mutagenicity evaluation of vegetables grown in diesel...
TRANSCRIPT
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
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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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
3836203 NUFIA{ : d11.1'ts1 : yl! tll8]Yn r011n:lln3 lnxutnrl : 'l1l.u. (t{u't11u11'n.101fi'l:
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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
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ttlalriffluflfl (heterozygous) xll^n improved high bioacrivarion (I]IB) cross (ORR;fl1/
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Copyright by Mahidol University
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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
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viiii.,
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J
3
5
13
l5
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19
2t
25
28
30
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Copyright by Mahidol University
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Table
1
2
3
4
5
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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
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3839
40
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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
Copyright by Mahidol University
.C
cm
LIST OFABBREVIATTON
degree celcius
centimetre
gram
hour
kilogram
miligram
mililitre
milimolar
number
h
kg
mg
ml
m I\,{
No.
Copyright by Mahidol University
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.
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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.
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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).
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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
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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
Copyright by Mahidol University
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).
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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
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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.
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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
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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).
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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
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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
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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
Copyright by Mahidol University
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
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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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
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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:
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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
Copyright by Mahidol University
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
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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).
Copyright by Mahidol University
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/.)
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
-_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'., -
Copyright by Mahidol University
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).
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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.
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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
Copyright by Mahidol University
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
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\
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/././
Copyright by Mahidol University
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.
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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.
Copyright by Mahidol University
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.
Copyright by Mahidol University
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
Copyright by Mahidol University
39
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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).
Copyright by Mahidol University
42
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Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
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Muller H. Aufnahme von 3,4-Benzpyren durch Nahrungspflanzen aus kunstlich
angereicherten Substraten. Z PfTanzetemafu Bodenkd 197 6; 7 6: 6g5-95.
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Shcherbak NP. Fate of benzo(a)pyrene in soil. Vopr Onkol 1969; 15: 7 5-9 .
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verschiedenen Gemusesorten und Saraten. Dtsch Lebensm Rundsch 1965;
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14. Grimmer G. cancerogene Kohlenwasserstoffe in der Umgebr.urg des Menschen.
Dtsch Apoth Ztg 1968; 108: 529-35.
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Copyright by Mahidol University
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.
Copyright by Mahidol University
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
'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
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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
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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.,
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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
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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