalResearchof the U. S. Departmentof Energy Under ContractNumber DE-ACO4-76EVOlO13.

ACKNOWLEDGEMENTS I gratefully

acknowledge the helpful

cology Research institute, thanks to Drs. Albert

P. Li,

and their

discussions

with my colleagues

role in conducting

at the Inhalation

much of the work I have reported.

ToxiSpecial

Thomas R. Henderson, John S. Dutcher, James D. Sun, Antone L. Brooks,

Rogene F. Henderson and Roger O. McClellan for their

critique

of this

review.

TABLEOF CONTENTS Title

Section

Page

1/2

EXECUTIVESUMMARY .......................................................................... I INTRODUCTION ...............................................................................

3

II ROLEOF MUTAGENESIS TESTNG.................................................................

4

General Description III

4

of Mutagenesis Tests ..............................................

9

SALMONELLA MUTAGENICITY TEST RESULTS .......................................................

9

General ............................................................................... Influence of Operating Variables on Mutagenicity of Diesel Exhaust Particle Extracts ...........................................................................

ii

Mutagenicity

of Gaseous Diesel Exhaust Components .....................................

17

Mutagenicity

of Heavy vs Light Duty Diesel Engine Emissions ...........................

18

Comparison of Diesel and Spark-lgnition (Gasoline) Mutagenicity ................................................. Bioavailability Limitations

Engine Exhaust Particle ......................

of Organic CompoundsAdsorbed to Diesel Particulate

Exhaust ...........

of Salmonella Bioassay Data ...............................................

IV OTHERSHORT-TERM BIOLOGICALTEST RESULTS ...................................................

V

18 18 21 23

DNARepair ............................................................................

23

Yeast .................................................................................

23

MammalianCell Mutagenesis Assays .....................................................

23

SUMMARY ................................................ REFERENCES .....

....................................

, ..... ~ ......................................................................

25 27

LIST OF FIGURES FigureNO.

Title

Pa~e

TypicalResultsof MutagenicEvaluationof Dichloromethane Extractsof Diesel ParticulateExhaust in 4 Strainsof Salmonellat~phimurium.Data points represent mean and standarddeviationfrom triplicateplates.Closed circles representresponsein absenceof S-9; open circlesindicateresponseafter adding aroclorinducedrat liver S-9 (fromClark and Vigil, 1980) ............................ 10 Distributionof Mass and Mutagenicityof HPLC Fractionsof ExhaustParticle Extractsfrom a 1980 OldsmobileDelta 88, Audi 5000, and Peugeot504 (TA-98, 10 No. S-9) .............................................................................. 3

12 ParticleSamplingSystem.............................................................. DrivingCycles Used in ChassisDynomometerTests of an OldsmobileDelta 88 and 16 VolkswagenDasher ..................................................................... Abilityof VariousSolventsto ExtractMutagenicComponentsfrom Diesel Exhaust 20 Particles.............................................................................

6

20 Extractionof 14C from 14C-LabeledDieselExhaustParticles...........................

LIST OF TABLES TableNo.

Title Page In Vitro BiologicalTests Used to EvaluateExtractsof DieselParticulate 6 Exhaust......................................................................... ChemicalClassesDetectedin HPLC Fractionsof Diesel ExhaustParticle 11 Extractsand Some SpecificChemicalsIdentified................................. Mutagenicityof ExhaustParticleExtractsfrom VariousDieselCars ..............13 Mutagenicityof Extractsof ParticlesCollectedfrom the Exhaustof an OldsmobileDiesel Cara Operatedon DifferentFuels .............................. 14 Compositionof DieselFuels ..................................................... 14 Influenceof Jet "A" and IsoparaffinicFuels on Mutagenicityof Exhaust 15 ParticleExtractsof an OldsmobileDiesel Cara .................................. Influenceof DrivingPatternon Mutagenicityof Diesel ExhaustParticle 17 Extracts........................................................................

8

Mutagenicity of Spark-lgnition and Diesel EngineExhaustParticleExtracts...... 19

9

Mutagenicity of Diesel ExhaustParticleExtractsin CHO/HGPRTAssay.............24

I0

Summaryof Influenceof OperatingVariableson Mutagenicity of Diesel 26 ExhaustParticles...............................................................

ii

EXECUTIVESUMMARY This document is a review of our current knowledgeof the genetictoxicity of diesel exhaust particle associated organics as derived From in vitro mutagenicity assays (principally the Salmonellaor Ames bioassay).In vitro mutagenicityassays, because of their reliablepredictive capabilityfor carcinogenicpotential,have played a useful role in helping to define the biologicallyactive chemicalclassesof diesel particleextractsand the influenceof operatingvariables on exhaust toxicity.They permit testing of a large number of samplesin a relativelyshort time period and provideuseful informationfor settingtesting prioritiesin more complexand Lime consuminganimal tests. A major Departmentof Energy funded collaborativeeffort betweenthe LovelaceInhalationToxicology Research Institute and the BartlesvilleEnergy Technology Center, and the Environmental ProtectionAgency’sHealth Effects Research Laboratoryare the source of much of the information presented.Based on the resultsof these in vitro tests we can concludethat: ¯

Organicsolvent extractsof diesel particulateexhaust containsubstancesthat are muta-

¯

genic in bacterialmammaliancell systems. The mutagenic response to diesel particle extracts is lower in mammalian cells than in bacteria,presumablydue to differencesin metaboliccapacity.

¯

The fate of the particle associatedorganic material, once deposited in the respiratory tract, remains a critical uncertainty.In vitro dissolution studies suggest that the organic materialsmay slowly leave the particlesin the biologicalenvironment,but reactive mutagens may be quickly detoxifiedby binding to non-specificmacromolecules,or

¯

via engulfmentby pulmonarymacrophages. There is no reason to suspectthat the genetictoxicityof diesel exhaustis markedlyinfluencedby engine design,in fact, the mutagenicityof exhaustparticleextractsfrom 12 diesel passengercars operatedunder the same conditionsis very similar.

¯

Driving pattern does not markedly alter the emission of mutagenic chemicals from new cars, but urban drivingmay produceexhaustwith a higher genetictoxicityin older cars.

¯

The mutagenicityof diesel exhaust particleextractsis not markedlydifferentfrom gasoline engine exhaust. However, the emission of genotoxic chemicals in the exhaust may be 10-50 times higher in diesel vehicles because of higher particle emission rates and larger amountsof organicmaterialassociatedwith the particles.

Included in this document is a section on the role of mutagenicitytesting in defining the genetic toxicity of diesel exhaust, a descriptionof the tests used and factors to be considered when interpretingdata from short-termbioassays.This informationwill provide the reader with a better perspectiveof how in vitro mutagenicitydata can be used in designinganimal studiesthat are more reliablypredictiveof potentialhuman health hazards.

1/2

SECTIONI INTRODUCTION While diesel vehicles representonly a few percent of the light duty passenger vehicles currently in use in the U. S,, it is estimated that as much as 20% of the light duty fleet may be powered by diesel engines within 15 years (Cuddihyet al., 1981). Significantadvances in diesel engine design have minimizedthe cost, size, weight,noise, odor and cold start difficultieswhich have traditionally preventedwide spread use of diesel enginesin passengercars. Chief among the diesel poweredvehicle’s disadvantagesare the significantlygreater exhaust emissionsof particles(soot) which have been reportedto be 50-I00 times greaterthan particulate exhaustfrom comparablespark ignitionengines (Springerand Baines,1977). The submicronsize the particlesmake them readilyrespirableinto the deep lung (Wolffe t a l t, 1981) and their chain aggregate, carbonaceous matrix provides a large surface area for adsorption of organic and inorganic combustionproducts.Solvent extractionof the particles collected on filters removes organicchemicalswhich are mutagenicin bacterialtests systems(Huisinghet al., 1978; Clark and Vigil 1980). Since genetic alterationshave been suggestedas a criticalstep in the inductionof cancer (Ames e t a l., 1979), concernhas been raised over the possibleimpact of increaseddiesel vehicleusage on publichealth (Barth and Blacker1978). A number of factors are known to influence the particulateexhaust emission rates of diesel engines includingfuel density, aromaticityand distillationproperties,fuel additives,and the car make and model (Hare et al., 1976; Springer and Baines 1977; Lipkea et al., 1978; Burley and Rosebrock 1979; Hare and Baines 1979). However, little informationis availableon factorswhich influencethe toxicityof organiccompoundsassociatedwith diesel particles.In vitro techniques for estimatingthe mutagenicpotentialof chemicalsand more complexmixtures,have played a useful role in helping to define the influence of certain operating variables, including engine design, fuel composition,driving pattern and temperature.They have also been instrumentalin characterizing the classesof chemicalsresponsiblefor biologicalactivityand in setti.gpriorities for furtherstudiesusing more expensiveanimal exposures. This documentsummarizesthe currentstatus of our knowledgeon the genetictoxicityof diesel exhaust particle associatedmaterials as related to in vitro test results. Much of the information presentedcomes from a major collaborativeeffort between the LovelaceInhalationToxicology ResearchInstitute and the BartlesvilleEnergy TechnologyCenter, funded by the U. S. Department of Energy, Office of Health and EnvironmentalResearch and the U. S. EnvironmentalProtection Agency’s Health Effects Research Laboratory.Findings reported in the scientificliteratureand in symposiumproceedingsare also summarizedhere.

3/4

SECTIONII ROLE OF MUTAGENESISTESTING The concern by genetic toxicologists over the presence of environmental mutagens has been amplified in recent years by evidence suggesting a strong relationshipbetween the ability of chemicals to induce mutations and their carcinogenicpotential.In view of the similaritiesof mechanismsby which chemical mutagens and most chemical carcinogensinduce genetic effects (i.e. molecular alterationof DNA), it has been postulatedthat mutagenic effects can be predictiveof carcinogenecity(Ames et al., 1975). Based on this postulate, a multitude of short-term tests have been developed for detecting potential carcinogens as mutagens and guidelines have been draftedby federalregulatoryagenciesfor the use of these assays. In November 1977, the United States EnvironmentalProtectionAgency issued a precautionary notice which reportedthat organic solventextracts of diesel exhaustparticleswere mutagenicin bacterialassays.Because of the demonstratedrelationshipbetween inducedmutationsand carcinogenesis,a primaryconcernwas the potentialfor increasedlung cancer risk from the inhalationof diesel exhaust. This concernprecipitateda major effort by scientistsfrom industry,government, academia,and private researchinstitutesto better define the potentialhuman health risks associated wth exposure to diesel exhaust. Much of the information now available has come from in vitro short-termmutagenicitytests. These tests, which use a variety of prokaryotic organisms and eukaryotic cells in culture, take advantage of the similar structure and function of DNA in all organisms. Thus, the demonstrationof geneticdamage by a chemicalin bacteria is reasonablecause for concernthat similar damage might occur in other organisms. Detection of mutagenic activity is complicated by the varietyof geneticlesions which can be induced.Mutationsare defined as a change in the genetic information of a somatic or germ cell. Base-pair substitutionand frameshift mutations result from changes in the nucleotidenumber or sequence in the codon. Brusick (1980) has termed these nonvisiblechanges or microlesions,as opposed to visible damage or macrolesions,which are detectable through cytologic analysis of chromosomes.Macrolesionsinclude changes in chromosome number (ploidy)and structuralchangesin the chromosome(deletions,rearrangements, breaks,etc.)~ The primarygoal of the genetictoxicitytestingof diesel exhausthas been the developmentof informationwhich can be used in predictingits carcinogenicpotential.Of more importancehowever from the standpointof the integrityof the human gene pool, is the potentialincreasein the genetic load or frequency of deleteriousmutations in man. The introductionof large numbers of mutations into the gene pool of any species can seriously compromise reproductivecapacity and survival.The impact of a gene mutation on the gene pool is determinedby the cell type affected and the expressionof the mutation. For example, in germ cells, a dominant lethal mutation has little impact on the transmissablegeneticburden since no offspringsurviveto pass on the mutant genes. In dominant, viable mutations, half of the offspring will be affected by the mutation. Recessive mutations are usually not expressed for several generationssince expressionrequires that the offspring receive mutant genes from both parents. This type of mutation is of most concern since recessive mutations can accumulaterapidly in the gene pool as phenotypicallynormal heterozygotes. GeneralDescriptionof MutagenesisTests Mutagenicitytests are usually classifiedby the types of damage which they detect. Many of the tests used to detect gene mutations and DNA damage induced by diesel particle extracts are listed in Table I. Bacterial tests are the most widely used test for detecting gene mutations because of their lower cost, high sensitivityand short time requirements.They generallyrely on

TABLE1 IN VITRO BIOLOGICALTESTS USEDTO EVALUATE EXTRACTS OF DIESEL PARTICULATE EXHAUST A. Gene Mutation Assays I. Reverse mutation in Salmonella typhimurium (Ames test). 2. Forward mutation in Salmonella t~(phimurium. 3. Forward mutation in Saccharomyces pombe, cerevisiae. 4. Mouse lymphoma L5178Y cells,

thymidine

kinase locus.

5. Chinese hamster ovary (CHO) and lung (V-79), 6. Mouse embryo fibroblasts 7. Diploid

cells,

HGPRTand Na/K ATPase loci.

(BALB/c 3T3), Na/K ATPase locus.

human lymphoblasts,

B. Primar~ DNADamageAssays I. Alkaline sucrose gradient

HGPRTlocus. sedimentation

analysis

for DNAstrand breaks.

2. Unscheduled DNAsynthesis. 3. Sister 4. Mitotic

chromatid exchange. recombination

5. DNArepair

in bacteria

C. Oncogenic Transformation I. Mouse embryo fibroblasts 2. Syrian hamster primary

and gene conversion in yeast. (differential

killing

of DNArepair

defective

strains).

(BALB/c 3T3). embryo (SHE) cells.

mutagen-inducedchanges in the nutritional requirement of the cells as a basis for selecting mutant organisms.Bacterial systems used for measuring primary DNA damage take advantage of the DNA modifying ability of most mutagens. DNA repair tests measure DNA damage and only presume to detect mutagens,and are used as complementary detectionsystemswith the gene mutationassays. The yeast Saccharomycescerevisiaeis a eukaryoticorganism containinga nucleus, cytoplasm and differentiatedorganellessimilar to other life forms. The organism can be cultivated as a haploiduseful for detectionof gene mutationsor as stable diploidsfor detectingmitoticrecombinations.The consequencesof mitoticrecombinationsare the generationof new genotypesvia ex= change of genetic material between homologouschromosomes and segregation of mitotic products. This leads to expressionof a recessive phenotypein the homozygousconditionwhich would otherwise go undetectedin the heterozygousstate. Mitotic gene conversionis regarded by some as an indicatorof repairof inducedDNA damage. In vitro mammaliancell mutationassays are used both in the detection of gene mutations and to confirm that a chemical positive in bacterial tests is active in more relevant mammalian cells. Bacterialcells used in mutationassays are optimizedfor their ability to detect mutagens and differ significantlyfrom mammalian cells in their membrane integrity,genome organization, DNA repair mechanismsand metaboliccapability.While mammaliancell assays are not as sensitive as bacterialsystems,they providerelevancein extrapolationto potentialhuman risk. The genetic endpointsin most mammaliancell mutagenesisassays are based on drug-resistance becauseof the ease with which resistance can be used for mutant selection. The most commonly used drugresistancemarkers are 6-thioguanineor 8-azaguanineresistance,ouabain resistance,and bromodeoxyuridineor trifluorothymidine resistance.

Another mammalian cell cinogenesis

is in vitro

damage, Cell

transformation

assay, the cells

(usually

posed to the test for

assay which may prove to be particularly

cell

transformed

transformation. is generally

While this

thought to simulate

rodent embryonic fibroblast

chemical for a predetermined

foci.

Normal cells

will

with chemical carcinogens tend to "pile of contact inhibition

divide

useful

as a predictor

assay does not directly

lines)

time period.

in vivo oncogenic events. are seeded in culture The cells

and form an orderly

up" with criss-cross

of car-

measure genetic

patterns

In this

flasks

and ex-

are scored after

5-8 weeks

monolayer while cells

treated

and have an an apparent lack

of growth.

Most of the tests provide an indication

described above include the use of a liver microsomal enzyme preparation to of the role of metabolism in the genetic toxicity of the test compound.

Before the inclusion

of this

which required use of rat

liver

biotransformation

procedure

it

was not possible

by metabolic

enzymes to exert their

homogenates (9000 x g supernatant

Aroclor

1254 to enhance enzyme activity

et al.,

1975).

to detect

although

fraction,

other tissues

718

indirect

activity.

acting

mutagens

Most commonis the

hence the name S-9) induced with and inducers

have been used (Ames

SECTIONIII SALMONELLAMUTAGENICITYTEST RESULTS General The Salmonellamutagenicitytest, commonly referredto as the Ames test, has found widespread applicationin defining the genetictoxicityof diesel particulateexhaust.It is a reversemutation assay using five different strains of Salmonella typhimurium (Ames et al., 1975). The strains contain one of three different mutations in the histidine operon which results in a requirementfor histidineenrichedmedia for survival.This nutritionaldeficiencycan be corrected by mutagenswhich reversethe originalmutationsand is the basis for selectionof mutant (revertant) colonies in the test. Special strains have been constructedwhich have more permeablecell walls and that lack the DNA excision repair system. This greatly facilitates the exposure of intracellularDNA which enhancessusceptibilityto damage.In additiontwo of the strainscontain R-factor plasmids which enhance mutagen detection presumably by additional alterations in DNA repaircapability. The major role of the Ames test in the overalleffort to predict the hazard of diesel exhaust has been in estimatingfactorswhich influencethe mutagenicityof the particleassociatedorganic materials.This informationis critical in estimatingthe likely exposure from various scenarios of diesel vehicle penetrationinto the light duty fleet. Using the Ames test as an indicator of differencesin biologicalactivity, diesel exhaust samples have been evaluated for mutagenicity investigatingvariables such as engine type, fuel composition, driving pattern, hot vs. cold start, environmentaltemperature,and turbocharging.Comparisonshave also been made between diesel and gasoline engine exhaust,new and in-use vehicles,and light vs heavy duty diesel exhaust. The Ames test has also been coupled with chemical fractionationproceduresto aid in identifying the classesof chemicalscontributingto mutagenicityand has been useful in lookingat the potential for artifactualformationof mutagensduring exhaustsampling. Since EPA’s initial cautionary notice on the potential hazard Of diesel exhaust particles, numerous investigatorshave confirmed the mutagenicityof organic solvent extracts of the particles in the Ames test. The available evidence suggests that a complex mixture of both direct and indirect frameshifttype mutagens are extractable from the particles. Figure l illustrates the typical responseof the particle extractsin four strains of Salmonella,with and without the addition of liver S-9 as a source of metabolicenzymes.The R-factorcontainingstrains TA-98 and TA-IO0 show the greatest sensitivity in response. The response in TA-1535 (not shown), which detectsonly base pair substitutiontype mutationsis consistentlynegative. Attention has been focused primarily on the "direct" mutagenicityof the particle extracts, i.e. that activity not requiring metabolic enzymes. High pressure liquid chromatographic(HPLC) separationof the extractsinto variousfractionsby polarity,coupledwith Ames test bioassayof the fractions, has been useful for studying the chemical nature of the mutagens. Schuetzle et al_~L.,(1980)have reportedthat greaterthan 90% of the totalmutagenicactivitycan be isolated fractions comprisinga small percentageof the mass of the crude extract. Unpublisheddata from this laboratoryhave confirmed these findings with particle extracts from 1980 Oldsmobile,Audi and Peugeotdiesel vehicles(Figure2). The most mutagenicfractionsconsistprimarilyof substituted polycyclicaromatic hydrocarbon(PAH) derivatives,including nitro-PAH species (Table In one of the fractions,l-nitropyrenewas positivelyidentified.It is clear from these studies that the biological activity cannot be traced to one or a few compounds but to many mutagenic species.

I00

500TA-153F

TA-1538

~

80

400-

6O

500-

40

200-

mr [,i > 0o I bd mr 12OO b0 rr hi m

800

I00 - ¯

=c~ 200

OO

400

125 250 12OO- TA’98

~

TA-IO0

_

?

s-9

400-

40C-

0

500

800

Soline-~

Z

0

II

I I I =u400 O0 125 250 500 DIESEL PARTICULATEEXHAUSTEXTRACT

200 ,u.g

Figure I. Typical results of mutagenicevaluationof dichloromethaneextracts of diesel particulate exhaust in 4 strains of Salmonellat x_phimurium.Data points represent mean and standard deviation from triplicate plates. Closed circles represent response in absence of S-9; open circlesindicateresponseafter adding Aroclorinducedrat liver S-9 (From Clark and Vigil, 1980).

DIESEL 100 o

.,., o

MASS

5O IZ ,,, 0 0 rim 100n

n , liJ O:

500 Lavage Fluid

/

____~ .... I

Saline

06..... s

.~

I

I 48 EXTRACTION TIME (hr)

I 72

24

Figure 5. Ability of various solvents to extract mutagenic components from diesel exhaust particles.

0100[ - I ]~

CH2CI2

/ rT’-~

,, ~~51/

// ,,=x ,I O0 6 12

-, ~f

24 48 EXTRACTION TIME (hr)

?2

Figure 6. Extractionof 14C from 14C-labeleddiesel exhaustparticles.

20

Limitationsof SalmonellaBioassayData The Ames test is generallythought to be most useful as a means for detectionof a chemical’s potential to induce mutations and is thus widely used in the initial testing phase in programs where large numbers of chemicals are being evaluated. Basic differences in the structure and organizationof prokaryoticand eukaryoticcells and their geneticmaterialcautionagainstdirect extrapolationof Ames test results to probable mammalian cell toxicity without confirmationin mammalian cell tests. Differences in the metabolic capability of bacterial and mammalian cells may play a key role in decipheringthe usefulnessof Ames test data in assessing the human health implicationsof inhaleddieselexhaust. The presenceof active nitroreductase enzymesin the Ames test bacteriamay be responsiblefor the potent mutagenicityof nitro-substituted PAH, a number of which have been positivelyor tentatively identified in diesel exhaust particle extracts (Table 2). One report estimated that l-nitropyreneaccountedfor as much as 30% of the "direct"mutagenicityobservedin an extract of diesel exhaust particles (Schuetzle et al., 1981). Since the mutagenicity of nitropyrene and other nitro-PAHis significantlylower in nitroreductase deficientSalmonellastrains,it has been suggestedthat the use of these strainsmay be more appropriatefor estimatingthe potentialmutagenic/carcinogenic risk of nitro-PAHcontainingcombustionproducts(NationalAcademyof Sciences, 1981). A number of studieshave demonstratedthat extractsof diesel exhaustparticlesare less mutagenic in nitroreductasedeficientstrains. Furthermore,mutagenicitywas increasedunder anaerobic conditons which favor reductive metabolism(Pederson and Siak, 1981b). When the extract was separatedinto unsubstitutedPAH, nitro-PAHand more polar aromaticcompounds,about one-thirdof the mutagenicityof the crude extractcould be recoveredin the nitro-PAHfraction. In studies at ITRI/BETC,extracts from five different diesel cars were 30-60% less mutagenic in a nitroreductasedeficient strain (Clark et al., 1982). These same extracts were negative or only weakly mutagenicin Chinesehamsterovary (CHO) cells (Li and Royer, 1981). Subsequentassay of the CHO cell homogenatesfor nitroreductaseactivity showed them to be essentiallydevoid of any activity.Known mammalianenzyme systems capableof catalyzingnitroreductionsincludemicrosomal NADPH-cytochromec reductase,cytosol DT diaphorase,xanthineoxidase and aldehydeoxidase. Another significant source of nitroreductasesare microflora present in the gastrointestinal tract. Unfortunately,the role of nitroreductasesin the toxicologyof nitro-PAHis poorly understood. (Interpretation is further complicated by the suggestions that there may be multiple nitroreductasesin Salmonellawhich may differ in their specificityfor nitro-PAH-McCoyet al., 1981.) The nitroreductasecapabilityof intestinalmicrofloramay be particularlyrelevantto the ultimatetoxicityof inhaled diesel exhaust since particlesdepositedin the upper airwaysmay be clearedby mucociliarytransportand subsequentlyswallowed. While the occurrenceof nitro-PAH in ambientair and fossil fuel combustionproductshas been well documented,the mechanismfor their formationhas been somewhatcontroversialsince Pitts et i al. (1978) showed that nitrated benzo(a)pyrene(BaP) could be produced by drawing 2 through a BaP coated filter. Pitts suggestedthat PAH adsorbedto particlesmay react with gaseous air pollutantsto convertindirectmutagens(requiringenzymic activation)to direct acting mutagens. also raised the possibilitythat oxygenatedand nitratedPAH might occur as a result of the procedures used to sample, i.e., trapping particleswith adsorbedPAH on filters and drawing reactive gases across the particles during air sampling. Subsequently,Lee et al. (1981) showed that the filter type influencedthe reaction of BaP with NO2; more degradationof BaP occurred on quartz fiber than on Teflon membranefilters. Two additional studies have been reported in which sampling of diesel exhaust on different filter types for varying time periodswas done to investigatethe occurrenceof filter artifacts

21

during sampling. The first demonstratedthat filter type did not influence the mutagenicityof the particles but did not rule out the occurrence of artifacts on all filters (Clark et al., 1981d). The second study (Gorse et al., 1981) showed that increasing the sampling period and, thus, the mass loadingof the filter,increasesthe extractablematerialassociatedwith the particles and the specific mutagenicityof the particle extracts (revertants/ugextract). The increaseswere attributedto reactionsbetween particlebound organicmaterialand reactivegases in the exhauststream. Whethernitro-PAHformationoccurs in the combustionprocess or during sampling, or both, still has not been resolved.

22

SECTIONIV OTHER SHORT-TERMBIOLOGICALTESTS DNA Repair Analysis for DNA strand breaks by sedimentation of isolated DNA in an alkaline sucrose gradientwas negativeafter direct incubationwith extractsof exhaustparticlesfrom three diesel cars (Casto et al., 1980). These studies used primary cultures of Syrian hamster embryo (SHE) cells treated for 18 hours with the extract. Similar extracts were positive in the B. subtilis comptest which measures the abilityof the test substanceto induce DNA damage or prevent DNA replicaton (Dukovichet al., 1981). While this assay measures induction of a repair system that is probably present only in prokaryoticcells the results do serve as an indicator that DNA damage has occurred (Brusick, 1980). It is noteworthy that particle extracts incubated directly with isolated DNA preparations failed to form adducts or react with the DNA (Pederson and Siak, 1981b). On the other hand, it has been suggested that human Xeroderma pigmentosum (xp) fibroblasts are more sensitiveto the cytotoxicityof diesel particleextractsthan normal human fibroblasts because of the formation of lethal DNA adducts for which the xp cells have little or no repair capacity(McCormacket alo, 1980). Yeast Detection of mitotic recombinationand gene conversionsare the primaryuses of yeast assays although some strains are used in assays for point mutations. Three diesel particle extracts failed to increase the observed number of recombinantsin S. cerevisiae D-3 at concentrations ranging from I00 to 2000 ug (Mitchell et al., 1980). The polar oxygenatedfraction of the crude extract(OXY) however,did result in a moderateincreasein mitoticgene conversionsand was positive in a S._S~ pombe forwardmutationassay. The transitionfractionwhich containsoxygenatedPAH and is the most active fraction in the Ames test, was negativein the yeast forward mutation and recombinationassays(Lopr!enoet al., 1980). MammalianCell MutagenesisAssays These assays, like the Ames test, are capable of detecting gene locus (point) mutations.The best validated of these is the Chinese hamster ovary (CHO) test which measures mutations at the hypoxanthineguanine phosoribosyl transferase (HGPRT) locus and the mouse lymphoma test which selectsfor mutantsthat are resistantto ouabaindue to inducedabnormalitiesin the synthesisof thymidine kinase. Most of the cell lines used in these assays require the addition of metabolic enzymesto detect promutagens. Diesel exhaust particleextractshave been shown to be negativeor weakly positivein Chinese hamster ovary cells (CHO/HGPRT) and usually require S-9 to elicit a positive response (Li Royer, 1982; Casto et al., 1981). Table 9 summarizesthe resultsof testing5 extractswhich were positive in the Ames test. The mutagenicityof diesel exhaust extracts in CHO cells was calculated to be only 0.5% of that of benzo(a)pyrene (Li et al., 1982). Li and Royer, (1982) reported that diesel exhaust particle extracts are "co-mutagenic,"i.e., when assayed together with a known mutagenthey enhance the predictedmutagenicresponse.Extracts from the particulate exhaust of an Oldsmobile 350 diesel were negative in Chinese hamster lung (V-79) cells (Rudd, 1980). Weak induction of sister chromatid exchangeswere induced by a diesel particle extract in the presenceof liver S-9 (Li and Brooks,1981). Diesel particleextracts were positive, with and withoutS-9, in L5178Y mouse lymphoma cells (Mitchell et al., 1981) and negative or weakly positive in BALB/c 3T3 cells (Current et al., 1980). Particle extracts of an Oldsmobile350 were positive in human lymphoblastsbut only when S-9 was added (Barfknechtet al., 1981).

23

TABLE9 Mutagenicityof Diesel ExhaustParticleExtractsin the CHO/HGPRTAssay Mutant Frequency (per lO6 survivors) Treatment

WithoutS-9

With S-9

2

7

Car A

7

28

Car B

3

6

Car C

2

II

Car D

15

9

Car E Benzo(a)pyrene (0.5 ~g/ml)

lO 2

25

MNNG (0.05 ~g/ml)

62

-

SolventControl(DMSO) ExhaustExtracts(60 ~g/ml)

56

From Li and Royer,1982.

Anothermammaliancell assay which may be predictiveof carcinogenesis is in vitro cell transformation. Extracts of diesel particulate exhaust have produced morphologically transformed colonies in BALB/c 3T3 cells at some concentrationsbut no clear dose-dependentrelationshipwas observed (Current et al., 1980). Two of four diesel extracts tested produced an increase in the number of virus-transformedloci in a viral enhancementassay using Syrian hamster embryo cells (Casto et al. 1981). This assay measures the ability of a chemical to enhance viral transformation in mammaliancells while the BALB assay measuresthe ability of a chemicalto directlytransform mammaliancells.

24

SECTIONV SUMMARY Research conducted understanding on better

in our capability it

¯

identify

specific

that the mutagenicity

extracts

effort

certain

classes.

to our

has been focused of the resultant advancements

with the exhaust parti-

chemicals responsible

can be attributed

for

the muta-

to one or a few compounds,

Nitro-substituted

PAH may contribute

From the evidence cited above we can conclude that:

of diesel

particulate

and mammalian cell

exhaust contain

substances that are muta-

systems.

The lower mutagenic response of mammalian cells, ferences in the levels

¯

Considerable

parameters on mutagenicity

are summarized in Table I0. Despite significant

to conclusively

genic in bacterial e

exhaust.

operating

to the observed mutagenicity.

Organic solvent

few years have added greatly

the complex array of chemicals associated

to numerous compounds within

significantly

in the last

of diesel

of various

These findings

to separate

It appears unlikely

but rather

term tests

toxicology

the influence

has been difficult

genicity.

short

of the genetic

defining

combustion products. cles,

with

of nitroreductase

compared to bacteria,

may be due to dif-

enzymes.

The fate of particle associated organic material, once deposited in the respiratory tract, remains a critical uncertainty. In vitro dissolution studies suggest that the organic materialsmay slowly leave the particles in the biologicalenvironment,but reactive mutagens may be quickly detoxifiedby binding to non-specificmacromolecules,or via engulfmentby pulmonarymarcrophages.

¯

There is no reason to suspectthat the genetictoxicityof diesel exhaustis markedlyinfluencedby enginedesign.

¯

Driving pattern does not markedly alter the emission of mutagenic chemicals from new cars, but urban drivingmay produceexhaustwith a higher genetictoxicityin older cars.

¯

The mutagenicity line

of diesel

engine exhaust.

10-50 times higher larger The results that require

exhaust particle

in diesel

vehicles

amounts of organic material of in vitro

further

extracts

is not markedly different

However, the emission of genotoxic

mutagenicity

investigation.

from gaso-

chemicals in the exhaust may be

because of higher

particle

emission

rates

and

associated with the particles. assays have also helped to better

define

research areas

These include:

¯

The extent to which engine age or malfunctioninfluencesmutagenicityof the exhaust.

¯

The fate of biologicallyactive chemicalsonce dissociatedfrom the particles.

¯

The role of mammalian, including human, nitroreductasesin the toxicity of nitro-PAH, particularlyin the lung and gastrointestinal tract.

¯

A more critical examination of the occurrence of pro-mutagensassociated with exhaust particles (i.e., those requiring activation by metabolic enzymes), especially with respectto differencesin diesel and gasolineengine exhaust.

¯

Determine how differences term tests may influence

in repair

mechanisms between man and organisms used in short

genetic toxicity.

A better understandingof toxic responses resulting from interactionsbetween chemicals in the exhaust and other xenobiotics that individuals may be exposed to (e.g. drugs, cigarettesmoke). e

Measurementsof cellularconcentrationsof particleextractsrequiredto producetoxicity in short-term tests and their relevance to concentrationsrequired to produce cellular damagein vivo.

¯

Identification of the potentialtumor promotingpotentialof diesel particleextracts.

25

I--

tel

cU ~J

"O

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(lJ

l_

0J

~-

o o

~

O (.J

E O L ¯~

k~--

O2 O~

{U ~0 ,r-

0- E~

~lJ

(J a.j S-

"--.-

-~

°~

0J cl. ~L 1--~J LL

"O 4--O

¢’,,J

O tn

~

0J O

cn~. ~

r-- ¢=

0

40 (U (U

(.j °t-,r--

O

(U 0.)

O~

(1~ qJ

O2 0 H

O

4-O~

O~ ¢O

Od O3

u~

~"

0J n~ OJ ~J 0J 0.J S- ,’~ 0 O

O

(l~

L)

O2

~lJ ~J to ,~ L)

L)

o

r~

~-~

,r,-

(U ¢p

~2

~j u

0 z

O z

O Z

0

¢O

~ oj od o~

O

ic-

tJ

E

o 4J

qj O-

¢O

¢O O0

(1) 0~

.t~ .t3

~0

0.J O tJ ,’-- Lt~

O .~

0J .~ 4.J

o ~_

~.O

-r-

,~

u_

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% ,,=

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"E

,=,

26

"~

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> ~ O

o

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27

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28

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30