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MUTAGENIC AND GENOTOXIC POTENTIAL OF NITRATED POLYAROMATIC HYDROCARBONS IN COMBUSTION BYPRODUCT MIXTURES

by

Blair Pritchett, B.Sc., LL.B.

Athesis submitted to the School of Graduate Studies in partial fulfilment of the

requirements for the degree of Master of Science

Biochemistry

Memorial University of Newfoundland St. Jobo' s, Newfoundland

September,1999

ABSTRACT

The combustion of hydrocarbon fuels generates a considerable amount of reaction byproduets., some of which. are mutagenicand DNA-reactive nitro-polyaromatic hydrocarbons. This mutagenic action is generallyindirect,suchthat thecompounds require metabolic activation for the exertion of their toxiceffect.

Used motor oil extracts prove more mutagenicthan crude oil extractsinthe Ames Salmonellaassay for mutagenicity. This mutagenic effect is enhanced by the presence of a rat liver microsomal and cytosolic protein preparation, as well as by the use of a bacterial strainenrichedin0-acetylase activity. The mutagenic response is diminished in a bacterial strain that is deficientinbacterial nitroreduetase. These data implicate nitrocompounds as principal agentsinthe overallmutagenicityof crankcaseoilextracts.

Crankcase oil extractsprove tobegoodsubstrates for nitroreduetaseenzymeactivity inwrro.Theeffectivenessas a substrate ofnitrorcducweenzyme(s) also seems to correlate with the mutagenicity as measuredinthe AmesSalmonella assay. Themeasured nitroreductase activity~bits00appreciable ditfcrencc when NAnH or NADPH is used as the reaction cofactor.

Theactivityof nitroreduaase enzymescaDproduce reactive oxygen species such: as hydroxyl radicals (OH), whichc:angenerate singlesttaDdnicksinDNA. Thiseffect can be initiatedby theInl:tIbotism ofaiumdpolyooclear.-omatichydrocarbonsbymammalianliver enzymes.t-nitropyreneandcranicc:ueoilextractsare shownhereto be capable of producing

such nicks intheDNA ofthe plasmid pBRJ22.

iii

In

Memory of

H. Roy Taylor (1916 - 1996)

and

Cecil B. Pritchett (1908 •

1999)

iv

ACKNOWLEDGEMENTS

I wishtoexpressmygratitude to my program supervisor Dr. A.D. Rahimtula for his guidance and support duringmyproject. as well as for providing mewiththe opportUnity to do this work. Ialsowish tothank theothermembers of my supervisorycomminee. Dr- D.H.

Heeley andDr.J.R.Robinson. for their assistance and advice. Thanks to Donna Jackman Thotben Bieger, and Vietor Drover for guiding meinmy struggleswithDNA. Also thanks to Drs. S. Ray and1.Payne of Fisheries and Oceans. for aUowing me to use their labs and materials, I amindebtedto Marie Codnerforthecoundess occasions whenshepulled me out of the proverbial fire,andfailing that, for making thelaba fun and interesting place tobe The help orall my lab-mates over the past two and ahalf years isyeatly appreciated, as is the companionship and entertainment offeredbythe other members of the Biochemistry Department. I also wish to thank my family foraUtheir suppon throughouttheperiodof my university studies. Finally, I would like tothankmy girlfticndltianceelwife Paula for her constant love and suppon throughoutthecourse of my work; her evolving title serves as a testamem to both the length army programme andthedepth ofher commitment.

ABSTRACT ACIC'OWLEDGEMENTS TABLE OF CONTENTS LIST OFTABLES LIST OF FlGURES LIST OF ABBREVIATIONS

INTRODUCTION

TABLE OF CONTENTS

xi xii xvii

t.I Hydrocarbon combustion

\.1.1 Formation of nitrated polyaromatic hydrocarbon t.2 Metabolismlactivation of nitroarenes

1.2.\ Therole of freeradicals

1.2.2 Free radicalsandnongenotoxic olridative damase 1.3 Nitro.PAHDNAadduetioD

1.4 Theetfectsofmetaboljc routes onnitro-PAHtoxicity 12

l.5 Nitro--PAM and tumorigenesis IJ

1.6 MixturesofN~-PAH 14

1.6.1 Theuse ofnp.post1abelling for identification of DNAaddu<:tspnaa!edbyNO,.PAHmixtures IS

vi

1.7 TheAmes-Sa/~11a assayandnitro-PAH 1.8 Rationale andobjecti"es oftbis study

11 19

MATERIALS AL'll> METIfODS 2.1 Materials

2.1. I Chemicals 2.2 Methods

20 20 20 21

2.2.I Animaltreatment 21

2.2.2 Suceinoytation ofcytochrome c 22

2.2.2.1 Determination ofcytocltrome c

concentration 23

2.2.3 E:rtraetion of crude oils and used crankcase oils 23

2.2.4 Nitroredueweacti\'ityassay 2.2.4.1 Testingof inhibitoryaction

ofDMSOextraet5

2.2.3.1 Extraction of soots 2S

2S

26

27 26

2.2.5.2 2.2.5.3

2.2.S AmesSa/motttIJamutagmicity usay

2.2.S.1 Preparationofrutrm-post·mitocbondriaJ fraction and NADPH-regeoeming system 26 Preparation ofbacterial lester stnin cuJtures 27

vii

2.2.6 J~P.PostlabeUingof ONAadducts 2'

2.2.6.1 Isolation of ONA 2'

2.2.6.2 Digestion of ONA 30

2.2.6.3 Nuclease PI enrichment and detennination

of nucleotide levels 30

2.2.6.4 Preparation of standards and blanks 31

2.2.6.5 np.postlabelling reaction 31

2.2.6.6 PEI..(eUulosethinlayer chromatography

ofnucleotides 32

2.2.6.7 Standard, blank and sample residue analysis J4

2.2.6.8 Analysis oftestsamples 35

2.2.7 Plasmid DNA strand cleavage experiments 35

2.2.7.1 Preparation of solutions 36

2.2.7.2 Preparation ofrat liver tractions 36 2.2.7.3 Generation of pBRJ22 plasmid 36 2.2.7.4 Incubationandanalysis of plasmid DNA

suandclea~ 3.

2.2.7.5 Statistical Analysis of Scanning

DensitometerData 3"

RESULTS 40

3.! Nitroreductue~vity 40

viii

3.1.1 Crude oil elrtraets as rutroreductase substrates 40 3. L2 Crankcase oilextnIctSas ruuoreduetase substrates 45 3.1.3 Sootextractsas nitroreductasesubstrates 51 3.1 AmesSalmonelladetennination of extract mutagenicity 54 3.2.1 Mutagenicity of crude oil elCtraets 54

3.2.2 Mutagenicityofsootextraets 54

3.2.3 Mutagenicity of crankcase oil extracts 68

3.3 np·postlabelling studies 75

3.3.1 In vivocrankcase oilexposure 75

3.4 pBR322 plasmid nicking studies 81

3.4.1 DNAstrandbreaks with I-nitropyrene exposure 81 3.4.2 DNAstrandbreaks with crankcase oil extract exposure 86

3.4.2.1 DNA strand breaks with crankcase oil

3.4.2.2

and1-nitropyreneexposure DNAstrandbreakswithcrankcase oil andDMSO exposure

91

91

4. mSCUSSION

4. 1 Nitroreduetaseactivity stUdies 4.1.1 Crudeoilstudies 4.1.2 Industrial SlaCk soot studies

ix

101 101 101 102

4.1.3 Crankcase oil studies 103

4.1.4 Identity of the nitroreduetase 104

4.2 AmesSalmonella mutagenicity studies 105

4.2.1 Crude oil studies 105

4.2.2 Industrial stack soot studies (05

4.2.3 Crankcase oil studies (07

4.3 J%p.postlabelling studies 108

4.4 pBR322 plasmid nicking studies 109

5. CONCLUSlONS 112

5.1 Conclusions 112

6. REFERENCES 114

LIST OF TABLES

Table}.l

DNA adduct levels generated by crankcase oil administration

xi

76

LIST OF nGURES

Fig.1.1 Metabolism (activation) ofnitrated-PAH

Fig.1.2 Generation of a hydroxyl radical by nitroreduetion reaction inthepresence of iron

Fig, 1.3 Alternative DNA-reactive pathway ofnitroarenes 11

Fig. 2.[ Schematic ofl2p·postIabeUing TLC plates 33

Fig. 3.1.1 Nitroreduewe activity generatedwithDMSO

extract ofPrudhoe Bay crude oil as enzyme substrate 42 Fig, 3.1.2 NitroreductaseactivitygeneratedwithDMSO

extractof Alberta sweetmixcrude oil as enzyme substrate 42 Fig.).1.3 NitroreduetaseactivitygeneratedwithDMSO

extruetof Hibernia crude oil asenzymesubstrate 44 Fig.3.IA N'"rtroreduetase activity generatedwithDMSO

extractofVcnezueJ.an crude oil asenzymesubstrate 44 Fig. 3.2.1 Nitroredueweactivitygeneratedwith DMSO

extractof craDkcaseoil/lIasenzymesubstmc 41 Fig. 3.2.2 N"ttroreduetaseactivity generatedwithDMSO

extractof crankcase oillf2 as enzyme:substrate 41 Fig. 3.3.1 Response of nitroredueweactivity(generatedwithDMSO

extractof crankcaseoil#1asertzyrbCsubstrate) tovarying xii

levels of cytosolic protein 50 Fig. 3.3.2 Response of nitroreduetase activity (generated with DMSO

extract of crankcase oil #2 asenzyme substnte) to varying

levels of cytosolic protein 50

Fig. 3.4.1 Nitroreductase activity generated with DMSO

e:<t.ract of industrial Slack soot #1 as enzyme substrate 53 Fig. 3.4.2 Nitroreductase activity generated with DMSO

e:<t.ract of industrial stack soot #2 as enzyme substrate 53 Fig. 3.5.1 Salmonella typhinrurlum TA98NR revenants produced

byincubation with Prudhoe Bay crude oil DMSOe:<t.ract 56 Fig. 3.5.2 Salmonella typhimurium TA98 revenants produced

by incubation with Prudhoe Bay crude oil DMSOe:<t.ract 56 Fig. 3.5.3 Salmonella typhimurium YGl024 revertants produced

by incubation with Prudhoe Bay crude oil DMSOextract 56 Fig. 3.6.1 Salmonella typhi"""ium TA98NR revenants produced

by incubation with Hjbemia etUde oil DMSOe:<t.ract 58 Fig. 3.6.2 Salmonella typhinnuilJm TA98revertantsproduced

by incubation with Hjbemiacrudeoil DMSOextract 58 Fig. 3.6.3 Salmonella typhintllrium YGI024 revenants produced

by incubation withHibernia. crudeoil DMSOextract. 58 Fig. 3.1.1 Salmonella typhintllriu", TA98NR revertants produced

xiii

by incubationwithAlbertasweet mi.'Ccrude oil DMSO extract 60 Fig. 37.2 Salmondla typhimurium TA98 revenants produced

by incubationwithAlbertasweetmixcrude oil DMSO extract 60 Fig. 3.7.3 Salmonella ryphirnurium YG 1024 revenants produced

by incubationwithAlbertasweetmixcrude oil DMSOextract 60 Fig. 3.8.1 Salmonella typhimurium TA98NR revertants produced

by incubationwithVenezuelan crude oil DMSOextract 62 Fig. 3.8.2 Salmonella ryphimurium TA98 revertants produced

by incubationwithVenezuelan crude oil DMSOextract 62 Fig. 3.8.3 Salmonella typhimurium YGl024 revertants produced

by incubationwithVenezuelan crude oil DMSO extract 62 Fig. 3.9 Salmonell4 typhimurium YGI024 revenants produced

by incubationwithindustrial stack soot #1 DMSO extract 64 Fig.3.I0.! Sa/mOllf!IJa typhimurium TA98NR revertants produced

byincubationwithindustrial stack soot #2 DMSOextract 67 Fig. 3.10.2 Sa/monelJa ryphimurium TA98 revertants produced

by incubationwithindustrial stack soot 1#2 DMSOextract 67 Fig. 3.10.3 Sa/monelJa ryphimuriJmt YGl024 revertants produced

by incubationwith inciusuWstack soot112 DMSOextract 67 Fig. 3.11 Sabnonella ryphimurium TA98m.revertantsproduced

by incubationwithcrankcaseoil#1 DMSOcxtrII:t 70 xiv

Fig. 3.12 Salmonella ryphimuriumTA98 revenants produced

byincubationwithcrankcase oil # I DMSO extract 72 Fig. 3.13 Salmonella ryphimuriumYOI024 revertants produced

by incubationwithcrankcase oil #1 DMSO extract 7.

Fig. 3.14 Adduct profile of crankcase oil extract treated rat livers 78

Fig. 3.[5 Adduct profile of control rat livers 80

Fig. 3.16.1 Formation of relaxed coil pBR322 plasmidwith

exposure to l-nitropyrene (agarose gel) 83

Fig. 3.16.2 Formation of relaxed coil pBR322 plasmidwithexposure

to I-nitropyrene 85

Fig. 3.17.1 Formation of relaxed coil pBRJ22 plasmidwithexposure

to crankcase oilextract(agarose gel) 88

Fig. 3.17.2 Fonnalion of relaxed coil pBR322 plasmidwithexposure

to crankcase oil extract 90

Fig. 3.18,1 Formation ofrelaxedcoil pBRJ22 plasmidwithexposure to l-nitr0p)Tmeandcranlccase oil extract (agarose gel) 93 Fig. 3.18.2 Formation ofreluedcoilpBRJ22 plasmidwithexposure

to l-nitropyreneandcrankcase oil extract 95 Fig, 3.19.1 Formation ofre:laxed coil pBR322 plasmidwithexposureto

crankcase oilextractin tbc:presence ofDMSO (agarose gel) 97 Fig. 3.19.2 Formation ofreJaxed coil pBRJ22 plasmidwithexposure

10crankcase oilextractinthepresence ofOMSO

xvi

99

Am,

ATP

B[a)P cc oil ddH,O DMSO DNA EDTA HPLC KP 3·MC

<Nn NADH NADP·

NADPH NaP J-NP PAH PEl

LIST OF ABBIU:VIAnONS

ampicillin adenosine triphosphate benzo[a]pyrene aanl<case

distilledanddeionized water dimethyl sulfoxide deolC)'ribonucleic acid

ethylenediaminetetraacetic acid~di.sowum salt highperformanceliquid chromatography potassium phosphate buffer )-methyltholantbrene minutes

nicotinamideadeninediDudeotide,reducedConn nicotinamideadeninedimJdcotidephosphate nicotinamideadeftiDc:dioocleotidephosphate,reduced form sodium phosphatebuffer

t-nitropyrene

polynuclear aromatichydrocarbons polyethy_imJlf'!lllOled

xvii

R.l\IA ribonucleic acid ROS reactive Olcygen species TLC lhinlayer chromatography

xviii

CHAPTER 1 1.IN'TRODUcnON

1.1 Hydrocarbon c:ombllstion

The use ofhydrocarbon mixtures as fuelhasbecome a staple in this industrial age of humankind. Hydrocarbons are used to power motorized vehicles. beating systems, and even electricity generating systems. as is done hereinNewfoundland. A conventional, and oversimplified, view of the process of hydrocarbon combustion has been that their burning leads to the production ofeamon diolcideandwater. While this theoretical picture is quite sanitary, the actualbusinessofbuming tlydtocarbons, especiallyfossil fuels. is vastly different.

Inreality.thecombustion ofhydtocarbons produces a plethora of compounds, some of which are quite toxic. The reason for this liesinthe fact that incomplete combustion is oftentherulewhencomlideringtheuse ofthesecompounds in modemsociety.For example, the intense heat and pressure geoenued inside the modem automobile engine leads to the presence ofmanypartial combustion productsinthe petroleum oil lubricant, as compounds which are fonned from fuel. combustion dissolveinthe:crankcase oil. Improper disposal of this used oil canthencontaminate boththesoil and aquatic environments.

The deleterious effects of exposure to combustion byproductshadbeen seen IS far back as 200 years ago,butwerepoorly undcrszood until recently.Theeighteenthcentury British physician Percival Pott noticed an abnormal incidence of scrotal cancerinyoung chimneysweeps.whicht.ecorm;dyattributed tothebonendousworkenvironments to which

these boyswere e:cposed. Sincetfwtime therehavebeencountless similar discoveries. some ofwhich involvetheformation ofcancers,m:Imanyoc:bm which relate genetic rruwionand gcnoloxicity[0hydrocarbon combusrionbyproductexposure.

l.I.1 Formation ofnitntedpoly..,..tithydrocarbellls

Considerable research has been focussedon such compoundsas benzo[a]pYTene (B[a]P), whichhasbeenshown tointeract withDNAbothin vitro(Nishimoto and Varanasi, 1985)and in vivo (Culp and Beland., 1994), and other polynuclear aromatic hydrocarbons (PA.H), which are commonly formedin combustionreactions. Whilemanyofthese compounds are quite [oxicm:Ioftenrrutagenic,thereisaclassof compoundsfoonedwhich are evenl'f)(nreactiveand poterIiaUy more genoloxicthanPAH.The:se compoundsarethe nimuedderivativesofPAH which include among them some of themostDNA·reac:riveand mutagenic compouoc1s yetdiscovered (RosenknnzaDdMermelstein. 1983).

NitratedpcXycydic

a:rommc

hydrocarbonswetbrmed,inthemreme temperatUreand pressureconditions ofcombustion reactions,bythe reICtion of combustion byproducuwith atmospheric nitrogcn dioxide(NOJ and nitricacid(HN'OJ(PinsetaI., 1918).Oneexample of this phenomenon would betheformation of mutagenicnitrar:ed 8[aJP molecules, which can occurincombustion reactions where8[a]PitseU'is fonned (Fuet.al., 1994). Similar examples ofNOl·PAH can be foundin manycombusr:ionresidualmixturesincluding petro1ewn.-ba.sed fuelexhaust(NewtonetaI., 1982; Moller. 1994), cigarette smoke (Jones

etaI..1993), industrialemissions(Khesinaetat, 1994) and grilled food (Feilonet ai.,1994) The environmental burden ofnitro-PAHis considerable.Inthe early 1980's. it was estimated that passengercarsin the United States would annually generate 15,000 kg of 1- nitropyrene by thetumofthe decade(Rosenkranz, 1982). This ominous prediction portrays only part of the story,asthere are much more mutagenic forms of nitro-PAH than 1- nitropyrene (McCoy et al., 1983a). h also does not reflect the lion's share of nitro-PAH, which comes from industry. [n the United States, a 1980 estimate placed the total mass of N01·PAH produced for commercial use at greater than 800 million kg annually (Hartler, 1985). This large scale manufacturing is principally directed toward pesticide production.

with minimal contributions from explosives and phannaceuticals. lndustrial activities also produce copious amounts of nitro-PAH in the form of emissions (White, 1985), the total of which is virtually impossible to quantify.

1.2 Themetabolism/.cautio. of Ditro-PAD

Nitroarenes must undergo metabolic activation to produce the ultimate mutagenicIDNA reactive species. This is iIlustntedbynitroreductase-knockout bacterial strains which are resistant tonitrocompound toxicity (MasonandIosephy, 1985). Williams and Weisburger (1991) illustrlletheprocessofnitro-PAH activation as showninFigure1.1.

This scheme showsthe conversion of theDitrogroup 10the N.bydroxyform.via a nitroso intermediate. Amine groups can also undergo conversion. tothe N-bydroxy moiety,

"' Ilol....,.11 ....

~HH, ~H"O" ~NHOR

}LQJ -~ -~ -..

Nilre COMIM""1l1s

~NGl

~H

~NO,

I

~ -

^~".O

~ -~

^{~N"OH ~NHOR}

Fig_1.1 MtUbotiIa (activatiOll) ohitnted-PAIL

spcci...thenrtremwni<xL(Tok.. &om,_IDdW... I99I)

whichineithercase canbeenzymatically conjugated ....ith a suitablefunctiooal group (i.e. a sulphale or acetyl group).Thesulphateoracetylgroup canthennonenzymatically remove itself fromthenitrogen &tom. under slightly acidic conditions.,resultinginthefonnarion of a rtitreniumion.Itisthenitn:niwn ionwbicb5IJbsequendyreactswith cellularmacromolecules and can cause mutagenic responsesinaffected cdls. This conclusionwasalsoreachedby Wild (1990).who suggested that the ratelimitingstepintheninoarene-induced mutagenesis in Salmonella Iyphimurillm is the reaction of the nitrenium ion with DNA.

1.2.1 The Role or Free Radiuls

In addition to the formation of DNA·teac:tWe efcarophilic compounds.

nitroaromaliccompounds can also~etoxic n:sponsesvia free radical production (as showninFigure1.2.below).The initialSleptnnitroreductionis1be twodectronreduction of the niuofunctionality 10yielda niuo50group.Electrons are removedfromNADH or NADPH and transferred tothe nitrogroup,via1be flavin eomponcm ofthe nitroreducwe (OmaandMason,1989).Whenthis reduction0CQ1tSwithonly one dectron, a nitro radical is formed,whichcanfonn~(O!-)

moo

radicalsinthepresenceof molecular oxygen (Mason and Josepby, 198.5).Thesuperoxideradicalis notinitself a major toxic threat. but it can reactwith other ~ecu.les to fonn. potentially I!az.ardouscompounds (HailiweU and Gutteridge, 1989). AlternatM:ty, superolOde canbeharmlesslyremoved via amino thiolssuchas g1uwbione(Eyer, 1994).

6

R.N~+Ie"----> R- "NO:.

R- ·No,-+0,- - > R·No,+.0,-

·O!- +

00.:.

- >BlO: +0₁ RIO:+Fe1°- - >Olf+'08+_F~

Figure 1.2 GeDeraDH. 01 •~dnJ:JlI'8dicaIby.itrwedllClioaracduia _ prt:HDct

.r

iro..

Two superoxide radicals can dismute (combine to produce a more inert molecule) to fonn hydrogen peroxide(H10Jand molecular oxygen(OJ,either enzymatically. via superoxide dismutase, or nonenzymatically. [nthe presence of metal ions such as iron(II), copper(I), manganese<m. chromium(V) or nickel(III),hydrogen peroxide can then be cleaved to fonn one hydroxide anion and one hydroxyl radical(OH)(GregusandKlaassen.

1996).The hydroxyl radical itself isahighly reactive molecule whichcanreadily attack cellular macromoleaJ.ies and,inthecase ofONA,can produce strand scission(RahmanetaI., 1989).

The effect of DNA strand scissioncanbe easily illustrated inasystem of analysis involving plasmid DNAThistechniqueinvolvestheincubation of plasmid DNA with various chemical or biological preparalions.Circularplasmids occur inasupercoiledformwhich have consistent mobilities whenNnonan agarosegel.Thebreakage of a DNA strand results in the relaxing of this supercoil formation, withthe: result being acircularpiece of DNAina conformation known as a relaxed coil.Therelaxed coil DNA is less compact than the supercoiled fonnandthereforedoesnotrun u &sf: through an agarose gel. Thismobiliryshift can serve asanindex ofthedegree ofstrand scission levels.Inextremecases., excessive strand scission can producelinearftagmcnts of ONA ofvaryingsizes,andtheappearance of these fragments wouldbe indicative of very high rates of hydroxylradicalproduction (Kukielka and Cederbaum, 1994).

In addition to the obvious DNA reactiveandgenoloxic mechanismsfornitroarene toxicity, a nongen-atoxicmechanismhasalsobeen identified.Theroot ofthistheofyis that ONAbindingandother senoloxic events cannote:~plainthe specificityseeninterms of species variation and targetorgans for given carcinogens. Assuming that DNA adduction is the critical eventin' initiation. the nongenaloxic effects may occur as promotion steps, whereby the growth and conversion to malignancy ofan initiated cell is encouraged.

Neumann et al. (1994) investigated theknowncomplete carcinogen 2·acetylaminolluorene in comparison withtwordatedcompounds whichW1:Rknown to be incomplete carcinogens.

They observed only one significant difference, that being the propensity of the cortq)lete

~forthest:izwIationofredox cyclingintherritochondria. possibly as a consequence of disruption of the electronrrans:pon:chain.11tiJ redolt cycling created asinwionof oxidativestressinaffectedcells andwaspresumedtoplay.via!roleinthe promotionof livercancers.

Theoxidative scressIrespirabon ittterferencecouldpromote cancen in anynwOOcrof ways,asthereductionofATPformation couJdreduce theactivityofmanydift"eremenzymes.

These enzymes would not necessarilybeIimiled to metabolic functions; theymigl:abe responsible for detoxification processes or couJdbeinvolvedin theexcision and repair of adductedldamaged ooc!cotides.

l.J Nitro-PAR DNA adduction

The binding of lCenobiotic or endogenous electrophiles to DNA has longbeen associated with mutagenicity. This adduction of DNAcantherefore lead 10 countless deleterious health conditions for the affiieted organism, including various forms of cancer (PitotandDragan, 1996). For this reason, the study oflhe propensity of compounds to form DNA adduets is of considerable value.

Various species of nitro-PAH havebeenshown tobepotent DNA addueting compounds. I-Nitropyrene. a commonly occuning nitro-PAli,hasbeenshown to fonn DNA adductsin Sprague·Dawley rats, CD-I mice andNJmice. when injected into the intra peritoneal (i.p.) cavity in tumorigenic doses (Smith eta1., 1990). Thesame effecthasalso been observedin844rats(El-Bayoumyetat.,1994) andSalmonella typhinrurium bacteria (Messier etal.•198t).1,6-Dinitropyrene,adisubstitutedrelativeof I.nitropyrene, hasalso been identifiedasa DNA adduct fOmUng compound whenadministeredviathe i.p. route to Wistar and Sprague-Dawley rats (Diurieetat.•1993; WolffeIaI., 1993). F344 rats also exhibit DNA adductsinlung tissue uponpulmonaryexposure of 1,6-dinitropyrenc (Beland etaI .• 1994).

The capability of nitro-PAH to fonn DNAadduets is not limited tothenitrated pyrenes, though thesearesome of the morepotentSpecies. 2·Nitroiluorene and 2,7- nitroiluorenehavebeenshowntoformbepatic DNA adduetsinmaleWistar rats. This effect canbeseenwitheither oral or intra peritoneal administration (MoUeretaI., 1993).

10 As discussedinsection \.2,thenitrenium ion istheusualultimatefe:actMspeciesin niuoareneDNAadduction.Itis.however. nO!theonly means by which DNAbindingcan occur. The presence of a nitro substituent can resultintheformation of an e1ectrophilic intermediate as portrayedinFigure U.as described by FuetaI.(1994).Inthis mechanism.

a nitro group atthethirdcarbon ofB(a}P can behydroxy\aled,and a nitreruum ion generated.

Thisnitrenium ion. throughelectronrearrangancnt,leadsto a cubocation atthesixposition.

which can readilyreactwilt! nucleophilic molecules such as DNA. TIUs mechanism., which leadsto a DNA adductwith&freeaminogroup,couldbethemeans bywhichN--acetyiaJed adducts are fonned by nitroarenes.

Asalludedtoabove,ithasbeendetermined thattwo distincttypesorONA adducu, N-acetylated andnonacetytaled.can result from in vivo exposure to nitro-PAH (Meerman andvan de Pou. 1994).Thesame work: alsoi1Iusuated thedifferentialdcasofthetwotypes ofadducts.TheN.-acety\aledadductsformed attheC8 position of dcoxyguanosine residues are capable of blocking DNA replication. The N-acetyl metabolites ofaitroatellCS arealso associatedwiththepromotional capacityofvariou.scan:inogcns. This couJdoccurbecause the suppression of DNA syalhesisprevents theDNAeditingIDIChinaylfomCCln'tCting mutated adduC'tS whileaIthe same time drcwnvenuthenormalprocessofceUularmitosis.

ThenOnaceryWedadductsfonnedat theC8position of deoxyguanosineandtheN6position of deoxyadenosine have been corrciued with the initiation ofpreneoplastic:c:dls.This may betheresultoftheactivarion ofaD

onc:osene

^{or some}otherdefcteriousefJ'u:tofroutIgt:DeSis.

Toe rdative inability of noQlCCt)'lued adduas toblockDNArepticationmaybe theresult

11

05?--[~]- [0/]

[~~] -~

FiB_ 1.3: Aher. .tivc DNA·ractiYcpamway.r.iIroaftDa

Thegenention ofa DNA-ractive e1ectrophiJc from 3-NO).B[a]P. without dinareaction:oftbemtroSlbstitucnI:group.(Tumfh:m:Fuet11.,1994)

12 of theelttfabulkiness of the aceylated adduct. which may restrict the action of DNA replication machinery

LA The effect! or metabolic routts on nitro-PAD toskity

MaUer (1994)found differentialeffectsinthe metabolism of nitroarenes administered by different routes. When 2-nitroBuorene is administered via the pulmonary route,theN- hydroxyl derivative is produced, anditintum is conjugated to a glucuronide moiety. The relatively harmless glucuronide conjugate can thenbeexcretedinthe bile. Cytochrome P450 metabolism is responsible for this pathway, asit metabolizesthenitro group to the N- hydroxyl fonn. Hence this metabolic pathway is dominant whenthelung; istheorgan of administration, as would often be the case in terms of environmental exposure 10 industrial emissions.Withthis route of de:livery the liver canmodifythe xenobiotic duringfirstpass metabolism.

In cases where large amounts oftheglucuronide conjugate are preserttinthe bile, however, significant amountS oftoldcanl canbeliberated intheintestinebytheenzyme 13- glucuronidase. thus leading to the scenario seenwithoral administration.Oralingestion of 2-rntrofluorene results in its reduction to 2·aminofluorenebythe intestinal microflora. 2- AminofluorenecanbeSlbsequently acetyiated to form2-acetylaminot1uorene. a very potent mutagenTheseresultsclarifyearlierwork:by thesame group where itwasdeterminedthat 2-rntrofluorene and 2,7-dinitrofluoreneexhibitedmore potency 11 forming DNA adducts

13 when administered orally, asopposedtoip. (Molleretat. 1993). The eumple of 2·

nitrofluoreneis useful as itillustl'1lte5howboth the dose and route of administtuion of nitroarenes mustbeconsidered in the analysis of nitrOlt'efle toxicity.

1.5 Nitro-PAD ..dC".oncnais

Thereactivity andDNA-binding capacity ofN0₂-PAH can,intheworstcase scenario.

leadto the folTl1ation of tumours. ThishasbeeniIlustraledwith1,6-dinirropyrene which can produce lung tumours in f344 ratswithitsingle 0.1 rng pulmonary dosage (fokiwaet&1.•

1990).Thesamesrudyfoundsimilar effectswiththeone rime pulmonary administrations of 3,7. and J,9-dinitrofluoranthene, whichproduced significant levels of lungtumoursupon exposure to 0.2mgoCtile respective nitro-PARThetumorigenicityof 1,6-dinitropyrene (with pulmonary administration)hasalsobeenshowntocorrefatt well withDNAadduct formation(BelandetaI., 1994),where thepereattIIeof tumourincidence within an experimental group of animalsincreasedfrom0-4 to 80'4withacontOmium:incR:asein adduct levels from zero totwo6noIaddUClsl~8DNA.Tbescdatasuggestthatnitro-PAH DNA reactivity(andhencemutagenicity) can transWc into the formation oflungtulIlOUn.

Although nitro-PAH are often.airtlomepollutants. malring pulmonary exposure quitt common,thelungs are nottheonly targetorgan intermsofcarcinogenicity. Hepatictumoun havebeen producedinnewbornmice15theresult ofinb'aperitoneal.exposureto I·

nitropyrene. I-nitrosopyrene.,and6-ritrochrysene(W"1SIockiet al., 1986). Interestingly, 1-

14 nitropyrcnewas only marginally more potentthanpyrene in generatin9:twnoun,wtule1- nitrosopyrenewassubstantially more nunorigenic than either pyrene or I·nitropyrene. This isinkeeping with the scheme of nitro metabolism outlined in sectiont.2. whereby reduction of the nitro group to a nitroso intermediateisan essentialStepinnitro-PAH activation.

Theestablishment of tumorigenic activity of nitro·PAH serves as the culmination of mutagenicity and DNAbindingsrudies. Theexistence of tumoursintreated animals strongly suggests that thisistheultimate lalOe manifestation oCtbe deleterious effects of nitro-PAM e:tposure.

1.6 Mi.J:tuns ofNO:.PAH

Nitrated polyaromaric hydrocarbons ace rarely seen in isolationin theenvironment but.instead.arepreserfin comp(cxmi'ttUreSof combustion byproducts. For thisreason,the investigation of puresamplesofthese compounds, while sriU avalidscicntik undertaking.

does oot neccssariJ.yexamineany interaction betweenthe constituents of themixrurc.

CombinatiON ofcompounds couldKt synergistically toenhancethetoxic:dfea.

Alternatively,the combined effects of mixture constituents couldactantagonistically and diminishthe toxic effects producedbytheindividualcompounds.For this reason, the study of these mixtures is imperati..-cintheinvestigation ofthetrueeffects of environmental exposure to nitfO..PAH..

Some combustion byproduc:t mixlurcs have been shown tobegenotOlcic.Tobacco

15 smokehas been linkedto DNA adduct formationinhuman lymphocytes (Jahnkeet:aI.•1990) andoraltissue (JonesetaL. 19(3).Thefeeding ofcoaltarto B60FImicehasbeenshown to form DNA adduetsintheliver,lung and forestomach (Culp andBeland.1994).Diesel emissions.inaddition tobeingbacterial mutagens (Rosenkranz. 1982a; Rosenkranz. 1982b).

have alsobeenshovm 00beDNAreactive.bothinm>oandin wrro(GallagheretaI.•19(3).

These findings.. althoughtheyare likelynotentirdydue totheaction ofnitro-PAR.cmainly mimic the action of various isolated nitro-PAH.

Inaddition to direct DNA reactivity, combustion byproducts are also capable of generating the reactive oxygen spec;ies(ROS)supcroJcideanion.Thishasbtcnclearly illustratedwith diesel exhaust(SapetaI.•19(3).ThesamestUdyalsofound that diesel particulale-induced mortality couldbediminished by the elevation of available superoxide dismuta:ie. The potentia!foroxidativestress,incombination withtheDNA-reectivity of combustion byproductmixtures,lay thefoundationforsubstantialtoxicitywithinvWo exposures.

1.6.1 The use or .AP-posdabeIIiq foridntiRaltiH ... DNA addllCU.,eMnted by N~-PAB

..m.ra

The np-postlabellirlgmethod for the identification ofONA adducu is wdlsuited for

monnoring_",""""""mixlwes.

Monysw<tieoofDNA-.et _ i n _ lhe bindingofradioaaivdyIIbeDedc:ompoundstoDNA.It woukI beimpossible to undertake

16 such a srudyusinga mixture ofunknowncomposition. It would alsobepractically impossible to accuratelyreconstructa complexmixturewithradioactively labelled constituents.Thenp.

postlabellingassay circumvents this problem by selectively labelling addueted nucleotides by a process known as enrichment.

Enrichment canbeperformedbyone of two different methods. BUlanol canbeused

[0extracthydrophillicadductednucleorides from a digested DNA sample, while at the same time selectively leavingbehindoonna! nucleotides(Gupta,1985).Whenthis step is performed prior to the labelling reaction. only the addueted nudeotides needberadioactively labeUed.

Nuclease PI enrichment achievestlte same goal via a different mechanism(Reddyand Randerath, 1986).Inthis system. DNAisdigestedto J'-nucleotides. The DNAdigest isthen subjected to treatment with nuclease PI. This enzyme cleaves the 3'.phospbate from each non-addueted nucleotide. The presence of a large hydrophobic molecule bound to a nucleotideSlerically inhibitsthe nuclease'sactivityand leaves thej'-phosphate group intact.

Sincethe polynucleotide kinaserequiredforthetabeUingreaction only recognizesRUcleotides witha3'-phosphate group.itselectively labels those oocleotides whose hydrophobic attachment preventedthenuclease PIactivity. Theend resultisagain theselective radio·

labeUingofaddueted nucleotides.

Byutilizingsdcc:tivc proccdURS of enrichment. invcstiguon are abletoresolve two daunting problems: (1) theincrediblenumber ofcompounds presentincombustionresidual mixtures;and (2) theenormousamountof nonnal DNAcompared totherelativefyfew nucleotides which form adductswithreactive compounds.

17 1.7 TbelulJ~"'JucI.itro-PAB

Thereis aCCll'lSiderabicbodyofpubisbcd wortonthemutagenicity ofni!:nrPAHand likecompounds, using theAmesSaJmone/la-mammaIian microsome assay(Amesetal.1915.

Maron andAmes.1983). The Amesusay usesSU'IiDsofthe bacteria$t:zJmo,w/Ja ryphimllri"m thuarc incapable of produciogb:istidiDe and. biotin. ThesmiDthave been genetically engineered tobKkIDJwe tothewildtypebKtcriawhich can produc:e both of these essentialcomponentsforgrowth. Tbis bKkauwioftcouldbeintheform of a fRmeshiftID.ltation,ISintheTA98.strain,or a bue-subsbtutionrmnatioa.ISin thecue of theTAtOOStI'liD(McCoyetaI., 1983b). Since the assay isdone onaprplaleswithlimiting quamities of biotinandhistidine, onlytheIJlUWlISwillproduce viable colonies. Thusthe number of colonies on anaprplaitisdircc:tlyproportional tothenI.Itqenicityofthetest compound.TheassayalsOallowsforthedetectionof~mutagens;by adding apreparatioll ofIDIImloI1ianliver mzymc:s. tompOUDds wbiebart DOtdRaty

!IlJtagaKbutare COIIYatcd to •ttmageDic: meW:JotitcillIDIlDDIJsCMIbeassayed..

Experi.-.Ievidmce bas _ thottheTA9I ... aCSaboone/iQ1JfI/O-ri- isgeneaDymorerapoasivetovariousDitroarares ladnitntcdpolyaromaticbydroca.rboas thantheTAlooSIRin(McCoy etaL1983.).impDcatiDltheprocess of&ame-sbift mutagenesis. These dataalsoshow •pattieu1ariySb'OD8rnut.lFicrespo_of TA98 bacteria to l,8-diDitropyrene,with272,000~producedpermicropmlofI,&- di,'utropyrene.Thispeper(1983.)alsoilluslratestheuteofIIIS.~ sttIiD

18 dcSgncdspecifically for workwilhailro<ompouDds. The

srnm

TA98NR.~.derivative of theparentTA98 SIninthat isde:ficicrjintheclassical bKterialaitrorafuctaerespoftSlblefor the metabolism,andICtivItioa.,ofmaoy*oarcDcs(ca.nitrofin:nsaod airidazo&e).The TA98NRstrain ishoweverpartiallyrespocsivetootbernitroaraIessuchasIIIOIIl).and di- nitropyrenes(Rosenkranz etaI.,1911,DjuricetaL.1986).

Another spec:iaJtymodifiedwnioaofTA98S.typNlJIIIIiuristheYQ1024strain.

whichis enrichedin O-aoerylrnnsfaue ICtivity(WataDIbeetaI., 1990). Thisstrainhas been shown tobemuch moreteDSitiveto~ecposutethinitsparentstrain.TA98 (Tokiwi etaI.,1994)due totheimpol'tlnCe of O-acetyWionilltheproductioaofthepresumed ultimate reactivespecies. thenitreaiumiOG,asoutliDedinsection1.2.

Ila<terialsaainslUCh as TA98NRaodYGI024gmdyOllllance thecapoIImtyofthe AmesassayfordiIgnosbcwork,espec:Wly intermsofuxfor complex:mixIureswbicbba¥e mutagenic potential."I'be5iemixIuresc:oaainIll.IbuDda:Dceofdif&tmtc:ompwDds,wbic:b makes theassignmentofthespeciesrapoasibIefor thea.zt.ageaicpoteatiII(If any is exhibited) quitedifficult. ByutiDgbKurial SIrIiDs wbicbaredcficicmIeDbIDccdill. c:auin area ofxcnobioOc mer.boliIDt, more canbe daamiDedas to wbic:bclassofcompouDdsare the primary mutagensin •mixture.

19 1.& Rationale.1tdobjKlivaof'thbstlldy

Considerable research has been directedatthemetabo6sm.. DNA·reactivity,and subsequent mutagenicity ofnitrated polyaromaric hydrocarbons. Considerably less informationisavailable onthesecharacteristics with respect totheirnormalenvironmental occurrence in complex mixtures of hydrocarbon combustion byproduet5.Thegoal of this thesis is to associate nitroreducI:aseactivity,bactcrial mutagenicity and DNA damageupan:

of the overall toxicity seenwithexposureto nitroarenes as constituents of complex mixtures.

20 CHAl'TERl

2.MATERIALS AND METHODS

2.1 Malerials

PEl-Cellulose TLC plates werepurdwcdfrom Fisher Scientific (Onawa. ON.

Canada).

TheS.typ#rimurirtnfstrain TA98 was obtained asitgiftfrom Dr. BruceAmes, University ofCa1ifomiaatBcn:dey. Berkeley, CA. USA.TheS.lyphitrlllriumstrainYGI024 was obtained asitgiftfromDr.M Watanabe. National tnstinne ofHygiene Sciences. Tokyo, Japan. The TA98NRstrainofS.typhimurium was agiftfromDr.Elena~cCoY.Case Western Reserve University, Cleveland. Off. USA.

Crude oiI5andusedcrankcaseoilswereobtainedfromDr.JeremiahPayne, Fisheries and Oceans Canada, St. lohn's., NF. Canada..

2.1.1 CMlIliWs

APl"UC,1i<me,calfthyrrwDNA,~ochrome.(typeVI,from honehart),OMSO, OTT, EDTA, J-met!lylchoIanl(Mqmiae<:OCC41...,l... NADH, NADP', NADPH, nuclease PI, pBr322 plasmid DNA.sodium dithioDite.spermidineaDdsuccUDc anhydride werepurchasedfromSigmaCbemicaJCo., SLLouis,MO, USA.TheenzymesII-amylase.

21 proteinasek.Ri'laseA.RL'lase T,andspleen phosphodiesterasewere obtained from Boehnn....Mann.1riem,Laval,PQ. Canada.[y-"PJ-ATP(specific activity 6000C~nunoI) wupu<chascd _ Amenhamc.n.daltd. • Oakville,ON.• Conada.AnaIytigIgradeKC~

KH!PO~. K~~and KOH werepurchasedfrom BDH Ltd.• Toronto, ON Canada.

Polynucleotidekinasewas purchased from US Biochemical Co.• Cleveland. Of{, USA.

Chelex 100resinwaspurchasedfromBio-Rad,Mississauga. ON, Canada. Aroclor 1254was purchased from Chern Services. West Chester, PA, USA All other chemicals were of the highest grade commerciaily available.

2.2 Metbocb

2.2.1 Animal Trat'lDnt

Male Sprague--Dawtey rus(2()()'2SOg) wereobtained from Memorial Univenity Animal Care Services.

For Araclor 1254 pretreat:mem. rauwere dosed once with Soomgperleg body weightofthepolychlorinated biphenyl (PCB) mixture, ArocIor 1254, suspendedincomoil (250mglmL).Maclor pretreatedratsweregiven chowandwateradlibitumfor fourdays prior[0sacrificing.Inthe case of3-methyt cholanthrene {J-MC)pretreatment., ratswere dnsedwM20ms ofJ-MCinaxnoil(10mglmL)perkgbodyweigh~daily. forthreeclays.

The3-MCpR!b1:I1ed1'IlSwere gjvmchow andwale-adlib;"""forthetinttwodays.After

22 theprescribed time,thefOod.butnot

me

walel'",was rerncM:d.The rats weref.a.sltdovernight and sacrificed(bycervicald.is1oation)therouowing; morning. Control rats., except where otherwise noted, received nopretreatment.but were subjected tothesamedietary regimen andsaaificingproceOJreasthe ecperimenulanimals. [Eartier experi.mc:ntarioninthis lab has illustratedthatrats treatedwiththecom oil vehicle only haveexhibitedno response.]

For the purposes of J:p.postlabelling analysis, four male rats were administered,by gavage, 0.5mLofOMSO crankcaseoilextractperkilogramofbody weight on alternate days over athiny-onedayperiod. Threecontrolrats werekeptforthesame timeperiod. butwere not given crankcase oil treatment. Forallbutthelast day of this trealfnentregimen.the rats were providedwithchow and'wuertz/libitum, thenfastedovernight priorandsacrificed the rouowing moming.Thebrains.hearts,livers.kidneysandstomachs ofthe:ratswere excised and subjected to thenp.postlabellingprocedure.

Partial$Llccinoylarion ofcytochromel:wasperfonncdas describedbyKuthaneraI.

(1982). 100mg of cytochromec.(horseheart; typeVI)wasdissolvedin40mLof ice cold )0mMKPbutTer, pH 7.6.Thestirringsolutionwuleft on iceand0.42mmoJof finely ground succinic anhydridewasadded. slowly, over aperiodof 30minutes, withCOnslant stirring.ThepHofthesolutionwascloselymooitoredandmaintainedat7.6bythe periodic addition of 2 M KQH. FoBowing the compietion ofthesuc:cinicanhydrideaddition, the

23 solutionwasstirredon ice for a further 20 minutes.

Thesuccinoylated cytochrome, solutionwas transferred to adialysisbag and dialysed for[6hours against 2 L 0.1mMEDTA.Thedialysis bag was placed in 2 L of deionized water for 4 hours, after which the solution was concentrated using a Diaflo ultrafiltration apparatus, used under nitrogen gas with a PM 10 filter.

2.2.2.1 Detcmlination or q-tocbrome, (ollcentratiOd.

ThelinaI preparation ofsuccinoylaledcytochromeIiwasassayed10determine its true concentration. Two viSl.ble light range cuvettes were identically prepared with SO mM KP buffer, pH 7.6. and a known volume ofsuccinoylated cytochromeIi.The two cuvettes were simultaneously monitored at550 nm,andthespectrophotometer zeroed.Thereference cuvette was lett unchanged while the sample cuvene received a 5 mg quantity of sodium dithionite to completely reducethecytochromeIi.The relative absorbance ofthesample cuvette was converted to a concentration value forthepreparationwinean extinction coefficient of2t mM·l.cm:l .

2.2.3 [nrutiOR

or

l:rude oils ••dused craaktueoils

ExtBctswere made offour differemaudeoils(PrudhoeBay crude. Hibernia crude, Albertasweetmixcrude,andVenezudan crude) and two separate pools ofusedcrankcase

24 oils Oils[0be extracted were combinedwithequalvolumes of DMSO in plastic tubes.

[DMSOwaschosen because is dissolves many hydrophobic compounds, is miscible with water, and does not appear toaffectedthebacteria used in the rnU[agenicity stUdies (Ames etaI.,1975).]Thetubes were gentlymixedby inversion., for 10 minutes., then centrifuged at 2000rpm (3000 x: 8)ina benchtop centrifuge. The DMSO phase was retovered by puncturingthe bottom of thetubeanddraining otTilie bottom layer.Therecovered extract was then combined with analtler equal volume portion of the oiland theprocess repeated.

The bottom layer of the secondcmacOonwas retained for future experimentation.

The above procedure was found tobe inadequate for the production of a crankcase oil e:ttract for plasmid DNA strand breakage studies.Asthehydroxyl radical is essential to this process, steps were required to removeDMSO.a hydroxyl radical scavenger, from the extract. For this preparation the DMSOextractwas preparedwitha triple extraction procedure, producing afinalvolume of 90mL.Thefinalextractwasthendistilled under

r

vacuum[0eliminate thebulkoftbc DMSO fromthepreparation.This condensedextract, 20 mLin volume, was combinedwith150mLwaterina separatory funnel andextractedwith chloroform(2 )( 100 mI.),dicbJoromethane(2x75mL)andtoluene(2 )( 75mL). The organic extractswerepooledandreducedinvolumeusinga rolary evaporator. The remaining 17.5mLoforganiccxnctwasaliquotedintothreeSmLportions and one 2.5mL portion,eachintoacappedtest tube.Priortouse,theextractswere evaporued 10 dryness under nitrogen andthenresuspendedintbeoriginalvolume of acetone.

25

2.2.3.1 [:Ktnction of soots

DMSOextractsof twoindustrial stack soots (A.T. Cameron and Gulf Star) were prepared. For theseextracts,DMSOandtheraw soot were combined in a I: 1 weight ratio.

Thesee~raetswere thenprepared as per section 2.2.3.

2.2.4 Nitroreduttase activity usay

Nittoreductaseactivitywasdeterminedas describedbyDjuricetaI.(1986). The assay wascarried outin SOmM KP buffer (pH 7.6), with JJ j.lM succinoylated cytochrome k. and atotalvo{umeof3.0mL\.IS,uLofDMSOoiVsootextraet and32-480j.lgofcytosolic protein (from3-MC pretreated nus) wereaddedto the sample and reference euveues. 100 JoILofeither10 mM NADHor10mMNADPH (330.uMIinalconcentration)wasaddedto stan the reaction.Thezero oilextrId.andzeroprotein levels of enzyme aaivity were determinedbyreplacingtbc oilextractwith10 j.lL DMSO,and theproteinwith10 JoILof50 mMKPbuff...(pH 1.6), respe<rively

The reduction ofthc strecinoyiatcd cytcchrome kwasmonitoredat550om. All assays wereperfonned induplicate,andtheaverage afme two rates rUen.

AJI nitrortduetioD assays wereperformed usinga commonratlivercytosol

26 preparation

2.2.4.1 Tenias oriallibitoryactioaofDMSOtnrKb

To evalua1eanynitroceducwe inhibitoryaction of variousextracts,nitroreduction of l·nitropyr~was assayedwiththepresenceandabsence of oilextrKt.usingthemethod outlinedinsection2.2.4.Assaysweredoneinaipljcate andtheresults aatistica1ly compared to determineifany changeinenzyme activity occurred.

2.2.5 Ames-SallffOM/laDUIUlftlicity UNY

This assay was pcrfonncd asdescribed byMaron andAmes(1983).

system.

Aroclor1254pretreatedruswerekiIJcd by ccMcaJ dislocation and pilcedon their backs. Thefuronthearueriorside ofeach an:imaIwasthoroughly swabbedwith95%ethanol.

and pointed scissorswereusedto o,ztthroughthe skiDwithout damagingtheundertyins muscle layer.TheskiDwaspulledbKkilIIdthe exposedrrusdetissue swabbedwith95%

ethanol. beforeitwasad.openwithSlCriJescissors.Theratliverswere excised.weighed.,and

27 placedina volume oficcCXlld 100mM KP buffer equallingthreetimesthewetweight aCme livers. Thismixturewas then komogemzed using a Polytron homogenizer andthehomogenate centrifugedat9000x g for 30 nUlutesat 4'C. Tbesupemwnt wustoredat.10·ein3mL aliquots for future use in theAmesassay.

On thedayoftheexperiment,an aliquot ofthefrozen supernatant(59)was used to prepare an NADPH-resencrating system This solution was a Iin10 dilution ofthe59. w;lh a final composition ofBmMKCI;

a

mMMgC~0.1 M KP. pH 7.4; 4mMNADP-;and5 mMg1ucose-6-phospbal:c.Theregenerating systemwassterilizedby$trairing lhtough a sterileOA5,urn filter.

2.2.5.2Preparation of.aerialtester strainadt...a.

Mastercultures ofTA9B, TA98NRand YGt024 were5I.0l'edat-70'Cwith~Ioof the culturevolwneofDMSOIddedforSlor1gC.Thenigbtbefore anexperiment.approximately 0.1mLofthe frozenmasurcultJ.RwasusedtoiDocuJIle10mLofOxoido.aItlR media.The inoculatedculture was leftat37'Covernight (16 bouts)ina.sbaJcing water bath after wtUch cultureswereleftoniceforthe cbabonaCmesampiepreparation

28 59-based NADPH-regenerating system.Thetestcompound (dissolved in acelOne or DMSO) was added to a sterileglassculture tube. followed by 2mLtopagar (0.6% DifeD agar; 0.5%

NaCl), and the appropriate bacterial tester strain

The combined mixture was briefly voncxcd, then aseptically transferred to an agar plate (1.5% Difea agar and 2% glucose in Vogel-Bonner medium E [Vogel and BOMer.

1956]).Theplares were left to solidifyin thedarkat 25°C, then inverted and kept at 31'C for 48 hours After the 48 hour incubationperiodthebacterial colonies on each plale were counted

2.2.6 uP-Poltl.bdliuc orDNA.dducb

l~P.Postiabellingexperiments were perfonned using the methods ofReddyand Randerath (1986),withmodifications.

2.2.6.1 Isollrioa or DNA

ApprolCimatdy 250mgoftissue were excised fromtestorgans and placedin 1.5mL plastic tubes.Inthe case of in vitro incubations. the entire volumewasused for extraction.

To each sample, 0.5mLofSETfSDS solution (100mM NaCl; 20mM EDTA; 50mM Tris base; 0.5%50S; lmglml proteinase K) was added.Thetubeswereincubated at3~Cfor threehawswith gentle mixingbyatuberotator. 0.5mLtris-saturated phenolwas then added

29 to the mixture and eachtubewas agitated for 5 minutes in a horizontal position. The tubes were then centrifuged at14,000rpm(12,8001Cg) for5minutes to separate the two phases.

The bottom phenol layer was pipette<! away, the tubes recentifuged at 14,000 rpm for I minute. and the top aqueous layer removed 10 a clean labelled tube.

The aqueous layer was extracted twice more asdescnoed above. with different organic solvents. Thesecondandthird extractions were performed with phenol: chlorofonn:

isoamyl alcohol (50:48:2), and chloroform: isoamyl alcohol (24: I), respectively.Thefinal aqueous extracts were combinedwith2 volumes of cold edloxycthanol. and gently mixed.

The samples were then placed at .20·C for at [east 2 hours to precipitatetheDNA, and centrifuged for 5 minutesatt4.000rpm.Thesupernatant was discarded. the pellet washed with Im.L70% ethanolandresuspendedin0.5m1SET buffer (100mMNaCl; 20mM EDTA; 50 mMTrisbase)withRNaseA(O.1!n&'ml).RNase Tl(1000 UlmJ) and "'·amytase (1mgtml).These mixtures were incubatedat37°C forthour.

10 J,lL of SET bufferwithproteinaseK (10 mg/mL) was added to each tube. after which all were incubatedat37'C for afurthertbour.Thesamples werethenextracted as before.withpheno~phenol: chloroform:isoamyla1coho~andch.lorofonn: isoamyl alcohol.

The remaining aqueous fractionwascombined.with2 volumes of coldetho"Yetbano~

incubatedat-20'C foratleast2hours..andcentrifuged at 14,000rpmfor 5 minutes to yieid aDNApeUet.

TheDNApeUetWlSwuhedwith70'10ethanol dissolvedin0.5mLSET bufferand combinedwith2 volumes of cold ethoxyethanol to precipitateanyremainingsmallRNA

30 fragments.Themixturewas again leftat-20'e.cerurifuged at 14,000rpm,andthepeDet washedwith 70"/,ethanolas before.Thefinal pellet was resuspended in distilled waler.and t~absorbanceaCme solutionmeasured11260 nm and 280 nm.Theabsorbanceat260 run wasusedto dttemiDethec:.ooc:entratioofONA.andtheAbs@26OiAbs@280ratiowastJSCd as an index orONApurity.

2.2.6.2 DigrsUoo orDNA

FromthepurifiedDNA samples..the voIwnecom:sponding to 2.5~gDNA was added to 1.5mLEppendorfcc:m:rifugetubes, and thesample evaporated todrynessunder vacuum.

S1Jg; (0.60U)miaococcalrlJdease and SJol!Jspleenphosphodiesterase were added.andeach was sample madeupto12.0 J.lLtoW vo(wne (20mMsuccinate, 10mMCOlO.).TheDNA digestion reactionwascarriedOUfat

3rc

for Jhoun.

EachDNAdigestionmixturewas combinedwith3 ,ul 0.25 Msodium acetate., 1.8 .uL 0.3 mM ZnCI2>and1.2,uL S.uWJ.lL nuclease PI. The enrichment reaction was allowed to occurfor1houru37-C.To neutralizetheenrichmcm reeaion, 2.4 JolL 0.5 M Tris buffer (pH 9.0)wasaddcd toad\tube. 2.0.ul ofeach solution was transferred to a sepaRlc tube containing18.0,uL ddH)O.Thismixturewas retained for futureHPLCanaiysis.whUc the

31 original enrichment mixture wasusedforthepostJabelling fuction.

HPLCanalysis of J'-tlJCleotide contentwas donew;thif.solvent system of 5%

melhanoV9S%tomMNaP buffer. pH 7.5. Thedcoxycytidine·)'·phosphate peakwas compared[0if.2nmoI~3''i'hosphaIestandard to determine J '-nucleotide Ievel:s.

2.2.6."Preparatio. or ,taad.rds ud bl. .ks

For eachsetofexperiments.two blank reactions and four nucleotide standards were prepared.Theblanks were 18.0~L ddH~O.andthestandards were 18.0,u.L ofa I :t 10.¹M deoxyadenosine.J'-phosphate solution.The standards andblanks weretreatedinthe same fashion in the postlabeUing reaaionISwere thetestsamples.

2.2.6.5 np-posdabdliaa racticHI

Akinase nixdJIionwaspreparedbythecombination of41 ,u.L ddH!O. 100 Jl.Ly- np_ATP solution (ImCi). 3,] ,u.L (SWlits)polynudeotide kinase., and56,u.LIcinuebuffer (0.2 Mbicine.. 0.1 MMA0.1 MDTI, 10mMspermjdinc).Tcn,uL oflhiskinasemixture wasaddedto each sample and incubated at37°efor 45 minutes. To thesetubes.4,u.L ofa bicine (20mM,pH 9.5)and apyrue (20U/mL)rn:ixtuRwasadded and the samplesli.arther inalbaredatJ70efor30trirucs.Alladditionswere made to oneblankandtwostandards.

thento the lest samples.aDdfinallyto theremaitUlg standardsand blank. Thisstepistaken

32

10ensurethat anydiJrinishingoflcina.seactivityoverthecourse ofthesample preparation can be detected.

2.2.6.6 PEl-Cehbtlbi.lIyu thnuaatop-apllyofDUdeoridts

PEl-Cellulose

nc

plales were washed with 100% methanol andddH10.then air dried prior to use.

The entiretestsample volumes. approximately 33 .uL, were spotted onto a PEl- Cellulose TLC plate attheorigin as indicated in Figure 2.1.Theplates were developed overnightinI.OMNaH~•• pH6.S.intheDIdirection(towardsthetop of Figure 2.1). The next morningthefiherpaperwicksandtop third aCme

TLe

plates were removed(CUtalong thelinemarked 01-inFigure 2.1)anddiscarded.Theremainder oCtile plates wererinsed twicewith ddH10andairdried.

The dryp1mcswerethendevelopedinS.3 Mlithiumformate,&.5 M

urea.

pH 3.5,in [he 03 direction(towanb thebottom ofFigure2.1).Theplatewasdevdopeduntilthe solvent front rcachedtheedgeaCme plate.ThelOp I em wuremoved(cutalongtheline marked 03· in Figure 2.1)anddiscarded. The cut and developed pWcs were rinsed with ddH~Oand then soaked for 10 minutesin10mM Tris-HCI. The plates were then furthcr rinsedwithd~O.andair dried.

Theplateswen:thendeveloped,in the.D4direction (towardsthe leftof Figure 2.1), again10theed~ofthepWe.in1.2 MUCl.O.S MTris-baseand 8.S Murea. pH8.0.The

33 Fig. 2.1 TLC plale markings and areas of removal in mullidimensional

chromalOCnlphy

+ - - - -

20cm

- - - +

DI"

DS"

D3"

i

+ - - - l'e..- - +

Scm

3cm

'" ^'"

i i~ r ^D3 ~

Dl

DS 'cm l

1

^{- 0 4}

20cm

• Denotes cut

i

DIVIDE

34 plates were twicerinsed inddH~Oandair dried.

Filterpaper ....ic:kswereanachedto thetop oflhc plates prior to theirfinal,overnight.

de...eIopmem.in 1.1M~~~.pH6.0.intbe05 direction (towards the tap ofFigure 2.1).

Thewiele. andthearea of attachment to the Tl.C plate were removed (cut along the line marked OS·). and the residual plates rinsedwithddH10airdried.

2.2.6.7 Staddard. blaak and samplemidHaaalysd

'~OlJOwing

the apyrase:bicineincubation. 970

~I

^ddH10wasadded to each of the standards and blanks.Ten~orthese~werethen sponedontOPEI-CeUulose plates.

Thetubes which. had contained lhetestsamples received100~I ddH~O.and 10 JJ.L of each of these residual solutionswerespottedonto PEI-CeUulose plates(sixsample residuesper plale) andtheplatts were devdopcdin0.3 M ammonium sulphate.

Thedevdoped plalcs weredriedand tbcnIabdJedwith •Ouorcscem:marker.The labelled TLCplates wereexposedfor 1U100000ography\ISingx-ray p1ucsat-70'Cfor one

hOUf.Thex-ray6lmsweredevelopedand theadenosinenucleotide standard radioactive spotsmarl:cdontheIilm. Theareasinthesampleblank \.aneswith

Rr

values colTesponding totheadenosine spots were alsomarked. Theareas on theTLCplales which corresponded to the marked. spots ontheautoradiogram were excisedandscintillation counted. The relationship between standard nucleotideadded andradioactivity counted wasusedto establish the specific activity oftheIabeI1ingreaction.

35 2.2.6.8 Analy5ls of test samples

FoUowing the final chromatographystep,the plates were labelled with a fluorescent marker and placed in autoradiography cassettes with x-ray film for 16 hours at .700C.

The x-rayfilmsweredeveloped after exposure and any visible radioactive spots were marked. In addition to the visible spots, a Icm~ponion of each film was markedinan area where no radioactivity was detected.This spot was used to calculate the background levels ofradioaetivity per square centimetre ofTLC plate.

The areas on the TLC plates whichCOlTespondedtothe markedspots on the film were excised and weighed 10 detennine their area as compared to a standard sample The radioactivity blanks as well as sample spots were then scintillation counted.

The radioactive counts detectedinthetestsamples were converted to a measure of the addueted nucleotidespresentusing thepreviouslyestablished specific activity oflabelling.

This number was then expressed as a molar ratio of the total 3'-nucleotides presentinthe sample. The means(+I-S.D.) oftriplieate samples were compared using a 2-way AJ.'4'OVA test

2.2.7 Plasmid DNA mud dtavap "perillllnilS

The investigation of piasnUd DNA nick productionwasdone according to the methods outlinedbyKukieikaandCederbaum (1994).withmodificatioDS.

2.2.7.1 Preparation ofsoiutions

The solutions used in preparation ofratliver fractions andinplasmid nicking incubations were prepared with deionized water (filtered through Chelex100resin) and analyticalgrade reagents- so as to minimize the presence offree metal ions in the solutions.

2.2.7.2Preparario. of rat liver fractions

Aroelor12$4pretreated and control rals were killed via cervical dislocation.The outer skin and the muscle layers were cut to expose the internal organs and the ponal vein was used to perfuse the livers with 0.9% KCI. FoUowing the perfusion. the livers were excised. weighed, and combinedwith a volumeequalling three timesthe liver weight ofsterile 50 mM KP buffer (pH 7.4).Thelivers weremincedwith clean scissorsandthemixture homogenized using a Polytron homogenizer.Theresulting homogenate was centrifuged at 9000J(gravity, the petiet cliscardedandthe supernatant retained as the post.mitochondrial fraction.

2.2.7oJGeneration of pBR312plasaid

Ftfty,uL Escherichia coli competent ceUs (DID« strain) was diluted with 450 ,ilL 0.1 M sterilecaC~.One,us pBr322 plasmid DNAwas combinedwith50,uL ofme diluted

37

£. coli cells, The mixture was kept on ice for 30 minutes and then heat shocked for 90 secondsat42'C.OnemLsterile LB broth was added to the plasmidlbaeteria mixture and the reaction tube was incubated at 31'C for I hour. The resultant culture was centrifuged at 14,000rpm for I minute to pellet the ceUs. the supernatant discarded., and the pellet spread on an LSI ampicillin plate. which was incubatedat37'C for 16 hours.

Colony scrapings from the overnight plate were used to inoculate 15mL sterile LB broth (with ampicillin), and the cultures were grown at3TCin a gyratory incubator for 24 hoursThe15mLcultures were used to inoc:uIate 500mLsterile LBIAmpbroth preparations.

which were also incubated at 31°C in a gyratory incubator for 24 hours.

The 500mLcultures were centrifuged at 4500rpm for 15 minutes [0 pellet the transfonned cells. Each culture pelletwasresuspended in 20mL sterile GTE solution (SO m.M:g1ucose, 25 mMTris, IOmM EDTA, pHS.O), containing lOO,uglmL RNaseA,to which 40mLsterile NaOH·SDS (0.2 N NaOH, 1% SOS) was added. This solution was gently mixed and left on ice for 5 minutes. after which 30mLsterileK.AcF(5 Mpotassium acetate, 6.7%ronnie acid)wasadded, the mixtureswirled,andleft on ice for a lUnher 10 minutes.

Theresultingslurry wasstrained through2 thicknesses of cheesecloth into sterile centrifuge rubes. The contents of eachtubewere combined with 0.6 x volume of isopropanoland centrifugedat10,000lCgfor 10 minutes.Theresultingsupernatantwas discarded and the pellets resuspended in4 mL sterile TE solution (10 mMTril, 1 mM EDTA. pH 8.0).

The TE solution was combined with 2 x volume of Tris-saturated phenoland thoroughlymixed.The mixture was centrifuged for 5 minutes at 2500 rpm (3000 x g)ina

38 benchtop centrifuge to separatetheorganicandaqueous phases., and the top aqueous layer was pipened into a clean glass tube.Theextraction procedure was repeated twice: once with Tris-sarura!ed phenol and once with chloroform:isoamyl alcohol (24:1). The final aqueous extract was combined with 7.5 M sterile ammonium acetate and incubated on ice for 15 minutes. The solution was centrifuged at 10,000 x g for 5 minutes and the pellet discarded The supernatant was combined with 2 x volume of 100-10 ethanol and keptat·20°C for 90 minutes. This suspensionwascentrifuged at10,000)( gravityandthe supernatant discarded.

The pellet wasdried under N2gasandresuspendedin0.5mLTE buffer.

The DNA content andpuritywas checkedbyrecordingtheabsorbance of the solution at260and 280 nm andcak:u1atingtheAbS@260/AbS@280 ratio. The DNA was also run on a0.5%agarosegelto verifYboththe absence oCRNA andthepurity afme plasmid.

2.2.7.4 Incubation and aulysis

.r

plasmid DNA Itnod cleavage

The test compounds (dissolvedinan organic solvent)wereadded to1.5mLplastic tubeswhich were left at J7°C to cvapotat:ethesample to dryness. Sterile 50mMKP buffer, pH 7.4,wasaddedtoeachNbein.quantity sufficicot to make the total reaction volume 50

;iL.2.5.uL of 2.5.uM FeSO. (final concentration'" 0.125.uM) wasadded to each reaction tube..foUowedbyI.u8ofpWmid.. 0 - 5 .uS postmitocl\ondrial fraction protein,and5 .uL 10 mMNADPH (ansaerile 50 mM KP buffer,pH7.4)to startthereaction. Capped sampletubes were incubatedat37'"Cfor I • 24boors.

39 Following the incubation, samples were removed from the incubator and briefly centrifuged to remove condensation from the cap and sides afthe tube. S,uL of6 x ttaeking dyewas added to each tube, foUawe'd by 5 ,uL of a solution I mstmLinprotease and RNase Tl .This sample was carefuUy mixed and incubated again at 37°C for a further 45 minutes.

The samples were t!len loaded onto a 0.5% agarose gel (in 0.5 x TBE buffer. with ethidium bromide) with the incubation samplesflankedonthegel, totheextremerightand lcft, by a molecular weight marker ofHind DlIEcoRI digested 1 phage DNA The gels were run at 20· 100 V for 4 to 16 hours. or sufficient time such that clear plasmid separation became visible on the gel. Gels wereviewedunder u.v. lightina darkroom. and photographed. The negative from the gel photograph was scanned using an LKB scanning laser densitometer. to reveal the relative intensities of the plasmid fonns evident on the gel.

Samples were comparedbyrelating the percentage of total plasmid DNA present inthe relaxed coil form.

2.2.7.5 StatisticalAaalysis olSta.aia. DeasitomdU Data

Statistical arWysiswasperformed.usingtheSigma Plot computer program.Thedata collectedwiththescanninglaserdensitometer were subjea.ed to apairedt-testStatistical analysis. Sample meansthat:were not different atthelevelp<0.05 were rejected as not significantly different.

40 CBAl'TER3

3. RESULTS

3.1 Nitroredue:tase a(twit)'

3.1.1 Crude 0" nlneu as .itroreductue.Mtntes

l-lltroreductase assaysshowedextremelylimitedenzyme activity with crudeoil extracts as substrates. The DMSO extract ofPrudhoe Bay crude oil exhibited no effect on nitroreductaseenzymeactivity(Figure3.1.1),asneithertested quantity ofextract,witheither NADHor~ADPHas a cofactor, produced abovecontrol levels of nitroreduetion.

Albertasweetmixcrude oil and Hibernia crude oil extracts both generated less than [ nmal/min niuoreductase activity, which were bothminimally,ifat all,greater than DMSO comrollevels (Figures 3.1.2 and 3.1.3).

Ofthe crude oils,theDMSOextractof Venezuelan crude oil exhibited the greatest potency as anitroreductase substr11e(Figure l.l.4). With 15~IaCme oilextractandNADH as the cofa.ctorinthereactionaMtte.a reaction rateoCO.937nmollmin. was exhibited. This was a 5 fold increase overthe DMSO control reaction rate orO,188 nmollrnin.

41

Fig.3.1.1 Nitrol"flluc:t&$t ac:tivity genenttel with DMSOeJ:tr.lc:to(Prudhoe Bay crude oil as enzyme substrate.

Fig. 3.1.2 Nitrortdactase activity generated wilb DMSO utraet

or

AJbertaSWett mix crude oil as eazyme substrate.

Cuvettescomainedatota!. volumeofJmLin50mMKP buffer, pH 7.6, with 330 JiM succinoylated cytochrome c. Cuvcucs also contained 10,ul cytosol (320/.lg protein),either 0 (IO,uL DMSO). 5. or 10.uL oilextract,and 100 .uL of 10 mM NAOlU NADPH tostartthe reaction. Reactions werecarried out at 37°C. Chart recordings of reactions were monitored foritminimum of 5minutes per assay.

'2 • NACH

~ ⁵ 0 NADPH

E 4

S

1

³

e

²

o 5 10

extract of prudhoe bay crude oil (fLl)

42

o 5 10 15

Albertasweetmix extract (JJ.L)

43

Fig. 3.1.3 NitrortduetastactmtypnenledwithDMSO utratt of Hibemia (rude oil as enzyme substrate.

Fig. 3.1.4 Nitroreductase atti"ity generated witb DMSO tltrac:t or Veaerumn crude oil U tllZ)'me substrate.

Cuvettes contained a totalvolumeonmLin50 mMI<Pbuffer, pH 7.6, with 330~Msuccinoylatedcytochrome c.Cuvettes also containedto,ulcytosol (J20,ug protein), either 0 (IO,uLDMSO).S, or 10,uL oiJextract. and100 .uL oflOmM NADHI NADPH to stan the reaction. Reactions were carried out at37°C. Chanrecordings of reactions were monitored for a minimum of 5 minutes per assay.

• NACI<

, 0 ^NAllPH

o 5 10 1S

Hibernia crude '1 extract (JJ.L)

• NACI<

, 0 NAllPH

o 5 10 15

Venezuelan crude extract(p.L)