Mutagenicity evaluation of vegetables grown in diesel ...mulinet11.li.mahidol.ac.th/e-thesis/scan/3836203.pdf · extract of leaves of sacred basil and green kuang futsoi grown in

MUTAGENICITY EVALUATION OF VEGETABLES GROWN INDIESEL EXHAUST CONTAMINATED SOIL USING THE WING

SOMATIC MUTATION AND RECOMBINATION TEST INDROSOPHILA MELANOGASTER

JANPEN SAI(SITPITAK

With comPlirnents, - of

f.,6,.1i,; q ':.,0irril, 'i r'Jl:tm.Ltuuuu uNi VEK)I I Y

A THESIS SUBMITTED IN PARTIAL FT]LFILLMENT OFTHE REQTIIREMENTS F'OR THE DEGREE OF

MASTER OF'SCIENCE(FOOD Ar{D NLnRTTTONAL TOXICOLOG9

FACI]LTY OF GRADUATE STT]DIESMAHIDOL T]NIVERSITY

1998ISBN 974-661-209-3

COPYRIGHT OF' MAHIDOL I]NIYERSITY

1t{

,i+ 1")

i' ,.qt

0C-rakryc

Copyright by Mahidol University

Thesisentitled

MUTAGENICITY EVALUATION OF VEGETABLES GROWN INDIESEL EXHAUST CONTAMINATED SOIL USING THE WING

SOMATIC MUTATION AND RECOMBINATION TEST INDROSOPHII-A MELANOGASTER

.i*xo....$t:'.ittlilt., ........

Janpen SaksitpitakCandidate

h*t /-

Anadi Nitithamyong, Ph.D.Co-advisor

LJl-^ k^^

Wannee Kusamran, Ph.D.Co-advisor*<f-Kraisid Tontisiriq M.D., Ph.DChairmanMaster of Science Program inFood and Nutritional ToxicologyInstitute of Nutrition

Kaew Kangsadalampai, Ph.D.Maior-advisor

,#*,1i /khu

ry UtvJrWAdulya Viriyavej akul,M.D., LL.B., F.R.C.P.DeanFaculty of Graduate Studies


Thesis

entitled

MUTAGENICITY EVALUATION OF VEGETABLES GROWN INDIESEL EXIIAUST CONTAMINATED SOIL USING THE WING

SOMATIC MUTATION AND RECOMBINATION TEST INDROSOPHILA MEI-ANOGASTER

was submitted to the Faculty of Graduate Studies, Mahidol University for the degree

of Master of Science (Food and Nutritional Toxicology)

on

May 22,1998

l)n".- \roLsilP,IaJ'l'

Janpen SaksitpitakCandidate

Anadi Nitithamyong, Ph.D.Member

Na. tl"-Wannee Kusamran, Ph.D.Member

Adulya Viriyavej akul,M.D., LL.B., F.R.C.P.DeanFaculty of Graduate Studies

Kraisid Tontisirin, M.D., Ph.DDirectorInstitute of Nutrition

Kaew Kangsadalampai, Ph.D.Chairman

fb-t-ilrt


ACKNOWLEDGEMENT

I would like to express my sincere gmtitude and deep appreciation to

my advisor, Associate Professor Dr. Kaew Kangsadalampai, for this encouragement,

valuable guidance and warm friendship thoughout my graduate program.

I wish to affirm my gratefulness to my co-advisor, Dr. Anadi

Nitithamyong and Dr. waruree Kusamran for their valuable comments and advice.

Also special thanks to l)rs" F.E. Wrrgler and U. Graf of Institute of Toxicology, Swiss

Federal Institute ofrechnology and University of Zurich for their stock cultures ofD.

melanogaster.

I would like to thank Miss Prapasri Laohavechvanich for her technical

instruction as well as Mr. Daokngam Sudjaom for his constant help.

Uttermost gratitude is expressed to my family for their warm

encouragement throughout the period of my graduate study.

Janpen Saksitpitak


IV

3836203 NUTX/M : MAJOR : FOOD AND NUTzuTIONAL TOXICOLOGY ;

M.Sc. ( FOOD AND NUTzuTIONAL TOXICOLOGY)KEY WORD : MUTAGENICITY/ DIESEL EXHAUST/ PLANT

CONTAMINATIONJANPEN SAKSITPITAK : MUTAGENICITY EVALUATION OF

VEGETABLES GROWN IN DIESEL EXHAUST CONTAMINATED SOIL USINGTHE WING SOMATIC MUTATION AND RECOMBINATION TEST INDROSOPHILA MELANOGASTER. THESIS ADVISOR : KAEWKANGSADALAMPAI, Ph. D., ANADI NITITHAMYONG , Ph. D. WANNEEKUSAMRAN, Ph. D. 77 p. ISBN 974-661-209-3

Larvae of the improved high bioactivation (IHB) cross Drosophilamelanogaster (ORR;ltl/TM3, Ser females mated with mwh males) were fed withmedium containing hexane extract of the edible portion of five vegetables grown inthree drfl'erent soil treatments (controi soil, soil treated with I g of diesel exhausL&g

soil and soil treated with 10 g ofdiesel exhaust/kg soil) for 48 h. The wing hairs ofthesurviving flies were analyzed for the frequency and size of single and twin spots. Itwas found that clone induction frequency ofthe wing hairs of flies treated with hexane

extract of leaves of sacred basil and green kuang futsoi grown in contaminated soilwas not statistically different from that of vegetables grown in control soil. Clearpositive results were obtained when larvae were brought up in the medium containinghexane extracts of lettuce and of water spinach grown in contaminated soils.

Interestingly, the extracts of multiply onion, both grown in the treated and untreated

soils, induced mutation in the wing spot test. It was concluded that some plants grownin diesel exhaust contaminated soil provoked mutagenic responses while some

showed negative results.


3836203 NUFIA{ : d11.1'ts1 : yl! tll8]Yn r011n:lln3 lnxutnrl : 'l1l.u. (t{u't11u11'n.101fi'l:

rrdi lnTu lntt)

frnvrri rriq : qmGnonnrun'uq/ rrur:orJuflfirryn I nr:rJurrlouqu'u

nuT:ry{tu f,nodyltflTnu : n t: u: t[!uqYlt n 0fld 1unuq10,lr.tnY| qn lufluLuu

rflouo'lumrr'lorf,unr nsrrdrryaq?o wtNG soMATIC MUTATIoN AND

RECOMBINATION TEST 1U PROSOPTTTT A MELANOGASTER (MUTAGENICITY

EVALUATION OF VEGETABLES GROWN IN DIESEL EXHAUST CONTAMINATED

SOIL USING THE WING SOMATIC MUTATION AND RECOMBINATION TEST IN

DRosopHrLA MELAN}GASTER) n ufl:: nr:n'rr-rqruivrorfinut' , ruir fi'raenaiirlfl,

pr,.p., orord u'sr^t::ra.:, ptr.o.,':::fi ndr:rq, rl.o. ,z rri'l ,ssN 97 4-661-209-3

{ ]U .)nflUflnUrillr5 fl0nd tt uql0.lrufl 5 xuoTUen [u0uytxll t:nuunnrryd!u

J^rilou ro. l ;o l0 n:-lfloou I filan:-rl lqulsuua{ niaroniuq Drosophita

1u Somatic Mutation and Recombination Tesr (SMART) nt:ylfidoljfitlnU rn''luuouto,t

ttlalriffluflfl (heterozygous) xll^n improved high bioacrivarion (I]IB) cross (ORR;fl1/

TM3, Ser females and mwh mates.l I 16'o tluo nr r:fifiat: arior6nrryuto{r..1-fllrnaryu^nu1u

+s frl!'r oufrrnuouro?tgrn'rlnuns:onfiinnara,flu,r ar d ,,airirflnrurn::ouryuô

AiU!01U?U1]O.rrcraaljuT na10flun x0ry0m loruor'llJl .)tfl:1r11lt1{doft 11]l.lt nyt :lua,r.innrnrfiuenrnq'rhrfiqrfr'nonnrort'uq d,rfirJqnlu6ufifinmrilurdouuar6unruql neu

rinnronorrra;pinr.1o,,ar,,qnr*nu n o rur[u q'rritrorJqnludufifirtl l]uuiourvrrriu u6yul-y_ - ) | :, , "J i , q a ^

jd , Id.rrnn rnur..r 0rJu do{ qrr n 0n a toy{uqyt.tmil q n tuoun'llqlrraroufrfurrJt urllou ,.t i< . ^ 4' -'"'i ,

dnu rfrudnurrinrrlyu-ndrtlr:ouao rqri r n o n n run'uq tnur'orJq nluGufifircJurilur{lou


vl

CONTENTS

ACKNOWLEDGEMENT

ABSTRACT

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATION

CHAPTER

r r/^'rrl.\\TI tr\ I t\.trlrrJL rr\ir\

II REVIEW OF LITERATURE

Environmental Mutagens in plants

Engine Exhaust

Diesel engine exhaust

Carcinogenicity studies in animals

Toxic effects

Effects on reproduction and prenatal toxicity

Genetic and related effects

Somatic Mutation and Recombination Test

Principle of SMART

Genetic basis ofthe effects detected

Approach of SMART

M MATERIALSANDMETHODS

N RISULTS

V DISCUSSION

REFERENCES

APPENDIX

BIOGRAPHY

Page

iii

iv

vii

viiii.,

1

J

3

5

13

l5

16

17

19

2t

25

28

30

36

47

52

70

77


vtl

Table

1

2

3

4

5

6

LIST OF TABLES

Agents identified in engine exhausts that have been evaluated inIARC Mono graphs volumesSome compounds and classes of compounds in vehicle engineexhaustPolycyclic aromatic compounds identified or tentatively identifiedin three light-duty diesel particulate exkactsConcentrations of some nitroarenes (pglg) in diesel particulateextracts

The yield of planls exlract lor mutagenicity testClone induction frequency after feeding of 72 h old larvae ofinaproved high bioactivation cross rvith medium containingdifferent concentrations of crude diesel exhaust for 48 hClone induction frequency after feeding of 72 h old larvae ofimproved high bioactivation cross with medium containingdifferent concentrations ofhexane extract ofdiesel exhaust for 48hClone induction frequency after fe eding of 72 h old larvae ofimproved high bioactivation cross with medium containingdifferent concentrations ofhexane extract of sacred basil (Ocimumsdnctum Linn.) grown in control or diesel exhaust contaminatedsoils for 48 hClone induction frequency after feeding of 72 h old larvae ofimproved high bioactivation cross with medium containingdifferent concentrations ofhexane extract ofofgreen kuang futsoi( Brassica chinensis Jlsl.) grown in control or diesel exhaustcontaminated soils for 48 hClone induction frequency after feeding of 72h old larvae ofimproved high bioactivation cross with medium containingdifferent concentrations ofhexane extract of multiply onion(Allium cepa yar aggregatum Don) grown in control or dieselexhaust contaminated soils for 48 hClone induction frequency after feeding of 72 h old larvae ofimproved high bioactivation cross with medium containingdifferent concentrations of hexane extract of lettuce (Lactucasativa Lirn.) grown in control or diesel exhaust contaminated soilsfor 48 hClone induction frequency after feeding of 72 h old larvae ofimproved high bioactivation cross with medium containingdifferent concentrations ofhexane extract of water spinach(Ipomoea aqudtic Forsk) grown in control or diesel exhaustcontaminated soils for 48 h

11

Page

13

3839

40

42

43

44

45

46


vlll

FIGURE

I

2

3

4

LIST OF FIGURES

Page

Marker mutations of wing surface to show clone of cuticle 24secreted by cells homozygous for multiple wing hairsGenetic schemes illustrating various ways of spot formatio n 26Normal half mesothorax showing the regions A-E of the wing 34surface scored for spotsTrichomes on the wing btade, a) normal, b) deviate trichomes 14not counted as mwh or flr, c) configurations indicative ofmwh, d) typical manifestations of flr


.C

cm

LIST OFABBREVIATTON

degree celcius

centimetre

gram

hour

kilogram

miligram

mililitre

milimolar

number

h

kg

mg

ml

m I\,{

No.


CHAPTER I

INTRODUCTION

It is well documented that our environment contains a wide range of different

types of physical and chemical contaminants. Polycyctic aromatic hydrocarbons

(PAHs) are wideiy distributed not oniy in the air, but also in tbodstuffs. Nitaya (l)

determined the total PAHs in some Thai foods and found that smoked fish, charcoal-

broiled meats and sausage, fried meats and egg, cooking oils and leafo vegetables

were contaminated by PAHs at the levels of 7.59, 1.61, 2.47,9.00,0.07 ppm (mg/kg),

respectively. These compounds composed of fused benzene rings and some of which

are known or suspected carcinogens/mutagens. In 1976 Muller (2) reported that the

carcinogenic PAH, benzo(a)pyrene, was found in vegetables and further investigations

were carried out to determine the content of other pAHs in plants from various parts

ofthe world. For instance, various Russian investigators reported the contamination of

plants by PAHs. Shcherbak (3, 4) also stated that pollution ofplants might occur from

sedimentation of atmospheric dust and soot or by migration of the carcinogens into the

plants from polluted soils while Shabad and cohan (5) concluded that the main soiuce

of contamination of soil was from air particulates. They indicated that migration or

resorption of PAHs into plants was dependent on the pAHs level in the soil and the

type of plant. However, studies on mutagenic activity of plants contaminated nith

these chemical compounds have not been reported so far.Copyright by Mahidol University

Environmental PAHs are generated mainlv by, incomplete combustion of

petroleum derived products (6) in various types of improper adjusted engines.

schuetzle (7) and Bartle et al (8) stated that nearly all ofthe chemical species ofhealth

significance identified in the diesel exhaust were PAHs. Therefore, the sample of

diesel exhaust should be a suitable source of mutagenic PAHs in the model study of

contamination ofthese compounds in edible plants.

In order to fulfill the lack of information about the mutagenicity of pAHs

contaminated plants mentioned earlier, the somatic mutation and recombination test

(SMART) was employed for detecting mutagenicity of the plants as such. This assay

is an efficient and versatile eukaryotic short-term in vivo assay which detects various

types of mutation including mitotic recombination in cells of the wing imaginal discs

of Drosophila melanogaster larva In addition the larva possess metabolic activities

that allow them to activate mutagenic PAHs and their derivatives.


CHAPTER II

REVIEW OF LITERATURE

Environmental monitoring revealed many uncalming results, including the

identification ofa list of persistent chemicals which were found over the entire globe,

not only in industrial areas but also in our food. pollution was primarily human waste

along with litter and smoke, but with the advance of modem chemistry, the scene

changed dramatically. The growing numbers of diesel automobiles in Thailand

expanded the effort to understand the health effects of air bome pollutants arising

from increased automotive emissions, especially when they became incorporated into

our food chain. Diesel exhaust was shown to be mutagenic in short-term bioassays

and carcinogenic in laboratory animals (9). These particulates contained hundreds to

thousands of chemical components (7, 8) and the majority of the chemical species of

biological signifrcance identified were pAHs or pAHs-derivatives.

2.1 Environmental Mutagens in plants

The presence of PAHs was demonstrated in a wide variety of plants from

diverse sources. Guddal (10) first reported the isolation of anthracene, pyrene, and

fluoranthene from chrysanthemum roots grown in contaminated soil near a gas works

plant and concluded that the PAHs were resorbed by the prant since follow-upCopyright by Mahidol University

investigations showing that roots grown in uncontaminated soils and not exposed to

smoke from the factory were not for:nd to be contaminated with the hydrocarbons.

Tilgner (11) noted that a strong relationship appeared to exist between air

pollution and the occurrence of benzo(a)pyrene in grain and vegetables. For example,

grain samples from the heavily industrialized Ruhr dishict in Germany were found by

Grimmer and Hildebrandt (12) to contain approximately l0 times more pAHs than

samples taken from Lower Saxony and the Holstein District remote from industry.

Grimmer and I Iildebre,ldt (13) also conducted sf.rdies on 4 difforent types of

vegetables grown simultaneously in the same field. The benzo(a)pyrene content found

varied considerably, e.g., tomatoe s 0.22 Stgkg,leeks 6.6 pglkg, spinach 7.4 lt glkg,

and kale 20 Stglkg. Salad greens grown close to Hamburg contained levels of pAHs

five to six times greater than that grown in a suburban area. In subsequent work,

Grimmer ( 14) expressed the opinion that " in the appraisal of the amount of

carcinogenic hydrocarbons ingested by man, quite new criteria are apparent. Neither

smoked foods or grilled meat but vegetables and salads contain the largest amounts of

PAHs."

Gunther et al. (15) reported the presence of about 125 mg/kg of anthracene

and six unidentified PAHs in the rinds of oranges grown in atmosphere-polluted areas

in the United states, but not in fruit harvested in uncontaminated locations. Bolling

(1964) studied the effects of location on the benzo(a)pyrene content ofcereals. wheat,

com, oats, and barley grown in industrial areas showed a 4-fold to 10_fold higher

contamination than crops from more remote areas.


Various Russian investigators reported the contamination of plants by pAHs.

Shabad et a/.(16) stated that, on the basis of available data, there were at least 3 routes

of PAHs passage into plants: air deposition, adsorption from soil, and synthesis. The

results of their studies led them to believe that air deposition is the principal route of

contamination. Shcherbak (3, 4) also stated that pollution of plants might occur from

sedimentation of atmospheric dust and soot or by migration of the carcinogens into

the plants from polluted soils. Shabad and Cohan (5) concluded that the main source

of contamination of soil was from aii particuiates. They indicated that rnigration or

resorption of PAHs into plants was dependent on the PAHs level in the soil and the

type of plant.

2.2 Engine Exhausts

Engine exhausts are complex mixtures containing thousands of chemical

compounds in the particulate and gaseous phases. Many components of engine

exhausts are found in tobacco smoke and other combustion products, Table 1 lists

agents that were identified in engine exhausts and that were evaluated by the

International Agency for Research on Cancer (IARC).


Table 1. Agents identified in engine exhausts that have been evaluated in IARCMonographs volumes

Evidence of carcinogenicity'

Humans Animals Group

AcetaldehydeAcridines

Benz(c)acridineDibenz(a,h)acridineDibenz(aj)acridine

AcroleinBenzene1,3-Butadiene1,2-Dibromoethane(ethylenedibromide) I1,2-DichloroethaneEthyleneFormaldehydeLead and lead compounds

InorganicOrganolead

MethylbromideNitroarenes

3,7-Dinitrofl uoranthene3,9-Dinitrofl uoranthene1,3-Dinitropyrene1 ,6-Dinitropyrene1 ,8-Dinitropyrene9-Nitroanthracene6-Nitrobenzo(a)pyrene3 -Nitrofluoranthene2-Nitrofluorene1-Nitronaphthalene2-Nitronaphthalene1-Nitropyrene

Polycyclic aromatic compoundsAnthanthreneAnthraceneBenz(a)anthraceneBenzo(b)fluorantheneBenzo()fluoranthene

NDNDNDIa

I

S

LS

S

IS

S

S

S

NDS

2B

-)

2B28J

1

2B2A28J

2A

2B3

J

J

J

3

2B2BJ

J

J

2B3

3

28

J)2A2B28

S

IL

LLLS

S

NDLIS

IIS

LIS

S

S

NDNDL

III

NDNDNDNDNDNDNDNDNDNDNDND

NDNDNDNDND


Table 1 (continued)

Agent Evidence of carcinogenicityu

Humans Animals Group

Benzo(k)fluorantheneBenzo(ghi)fl uorantheneBenzo(a)fluoreneBenzo(b)fluoreneBenzo(ghi)peryleneBenzo(c)phenanthreneBenzo(a)pyreneBenzo(e)pyreneChryseneCoronene

Cyclopenta(cd)pyreneDibenz(a,h)anthraceneDibenzo(a,e)pyreneDibenzo(a,h)pyrene1,4 -DimethylphenanthreneFluorantheneFluoreneIndeno( 1,2,3 -cd)pyrene2-Methylchrysene3-Methylchrysene4-Methylchrysene5-Methylchrysene6-Methylchrysene1-MethylphenanthenePerylenePhenanthrenePyreneTriphenylene

Propylene

2B3

3

J

J

)

2A3

J

J

J

2A2B2BJ

3

J

283

3

J

28J

J

JJ

3

J

J

S

IIIIIS

ILILS

S

S

IIIS

LLLS

LIIIIIND

NDNDNDNDNDNDNDND

NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND

uFrom supplem ent 7 (17), unless otherwise indicated; I, inadequate evidence; L,

limited evidence; ND, no adequate data; S, sufficient evidence; 1, Group 1 - theagent is carcinogenic to humans; 24, Group 2A- the agent is probably carcinogenicto humans; 28, Group 28

-the agent is possibly carcinogenic to humans; 3, Group 3

-the agent is not classifiable as to its carcinogenicity to humans


The major products of the complete combustion of petroleum-based fuels in an

intemal combustion engine are carbon dioxide (13%) and water (13%), with nitrogen

from air comprising most (73%o) of the remaining exhaust. A very small portion of the

nitrogen is converted to nitrogen oxides and some nitrated hydrocarbons.

Incomplete combustion results in the emission of carbon monoxide, unbumt

fuel and lubricating oil (18) and of oxidation and nitration products of the fuel and

lubricating oil. These incomplete combustion products comprise thousands of

chemical componenis plesent iri the gas and particulate phases (19); some specilic

chemical species and classes fourd in engine exhausts are listed in Table 2. The

concentration ofa chemical species in vehicle exhaust is a function of several factors,

including engine type, engine operating conditions, fuel and lubricating oil

composition and emission control system (20).


Table 2. Some compounds and classes ofcompounds in vehicle engine exhaust^

Gas phase

AcroleinAmmoniaBenzene

1,3-ButadieneFormaldehydeFormic acidHeterocyclics and derivativesbHydrocarbons (Cr-Crr) and derivativesbHydrogen cyanideHydrogen sulfideMethaneMethanoiNitric acidNitrous acidOxides of nitrogenPolycyclic aromatic hydrocarbons and derivativesbSulfi.r dioxideToluene

Pariculate phase

Heterocyclics and derivativesbHydrocarbons (Crq-C:s) and derivativesbInorganic sulfates and nitratesMetals (e.g., lead and platinum)Polycyclic aromatic hydrocarbons and derivativesb

"From National Research Council (21)b Derivatives include acids, alcohols, aldehydes, anhydrides, esters, ketones, nitriles,quinones, sulfonates and halogenated and nitrated compounds, and multifunctionalderivatives

2.2.1 Diesel Engine Exhaust

Diesel engines produce two to ten times more particulate emissions than

gasoline engines (without catalytic converter) of comparable power output and two to

forty times more particulate emissions than gasoline engines equipped with a catalytic

converter (9, 22, 23, 24, 25). Diesel particles are aggregates of spherical primary


l0

pa(icles of 0.1- 0.5pm (26). Smaller primary spheres, formed within the combustion

cylinder, grow by agglomeration and by acting as nuclei for the condensation of

organic compounds (27). The particles consist primarily of 60-g0% elemental carbon

(19,28),2-7Yo srlfuic acid (29) and some metallic species, e.g., iron from the engine

and exhaust system (25), barium from fuel (30) and zinc from lubricating oil (31), and

adsorbed organic compounds (26).

A variety of solvents were used to extract organic compounds from diesel

particles (32). The soluble organic fraction of dlesel particles usually accounts for l5-

45oh of the total particulate mass. The nonpolar fractions containing hydrocarbons

derived from unbumt fuel and lubricating oil. In addition, many pAHs in the

molecular weight range of 178-320 were identified. Some pAHs and thioarenes

identified in diesel engine exhausts are listed in Table 3 (33). The alkyl substituted

derivatives ofat least some PAHs are more abundant than the parent hydrocarbons. Ifthe amount of dimethylanthracenes or dimethylphenanthrenes is taken as 1.00, the

relative abundance of anthracene or phenanthrene is 0.27, that of the methyl

derivatives, 0.54, and that of the trimethyl derivatives, 0.37 (34). Since moderately

polar fractions have been found to contribute a significant proportion of the

mutagenicity of the total soluble organic fraction, much effort has been expended to

characterize them.


Table 3. Polycyclic aromatic compounds identified or tentatively identified in threeight-duty diesel parti extractsa

Compound Molecularweight

Concentration(pgig ol extract)

AcenaphthyleneFluoreneTrimethylnaphthaleneAnthracenePhenanthreneDimethylbiphenylTetramethylnaphthaleneDibenzothiophene4H-Cyclopenta(def)phenanttrene2-Methyianthracene2-Methylphenanthrene3 -MethylphenanthreneTrimethylbiphenylMethyldibenzothiopheneBenzacenaphthylene

FluoranthenePyrene2-PhenylnaphthaleneDimethylphenanthrene2- or 9-EthylphenantlreneDimethylphenanthrene or-anthraceneBenzo(def)dibenzothiopheneEthyldibenzothiopheneBenzo(a)fluoreneBenzo(b)fluoreneMethylfl uoranthene or -pyreneI -MetylpyreneEthylmethylphenanthrene or -anthraceneBenzo(ghi)fl uorantheneCyclopenta(cd)pyreneBenz(a)anthracene

Chrysene or triphenyleneB enzonaphthothiopheneBenzo(b)naphtho(2, 1 -d)thiopheneMethyl benz(a)anthracene3-MethylchryseneBenzo(b)fluoraritheneBenzo$fluoranthene

152

166

170178

178182184

184

190

192

192t92196198

202

202202204

206206206208212

216216216216220226

226228228234234242242

252252

30

100- 168

30-50155-3 562l 86-488330-91

50-1s2129-246517-1033517-t5221099- 1481

929-128750101-323t9t-16433399-73213532-8002650-1336443-1046388-46486-s85254-3331st-179541-990175-538224-552144-443286-432217-418869-167t463-t076657-152930-12630-5330-5050-192421-109492-1367


12

Table 3 (continued)

Compound Molecular Corcert."tio;6--weight (pglg of extract)

Benzo(k)fluorantheneBenzo(e)pyreneBenzo(a)pyrene1,2-Binaphthyl2,2-Binaphthyl1-Phenylphenanthrene9-PhenylphenanthrenePhenylphenanthrene or -anthracene

Benzo(ghi)peryleneIndeno( 1,2,3 -cd)pyreneDibenz(a,h)anthraceneCoroneneDibenzopyrene or -(def,p)chrysene

91-289487-946208-55830-5089-28389- 163

30-9430-116443- 1050JU-YJ

50-96301-521

89-2s4

252

252252

2542542s4254254276

278300

302

Trom Tong and Karasek (33)bConcentrations

ofless than 50 pglg extract were obtained by approximate calculation

More than 50 nitrated derivatives of PAHs in diesel vehicle particles were

identified tentatively and 23 have been identified positively. These compounds occur

in very low concentrations in comparison with the other PAHs derivatives (35). The

concentrations of nitroarenes measure in a lighfduty diesel particulate extract by a

method with a 0.3-ppm (pglg) limit of detection are given in Table 4. Overall, about

40%o of the direct mutagenicity ofdiesel particulate extracts could be accounted for by

l -nitro-3-acetoxypyrenes, dinitropyrenes and I -nitropyrene (36).


l3

Table 4. Concentrations of some nitroarenes (pglg) in diesel particulate extracts

Compound Concentration Reference

I -Nitronaphthalene2-Nitronaphthalene2-Nitrofluorene1-Nitropyrene3 -Nitrofluoranthene8-Nitrofluoranthene6-Nitrobenzo(a)pyrene1,3-Dinitropyrene1,6-Dinitropyrene1,8-Dinitropyrene2,7-Dinitrofluorcne2,7-Dinitro-9-fl uorenonel -Nitro-3 -hydroxypyreneI -Nitro-3 -acetoxypyrene

0.9s0.3 5

1.2

75

3.5

1.3

4.2

0.300.40053

3.0,8.670

6.3

a

a

a

a

a

a

a

a

I'

b

c

c

a : from Paputa-Peck et al. (37)b = from Schuetzle (38)c = from Manabe et al. (36)

2.2.1.1 Carcinogenicity studies in animals

Nesnow e/ c/. (39) gave skin applications to groups of40 male and 40 females

SENCAR mice, seven to nine weeks of age, of 0.1, 0.5, 1.0, 2, or 10 mg of

dichloromethane extracts ofparticles obtained from the exhausts of hve diesel engines

A, B, C, D and E (E being a heavy duty engine) in 0.2 ml acetone; the 10 mg dose was

given in five daily doses. The benzo(a)pyrene content started from 1173 ngimg in the

exhaust from engine A down to 2 ng/mg in that from engines B and E. One week

later, all mice received 2 pg l2-0+etradecanoylphorbol- l3-acetate (TpA) in 0.2 ml

acetone twice a week for 24-26 weeks. A control group was treated with rpA only.

The sample from engine A produce a dose-related increase in the incidence of skin


t4

papillomas, with 5.5 and 5.7 papillomas/mouse, 31 % of males and,36 %o of females at

the highest dose having skin carcinomas. with samples from engines B, c and D,

responses of 0.1-0.5 papillomas/mouse were observed compared to 0.05_0.0g

papilloma,/mouse in TPA controls. The sample from engine E produced a response

similar to that in controls.

Heinrich et al. (40) exposed two groups of 96 female NMRI mice, eight to ten

weeks old, to filtered or unfiltered exhaust from a 1.6-1 displacement diesel engine

operaied acoordirrg r.o the US-72 test cycle to simulaie average urban driving, or to

clean air, for 19 h per day on five days per week for life. The unfiltered and filtered

exhausts were diluted 1:17 with air and contained,4.24 mglm3 particles. Exposure to

total diesel exhaust and filtered diesel exhaust significantly increased the number of

animals with lung tumors (adenomas and carcinomas) to 32 %o and 31%, respectively,

as compared to 13 %o in controls. when the incidences of adenomas and carcinomas

were evaluated separately, significantly higher numbers of animals in both diesel

exhaust-exposed groups had adenocarcinomas (17 yo and 190%, respectively) than in

controls (2.4 %).

Iwai et al. (41) exposed two groups of 24 female specific_pathogen_free

Fischer 344 rats, seven weeks ofage, to either diluted diesel exhaust ofdiluted filtered

diesel exhaust for 8 h per day on seven days a week for 24 months, at which time

some rats were sacrificed and the remainder were returned to clean air for a further six

months of observation. The diesel exhaust was produced by a 2.4-1 displacement

small truck engine; it was diluted ten times with clean air and contained 4.9 + 1.6


mg/mr particles , 3.6 + 3.6 mgim3 nitogen dioxide and 30.9 + 10.9 ppm nitrogen

oxides. Another group of 24 rats was exposed to fresh air only for 30 months.

Incidences of lung tumours, diagnosed as adenomas, adenocarcinomas, squamous-cell

carcinomas and adenosquamous carcinomas, were signihcantly higher in the group

exposed to whole diesel exhaust, with or without a subsequent observation period than

the control group. No lung tumor was observed in the group exposed to filtered

exhaust. Incidences of malignant lymphomas and tumors at other sites did not differ

among the *ree groups.

Kawabata et al. (42) exposed four groups of female specific-pathogen_free

Fischer 344 rats, six weeks of age, received ten weekly intrapulmonary instillations of

1 mgianimal activated carbon or 1 mg/animal diesel exhaust particles in phosphate

buffer with 0.05 % Tween 80, 2 ml of buffer alone or were untreated. Rats surviving

18 months constituted the effective numbers. The experiment was terminated 30

months after instillation. The numbers of animals with malignant lung tumors were

significantly higher in the groups treated with activated carbon and with diesel

particles than in untreated or vehicle controls. Similarly, the numbers of animals with

benign and malignant lung tumors were also significantly increased in the groups

treated with activated carbon and diesel particles.

2.2.1.2 Toxic effects

Toxic effects of diesel exhaust had been .studied in animals. After about 4g0

days, NMRI mice exposed to unfiltered, diluted (l:17) diesel exhaust (particles,4

mg/m3; carbon monoxide r4.3 + 2.5 mg/m3) had lost body weight in comparison with


l6

animals exposed to filtered exhaust (carbon monoxide, 12.7 + 2.2 mgim3) or with

controls. Under the same circumstances, rats had a lower weight increase (40). The

livers of Syrian golden hamsters exposed for five months to diesel exhaust diluted 1:5

and l:10 in air had enlarged sinusoids with activated Kupffer's cells. Nucleoli were

frequently fragmented or inegularly shaped. Fat deposition was observed in the

sinusoids. Mitochondria from animals exposed to the 1:5 dilution had frequently lost

cristae. Giant microbodies were observed in hepatocytes, and gap junctions between

hepatocltes were disfxbed (43). Shortterm exposure to riiesel exhaust (28 days) led

lo a 35 %o increase in pulmonary air flow resistance in Hartley guinea-pigs (44) but

increased vital capacity and total lung capacity in Sprague-Dawley rats (45). In

Fischer 344 rats, DNA synthesis in lung tissue was increased four-fold after two days

of continuous exposure by inhalation to diesel exhaust. DNA synthesis retumed to

control levels one week after exposure. The labelling index of type II cells was

significantly greater than that in controls after two and three days of exposure to diesel

exhaust. After one day of exposure, palmitic acid incorporation into

phosphatidylcholine in lung tissue increased by three fold when tissue palmitic acid

content decreased. Total lung fatty acid content decrease d by 23 % after one day of

exposure (46).

2.2.1.3 Effects on reproduction and prenatal toxicity

A three-fold increase in sperm abnormalities was observed in Chinese

hamsters exposed to diesel engine exhaust (dose unspecified) for six months, as

compared to controls exposed to fresh air (47). A statistically significant dose-related


increase in sperm abnormalities was observed in males (C57Bll6 x C3H) F1 mice

receiving 50, 100 or 200 mg/kg body weight diesel exhaust particulates by

intraperitoneal injection for five days. An eightfold increase in sperm abnormalities

over the spontaneous level was observed in mice receiving the highest does; testicular

weight was not affected (48).

2.2.1.4 Genetic and related effects

The soluble organic matter extracted from diesel particles obtained from the

exhaust ofseveral types ofdiesel engines induced DIIA da.age in Bacillus subtilis in

the absence of an exogenous metabolic system at doses of 60-500 pglml (49). The

majority of studies on the mutagenicity of diesel exhaust were conducted in

salmonella typhimurium on soluble or extractable organic matter removed from soot

particles. The dichloromethane extractable organic matter from soot particles

collected fiom two diesel engines was mutagenic to Salmonella typhimurium TA1537,

TA1538, TA98 and TA100 in the presence and absence of an exogenous metabolic

system from Aroclor 1254-induced rat liver. In the presence of activation, one soot

exhact was weakly mutagenic to TA1535 (50). other studies of particulate extracts

from the exhausts of various diesel engines and vehicles also induced mutation in

Salmonella typhimurium T A1537, TA1538, TA98 and TAt00 with and without an

exogenous metabolic system, but not in TA1535 (49, 51, 52, 53, 54,55). Diesel

engine exhaust particulate extracts were mutagenic in salmonella typhimurium TM

677 in a forward mutation assay using 8-azaguanine resistance (54, 56) and in

mutagenesis assays in Escherichia coli WP2 and Kl2 (57, 58).

t7


Chemical characterization by the use of bioassays was reviewed (59). Such

studies showed that nitrated pAHs contributed to the mutagenicity of diesel

particulate extracts. The first evidence for the presence of nitroarenes in diesel

particulate extracts was provided when a decrease in mutagenicity was observed in

nitroreductase-deficient strains of Salmonella typhimurium (54, 60, 61, 62, 63). The

contribution of mono- and dinitro-pAHs to the mutagenicity of these extracts (20-

55%) was estimated by measuring both nitro-pAH and mutagenicity in sarmonela

typhitnurium TA98 rn thc seuno dicsci piuticulate cxtracts (3g, €,q,55,66,67). Other

oxidised PAHs in diesel particulate extracts, such as pAH epoxides (6g), pyrene-3,4-

dicarboxylic acid anhydride (69) and 5 H-phenantko(4,5-bcd)pyran_5-one (63), were

shown to be mutagenic to ,Sd lmonella typhimurium.

The urine of female Swiss mice exposed for g h per day on five days per week

to whole diesel exhaust (dilution, l:1g; particles, 6-7 mgrm3) for seven weeks (47) or

of Fischer 344 rats exposed to diesel exhaust particles (1.9 mg/m3) for three to 24

months (70, 71) was not mutagenic to Salmonella typhimurium. However, positive

responses were obtained with the urine of Sprague-Dawley rats given 1000_2000

mgikg bw diesel exhaust particles by gastric incubation or by inhaperitoneal or

subcutaneous administration (55, 72).

Extracts from the emissions ofdiesel engines (up to 250 pglml) did not induce

DNA damage in cultured Syrian hamster embryo cells, as determined by atkaline

sucrose gradient centrifugation (73). However, diesel exhaust particles (1 and 2

mg/ml) induced unscheduled DNA synthesis in trachear ring cultures prepared from


19

female Fischer 344 rats (42). Diesel engine emission particles and particulate extracts

were more cltotoxic for excision repair-deficient xeroderma pigmentosum fibroblasts

than for normal human fibroblasts (74).

when whole diesel exhaust was bubbled through cultures of human peripheral

lymphocytes from four healthy nonsmokers, sister chromatid exchange was induced

in two of the samples (75). Sister chromatid exchange was also induced in cultered

human lymphocytes by a light-duty diesel particulate cxtract (5-50 pglml; 76) and by

Ciesel pa:ticulate extracts (10-200 pgiml) from emissions of lighiduti, and heavy_

duty diesel engines (77). In the last study, light-duty samples were more potent in

inducing sister cfuomatid exchange than heavy-duty samples.

2.3 Somatic Mutation and Recombination Test

considerable efforts were spent to develop somatic mutation systems on

Drosophila and they were highly successful. Rapid, sensitive and cheap methods were

worked out (78). These methods measure both mutations and recombination and they

had therefore been named somatic mutation and recombination tests (sMART). The

use of SMART assays is based on the treatment of larvae, and besides the number of

mutated spots appearing in the adult flies, indicating the frequency of genetic events,

the size of the spots indicates the time of action during embryogenesis. The SMART

provides a suitable substitute or at least complembntary in vivo methodto mammalian

in vivo investigations. Drosophira has detoxication-activating systems in many

respects closely resembling the corresponding systems in mammals (79, g0), which


20

makes it possible to extrapolate data to mammals. The wing spot test was already used

in a study for the evaluation of genotoxicity of extracts from airbome particulate

matter from building ventilation filters (81). In addition, there were many of

publications reported that PAHs were mutagenic if larvae were treated (82, g3, 94. g5,

86).

Mollet and Wurgler (87) suggested to use somatic cells of Drosophila for

mutagencity testing. In individuals heterozygous for so-called visible marker

rnutations, ce*ain mutagenic even'r.s can leaC to the lcss of the dominant wild-type

allele opposite to the marker mutation, with subsequent expression of the recessive

marker allele in a clone ofensuing mutant cells. Loss of the dominant allele can occur

by a variety of mechanisms: homologous mitotic exchange or mutation in the

dominant allele leads to clones of euploid cells, while nonhomologous mitotic

exchange or deletion generates clones of aneuploid cells. Mollet and Wurgler (g7)

showed that alkylating agents induced clones in the eye imaginal discs of treated

larvae. In the subsequent years, the test was not further developed, because at that

time it appeared that somatic events which could not be studied further by genetic

analysis were of minor interest compared with the sex-linked recessive lethal assay in

which genetically transmissible damage was detected. In the meantime, a number of

developments took place which reactivated their interest in somatic mutation assays in

Drosophila:


2t

l. The correlation between mutagenic and carcinogenic activity of chemicals

generated much interest in the genotoxic activity of chemicals in somatic cells. In

Drosophila about 85% ofthe carcinogens tested were mutagenic in germ cetls (88).

2. Not only mutagenic but also recombinogenic activity of chemicals was

discussed in connection with their carcinogenicity (89, 90).

3. Drosophila larvae possess metabolic activities that allow them to activate a

large variety of xenobiotics. It was shown by Vogel and co-workers that a number of

polycyclic hy'drocarbcns and a.romatic amines v,,ere mutagenic if lar,,ae u'ere treated

(88, e1).

4. The test is advantageous because permanent preparations of the wings can

be made. Therefore, verification and reconsideration are always possible on the basis

of the original material. The second advantage compared with the eye system is the

possibility to store the treated flies in 70% ethanol for mounting ofthe wings at a later

time. The third advantage of the wing system is that in every exposed imaginai disc,

Iiteraily thousands of target cells are potentially at risk.

2.3.1 Principle of SMART

Drosophila melanogaster as a dipteran insect develops through successive

developmental stages of different duration (92). Drosophila undergoes complete

metamorphosis: (duration 1 day at the optimal culture temperature of25"c ), l't larval

instar (Ll, 1 day), 2nd larval instar (L2, 1 day), 3'd larval instar (L3, 2days),

metamorphosis in pupal stage (prepupa 4 h, pupa 4.5 days) and adult stage (imago, up


22

to 40 days). During embryogenesis primarily larval tissues (cuticle, gut, fat body,

nervous system, etc.) are formed, and during the rarval period these tissues enlarge

and finally form body of a rarge L3 larva ready for pupation. The adult structures

(wings, legs, eyes, etc.) are formed in pupal stage from so-called imaginal discs. Such

discs grow during the larvar period by cell proliferation. The developmentar

parameters of this type of imaginal disc were described by Garcia-Bellido (93). The

cells of the imaginal wing disc derived from a sample of about 50 nuclei of the

primitivc cgg syncytium, which happen to miglate to a given region of egg conex.

After these nuclei have been surrounded by cytoplasm and membranes, the

corresponding cells become deveropmenta[y segregated from the neighboring

ectodermal cells. They do not divide during embryonic development but can already

be detected histologically, grouped in a discrete wing imaginal disc, in the newly

hatched larva. Proliferative growth starts in the first instar and continues throughout

the larval period. cell proliferation is logarithmic in all the presumptive adult

cuticular cells, although the number of cells per clone deviates from 2n. on average,

cells divide every 8.5 h and growth is complete after 9-10 ce divisions. After

pupation, mitoses are still detectable, but somatic crossing over initiated at this time

results in single marked ce s. Twenty-four hours later, mitosis ceases altogether and

visible cell differentiation begins. During differentiation about 50,000 cells give rise

to single identifiable cuticular processes organized in the typical adult pattem.

If during disc growth in the rarval stage a wing imaginal disc celr is genetically

altered into a mutant form, a group of mutant cells will resurt from clonal expansion


during disc growth. After pupation, in the course of metamorphosis of the imaginal

disc into an adult wing the mutant phenotype will be come expressed. The mutant

clone will be recognizable as a group of phenotypically altered wing blade structures

(called a "spot") , that can show multiple hairs instead of the single hair formed by

each wing cell (Figure 1).


Figure 1. Marker mutations of wing surface to show clone of cuticle secreted by cellshomozygous for multiple wing hairs, a) small single spots, b) flare on wing vlin, c)twin spots, d) large single spots.

b',/ './'/ - / J."ri --/ /=-H

'*-*-t.--ry,*?- ^-{*l;*.? -)< '

{t:,l/.)


25

2.3.2 Genetic Basis of the Effects Detected

The assay consists of exposing to a mutagen populations of cells that are

destined to multiply in relatively fixed configurations so that an induced mutation in

one of the exposed cells will give rise to a detectable clone. To ensure the clone is

identificable on the surface of the adult fly, one chooses genetic markers that are

expressed autonomously in wing cells. The exposed cells of the larval wing imaginal

discs are trans-heterozygous for two recessive markers located on the left arm of

chromosome 3. Ir4ultiple wing hairs (mwh) is at position 0.0 cM and fl:r:e (ftr) at 39.0

cM, while the centromere is located at 47 .7 cM (94, 95).In all the experimental series

analyzed, the occurrence of the various types of spots was as follows: most frequent

were single spots expressing the mwh phenotype, less frequent twin spots with both a

mwh and a/r sub-clone, and quite rare single spots with the/r phenotype.

There exist several mechanisms that lead to genetically marked clones (see

Figure 2). An important possibility is a mitotic recombination event between two non-

sister chromatids. Twin spots are expected if recombination occurs between /r and

the centromere (96). A recombination event betw een mwh arrrdflr may result in a mwh

single spot. If both types of recombination event (one between flr and the centromere

and a second between mwh arrd flr) take place within the same cell, a/r single spot

may result.

The appearance of a single spot can, theoretically, have a number of other

genetic causes as well: a gene mutation or gene conversion in the wild+ype allele, as

well as the deletion (small or large) of a chromosomal segment involving the wildtype


allele may result in a single spot. Phenocopies might mimic genetically determined

spots. Nondisjunctional or other loss of the chromosome carrying the wild type allele

represents another mechanism that may lead to single spots.

I Ca 2 Ci .

'.,,itwh

_ spot

Figure 2. Genetic schemes illustrating various ways of spot formation in the somaticmutation and recombination test with the wing cell markers multiple wing hairs(mwt) and flare (flr). Twin spots are obtained by recombination proximar to-the flrmarker (b), while more distal recombination produces mwh sinile spots only (d).Deficiencies (c), point mutations (e) and nondisjunction events (fl give rise to mwhsingle spots, or in analogous ways to flr single spots (not illustrated) (97)

/.\x; --r-1]

;.{

Delelron

-" ---{); ;-r

-

1-,;.r

't ! i' singlespot


-_4{Jl- -- 7, {

*o;; ,,,--{

--- -+-)- -- -- -{)--a-.

Figure 2. (continued) Genetic schemes illustrating various ways of spot formation inthe somatic mutation and recombination test with'the wing cell markers multiple winghairs (mwh) and flare (flr). Twin spots are obtained by recombination proximal to theflr marker (b), while more distal recombination produces mwh single spots only (d).Deficiencies (c), point mutations (e) and nondisjunction events (f) give rise to mwhsingle spots, or in analogous ways to flr single spots (not illustrated).

-//_i-----1\)

--J{J-

tJ :t ,'

:1" ,l 4'\ssiEe

I spol

27

1 Cel [1il o s LS 2 C::s Ens!inq clones

. _J-1,: -;,:-rRecombrnalicn

Po nl mulalron

/ /i,

ir "7 { ."'

side./ / spot

t'., -


28

2.3.3 Approach of SMART

All 3 crosses produce larvae which are heterozygous for the 2 marker genes

mwh and, flr3 on chromosome 3 and required for the wing spot test. The following 3

crosses of flies carrying markers on the left arm of third chromosome were:

l. Standard crossz yr3/ln13LR)TM3, ri f sep bx3a" e, Ser vergin

lemales mated lo mwh males. This is the reciprocal cross of the standard cross used

previously (91, 98, 99).

2. High bioactivation (HB) cross: ORR; ltr3 /T1tI3 fcmalcs crossed

with OrRR; mwh males This is the reciprocal cross of the one described by Frolich and

Wurgler (100).

3. Improved high bioactivation cross: ORR; flr3,[M3 females

crossed with zwft males (101).

The hybrid larvae of the improved high bioactivation (IHB) cross show

P450-dependent bioactivation capacity equal to or even slightly higher than those of

the original high bioactivation (HB) cross. This was demonstrated by measuring the

genotoxic activity of the promutagens diethylnitosamine, 7,12-dimethylbenz(a)

anthracene, N-nitrosopynolidine and urethane (l0l). In addition, the IHB cross is

more sensitive than standard cross (sr) to the genotoxic activity of pAHs and their

nitro derivatives (82).


29

STATEMENT OF THESIS

Plants growing in soil can potentially receive mutagens such as PAHs via

several processes: (i) uptake of PAHs in the soil solution through root tissues, (ii)

absorption of PAHs to the root surfaces, (iii) foliar uptake of PAHs that have

volatilized from the soil surface, (iv) absorption of PAHs from the atmosphere

through leaf surfaces. There was no information on the mutagenic expression of such

contaminants absorbed into any plants. It was also of interest to know whether plants

currently consumed in Thailand could possess this environmental hazard. Therefore,

five vegetables namely, sacred basil (Ocimum sanctum Linn.), lettuce (Ldctuca sativa

Linn.), water spinach (lpomoea oquatica Forsk.), green kuang futsoi (Brassica

chinensis Jusl.) and multiply onion (Allium cepa var. aggregatum Don.) were chosen

to grow in diesel exhaust added soil and use as a model to determine if the hexane

extract, which could extract PAHs, of edible portions were mutagenic in the SMART.

SPECIFIC OBJECTIVE

To investigate the mutagenicity of commonly consumed vegetables grown in

diesel exhaust contaminated soil using somatic mutation and recombination test.


CHAPTER III

MATERIALS AND METHODS

3.1 Chemicals and Diesel Exhaust

Mitomycin C was purchased from Fluka AG (Buchs, Switzerland). yeast-

glucose-agar Drosophila medium was prepared according to the method of Roberts

(92) (see in Appendix A). other chemicals were of laboratory grade. Diesel exhaust

was collected from healy duty diesel engine vehicles, namely 45 buses and 52 trucks

in Bangkok. The collected particulates were ground and mixed well by a Norton pair-

roller (Ohio, U.S.A.); then, subdivided into two portions. The first portion

(unprepared sample) was tested for its mutagenicity by mixing exhaust particulates

(0.01, 0.02 or 0.05 g) with 2 ml freshly prepared Drosophira culture medium in a

glass tube. The second portion (1, 2 or 4 g) was stirred with 300 ml hexane for 2 h in

order to extract nonpolar compounds especially polycyclic aromatic hydrocarbons and

their nitro-derivatives. The solution was filtered through glass wool to obtain clear

solution which was evaporated to dryness under reduced pressure (40oc). Each extract

was weighed and suspended in the prescribed volume of 95 o/o ethanol and

consequently diluted with distilled water to obttrin 4 ml of 5% ethanol suspension.

The suspension was prepared for two consecutive serial dilutions (original

concenftation and 2 diluted concentrations) and subjected to the SMART assay by

mixing the 2 ml of each dilution with Drosophila medium prepared with sugar (34.0Copyright by Mahidol University

3l

ohwlw), agar (4.8 Yo wlw), com meal (42.5 %wlw), yeast (17.0% w/w) and propionic

acid (1.7 o/o wlw).

3.2 Preparation of Soil, Vegetable Planting and Hexane Extraction

SandyJoam soil was sun dried for 5 days and then it was loosen. Three

different type of soil were prepared in four replicates. The control soil contained no

diesel exhaust and other two were mixed with two different amount of diesel emission

particulates in order to obtain I and l0 g particulate/kg soil. For each preparation 1 kg

(dry weight) of soil was thoroughly mixed with diesel exhaust and water; then, it was

air dried for I day. One kg of treated soil was filled into a clay pot (20 cm x 17 cm)

with cloth lining to prevent soil leaching.

Five vegetables namely, sacred basil (Ocimum sanctum Liwt.) (nytfl:t), lettuce

(Lactuca sativa Liwt.) (?inntn ou), water spinach (Ipomoea dquatica Forsk.) trinrlJ t,

green kuang futsoi (Brcssica chinensis Jusl.) (fl'nnrorfifl?nit{d{) and multiply onion

(Allium cepa var. aggregatum Don.) lelunoly were used in this study. Before sowing

the seeds of the first four vegetables and the bulb of the fifth one, the soil was added

with commercial chemical fertilizers (15-15-15). The number of each species grown

in each pot was as followed: one for sacred basil; two for green kuang futsoi, lettuce,

water spinach; and four for multiply onion. The plants were watered twice a day until

edible portions were collected. The samples were washed with tap water and dried at


32

40"c. The replicates of each vegetable were pooled and homogenized in a home-use

electrical blender and stored in a dessicator. Each sample was extracted with 300 ml

hexane for 2 h. Solid materials were removed by filtering through glass wool. The

filtrate was evaporated, weighed, suspended in 5 % ethanol and incorporated into

Drosophila medium for mutagenicity assay. The yield of plant extract is shown in

Table 5 (see in result).

3.3 Mutagenicity Assay

The mutagenesis assay was carried out according to the method of Graf et al.

(97). Virgin females of OXR; flr'/TM3, ,Ser mated to males of mwh/mwh were used to

produce larvae of improved high bioactivation cross (IIIB). The three-day old larvae

(72 h) were collected and washed with water and transferred (with the help of a fine

artist brush) to the glass tubes with medium containing the tested sample or control.

Milomycin C (0.625 mM) was used as a positive control. Larvae were maintained at

25 + loc for 48 h. After metamorphosis, the surviving hatched adults were collected

from the treatment tubes between days 10 and 12 after egg laying. The flies were

stored in 70% ethanol. Flies of the trans-heterozygo ls (mwh +l+ Jll) genotlpe were

collected and mounted on micrcscope slide (described below) and were examin:r for

the mutant spots.

3.4 Preparation and Microscopic Analysis of Wings

Flies were washed with distilled water and brought into a drop of Faure,s

solution (30 g gum arabic, 20 ml glycerol, 50 g chloral hydrate and 50 ml distilledCopyright by Mahidol University

33

water). The wings were first separated from the body and were then, with a fine paint

brush, lined up on a clean slide. They were allowed to spread out. The wings arranged

on the slide were kept in a petri dish for at least 24 h. A droplet of Faure's solution

was dropped on a cover slip, which was then, with the hanging drop, lowered on top

ofthe wings. A permanent preparation was obtained by sealing the cover slip with per

mount. The wings were aialyzed under a compound microscope at 400 x

magnification. The position of the spots was noted according to the sector of the wing

(see Figure 3). Different tlpes of spots were recorded separately, namely single spots

showing either the multiple wing hairs (mwh) or the flare (/r) phenotype, and twin

spots showing adjacent mwh and flr areas. The size of a spot was determined by

counting the number of wing cells (hairs) exhibiting the mutant phenotype. The spots

were counted as two spots if they were separated by three or more wild{ype cell

rows. Multiple wing hairs (mwh) were classified in which a wing cell contained three

or more hairs instead of one hair per cell as in wild-type (see Figure 4). Flare wing

hairs (//r) exhibited a quite variable expression, ranging from pointed, shortened and

thickened hairs to amorphic, sometimes balloon-like extrusions of melanolic

chitinous material.

40671


\

Figure 3. Normal half mesothorax showing the regions A-E of the wing surface scored

for spots (97).

Figure 4. Trichomes on the wing blade, a) normal, b) deviate trichomes not counted as

mwh or flr, c) configurations indicative of mwh, d) typical manifestations offlr (97).

., ,, or, r,'7, a tu i(/b)

ltt}U lbuI

^4,PJl udc)t

\U,,l/ Jd'

//0._ a/l .r

/^.1 o I(,1;,/^ \,/[ "'l/././


35

Data Evaluation and Statistical Analysis

The wing spot data were evaluated by the statistical procedure described by

Frei and Wurgler (102). The data were grouped into three different categories: small

single spots (one or two cells in size), large single spots (three or more cells) and twin

spots. The estimation of spot frequencies and confidence limits of the estimated

mutation frequency was performed with significance levels o = p : 0.05. Statistical

consideration and calculations step by step is shown in Appendix B.


CHAPTERIV

RESULTS

The aim of the present study was to determine the mutagenic activity of

extracts of the edible portions of plants grown in diesel exhaust contaminated soils.

The results obtained \,\rith the extracts are shown with their concurrent controls (Tables

6-12). All experiments were chronic feeding studies (48 h) starting with 3-day-old

larvae and ending with pupation when the larvae left the medium and stopped feeding.

Schuetzle (7) and Bartle et al. (8) revealed that diesel exhaust particulates contained

hundreds to thousands of chemical components and the majority of the chemical

species of biological significance identified were PAHs or PAHs-derivatives. It is well

known that the PAHs require metabolic activation to exert their genotoxicity;

therefore, a cross of flies with improved high bioactivation capacity (an increased

presence and activity of cltochrome P450) was used as suggested by Frolich and

Wurgler (100) and Graf and van Schaik (101). The spontaneous clone induction

frequency expressed as total mutant spots per wing of 0.22-0.29 in the present

experiment are within the usual range of Alonso-Moraga and Graf (9s) and Graf et al.

(86). The majority were small single spots.


37

The Yield of Plant Extract for the Mutagenicity Test

Growing each plant in different contaminated soils resulted that there was no

difference on the percentage of solid matter obtained after evaporation of hexane

(Table 5). sacred basil gave the highest percent yield while green kuang futsoi gave

the lowest one based on dry sample.

Mutagenicity of Crude Diesel Exhaust

Crude diesel exhaust gave rise to the clone induction frequencies of spots

mostly as small single spots rather than large spots (Table 6). The positive results

obtained in this experiment showed dose-response relationships.

Mutagenicity of Hexane Extract of Diesel Exhaust

An attempt was made to verifu if the toxic substances in the diesel exhaust

such as PAHs and their nitro derivatives could express their mutagenicity in the

hexane extract fraction since hexane was used as the extracting solvent for the plant

sample. It was shown that hexane extraction of diesel exhaust samples increased the

frequency of wing hair aberration of treated larvae (Table 7). Better dose-response

relatior-^sirips in this trial were present by both small and large single spots compared

to the results of the crude exhaust. It was, thus, assumed that hexane was a suitable

solvent for sample extraction ofwhich the results are reported hereafter.


38

Table 5. The yield ofextract ofdifferent plants grown in diesel exhaust contaminatedsoils for mutagenicity test

Common nameofplant

Amount ofdiesel

exhaust

Wetweight

(e)

Dryweight

(e)

Drymatter

(g)

% Yield of theextract based

on dry weightsoil

Sacred basil

Green kuangfutsoi

Multiply onion

Lettuce

Water spinach

7.5

7.4

01

10

01

10

0i

10

0

1

10

01

10

130.81

99.23114.85205.14230.17

189.24180.87155.1 5

129.22250.93

184.552t6.20207.32

181.83204.45

10.54

8.868.898.148.247.67

9.40a o/:

8.848.01

6.91

6.99t2.20t2.52t2.39

2.041.94

2.030.240.23

0.1 8

0.900.85

0.870.s00.400.4i0.880.940.92

19.4

2r.9))92.92.82.3

9.6

9.5

9.86.25.8

s.97.2


39

0)

d(J

(l

o

C)

,.i.:

au0)

(l)

.od-o<

o'n.hd

otr9ooll :oo-Z-bo/tr9

E.o9!,

ll *+*

l^looloot6\ti.t:-toluD

5-dc.:otJioh0

oo

E

0)oo

o

ooo

o,r

o

ooo

o

ooo

dE

(.)

b0

ooo0

(no

a.r

-r.)r-+l'<faarnoJH

+oo

N

.+

oo

*o

E:

o

.1

c.t

:f,oo

o9

IoI

o00)z

+oooqO

\o

c.l

+tt'

c-n

I.-'

+

o\

bo

oo.

F

(.)aN

(,oN

.5*oX30H6er

(-)

oa

BF

o.(.)

b0

ooo(g.l

oo.ooo

dEa

b0

t-l

o Ei)

zv

_Ea.=ard0)(DEtr(L]t r\!=', -s6$3q< Pi- ibDoLl ts'a -2s.=:d,o(g

:q

a

o0

o

.ooE

B

Io

d

o

ph0

!I

aE

o0)al

d!

Nr-i.\6-=fOOktr_o

=e,o5-dL<OJXfi0)

d)

i.'llt8-o50)i.r. E

9:

9U=ou3\oc

{D

-oCi.dEF.d


40

Q

ax

boE

(\

I

*t+

I+t-tÎ6l€lo,l=t*t0)lhDl!t,-t,:IEl.-

lLr.toI bI)lltEdli.,lu slo -l(g (E

la.l2

15sl(J .,')

lE-ql'o.:lll 6)

t-dlC tlldo1o,;lor vl'6lE6I o-, -l= ll

lS o

l: o)t.Y >t! >,

lo,€l5 €

c, ,,li

vo)

.=a

coE3trE'is9E&€.99a'i=au)d*ll

+coO

N

:

:lEIol

:lolo. I

'-l-l-O\\O rn*a!li"1 ..j nq"l:

C)t<(\ FrO..-

C'l al :j' C.l + f'- ..

oO O, t_- \O (n O\ oO*.a <f N C.t v) C{

+-N .+ cs,n N\ot-v \o..$=

(6

9a)oo ,o.t I o. og r,o\6s!8.d,-g:

dJEIJ (Jooo

x bI)

EZ

' '+n c! vi

OHN

\O I'- *

OFr+F- \O \O

=4.):-con P

T.i+odt(l)(L)v) chq) 0-)

a0 bo(\

ooA !.r

coo.: - -i-r c ;J;tuO.= I O.

; oJ-

r./ F

a

BF

oo.ô='to !iC-

o.l ,\EOv(B

'.J

a.o1 G'o-bD !2

6 c,r

(!-

a

bI)

(!

xoa)

(!xo

sQoo

c-)

o0

<d

o

0)

(.)

(!

o(6osb0

I

o.E

o0)(d

(d3co

=$Or--o

N9'dqEOX

Ob0_troE 0.)

6'o!O(E()

oo

UX,! :a

.= r<

ontsCJrr Ovor-Cor-

F -al

eoD

zB

bI)

)oE

s-oo


4l

Mutagenicity of Hexane Extracts of plants

Grown in Contaminated Soils

Tables 8 - 12 show the data obtained from the test of five vegetables grown in

three different soil treatments (control soil, soil treated with 1 g of diesel exhausL&g

soil and soil treated with 10 g of diesel exhaust/kg soil). It was found that clone

induction frequency of the wing hair of flies treated with hexane extract of leaves of

sacred basil and green kuang futsoi grown in all three different treated soils was not

statistically different from that of control (Tables 8 and 9).

The extract of multiply onion was the only sample that induced mutation in

the wing spot test both in the treated and untreated soils (Table 10). The resutts

obtained from the extract of multiply onion grown in diesel exhaust treated soils

showed an apparent dose-response relationship in this assay.

Larvae treated by the hexane extract of lettuce grown in untreated soil showed

negative result in the wing assay (Table 11). The two higher doses of sample grown in

10 g of diesel exhaust/kg soil significantly increased in the frequency of small single

spots.

The hexane extract of water spinach grown in control soil did not affect on the

clone induction frequency of the tester organism. However, the highest concentration

of sample extract ofplant grown in diesel exhaust contaminated soils (l and r0 g/kg

soil) markedly induced the frequency of small single spots to become statistically

different from that ofcontrol sample (Table 12).


42

9oooo*rz o0cboor -9= (.)

trdx

(-r(!:trx6iE 8.9-89N .E ':

v;o_::ooo'6.oo(JOto(!in

^-E:i OU

l-llcalo,t-t-t-loJlhol5t>t:l.dt.-lorItr.t-l!loo14It.hl= 0)

lO cir1() oIH.Ela tItr i,l=.ols #Itr u)

t€dItr LJloIo

IdolE,,t ;'i o

lq llo .-19.!e (,)

EB'E-ets)tr-o.F C!oPco

!s)c tt,

0)ooll

EioJ6?: 6Do c.,

b0,,(! ll

€,"3.:bo-v) ll*+

+C\l\

an

oo

cJlzt

:tctolUI

OIa-I

ac!o

O

N

N

oo

o(tl

o6\

g

ooo

(goo(l)

z

c--.1O

N

\o

c.l

o

O

F-

N

ct

a.-qO

N

++

a-C.l

o.l c\ .a

.^ - a'n

oat-

'-: Ci V1 -c\ rt qo

-N*:= o

ru(tt

(')

c'! qY

ooooan

^coa\

oo \O \O

ao|.)oc!r.)--*Ntr=oa.60)

al;

otr

QO

cb0

ForO

H d-

\J l-

op.

F

o^5o !lc-'6 c'lonb0v(n

,.1

EoaGib0Y'6 cJ

c!-Ea

aoU,

cdcd ,o=d)o(!otrJ -::

E5tre

90)53o!

()E

.: o'otro0tr-cldooI --:

dc

(]g

.i S

c'tvF-+

9P 'o.: .o)

o(!

o(tld

g6iood:. (!0)X

lqi

e5rJc

oo l<,9co

F'o d

t!o(,bo

zt

;' bo

XF

or.I(! o-)Xtrr- rie-s

tiA

o .=

rJ ,::p.


41

o(B

0)

o(J

,r;

lt

d 9oooa d)<6 )l oot tDoh ol9

.= .":r (J

EEHEEv e.o

>trxei3.!; ToAl2..,o ooE "o tr,!'X = E.E.-ddroieE6.2s5"bI)o==(Drrir^c-.6dll o.d E, ogll-CrG!dq>cE

.4.= c e'=;{d.d6d; ts tsA.* 6 6II ;. C)U

t1laloolqtvlLlolEoIEt:15t-dlctdt.-lorIEleloa

t.d

l3l3laIEl=l0)lol(L]rl-dq=

oo.dtrdoo(.)

o),.!

o

o

a

o

E

ob0

(l)

ou0(d

=o

=a*

'Lo t'-. o oo.rr oooo!f ^,- - a.l - -

..J \': ;_.ooo ddd oooci

OOO OOO OOOOi

3-- co \o ce OrOco!5

<f o|.O -"-r-- oO o\\ '.t .-- oot.) N si- +.aN ca.agro-

E,q*

t/)OOr<.oor--E-..',-o\o=-N,^=-^*f!ooatn'o.dtrcQ dJ EEcl .iE .H :3tr c >oA k 5.=5 E E'EO U Zd

cbo

3();.ii e ij=oro9 B:: .r. (i:-:oL,F

oa.

F

oo.ô=50 !l

oAaov(!:.]

bI)

ooo

z

oO

OEU}(tJEo-=AEorE

e'sc\

EAococ

o.

aG.o=50 92

.a c,.t

=r-U)

9p;;d(nxEoootr0)

ooo

AC,o.:otr

tsb0)-65o-

ooS

Br,,

Q.qE3qaQ

ot

>Z(g5

il on

c.lF-d_(DEgo0

b0_

Eoti_o (,

0)(ttcdx- (.)

q o.)EEod=x('o)rD _g

^6.; E+tri:r,/)o(Ll;Eo-',o ct: .-ooLJ oEo\tr=.9E"= ,lD ;:

.rq .:-: Ol-< 'r, o


44

l;{loold\t-l,.ldrto0

t4l'ulclcdt.-talfr.totool= t^lY o)tb -lY o

Itr dt-_l;0.)lE'ol!,)';I'El r

60oXnthOID,,(, --Eqr

=

f,u e S& U oo*cT ;v bI)

-o i doo5 >. n, o'o.: = o

EE€ F E.s 9u;6e oi xt EE.: E X i''E :i!- tiftl =

.r {u r,/)

E 9 E 3.9E 6 oo5 cU 3 E'oEep': - E .g

="?,^c;!rYNQo

E b00==obg ! o o6!icLaa; I O -d.rf-.()oc,(,^.)adi6oo>cc'; .r .:'E EH?ôSS.D A' = =* rr i Od

.l+: irl ;fiXrclq\o .ln\o .1 ,.1iOCO CCO c=O-'i

Oc..l+ N.a.a C)*cai-

\oo\@

Nc{C\a.l-

+OO\v-)N:c\.l:-:

\o(.)l.)o\oo+$.a\O-

E*o l,

=*Pr:)E;; -r- a.l v >9 =---' E i')d i=S: o!?tr >o(B e.ir

Oo-rxu zd

*XX

a-oo*

.n\*,.,yF.^ aa rat;=N=oa.dg

4.,5l!ooOLI

cbo

39-:b6: o-

F

F

oaô=F3o.)Abov(tl

E]

6aa?;d.)-50E'4.,.r

EU)

!oL' hD

z>

o0E

E

0)

a.o

r-l

oo

xo)c.)

GIxo

o

ooo(,

9;

€xEOJo-oo

-ct>oa.=o

tro.: bo

>.jux-oS.10 do<93ffIE>tss>!

"r!r\voEb0=

EEq9 .*

o

'E(!

o0,Eco.:\is6=,^-.: - oo

cE-E-o;'=E6;

-3E. ois

e=.=

o,9E5e:.(Bts 5F'O o


45

o

=

(B

@

0)

,.i

ca-ll :'6

3 oo{-o _V oO

o ou iJ

(lCrr.d€{ x ad=--2I !-,) E.d'aEX6i=.Y- o)

ctr9!o)9 99! ".: E.d Hrr -9E.- r_r'= (!l

e3H8

ll o t.6iOEE-o) (E (Eo>cc>'E'='=

'= ;{ d d5dEEil I --

+

ooooo,a*

0-,

so

RI

q)

f!

60

Eooo

E

ooo.6

o

(tt

oo0)

(l)

ooo

CB

o50

o)

obI)

!do

da'l+

.-+ rr-<.+ -oo s^,.-r i.rii .rYi ifi3l.rBOOO OOO OCO"-j

N i c..l c.l C'l

:<Fa.roo\orNNi*

oo € ra) ra) @.?) <f <f t-. H

o t.H,.o*1P)5=..t,.):VS:; g='O2tig xEq 60!_o'A 9!,9(EP

ooû zd

*OO OO+

I9o. ,-N=

\o \o oo

Hra)o01-ol(nO\o.dod

Q

;ET

.. b0

'E* >o i<H;E: orO

x d)-

-oIJF

oq

BF

boX.;i ...t

o, .n60 \J

,l

!o.thG(.) =Eo 9?

'aq

Ea

obI)

bo

os

i(B

'a 6

Oo9=t

.YX!0J

'i (ho(l)ovsob0i

a!o9-\)_o->

oz)o:

(\:r-- b(kBorlbo -..: (,)

()E.oo

=o()Xl0)9 o.r

(tJJ,.ii 6i

Ec*E25!

otsi?ovo

.o

!).= -O-c! tsFd

'rarr bo

z,

EbI)E

0)

sor-a

(t!

&

o(B

xq)

o

Xo

o

(!

oo

U


46

+;{oo€

oo0:

E(!oIl.oo0

ts-

a.9

OJ;

E(Jotroo.6ll/)

orrE-oJ-

=Ss 9 oo& ;; oo{- = -:z bI):o\^v > o0 (,U.: - o_r€h gze€-E E gt:5 /: l+e ^\, - --7,-^'a';tE 6 d Fu d >r -< ?c>txai.9 '6 5 '.^-E:'E fr B

'E ^

6D; e6 n d -to E c! 6.E I cl .= 6=^:,nqE3.- I3.I E'-E,

c,r-,iCoo:= I o.d.d-.q(Dd)d.(Dadanoo)>ccr,i.lt-l-'EaEaaa a-.. A E* rr i dri

, | + , .- + * +O\tv1 ca * +,i, ca..! c! * 6lah_rF--^------.ie--l

*Oi OrN.iN

,^ ^ \O - + O\ O\ a!

+oooo ao+*aloo.a ca ca <t+<tO\*

o

3*or 1t

'' ('']a€rE*trlc).,o\5;i=R:-=Reg

a 9=EYY ;.=.Y Atr:: ()9.= dr "tr > -i,)((l e.i-rtr oo'=O orx\J ZO"

s: F;U)

ID

(JL)

o \o rara.l r (.1

F-a{N

O, tal oO

aU)

'E* s9 a<ioba 1-- !)=: .-o(-, F

o.

F

o=Eo !lc-.;l .\

oA00 \-,

,.1

oo-a;o-on !.1

.a c,r

Ea

hI)cn

IJ

bo 'Eco)

6Z.,oa=6a=

X-E qJ

-.9o'ooosJ

>o,n o0-o

.9 Fo-cE-vÔa.=Et::rJ st

>da9Er*.!9

O0!c0)d'

,,o '_orf,()

>,x()(Dcc996oo

o5

N-_-

o-,9d,'v .- o

uoo

zt

:;o*.-atro-:(EOxeo, r.i-trxe-s

:A!?tr

o.


CHAPTER V

DISCUSSION

The Yield of Plant Extract for the Mutagenicity Test

The tested concentrations of diesel exhaust particulate incorporated into soils

did not appear to have any effect on the yield of solid matter in the hexane extract of

all vegetables. However, species difference seemed to be the determinant of such

yield.

Mutagenicity of Diesel Exhaust

The results of this study indicated that diesel exhaust and its hexane extract

were cleary positive in wing spot mutation of the Drosophila wing somatic mutation

and recombination test. Normally, the positive genotoxic effects observed are

restricted to an increase in the frequency of small single spots. The small single spots

are indicative of clones of mutated cells which either did not divide at all or divided

only once. However, it was found that positive result was obtained fiom the hexane

extract at the concentration of 7.6 mg/ml while crude diesel exhaust showed positive

result at the concentration of 5 mg/ml. It was proposed that the hexane extraction did

not extract all mutagens of diesel exhaust. While the non-polar compounds ofhexane

extract of diesel exhaust might contain only promutagens, the crude diesel exhaustCopyright by Mahidol University

48

may contain some direct-acting mutagens in addition to indirectacting ones thus

produced greater. mutagenicity than that of the hexane extract. The results ol Tabte 7

also confirm that hexane extraction provided a good precision in isolation of mutagen

in this experiment.

Diesel exhaust was shown to be mutagenic in short-term bioassays and

carcinogenic in laboratory animals (9). These particulates contained hundreds to

thousands of chemical components (7, 8). Nearly all of the chemical species of

biological significance identified to date in environmental samples are polycyclic

aromatic hydrocarbons (PAHs) and PAHs-derivatives. Numerous aromatic and nitro

aromatic compounds were shown to be mutagenic in Salmonella typhimurium as well

as carcinogenic in experimental animals (103, 104, 105, 106, i07, 108, 109, 110, 11i,

t12,113, 1t4).

Mutagenicity of Hexane Extracts of Plants Grown in Diesel Exhaust

Contaminated Soils

The mutagenicity of five vegetables grown in three different soil treatments

(control soil, soil treated with 1 g of diesel exhausL&g soil and soil treated with 10 g

ofdiesel exhaust/kg soil) was different. The hexane extracts of sacred basil and green

kuang futsoi grown in all three different (treated and unheated) soils were unable to

induce somatic mutation and recombination in Drosophila melanogaster. Possible

factors in suppressing their genotoxic will be discussed later.

The extract of multiply onion was the only sample that induced mutation in the

wing spot test both in the treated and untreated soils. The mutagenicity of the extractCopyright by Mahidol University

49

of the sample grown in untreated soil may due to some naturally occurring

compounds. The instinctive mutagenicity of shallot (Allium ascalonicum), a very

close variety in taxonomy to multiply onion (Atlium cepa var. aggregarum Don.), was

shown to be positive in Salmonella typhimuriumlmammalian microsome assay (l I5).

Their preliminary study in purification of mutagenic extracts of shallot resulted in the

isolation and identification of natural mutagens namely, quercetin and unknown

glycosides (unpublished observation). Quercetin was also shown to be mutagenic in

SMART by Graf et al. (116).

The results obtained from the extract of multiply onion grown in diesel

exhaust treated soils showed an apparent dose-response relationship in this assay.

Conclusive results were also obtained when larvae brought up with the medium

containing the hexane extracts of lettuce and of water spinach grown in the

contaminated soils. It was suggested that the extracts of vegetables grown in diesel

exhaust contaminated soil significantly increased the flequency of small single spots

compared to those of the samples grown in control soil. It was, thus, postulated that

some PAHs in the exhaust played an important role in increasing the mutagenicity.

Various investigators reported the contamination of plants by PAHs. Shabad et a/.

(16) stated that the PAHs penetrated in to the soil mainly from air spread across the

layers into the water and passed into plants, fodder, and finally into human food.

Shcherbak (3, 4) also stated that pollution ofplants might occur from sedimentation of

atmospheric dust and soot or by migration of the carcinogens into the plants from

polluted soils. Shabad and Cohan (5) concluded that the main source of contamination


of soil was from air-bome particulates. They indicated that migration or resorption of

PAHs into plants was dependent on the PAHs level in the soil and the tlpe of plant.

The present experiment showed that some plants grown in diesel exhaust

contaminated soil provoked a mutagenic response while some showed negative

results. It is likely that the mutagenicity of each plant extract may be reflected by the

indigenous difference of plants. Furthermore, most of the extracts contained some

green pigments probably chlorophyll (117). The weak mutagenicity or absence of

mutagenicity of some samples may due to the antimutagenicity of chlorophyll against

diesel exhaust mutagens absorbed by such plants. Since chlorophyll was demonstrated

to suppress mutagenicity of many toxicants (118, 119, 120, 121) and its content was

different in various vegetables (122).

In addition, plants are generally able to metabolize organic chemicals that are

foreigrr to them (xenobiotics). Cell cultures were of special value to demonstrate that

plants could metabolize a great number of xenobiotics (123) ranging from highly

polar xenobiotics to highly nonpolar chemicals. Harms and Langebartels (124) found

that the percentage of metabolites of seven xenobiotics, including benzo(a)pyrene,

was markedly higher (35 + 9 Yo) in soybean than in wheat (5 ! I %). It is concluded

that difference of mutagenic potential of different type of plants grown in diesel

exhaust contaminated soil may depend on the content of chlorophyll and xenobiotic

metabolizing ability of each plant.

The other possibility to explain the negative results found in some samples

was the accumulation of toxic compound in inedible portion of plant. For


instance, sacred basil might absorb some mutagens to accumulate in the stem but the

part that was used to test for mutagenicity was the leaves.


REFERENCES

1. Nitaya P. Levels and risk assessment of potentiallu toxic polycyclic aromatic

Nutritionl. Bangkok:hydrocarbons in some Thai foods [M.S. Thesis in

Faculty of Graduate Studies, Mahidol University, 19g5.

Muller H. Aufnahme von 3,4-Benzpyren durch Nahrungspflanzen aus kunstlich

angereicherten Substraten. Z PfTanzetemafu Bodenkd 197 6; 7 6: 6g5-95.

3. Shcherbak NP. Effect of oil products refinery ejections on soil and plant

contamination. Gig Sanit 1968; 7 : 93-6.

Shcherbak NP. Fate of benzo(a)pyrene in soil. Vopr Onkol 1969; 15: 7 5-9 .

Shabad LM, Cohan YL. The contents of benzo(a)pyrene in some crops. Arch

Geschwulstforsch 1972; 40: 237 -43.

6. Pitts k, van Cauwenberge KA, Grosjean D, Schmid JT, Fitz WL. Atmospheric

reactions ofpolycyclic aromatic hydrocarbons: facile formation of mutagenic

nitro derivatices. Science l97B:202: 515-9.

7. Schuetzle D. Air pollutants. In: waller G, Dermer o, editors. Biochemical

applications of mass spectrometry. New york: John Wiley and Sons, 19g0:

969- 1005.

8 Bartle KD, Lee ML, wise SA. Modem analytical methods for environmental

polycyclic aromatic compounds. Chem.Soc Rev l9g1; 10: 113-15g.

9. Holmberg B, Ahlborg U. Consensus report: mutagenicity and carcinogenicity of car

exhausts and coal combustion emissions. Environ Health perspect l9g3;47:

1-30. Copyright by Mahidol University

53

10. Guddal E. Isolation of pAH from the roots of Chrysanthemum vurgare. Bemh Acta

Chem Scand 1969; 13: 834-5.

1 1. Tilgner DJ. Food in a carcinogenic environment. Food Manuf 1970; g7: 47 -50.

12. Grimmer G, Hildebrandt A. Der Gehalt polycyklischer Kohlenwassenstoffe in

verschiedenen Gemusesorten und Saraten. Dtsch Lebensm Rundsch 1965;

61:237-9.

13. Grimmer G, Hildebrandt A. Der Gehalt polycyklischer Kohlenwassenstoffe in

Brotgetreide versliiedener Siandorte. Z Krebslursoli 1965 ; 67 : 2i2-i .

14. Grimmer G. cancerogene Kohlenwasserstoffe in der Umgebr.urg des Menschen.

Dtsch Apoth Ztg 1968; 108: 529-35.

15. Hallstrom I, Blanck A, Atuma S. Genetic variation in cytochrome p-450 and

xenobiotic metabolism in Drosophila melanogaster. Biochem pharmacol

1984;33: 13-20.

16. Bolling H. carcinogenic substances in cereals dried with combustion gas. Tec

Molitoria 1964; 15: 137 _9.

17. IARC. overall evaluations of carcinogenicity: an updating of IARC monographs

volumes 1 to 42. Lyon, 1987 .

18. Yamaki N, Kohno T, Ishiwata S, Matsushita H, yoshihara K, Iida y, Mizoguchi T,

Okuzawa S, Sakamoto K, Kachi H, Goto S, Sakamoto T, Daishima S. The

state of the art on the chemical characterization of diesel paticulates in

Japan. In: Ishinishi N, Koizumi A, McClellan RO, Stober W editors.


54

Carcinogenic and mutagenic effects of diesel engine exhaust. Amsterdam:

Elsevier, 1986: 17-40.

19. Zaebst DD, Blade LM, Monis JA, schuetzre D, Bulter J. Elemental carbon as a

surrogate index of diesel exhaust exposure. In: proceedings of the

American Industrial Hygiene conference; 19gg May l5-20; San Francisco.

San Francisco: National Institute for occupationar safety and Health, l9gg.

20. Johnson J. Automotive emissions. In: watson Ay, Bates RR, Kennedy D, editors.

Air pollution, thc automob e, and public health. washingto DC: National

Academy Press, 1988.

21. National Research council. Feasibility of assessment of health risks from vapor-

phase organic chemicals in gasoline and diesel exhaust. Washington DC:

National Academy of Sciences, 19g3.

22. Schuetzle D, Frazier JA. Factors influencing the emission of vapor and particulate

phase components from diesel engines. In: Ishinishi N, Koizumi A,

McClellan RO, Stober W, editors. Carcinogenic and mutagenic effects of

diesel engine exhaust. Amsterdam: Elsevier, 19g6:41_63.

23. zweidinger RB. Emission factors from diesel and gasoline powered vehicles:

conelation with the Ames test. In: Lewtas J, editor. Toxicological effects of

emissions from diesel engines. Amsterdam: Elsevier, l9g2: g3_96.

24. Williams RL, Swarin SJ. Benzo(a)pyrene emissions from gasoline and diesel

automobiles. Wanendale (PA): Society of Automotive Engineers, 1979.


55

25. Lang JM, Snow L, Carlson R, Black F, Zweidinger R, Tejada S. Characterization of

particulate emissions from in-use gasoline-fuelled motor vehicles.

Warrendale (PA): Society of Automotive Engineers, 19g1.

26' National Research council. Diesel technology impacts of diesel-powered light-duty

vehicles. Washington DC: National Academy of Sciences, 19g2.

27 . Duleep KG, Dulla RG. Survey and analysis of automotive particulate sampling. In:

Pepelko WE, Danner RM, Clarke NA, editors. Health effects of diesel

cngine emissions. Proceedings ofan Internaiional Symposium; 1979 Dec 3_

5. Springfield (VA): US Environmental protection Agency, 1990: 93_112.

28. Ball DJ. Particulate carbon emissions and diesel vehicles. In: proceeding of the

Institution of Mechanical Engineers: Vehicle Emissions and their Impact in

European Air Quality; 1987 March; Surrey. Surrey: Automobile Division

ofthe Institution of Mechanical Engineers, 1997:93_7.

29. Pierson wR, Brachaczek ww. particulate matter associated with vehicles on the

road. Aerosol Sci Technol l9B3;2: l-40.

30. Hampton, CV, Pierson WR, Schuetzle D, Harvey TM. Hydrocarbon gases emitted

from vehicles on the road: deterimation of emission rates from diesel and

spark-rgrution vehicles. Environ Sci Technol 19g3; 17:699-70g.

31. Hare cr, Baines TM. characterization of particurate and gaseous emissions from

two diesel automobiles as functions pf fuel and driving cycle. Warrendale

(PA): Society of automotive engineers, 1979.


56

32. Bjorseth A, editor. Handbook of Polycyclic Aromatic Hydrocarbons. New york:

Marcel Dekker, 1983.

33. Tong HY, Karasek FW. Quantitation of polycyclic aromatic hydrocarbons in diesel

exhaust particulate matter by high-performance liquid chromatography

fractionation and high-resolution gas chromatography. Anal Chem 1984;

56:2129-34.

34. Schuetzle D, Lee FSC, Prater TJ, Tejada, SB. The identification of polynuclear

aromatic hydrocarbon (PAH) derivatives in muiagcnic fiactions of diesel

particulate extacts. Int J Environ Anal Chem 19811'9: 93-144.

35. Schuetzle D, Jensen TE. Analysis of nitrated polycyclic aromatic hydrocarbons

(nitro-PAH) by mass spectrometry. In: White C, editor. Nitrated polycyclic

aromatic hydrocarbons. Heidelberg: A. Huthig Verlag,1985: 12l-67 .

36. Manabe Y, Kinouchi T, Ohnishi Y. Identification and quantification of highly

mutagenic nitrohydroxypyrenes in diesel-exhaust particles. Mutat Res

1985; 158: 3-18.

37. Paputa-Peck MC, Marano RS, Schuetzle D, Riley TL, Hampton CV, prater TJ,

Skewes LM, Jensen TE, Ruehle PH, Bosch LC, Duncan W?.

Determination of nitrated polynuclear aromatic hydrocarbons in particulate

extracts by capillary column gas chromatography with nitrogen selcctive

detection. Anal Chem 1983; 55: 1946-54.

38. Schuetzle D. sampling of vehicle emissions for chemical analysis and biological

testing. Environ Health Perspect 1983;47: 65-80.


57

39. Nesnow S, Triplett LL,, slaga TJ. comparative tumor-initiating activity of complex

mixtures from environmental particulate emissions on SENCAR mouse

skin. J Natl Cancer Inst 1982;68:829-34.

40. Heinrich U, Muhle H, Takenaka S, Emst E, Fuhst R. Chronic effects on the

respiratory tract of mice after long-term inhalation of high concentrations of

filtered and unfiltered diesel engine emissions. J Appl Toxicol 19g6; 6: 393-

39s.

41. Iwai K, Udagawa T, Yamagishi IvI, Yamada FI. Long-term inhalation studies of

diesel exhaust on F344 SPF rats. In: Ishinish N, Koizumi A, editors.Incidence

of lung cancer and lymphoma. Carcinogenic and Mutagenic Effects of Diesel

Engine Exhaust. Amsterdam: Elsevier, 1986: 349-60.

42. Kawabata Y, Iwai K, Udagawa T, Tukagoshi K, Higuchi K. Effects of diesel soot on

unscheduled DNA synthesis of tracheal epithelium and lung tomor formation.

In: Ishinishi N, Koizumi A, McClellan RO, Stober W, editors. Carcinogenic

and mutagenic effects of diesel engine exhaust. Amsterdam: Elsevier, 19g6:

213-22.

43. Meiss R, Robeneck H, Schubert M, Themann H, Heinrich U. Ultrastructual

alterations in the livers of golden hamsters following experimental chronic

inhalation of diluted diesel exhaust emission. Int Arch Occup Environ Health,

l98l:.48: 147-57.

44. Wiester MJ, Iltis R, Moore W. Altered function and histology in guinea pigs after

inhalation ofdiesel exhaust. Environ Res, 1980;22: 285-97 .


58

45. Pepelko, wE. Effects of28 days exposure to diesel engine emissions in rats. Environ

Res, 1982; 27: 16-23.

46. Wright ES. Effects of short-term exposure to diesel exhaust on lung cell proliferation

and phospholipid metabolism. Exp Lung Res, 1986; 10: 39-55.

47. Pereira MA, Connor TH, Meyne J, Legator MS. Metaphase analysis, micronuclei

assay, and urinary mutagenicity assay of mice exposed to diesel emissions.

Environ Int 1981; 5: 435-8.

48. Quinto.l, De l"{aiinis E. Spcrm abnonnaiities in mice exposed to diesel particulate.

Mutat Res, 1984; 130: 242-9.

49. Dukovich M, Yasbin RE, Lestz SS, Risby TH, Zweidinger RB. The mutagenic and

SoS-inducing potential of the soluble organic fraction collected from diesel

particulate emissions. Environ Mutagenesis 1981 ; 3: 253-64.

50. Huisingh JL, Bradow R, Jungers R, Claxton L, Zweidinger R, Tejada S, Bumgamer

J, Duffield F, Waters M, Simmon VF, Hare C, Rodriguez C, Snow L.

Application of bioassay to the characterization of diesel particle emissions.

In: Waters MD, Nesnow S, Huisingh JL, Sandhu SS, Claxton L, editors.

Application of short-term bioassays in the fractionation and analysis of

complex environmental mixtures. New York: Plenum, 1978: 381-418.

51. Clark CR, Vigil CL. Influence of rat lung and liver homogenates on the mutagenicity

ofdiesel exhaust particulate extracts. Toxicol Appl Pharmacol 1980; 56: 110-

5.


59

52. Clark CR, Royer RE, Brooks AL, McClellan RO, Marshall WF, Naman TM,

Seizinger DE. Mutagenicity of diesel exhaust particle extracts: influence of

car type. Fundam Appl Toxicol 1981; 1:260-5.

53. Claxton LD. Mutagenic and carcinogenic potency of diesel and related

environmental emissions: Salmonella bioassay. Environ Int 1981; 5: 389-91.

54. Claxton LC, Kohan M. Bacterial mutagenesis and the evaluation of mobile-source

emissions. In: Waters MD, Sandhu SS, Lewtas Huisingh J, Claxton L,

Nesnolv S, editors. Bioassays in the analysis of complex environmental

mixtwes. New York: Plenum, 1981:299-317.

55. Belisario MA, Buonocore V, De Marinis E, De Lorenzo F. Biological availabiity of

mutagenic compounds adsorbed onto diesel particulate. Mutat Res 1984;

135: 1-9.

56. Liber HL, Andon BM, Hites RA, Thilly WG. Diesel soot: mutation measurements in

bacterial and human cells. Environ Int 1981; 5: 281-4.

57. Lewtas J. Evaluation of the mutagenicity and carcinogenicity of motor vehicle

emissions in short-term bioassays. Environ Health Perspect 1983;' 47: 141-

52.

58. Lewtas J, Williams K. A retrospective view of the value of short-term genetic

bioassays in predicting the chronic effects of diesel soot. In: Ishinishi N,

Koizumi A, McClellan RO, Stober W, editors. Carcinogenicity and

mutagenic effects of diesel engine exhaust. Amsterdam: Elsevier, 1986:

t19-40.


60

59. Schuetzle D, Lewtas J. Bioassay-directed chemical analysis in environmental

research. Anal Chem 1986; 58: 10604-1075A.

60. Lofroth G. comparison of the mutagenic activity in carbon particulate matter and in

diesel and gasoline engine exhaust. In: Waters MD, Sandhu SS, Lewtas

Huisingh J, Claxton L, Nesnow S, editors. Short-term bioassays in the

analysis of complex environmental mixtures. New York: plenum, 1981 :

319-36.

61. Pederson TC, Siak JS. Thc role of nitroaiomatic compounds in the dircct-aciirig

mutagenicity ofdiesel particle extracts. J Appl Toxicol 1981; I : 54-60.

62. Rosenkranz HS, McCoy EC, Mermelstein R, Speck WT. A cautionary note on the

use of nitroreductase-deficient strains of Salmonella typhimurium for the

detection of nitroarenes as mutagens in complex mixtures including diesel

exhausts. Mutat Res 1981;91: 103-5.

63. Pitts JN, Lokensgard DM, Harger W, Fisher TS, Mejia V, Schuler JJ, Scorziell GM,

Katzenstein YA. Mutagens in diesel exhaust particulate: identification and

direct activities of 6-nitrobenzo(a)pyrene, 9-nitroanthracene, l-nitropyrene

and 5I1- phenanthro(4,5-bcd)pyran-5-one. Mutat Res l9B2 103: 241-9.

64.Nishioka MG, Petersen BA, Lewtas J. Comparison of nitro-aromatic content and

direct-acting mutagenicity of diesel emissions. In: Cooke M, Dennis AJ,

Fisher GL, editors. Polynuclear Aromatic Hydrocarbons; 6th Intemational

Symposium: Physical and Biological Chemistry. Columbus (OH): Battelle,

1982:603-13.


61

65. Salmeen I, Durisin AM, Prater TJ, Riley T, Schuetzle D. Contribution of 1-

nitropyrene to direct-acting Ames assay mutagenicities of diesel particulate

extracts. Mutat Res 1982: 104: 17-23.

66. Nakagawa R, Kitamori S, Horikawa K, Nakashima K, Tokiwa H. Identification of

dinitropyrenes in diesel-exhaust particles: their probable presence as the

major mutagens. Mutat Res 1983; 124: 201-11.

67. Tokiwa H, Otofuji T, Nakagawa R, Horikawa K, Maeda T, Sano N, Izumi K, Otsuka

II. Dinitro derivatives of pyrene and fluoran-Jiene in diesel emission

particulates and their tumorigenicity in mice and rats. In: Ishinishi N,

Koizumi A, McClellan RO, Stober W, editors. Carcinogenic and

mutagenic effects of diesel exhaust. Amsterdam: Elsevier, 1986: 253 -7 0.

68. Stauff J, Tsai WL, Stark G, Miltenburger H. Chemiluminescence and mutagenic

activity of exhaust gas after combustion. Staub-Reinhalt Luft 1980; 40:

284-9.

69. Rappaport SM, Wang YY, Wei ET, Sawyer R, Watkins BE, Rapoport H. Isolation

and identification of a direct-acting mutagen in diesel-exhaust particulates.

Environ Sci Technol 1980; 14: 1505-9.

70. Green FHY, Boyd RL, Danner-Rabovsky J, Fisher MJ, Moorman WJ, Ong TM,

Tucker J, Vallyathan V, Whong WZ, Zoldak J, Lewis T. Inhalation studies

ofdiesel exhaust and coal dust in rats. Scand J Work Environ Health 1983;

9:181-8.


62

71. Ong T, Whong WZ, Xu J, Burchell B, Green FHy, Lewis T. Genotoxicity studies of

rodents exposed to coal dust and diesel emission particulates. Environ Res

1985; 37 : 399-409.

72. Belisario MA, Farina C, Buonocore V. Evaluation of concentration procedures of

mutagenic metabolites from urine ofdiesel particulate-treated rats. Toxicol

Lett 1985; 25: 8l-8.

73. Casto BC, Hatch GG, Huang SL, Lewtas J, Nesnow S, Waters MD. Mutagenic and

carcinogenic potency of extracts of diesel arrd relalcd environmcntal

emissions: in vitro mutagenesis and oncogenic transformation. Environ Int

1981; 5: 403-9.

74. McCormick Jl, Zator RM, DaGue BB, Maher VM. Studies on the effects of diesel

particulate on normal and xeroderma pigmentosum cells. In: Pepelko WE,

Danner RM, Clarke NA, editors. Health effects of diesel engine emissions.

Cincinnati (OH): US Environmental Protection Agency, 1980: 413-5.

75. Tucker JD, Xu J, Stewart J, Baciu PC, Ong TM. Detection of sister chomatid

exchanges induced by volatile genotoxicants. Teratog Carcinog

Mutagencsis 1986; 6: 15-21.

76. Lockard JM, Kaur P, Lee-Stephens C, Sabharwal PS, Pereira MA, McMillan L,

Mattox J. Induction of sister-chromatid exchanges in human lymphocytes

by extracts of particulate emissions fiom a diesel engine. Mutat Res l9g2l

104: 355-9.


ffi 63

77. Morimoto K, Kitamura M, Kondo H, Koizumi A. Genotoxicity of diesel exhaust

emissions in a battery of in-vitro short-term and in-vivo bioassays. In:

Ishinishi N, Koizumi A, McClellan RO, Stober W, editors. Carcinogenic

and mutagenic effects of diesel engine exhaust. Amsterdam: Elsevier

1986:85-101.

78. Wurgler FE, Vogel EW. In-vivo mutagenicity testing using somatic cells of

Drosophila melanogaster. In: de Serres FJ, editor. Chemical mutagens.

Iiew Yolk: Plenum, 1986: 1-72.

79. Hallstrom I, Grafstrom R. The metabolism of drugs and carcinogens in isolated

subcellular fractions of Drosophila melanogaster. II Enzyme induction

and metabolism of benzo(a)pyrene. Chem-Biol Interact 1981; 34 145-59.

80. Hallstrom I, Sundvall A, Rannug U, Grafstrom R, Ramel C. The metabolism of

drugs and carcinogens in isolated subcellulax fractions of Drosophila

melanogaster. I. Activation of vinyl chloride, 2-aminoanthracene and

benzo(a)pyrene as measured by mutagenic effects in Salmonella

ty p himur ium. Chem-Biol Interact 1 98 1 ; 3 4: 1 29 -43.

8l . Graf U, Singer D. Somatic mutation and recombination test it Drosophila

melat,Jtaster (wing spot test): Effects of extracts of airborne particulate

matter from fire-exposed and non fire-exposed building ventilation filters.

Chemosphere 1989; 19: 1094-7.

82. Delgado-Rodriguez A, Ortiz-Marttelo R, Graf U, Villalobos-Pietrini R, Gomez-

Anoyo S. Genotoxic activity of environmentally important polycyclic

40671


64

aromatic hydrocarbons and their nitro derivatives in the wing spot test of

Drosophila melanogaster. Mutat Res 1995;341:235-47 .

83. Graf U, Singer D. Genotoxicity testing of promutagens in the wing somatic mutation

and recombination test in Drosophila melanogaster. Rev Int Contam

Ambient 1992;8: 15-27.

84. Frolich A, Wurgler FE. Drosophila wing-spot test: improved detectabitity of

genotoxicity of polycyclic uuomatic hydrocarbons. Mutat Res 1990;234:

71-80.

85. Zordan M, Osti M, Pavanello S, Costa R, Levis AG. Relationship between benz(a)

pyrene-DNA adducts and somatic mutation and recombination in

Drosophila melanogaster. Envion Mol Mutagen 1994;23: 171-8.

86. Graf U, Frei H, Kagi A, Katz Al, Wurgler FE. Thirty compounds tested in the

Drosophila wirrg spot test. Mutat Res 1989;222: 359-73.

87. Mollet P, Wurgler FE. Detection of somatic recombination and mutation in

Drosophila: a method for testing genetic activity of chemical compounds.

' Mutat Res 19741'25: 421-4.

88. Vogel E, Blijileven WGH, I(apwijk PM, Zijlstra JA. Some current perspectives of

the application of Drosophila in the evaluation of carcinogens. In:

Williams GM, editor. The Predictive value of short-term screening tests in

carcinogenicity. Amsterdam: Elsevier, 1980 125-47 .

89. Caims J. The origin of human cancers. Nature 1981; 289: 353-7 .


65

90. Radman M, Kinsella AR. Chromosomal events in carcinogenic initiation and

promotion: Implications for carcinogenicity testing and cancer prevention

strategies. In: Montesano R, Bartsch H, Tomatis L, editors. Molecular and

cellular aspects of carcinogen screening tests. Intemational Agency for

Research on Cancer, 1980: 75-90.

91. Vogel EW, Zijlstra JA, Blijleven WGH. Mutagenic activity of selected aromatic

amines and polycyclic hydrocarbons in Drosophila melanogaster. Mr;f.aI

Res 1983; lC7: 53-7''.

Roberts DB. Basic Drosophila care and techniques. In: Roberts DB, editor'

Drosophila: A practical approach. Oxford: IRL Press, 1986: 17-9'

93. Garcia-Bellido A. Genetic control of wing disc development in Drosophila. Cell

Patteming: Ciba Foundation Symposium 29, 1975: 161-82.

94. Lindsley DL, Grell EH. Genetic variations ofDrosophila melanogaster. Camegie

Inst Wash Publ 1968; 627 : 472-80.

95. Garcia-Bellido A, Dapena J. Induction, detection, and characterization of cell

differentiation mutants in Drosophila. Molec Gen Genet 1974; 128: 117'

30.

96. Decker HJ. Nilitotic recombination. In: Ashbumer M, Novitski E, editors. The

genetics and biology of Drosophila. London, New York, San Francisco:

Academic Press, 1976: 1019-87.


66

97. Graf U, Wurgler FE, Katz AJ, Frei H, Joun H, Hall CB, Kale PG. Somatic mutation

and recombination test in Drosophila melanogaster. Environ Mutagen

1984; 6: 153-88.

98. Alonso Moraga A, Graf U. Genotoxicity testing of antiparasitic nitrofurans in the

Drosophila somatic mutation and recombination test. Mutagenesis 1989;

4:105-10.

99. Van Schaik N, Graf U. Genotoxicity evaluation of five tricyclic antidepressants in

tlie rring somatic mutaiion and resombination test in Drosopliila

melanogaster. Mutat Res 1991; 260: 99-104.

100. Frolich A, Wurgler FE. New tester strains with improved high bioactivation

capacity for the Drosophiia wing spot test. Mut Res 19&9;216: 179-87.

101. Graf U, van Schaik N. Improved high bioactivation cross for the wing somatic

mutation and recombination test in Drosophila melanogaster. Mutat Res

1992;27l: 59-67 .

102. Frei H, Wurgler FE. Statistical methods to decide whether mutagenicity test data

from Drosophila assays indicate a positive, negative or inconclusive

resuit. Mutat Res 1988; 203:297-308.

103. Miller JA, Sandin RB, Miller EC, Rusch HP. The carcinogenicity of compounds

related to 2-acetylaminofluorene. IL Variations in the bridges and the 2-

substituent. Cancer Res 1955; 15: 188-95.


67

104. Deichmann WB, MacDonald WM, Coplan MM, Woods FM, Anderson WA. para-

nitro-biphenyl, a new bladder carcinogen in the dog. Ind Med Surg i958;

27:634-7.

105. Cohen SM, Ertuck E, von Esch AM, Crovetti AJ, Bryan GT. Carcinogenicity of 5-

nitroimidazoles, 4-nitrobenzenes, and related compounds. J Natl Cancer

Inst 1973; 51:403-17.

106. McCann J, Choi E, Yamasaki E, Ames BN. Detection of carcinogens as mutagens

in the Sakncnella./microsome test: assay of 300 chcmicals. Proc Natl Acad

Sci 1975; 72:5135-9.

107. Rosenkranz HS, Speck WT. Mutagenicity of metonidazole: activation

mammalian liver enzymes. Biochem Biophys Res Commun 1975;

520-5.

108 Yahagi T, Shimizu H, Nagao M, Takemura N, Sugimura T. Mutagenicity of 5-

nitroacenaphthene in Salmonella. Gann 1975; 66: 581-2.

109. Clayson DG, Gamer RC. Carcinogenic aromatic amines. In: Searle CE, editor.

Chemical Carcinogens. Washinglon DC (USA): American Chemical

Society Monogaraph, 197 6: 366-461.

1 10. Chin JB, Scheinin DMK, Rauth AM. Screening for the mutagenicity of nitro-group

containing hypoxic cell radiosensitizers using Salmonella typhimurium

strains TA100 and TA98. Mutat Res 1978; 58: 1-10.

by

66:


68

1 1 1. Chiu CW, Lee LH, Wang CY, Bryan GT. Mutagenicity of some commercially

available nitro compounds for Salmonella typhimurium. Mutat Res l97g;

58:11-22.

I 12. Wang YY. Rappaport SM, Sawyer RF, Talcott RE, Wei E. Direct-acting mutagens

in automobile exhaust. Cancer Lett 1978; 5: 39-47.

113. Takahashi K, Huang GF, Amki M, Kawazoe Y. Carcinogenicity and mutagenicity

I 14. Grirnmer G. Chemistry. In: Grimrner G, editor. Environmenta!

CRC Press,polycyclic aromatic hydrocarbons. Boca Raton FL (USA):

1983:27-60.

I15. Usanee V, Juntipa P, Taijiro M. Mutagenicity of Thai food plants in Salmonella

mutation assay. Thai J Toxicol l99l1'7:17-22.

I 16. Graf U, Alonso Moraga A, Castro R, Diaz Carrillo E. Genotoxicity testing of

different types of beverages in the Drosophila wing somatic mutation and

recombination test. Food Chem Toxicol 1994; 32: 423-30.

I 17. Vekiari SA, Tzia C, Oreopoulou V, Thomopoulos CD. Isolation of natural

antioxidants from Oregano [abstract]. Rivista Italiana Delle Sostanze

Grasse 1993;70:25-8.

I 18. Arimoto S, Ohara Y, Namba T, Negishi T, Hayatsu H. Inhibition to the

mutagenicity of amino acid pyrolysis products by hemin and other

biological pyrrole pigments. Biochem Biophys Res Commun 1980; 92:

662-8.


119. Terwel L, van der Hoeven JCM. Antimutagenic activity of some naturally occuring

compounds towards cigarette-smoke condensate and benzo(a)pyrene in the

Salmonella./microsome assay. Mutat Res 1985; 152: 1-4.

120. Ong T, Whong WW, Stewart J, Brockman HE. Chlorophyllin: a potent antimutagen

against environmental and dietary complex mixtures. Mutat Res 1986;

173: l1l-5.

121. Negishi T, Arimoto S, Nishizaki C, Hayatsu H. Inhibitory effecr of chlorophyll on

the gcnotoxicity of 3-amino-1-nethyl-5li-pyri,io(4,3-b)indole (Trp-P-2).

Carcinogenesis 1989; 10: 145-9.

122. Gross J, editor. Pigments in vegetables: chlorophylls and carotenoids. New York:

Van Nostrand Reinhold, 1991 .

123. Sandermann H, Diesperger H, Scheel D. Metabolism of xenobiotics by plant cell

cultures. In: Barz W, Reinhard E, Ze* MH, editors. Plant tissue culture

and its biotechnological application. Berlin: Springer-Verlag, 1 977.

124. Harms H, Langebartels C. Standardized plant cell suspension test systems for an

ecotoxicologic evaluation of the metabolic fate of xenobiotics. Plant Sci

1986; 45: 157-65.

125. Sachs L, editor. Applied statistis: a handbook of techniques. NewYork: Springer,

1982.

126. Sachs L, editor. Angewandte statistik. 6th ed. Berlin: Springer, 1984.


APPEI\DIXA

Preparation of Standard Culture Medium

Ingredient

Com flour

Sugar

Yeast

Agar

Propionic acid

Distilled water

12s

100

50

t4

5

1000

ml

Steps of preparation of standard medium for Drosophila melanogaster stocks

1. Boil and blend sugar, agar, yeast and com flour in 1000 ml water until

2. Add propionic acid.

3. Fill each 125 ml-erlenmeyer flask with 50 ml of the medium.

4. Close off the flask with a plug (made of gauze and cotton cover with

aluminum foil).

5. Sterile the flasks in an autoclave to avoid microbial contamination that can

harm the flies.


7t

APPENDIXB

Statistical Consideration

In experiments designed to assess the mutagenicity of a chemical, most often

treatment series were compared with a control series. One might like to decide

whether the compound used in the treatment should be considered as mutagenic or

nonmutagenic. The formulation of2 altemative hypotheses allowed one to distinguish

among the possibilities ofa positive, inconclusive, or negative result ofan experiment

(102).

In the null hypothesis one assumes that there was no difference in the mutation

frequency between control and treated series. Rejection of the null hypothesis

indicated that the treatrnent resulted in a statistically increased mutation frequency.

The altemative hypothesis poshrlated a priory that the treatment results in an increased

mutation frequency compared to the spontaneous frequency.

This altemative hypothesis was rejected if the mutation fiequency was

significantly lower than the postulated increased frequency. Rejection indicates that

the treatment did not produce the increase requires to consider the treatment as

mutagenic. If neither of the 2 hypotheses was rejected, the results was considered

inconclusive as one could not accept at the same time the 2 mutually exclusive

hypotheses. In the practical application of the decision procedure, one defines a

specific altemative hypothesis requiring the mutation frequency in the treated series


72

be m times that in the control series and used together with the null hypothesis. It

might happen in this case that both hypotheses had to be rejected. This should mean

that the treatment was weakly mutagenic, but led to a mutation frequency which was

significantly lower than rr times the control frequency.

Testing against the null hypothesis (H6) at the level cr and against the

altemative hypothesis (Hj at the level p led to the error probabilities for each of the

possible diagnoses: positive, weak but positive, negative, or inconclusive. The

follolving four decisions were possible; 1) accept both hypothcses; those can not be

true simultaneously, so no conclusions can be drawn--inconclusive result; 2) accept

the first hypothesis and reject the second hypothesis--negative result; 3) reject the first

hypothesis and accept the second hypothesis-positive result; 4) reject both

hypotheses-weak effect.


'73

Calculations step by step

Estimation of spot frequencies and confidence limits of m.

Particularly in the case that both hypotheses, H6 as well as Ho, had to be

rejected, one might be interested in knowing the confidence interval of m., i.e., of the

estimated multiple by which the mutation frequency in the experimental series was

larger than the spontaneous frequency. The estimated value was

m.: (n,/ n)N"

where N" and N, represented the respective sample sizes in control and treatment

series, n. and n the respective numbers of mutations found, and n the total of

mutations in both series together. Exact lower and upper confidence limits p1 and pu

for the proportion n"/n on one hand, as well as Qr and gu for the proportion n,/n on the

other hand, may be determined according to Sachs (125, 126). He gave an easy

method to calculate these values using an F-distribution table. To determined q1 and

p6 one-sidedly at the levelcr, and qu and p, also one-sidedly at the levelp. In this way

and in agreement with the foregoing section, a confidence limit mt>l led to rejection

of Ho, while a confidence limit mu<m led to rejection of H1.

In the first step, F-distribution according to Sachs (125, 126) were used to

determine the value F,7,,2 at the level o = 0.05, where the degrees of freedom ( vr,v2)

were given by the equations

(n. / n)N,

v1 :2 (n-n, + 1) and v2 = 2q


74

In the second step, the F-value so obtained was used to calculate the lower

confidence limit (qq) for the proportion of spots in the experimental series

Qr = nt / [nr + ( n-n +1 F,,.,r]

This gave a lower confidence limit for the frequency of spots per wing in the control

which was equal to

f.r: q1n /N,

This was the following complementadty, namely that the lower confidence

liinit for the number of spots in ihe experimental series (q,n) plus the uppei'

confidence limit for the number of spots in the experiment (pun) was equal to the total

number ofspots (n) found in experimental and control series together, i. e.,

p,n= ( l -qr)n

This gave an upper limit for the frequency ofspots per wing for the control which is

f., = q,n/N.

The lower confidence limit m, of the multiple m" can be determined as the

ratio between the lower confidence limit for the frequency in the treated series and the

upper confidence limit for the frequency in the control, i.e.,

qr n/Nt

Pu n'Nc

Only in the case that m1, the lower confidence limit of rn , was larger than 1.0 would

reject H6. Since this was not the case, H6 remain accepted.

In the same way, the lower confidence limit of the spot frequency may

determine in the controlf,.l which will givef.u ,the upper confidence limit of the spot

J r.lInl


i

75

frequency in the experimental series. This is also done one-sidedly, at the level p =

0.05. The inverse ratio of these values will provide the upper 5%o confidence limit mu

for the multiple m".

Again, the F-distribution according to Sachs (125, 126) was used and

determined the value Fvr,v2 at the level B : 0.05, where the degrees of freedom (v1.v2)

were this time given by the equations

r'1 :2(n- n" +l) and v2= 2n"

The I-valuc so obtained u'as used to calculate ihe lower confidence lilnit (pt) for the

proportion of spots in the control

P1 = n"/ [n"+( n - & + 1) F,r,,z]

This gave a lower confidence limit for the frequency of spots per wing in the control

which equal to

f,.1 = plnA{"

Again, There was complementarity, in that the lower confidence limit for the

number of spots in the control (p1n) plus the upper confidence limit for the number of

spots in the experiment (qun) was equal to the total number ofspots (n), so that

q,n : (1- P1 )n

This gave an upper limit for the frequency of spots per vring for this series which is

f,u = qon/N,

The upper confidence limit m, of the multiple m" can be determined as the

ratio between the upper confidence limit for the frequency in the treated series and the

lower confidence limit for the frequency in the control, i.e.,


76

I

)

mu=7.u=g,flrI.r plnNc

Ha was rejected if mr, the upper confidence limit of me, was less than m (m=2

for the total of all spots and for the small single spots, and m:5 for the large single

spots as well as for the twin spots). Substitution of m. by m1 or m, in the above

formulas provided the respective exact upper and lower confidence limits for the

frequencies estimated.

I


BIOGRAPHY

NAME

DATE OF'BIRTH

PLACE OF BIRTH

INSTITUTIONS ATTENDED

Miss Janpen Saksitpitak

20 November 1972

Bangkok, Thailand

King Mongkut's Institute of Technology

Ladkabang, 1991-1994: Bachelor of

Science (Agriculture)

Mahidol University, 1995-1 998: Master

of Science (Food and Nutritional

Toxicology)

5tt

406?1Copyright by Mahidol University

Documents

Mutagenicity evaluation of vegetables grown in diesel ...mulinet11.li.mahidol.ac.th/e-thesis/scan/3836203.pdf · extract of leaves of sacred basil and green kuang futsoi grown in