Copyright by Mahidol Universitymulinet11.li.mahidol.ac.th/e-thesis/scan/3936164.pdf · g,anode&ma ltjcidum (fk) ruwt on niirosated prodi,'ct of amit{opyrene.niiriie MODEL IN AMES

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PRAPATSORN PAKDEE

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e TT#STS SUBIVTTTEI}IN PARTIAL fl]LflLLMENT OF

TIIE REQUIREMENTS FOR

TIIE DEGREE OF MASTEROF SCIENCE

(FOOD Ar\D r{UTRTTTONAL TO)ilCOLOGY)

FACTTLTY OF GRADUATE STIJDTES

MAHII}OL T]NIYERSITY

2001

ISBN97+0+0392-1

COPYRIGIIT OF MAHIDOL T]MYERSITY

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Copyright by Mahidol University

Thesis

eotitled

EFFECTS OF HOT WATER E)ffRACT OB GANODERMA

LUCTDUM (Fn,) r/nSTONMTROSATED PRODUCT OF

AMINOPYREI\E.NITRITE MODEL IN AIVIES TEST AND ON

T]RETIIAI\IE INTIIE SOMAflC MUTATION AI\ID

RECOMBINATIONTEST

ASING DROSO PHILA MEI-/INOGASTER

....trJ.'*r....?:!4.Miss Prapafsom Pakdee

Candidate

b* [ry-^*Assoo.Prof, Kaew Katgqadalamtei, Ph-DMajor-Advisor

...$_,ww..Assist.Prof Somsri Charoenkiatkul, D.ScCo-Advisor

h^,1,*1,,,- l^11,u^,--. -. ---..t I. -. -. --

Asst.Prof. Anchalee P6ngpunM.A. (Applied Linguistics)Acting DeanFaculty of Graduate Studies

ChainnaoMaster of Science, Prcgramme io Foodand Nutitional Toxicolo gyInstitute of Nutrition


Thesisentitled

EFFECIS OF HOT WATER EXTRACT OF G,ENODENUA

LUCTDUM @R-) K/.RSr ON MTROSATED PRODUCT OF

W MODEL IN AMES TEST AI\D ON

URETIIAI\IE IN TIIE SOMATIC MUTATION AI\D

RECOMBINATION TEST

USING DXOSOTTZII M E L"AN O G A S TE R

was subrnitted to the Faculty ofGraduate Snrdies, Mahidol University for the degree

of Master of Science @ood and Nutritional Toxicology)

on

July 10,2001

A",l^"1,* fr1p" *;;; ;; ;: ;;;;; Ji,; ;;; ;;M.A. (Applied Linguistics)Acting DeanFaculty of Graduate StudiesMahidol University

Miss Prapatsorn PakdeeCandidate

Chairman

8 {l"r-*/,rxtfra/.-Assist.Prof.Somsri Charoenkiatkul, D.ScMember

.N.r:::...H,.....Mrs. Wannee KusarnrarL PtuD.Member

DirectorInstitute of NutritionMahidol University

....&e.].*:....!il#.,'-.........

lt/fa-, b*r-Z*-rr-"'

" '("" """" ""')/""""""t'Assoc.Prof.Kaew Kangsadalampai, Ptt"D.


ACKNOWLEDGEMENT

I woutril like to express my appreciation and sincere gatitude to Ik Kaew

fangsaaafampai, my advisor frr his valuable advice, suggestion and encourage, ert

throughout this wort, which enable me to carry out my shrdy successfirlly. Special

appreciation is also exterded to my co-advisors, Dr. Somsri Charoenki*ikul, Dr.

Wannee Kusaoran and lvlrs. ChaniFhun Butryee for theh kindness, conrments and

valuable suggestiotrs. AIso special thgnks to Dr. F.E. Wwgler and U. Graf of Institute

of Toxicology, Swiss Federal lostitute of Technobgy aod University of Zurich for

their stock culnnes of Drosophila melanogast*.

I would like to acknowledge my gratitude to Miss Prapasri laohavechvanich

aod Mrs" Kanokwan Poerawong for ber help, teachiog, assistance with the technical

labordory instructioos as well as Mr. Daoldmgsm Sudjaom for his he$.

Uttermost gptitude is eryressed to my family for their love, care, support and

much encouragemed throughout the period ofmy graduate study.

Prapafsorn Pakdee


Fac ofGrad Studies' Mahidol l-lniv. Thesis / iv

3936164 NLJFT/]VI : MdIOR FOOD AltID NUTRITIONAL TOXCOLOGY; M.Sc.(FOOD AND NUTRITIONAL TOXCOLOG9

KEY WORDS : ANTIMUTAGENICITY / GANODERMA EXTRACT/MUTAGEN

PRAPATSORN PAKDEE: EFFECTS OF HOT WATER EXTRACT OF

GANODERMA LUCIDUM (FR.) KARST ON NITROSATED PRODUCT OF

AMINOPYRENE-I{ITRITE MODEL IN AMES TEST AND ON I]RETHANE INTHE SOMATIC MUTATION AND RECOMBINATION TEST USINGDROSOPHILA MELANNASTER TTmSIS ADVISORS: KAEWKANGSADALAMPAI, Ph.D., SOMSRI CHAROENKIATKUL, D.Sc. 127p. ISBN97+0+0392-1

The mein objective of this study was to determine the antimutagenicity of hotwater ercract fiomGanoderma lucidum or GL eftarl- (final correntrcion were 1,000,

2,000, 4,000 aod 8,0tX) pglml) against 100 pl ofproduct that fomrd dudng 10 pl (fortesting on Salmonella typhimuriwt TA98) or ,tO pl (for TA100) of aminopyrene(0.0375 mg/ml) treded with nitrite (final conceffiation was 500 mM) in a totalvolume of 1000 pl acid sohrtion (pH 3) for 4 boa Salmonella typhimurium stainsTA98 axd TA100 in tk abserce of metabolic activatiotr AIso the antimutagenicpotedial agpinst rnethafle induced wing qot of Drosophila melanagaster in somatic

mutation and recombination t€st was performed. The second test was conducted todetemine the nodifying ef[ects on iz vivo induction of mttdion and mitoticrecombinarion in somatb cells of Drosophila melanogaster (SMART asmy) induced

by 4500 ppm urethare (U\p). Three{ay old larvae oftrans-heterorygous obtained bymating tbe vnglm ORR;IF ferrales and nwh llrales were tansferrcd to ttre mediumcontaining GL oaract (10,000, 100,000 or 1,000,000 ppm) aod URE (a500 ppno) umilthey became aduh flies. A pretretmsnt study was done by mating the parent flies ontie medium cotrainiry GL extract (1q000, 100,000 or 1,000,000 ppm) to obtain 3-

day old larv'ae wtich were transfetred to fresh medium containing LiRE until theybecame aduh flies, Tbe wings of tbe survived flies were aazlyzrd for the occurrcnceof mutant wing spos.

The resufts from Ames test showed that GL extract was rrutagenic only afternitrite ffitmed. However, it was antimutagenic towards the product of AP-nitriter€action at all doses errcept the lowest dose (1,000 pglml) showed potemiating effecton strain TA 98. Working with the Drosophfu it was foud tht GL exhct was notmutagenic. Interestingly, co-administration of GL e>aract with urethane to tlrc larvaereduced the tequemy of induced wing spots of tie flies (52-78Yo) with thegoup fed urethane, GL eraract prctre&eot reduc€d the ftequency of mutaff spots

indu&d by urethane $+85Yo); however, the reduction was less tban thal ohainedfiom the study on co-administration of urcthane with GL extract. The data tdicatedtbat bot w*er o<tract might be beneficial br consunrr in terms of cancer pr€ventionHoweve,r GL has mnrtagen when inceracted with nitrite. The consumer should avoidconsuming GL extract with food containing nitrite.


Fac. ofGrad- Studieg tvlahidol I-Ltiv.

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G,ANODE&MA LTJCIDUM (FK) ruWT ON NIIROSATED PRODI,'CT OF AMIT{OPYRENE.NIIRIIE

MODEL IN AMES TEST AND ON URETIIANE IN TIIE SOMATIC MUTATION AND

RECOTTGINATION TEST T SING DRNOPEII.A r,H.4NrcA,9IER). aozrrrunr:a:lqlirurfirnrf,:

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firu tooo, 2mo, 4cxn r.ffi 8,000 Fslptarc) io roo pt to.r einrfuq( iirfiarrnr.liiffirrrwirl to rr trdo

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CONTENTS

ACKNOWL,EDGEMENT

ABSTRACT

IISTOFTABLES

LIST OFFIGI]RE'S

LISTOFABBREVATIONS

CHAPTER

Page

lll

iv

viii

ix

xi

I

3

J

6

8

24

25

27

29

38

54

I INTRODUCTION

II LITERATT'RE REVIEW

2.1 Antimutagenesis and cancer prevention

2.2 Antir:arcinogenic and Antimutagenesis Mechanism

2.3 Ganoderma lucidum (fu.) Y,ar*

2,4 Dietary nitrosating Compound: Nitrate and Nitrite in

Foods

2.5 Mdagenicity and Carcinogenicity of Nitrite

2,6 Aminopyreoe-Nitrite Model for Inhibition

2.7 Tfu Salmonella Mrlragenic Assay (Ames Test)

2.8 Sornatic Mutation and Recombination Test

(sMARr)

Itr MATERIAI-SAI\DMETHODS

3.1 Chemicals 54

3.2 SarrErle Preparation 55

3.3 Experimental Design 56

3.4 Ames Test 5'7

3.5 Somatic Mutation and Recombhation Test 62

(sMARr)


cor{TENTS (CONT.)

RDSULTS

4.1 Mrtagenicity of Nitrite treded Exhact from

Garudermahrcidum

4.2 Adimutagenicity of E:<hact fiom Gobderrna

lucidum fur Ames test

4.3 Suvival of Larvae aod Aduh Flies in Somatic

Mutdion and Recombination Test

4.4 Efrect of Gataderma lucidtm Co-adminishation

with Uretbane on Induced Wing Spots

4.5 Etrects of Feeding Gonodenna lucidum to Newbom

Larvae for Thee Days on Wing Spot InArced by

Uretime

V DISCUSSION

5.f Mrnagenicity of Hot Waler E ffact fiom Gotoderma

luci&tm

5.2 Antimrdagenicity of Hot Water Extract tom GL in

Ames test

5.3 Study in Somatic Mutation and Recombination Test

YI CONCLUSK)N

BIBLI(rcRAPEY

APPENDIX

BIOGRAPEY

68

68

74

78

79

8t

82

84

85

lt3t27


TABLE

t,'

J

4

LIST OF'TABLES

Categorbs of foods with the mst prominent ckmoprevent€r

Mechanisms of action of cbmopreventers

Genotypes of tbe Salnonella typhimurium strains used for

mutagmesis testing

Mutagenicity of the bot uder qdract of Ganoderma lrcidum

treated with oide in acid condition pH 3.0-3.5 on S, tltphimwitmt

TA98 ad TAl00 Dda are expressed as tHIl and shdrddeviation of four plates fiom two erperimeots.

Eftect of the hot wder s<tract from Ganodentu lucldtn on tfu

histiline rc\rcrtants of S. ltphiruriun sains TA98 ad TAl00

induced by aminopyrene.nitrite product.

Number of srrvival trans-heterozygous Qnwh+/+tF) aduft flies

fed on e:dract fromGorcderma luci&m and urahans

Number of survival trans-heterozrygous Qnth+t+1t{1 aduh flies

from larvae pre&d with tbe medium containing the exbact from

GL for 3 days before codinue brhging up on normal medium

containing urethme

lnhibftbn,of Ganodentuhtcidum onWing Spot induced by 4500

ppm Urethalr in tars-hete,rorygous (mwh+/+Jf) aduh flie's

Inhibition of Ganoderma lucifur, pr€treatment on Wing Spot

induced by 4500 ppm Urethane in trans-kterozygous

(nwh+/+fli) a^tltflies


FIGTJRE

I

2

3

4

5

6

7

9

l0

LIST OBF'IGI]RES

Chemical structure,s of Triterpenes in Garcderma lucidum

Possible repeating rniis of Gotoderna htcifum ghrcare

Possible structual fragments of the polysaccharide mrety in

gaooderan B

Possiblc structural fragments of tbe potysaccharide moiety in

ganoderan C

The conversion ofnitrate to nitrite by niuifying bacteria

Suggested pathway for activation of l-ninopyrene

Gei:etic scheme.s illustrating various ways of spot formtion in

the somatic nnrtation and recombination test with tbe wing cell

ma*€rs mutiple wing hairs (nwh) aldfl*. $fi. Twin spots

are ottaircd by recombination proximal to tfu /F maker (b),

while more distal recombination produces nrw& single spots

only (d). Deficierrcies (c), poitr mutations (e) and

nondisjunction events (f) give rise tn moh sngle spots, or in

analogous ways toll singte spots (not illusrated)

Marker nartations of wing surfrce to sbw clone of cuticle

secreted by cells homzygous for multiple wing hairs' a) small

single spots, b) flre on wing vein, c) twin spots, d) large single

spots

Urethale, Vinyl carbamate and Vinyl carbamate epoxide.

Known and probable activation and inactivation pathways of

metabolism of rnetbane (ethyl carbamate), vinyl carbamate advinyl carbamate epoxide. (e) Mouse fiver microsomes + ethyl

ctrbamat€ or vinyl carbamate + adenosinet l"f-ethenoadenosine. (b) Human liver microsomal cytochome

P450 IIEI, (c) Vinyl cartamate epoxide + adenosine+ 1"f-ethenoadenosine. 6911 = ghrtathione

Page

il14

2t

22

25

28

42


tr'IGI]RE

ll12

r3

t4

15

l6

t7

l8

Lrsr oF flGrrRES (coNT.)

Page

heparation of hot wat€r errtract of Gmod*mo lucidum 55

Overall experimental design 57

Preparation of stadard direct mutagen 59

Steps in *udying the effect of GL oftact on the rnrtagenicity of 6l

4 bours incubatbn product of aminopyrene-nitrite rnodel

Mutagenicity testing and aatimrtagenicity testing 63

Pretreatment sudy of GL extract on mrtragenicity of urcthane in 64

Drosophila melanogaster

Nomlal half mesothorax showing tle regbns A-E of tbe wing 66

snrhce scored for spots according ta Grat et al. (1984)

Trichoes on the wbg blade, a) normal b) deviate trichomes 66

mt couted as znsft ar Jtl, c'S coafigrfre/rions indicative of mwk

d) q/pical manifesatbn ofll


AP

oc

pc

pl

mg

No.

h

mg

M

min

ml

LIST OFABBREVIATIONS

Aminopyrere

Degree celsius

Microgram

Microlitr,e

Milligram

Gram

Number

Hour

Milligram

Mohr

Minrre

Milliliter

S.tlphinurium Salmorclla gryhimurium

Glutathiore

Gfutathiono..9tramferase

G anoderma lucidum Fr.(Karst)

Part per million

GSH

GST

GL


Fac. of Grad- Studies, Mahidol l-hiv. lvLSc. (Food andNutriticnal Toxicolory / 1

CIIAPTERI

INTRODUCTION

Since the early postulare that abot* 35% of human cancer incidences of the

b,reast, pancreas, stornch and colon may be related to dietary factors (Doll' 1992;

Doll ard Peto, 1981; Willelt, 1995). Irrcreasing attention is being paid to tlre role of

di€t in tlrc initiation and pronrotion of cancer on the one ttan4 and in cancer

prevention on the other. Food may contain small arnounts of mutagenic and/or

carcinogenic coryounds, whereas also protective, antimutagenic/anticarcinogenic

compounds may be presenc. Exposure to diet related mutagens is diffrcult to avoid,

but our dietary habits, as well as dbt prepardion and composition, can limit the

exposure to mutagenic and carcinogenic food components. In addition, it appears

worthwhile to investigate tlre possibilities to cormterbalance the mutagenic potency of

food by inclusion of antirnutagenic coryounds in the diet.

The coosumpion of antimutagers ald carcinogens has been suggested as

and eflective strdegy for either preventing or minimizing the possible occurrence of

deleterious efects r€suhisg from exposure to an irrreasing number of mutagenic and

carcinogenic agents in the environmeff (Fergusoq 1994; Morse and Stoner, 1993;

Ramel ef al., 1986; Rogers eI al., 1993; Wanenberg, 1983 ad 1985). Many of the

corpounds with antimutagenic and carcinogenic properties ae known to occrr

naturally in fiuits, vegetables, spices, ard herbs (Fergusorl 1994; Ramel et al., 1986;

Wattenberg, 1985). Hence food ard trerbs can be considered as sorrces of potantial

inhibitors of environmental mutagenesis and carcinogenesis. By increasing the intakeCopyright by Mahidol University

Prapasom kkdee Introductiur / 2

of such cherropreve[tive food components aod lrcfts, it rnay be possible to strengthen

the defence agains damage caused by mrtagens and carcinogens.

Ganoderma lucidun (GL), an oriental herb has been widely used as a

rernedy for prornotion of heafth and longevay in China and other Asian countries

(lrttieu, 2540; qrfia,25,+0). Tlrc cultured mycelia and fruiting bodies of GL have

been used in rhe rcatment of chronic hepatopahy, hypertensioq hyperlipidemia

angina pectoris, chronic trrorchitis, rhumatoid arthits, gastitis, reurasthenia,

leukopeni4 and neoplasia (ilttirl, 2540; Elra;2540; Berry et al., 1993; Huarry,

1993). This fungus has attracted g€d dtention due to th€ fact that its polysaccharides

have affitumor activity (Miyazaki ad Nishijim4 l98l; Sone et al., 1985). Many

studies have reported that polysaccharides ofGL could itrhibit the growth of turnors

tnth in vivo d in vito (Sone er al., 1985; Ywr et al., 1995). In addition, crude

polysaccharide of GL significantly incr€asd the life span of tumor-implanted mice

when administered alooe or in combination with cytotoxic amitumr drug (Furusawa

et a1.,1992).

However, it is of geat futercst to daetmine the antimutagenicity of

Ganoderma extract on nitrosated product occurred in somach digesion pH using 4 h

aminopyrene-nitrite r€actbn as a nrcdel and ttrcir effect on the mutagenicity of

aminopyrene-nitrite pmduct were invesigated by in vitro assay by Ames test and

investig:ate whether co-administration of Ganoderma extract along with genotoxins

could also ld u in vruo modifring etrects by the somatic mutation and

recombination test (SMART) using Drosophilla melanogaster.


Fac. of Grad. Studies, Maftidol Univ. M.Sc. (Food and Nutritional Toxicology) / 3

CIIAPTER II

REVTEW LITERATTJRE

2.1 Antimutagenesis a.nd cancer Prevention

It has been estimated that about one tiird ofall human cancer may relate to the

diet. Although it would theoretically be possible to avoid exposure to all dietary

carcinogens, it is becoming increasingly clear that these are many and varied, and that

complete avoidance would be difficrdt (Fergusoq 1994). In later years the recognition

of chemicals, which function as anlimutagens and anticarcinogens by inhibitory

action at different levels of mutagenesis and tumor induction has lead to a rapidly

expanding research field-chemoprevention of mutations and cancer (Ramel, I99l),

Chemoprevention of cancer is a mean of cancer control in which the occurrence of

this disease is prevented by administration of one or several chemical compounds

(Wattenberg 1985). There is a wide array of chemicalq which have been shown to

function as antimutagens and anticarcinogeng many of them occrrrring as natural

components in our food and others made synthetically (Ramel 1991; Stawic, 1994).

Some are &:nown 1o have important roles in the preservation of human healttq whereas

other such as fiber or flavonoids, which were considered for a long time to be "inert"

components of foods, have not received much attention with regard to their subtle

effects on human health. Compound such as vitamins d B, C or E, already known to

possess distinctive physiological and pharmacological aclivities, until recently

received only scant attention for additional roles as chemopreventers (CP) (Stawic,Copyright by Mahidol University

Prapatsom Pakdee Literature Review / 4

1994). Morse and Stoner (1993) suggested tlnt a CP agent should have 5 qualities,

which they distinguished as: (i) little or no untoward side effects; (ii) high efiicacy;

(iii) capability of oral administration; (iv) a known mechanism of action and (v) low

cost. Many antimutagens may fulfill these criteria (Osawa, 1990). The most

thoroughly investigated CP in foods are fibrg polyphenolic compounds, flavonoids

(quercetin, ellagic acid, chlorogenic acid), conjugated isomers of linoleic acid, d-

limonene, epigallocatechin gallatq soybean proteins, isoflavones, vitamins (A B, C,

E), tocopherolg calciunr, seleniunL chlorophylline, aliphatic sulfides, catechirq

tetrahydrocurcumin, sesaminol, glutathionq coumarins, uric acid, indoles,

thiocyanates, and protease inhibitors. Over 25 diflerent classes of chemicals have

been found to possess antimutagen and anticarcinogen capacities. Chemopreventers

are found in all categories of foods, fruits and vegetables being the main source as

illustrated in Tablel. The amount of CP in different categories of food can, however,

vary considerably. Even the same type of food products, obtained from different

regions, may sometimes contain different levels of a particular CP (Stawic, 1994).

Wattenberg therefore suggested 'the composition of the diet could be an important

factor in determining the response of individuals to carcinogenic agents to which they

were exposed" (Wattenberg 1990). Mury micro nonnutrients in the diet were isolated

and identified and were demonstrated to block different stages of the carcinogenic

process in several animal models. Chemicals that are able to prevent the formation of

carcinogens from precursor substances or to prevent carcinogens from reaching with

critical target DNA sites in the tiszues are "blocking agents". Chemicals that act by

suppressing the expressing of neoplasia in cell previously exposed to doses of a


Fac. ofGrad. Studies, I\4ahidol Univ. M.Sc. Good and Nutritional Toxioology) / 5

carcinogenic €ent are "suppressing agents' (Wattenberg 1983; Wattenberg 1985;

Wattenberg 1992).

Table l. Categories of foods with the most prominent chemopreventerx

Type offood Chemopreventer

Fruit

Vegetables

Cereals

Meatq fislq eggg poultry

FaVoil

Milk

Nuts, beang grains

Spices

Tea

Coffee

Wine

Water

Vitamins, flavonoidg polyphenolic acids, fiber,

carotenes, monoterpenoids (dJimonene)

Vitaming flavonoids, plant phenolics, chlorophyll,

fiber, aliphatic sulfides, carotenes, aromatic

isothiocyanates, dithiolthiones, phytic acid, calcium

Fiber, a-tompherol, phytic acid, selenium

Conjugated isomers of linoleic acid vitamins (d E),

seleniles

Fatty acids, vitamins E, tocotrienols

Fermenlation products, calciurq free fatty acids

Polyphenolics, fiber, vitamins E, phytic acid,

coumarins, proteins

Coumarins, curcumiq sesaminal

Plant phenolics, epigallocatechin

Polyphenolic acids, diterpene, alcohol esters,

melanoidins

Flavonoids

Selenium

'From Wattenberg (1990)


Prapasorn kkdee LiteraturE Review / 6

There are numerous examples of antimutageniclanticarcinogenic substances

derived from nonnutrients. According to the terminology of Kada et al. (Kada, 1982\,

desmutagens are antimutagenic agents acting outside the cell by acting directly on

mutagens or their precursors and inactivating them while bio-artimutagens are

antimutagenic agents acting inside the cell by interfering on the process of

mutagenesis or repairing of damaged DNA5 thereby resulting in decreasing mutation.

It is now clear that many antimutagens function as antioxidants; primary examples of

antimutagens that may function in this manner include vitamins C and E. It is also

clear that a number of antimutagens function as interceptorddesmutagens that

intercept, bind or physically destroy mutagens and carcinogens. These substances

were extensively reviewing by Hartman and Shankel (1990). It is also evident that

there are "extracellular" inhibitors tfiat can be classified according to their

mechanismq and there are "intracellular" inhibitors that function only within.the

potentially damaged cells. The mechanisms by which antimutagens/anticarcinogens

may act were summarizing in an extensive review by DeFlora and Ramel (1988)

2.2 Anticarcinogenic and Antimutagenesis Mechanism

The antimutagenic and anticarcinogenic mechanisms of chemopreventers

appear to be complex and, in some cases, established. The beneficial activity of

chemopreventers depends on nrany unrelated factors and conditions. This effect can

be tlre result of a single evert or the simultaneous action of several factors acting in

concert. In a very artitrary way, the mechanisms ofaction of the chemopreventers can

be separated into two main cateBories and several subgroups according to the site or

ambient at which they exert their influencg as illustrated in Table 2. Any of theseCopyright by Mahidol University

Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food and Nuhitional Toxioology) / 7

actiong either alone or in combination, will reduce the hazardous activity of the

mutagens and/or carcinogens species in the body.

Even if the inhibitory agents or steps are identified, the molecular mechanism

underlying the inhibition is often not explained. This should not come as a zurprise,

since the science of chemoprevention is a relatively new area of research. Although

the mechanisms appear to be very heterogeneous, the antioxidative characteristics of

chemopreventers seem to play the most significant part in their protective activity

(Stawic, 1994).

Table 2. Mechanisms of action of chemopreventers n

EXTRACELLULARLY

During t}le preparation offoods, by:

. reducing (inhibition) the formaion of IWC

Effects in the intesline, by:

o formation of non-lWC complex

o reducing bioavilability

o diluting with diaary fibers

o increasing adsorption on other food components

. accelerating intestinal transit

o protecting the mucosal barrier

o modirying intestinal microbial flora

o inhibiting the penetration ofcells by M/C

INTRACELLULARLY

At cellular level, by:



. enhancing the activities of enzymes involved in detoxification oflvl/C

o inhibiting the activities of enzymes involved in formation of M/C metabolites

r trapping of electrophiles

. scavenging reactive orygen species

. inhibiting metabolic activation

o protectingnucleophilicactivalion

. inhibiting the detrimental effect of prc.carcinogens on DNA

IWC : mutagens and/or carcinogen

" From Stavric (1994).

2.3 Ganodetma lncidttm (Br.\ Karst

Ganoderma hrcidm (Fr.) Karst (GL) is a medicinal plant belonging to the

family Polyporaceae. It is well known in China where it is called Lirglhi or Ling zhi

cao that means "spirit plant'' and very popular in Japan under the name of Reishi.

English common names of GL are Lacquered mushroom and Holy mushroom

(Ir:t , 2540; Ertn, 2540; Berry et a/, 1993). GL is listed as a superior medicine to

ward off diseasg pres€rve healttr, and promote longevity for several thousands years

in Oriental material-medicus. Nowadays, GL is used for a variety purposes such as

protects against some types of cancer, increase vitality and strengthens intemal

organs, relieves neurasthenia and stress, improves conditions of viral hepatitis,

protects the liver against chemical damage, helps to normalize body functions,

relieves insomnia by enhancing muscle relaxation, improves the coronary arteries and


Fac. of Grad. Studies, Mahidol Univ. It {-Sc. (Food and Nutritional Toxicology) / 9

reduces excessive levels of cholesterol in the blood @erry et al, 1993; Huang, 1993;

Powell, 1990; Shiao et al., 1994).

GL is widely distributed as a wooddecaying fungus. They grow on stumps

and roots of deciduous trees in most forests, particularly in the temperate zone and

tend to be large or bright colorfirl. GL has an intensely shiny cap, orange, later

chestnut red-brown or purplish-brovrrn situated on a lateral or eccentrig equally

lustrous salk of the same color @erry et al, 1993; Powell, 1990; Svceh 1988;

Wassoq 1968). A stalk is usually ranging ftom 2 to 6 inches in length. The fruiting

body is hard, and the surface is glossy, looking as if it is painted with lacquer. The

bottom of the cap is tan to buff and covered with tiny pores @erry et al, 1993;

Powell, 1990; Svcelg 1988; Wasson, 1968). The spores of GL are flat and ovoidal.

Their size is 6.3- 7.1x3.54.3 x23-25pm long (-ee et a1.,1986'1.

Recently, GL is one of the main health food items in Japaq China and USA.

Exacts of Ganoderma are a major worldwide, 'health shop' commodity where various

health benefits are displayed @efty et al, 1993). Several preparations ofGL are used.

Common general preparations are synrps and tinctures. It is also available in powder,

tablet and capsule. The powder preparation are used as an hertal tea and added to

soup or other foods (Huang 1993; Blumenthat 1993).

2.3.1 Chemical constitueDts of GL

The constiluenls of dried mycelia and fruiting bodies of GL were amlyzel

(qfla, 2540; Lee et al., 1986; Kawamat a et al., 1987; Chung e/ al., 1986; Nagaraji

and Kumar, 1987.Tserng et al., 1984). The contents oftotal ashes, total carbohydrates,

crude lipid, crude protei4 crude fiber, crude cellulose, ergosterol, germanium andCopyright by Mahidol University

Frapatsom Pakdee Litenhre Rwiew / l0

resin were 0.80-4.85, 2O-3O,3.34.6,8.3-23.6,50-65, 59.0, 0.3-0.4, G'16 allLd I.5yo

respectively. Among reducing sugars, maltose was the most abundant. Seventeen free

amino acids were deteclod, with glutamic acid, aspartic acid, and alanine having the

higkest vatue- GL also contairs vitamins 81, 82, 83, C, D, and pantotlrcnic acid aiong

wittq potassiunl calciurq iroq and manganese.

GL contains many chemical @mpourds that have various pharmacological

activities. Several anlitumor polysaccharides were isolated from GL (Iluang 1993).

Adenosine and its derivative (Shimizr' et al., 1985; Kawagishi et al., 1993), xeroid,

derivatives (Mia.rshina et al., 1998), and some enzyme such as fungal lysozyme,

protieruse Gtuang, 1993), and endopolygalacturonase (Kumari and Sirsi, 1971) were

obtained from the GL isolation. Lanostan type triterpenes @igure l) such as

ganoderic acids, ganoderenic acid, ganoderiol, ganoderd lucidenic acids, and

lucidones were lnown to be the main chemical; entity found in many parts of GL

(Lee and Rhee, 1990; Hrotani et al., 1987; )(tclo et al., 1993; tflrotani and Furuya,

1986; Kikuchi et al., 1986; Hirotani et al.,1986; Kikuchi et a1.,1985 lniszwa et al.,

1986). Peptidoglycan ganoderan (Tomoda et al., 1989) and Ling Zhi-8 protein

(Tanaka et a1.,1989) was also obtained from GL.


Fac. ofGrad. Sndies, l{ahidol Univ.

Gmodcric acid B

M.Sc. (Food and Nutritional Toxicology) / I I

Gemdcric rsid Z

cll2oHsl

Gendqenic ecid G Gmoderiol A

Goodcrid E hcilcnic rcid A

Figure 1. Chemical structures of Triterpenes in Ganderma lacidum

-\-!, -cF.*

'z "qt

Garodcric-itlX


Prapalsom Pakdee LiteraEre REview / 12

Lscid.nicecfol D LucidoncA

Ganodcric acid R

Trideacetytgaaoderic acid T Ganoderic acid A

Gaaodcrkecid C Gerodcric acid D

Figure 1. Chemicat structures of Triterpenes in Gonoderma lucidum (continued)


Fac. ofGrad. Studies, Ifahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 13

Ganodtral A Genoderot A

Ganodcrcl B Glnodsic acid K

Ganodrrh acid F Ganodcric acid E

G!trodctfu *id G

Figure t. Chemical structures ofTriteqpenes in Gonoderma lucidum (continued)


PrdpdsGtr kkdee

23.2 Phamacological Activities

Literature Rsriew / 14

Many pharmacological activities of several compornrds extracted from GL

were reportd. They could be grouped as follow:

2.5.2.1 AntihrmorActivity

GL atnac*ed great attention recently due to it could produce cornpounds

having aotitumor activity. Several polysaccharides isolated from GL were reported to

possess affitumor properties. The antitumor polysaccharides contain brancM 0-(l-+

3)-D-Glucan core in (l-3)-p-, (14)-P- and (1-6)-p-linkages 6l{iyazati ana Nishijimq

1981; Shiao et al., 1994;Etsr et al., 1987) (Fig:re 2).

iD

Figure 2, Possible repeating vnits of Ganodermo lucidum glucans.

In genera[ higtly branched fuagal glucan having p-( I -+6)-D-glucopyranose

units on a p-(l-+3)rD-glucao backbone was suggested to be amitumor active'

(Whistl€r et al.,1976;Miyazaki, 1983). Branching fiequency seems to be importart

for the activity (Miyazaka 1983). Th€ antitumor properties of tlese polysaccharides

were based on the inhibition of growth of Sarcoma 180 solid turror and many arltured

"rI


Fac. of Grad. Studie+ Ivfahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 15

tumor cell lines. The polysaccharides B-D-glucan isolated from water extract of GL

had antitumor effects in mice against sarcoma 180 (Miyazaki, 1983, Etsu et aL,7987;

Mzuno et al., t9&a;Mizrno et al., 1986). Modification of D-glucosyl groups of side

chains to polyol groups enhanced significanlly its tumor inhibiting activity (Sone e,

a/., 1985; E,tslr et al., 1987). The alkaline-soluble polysaccharides of GL were also

reported to have the tumor inhibiling activity against the sarcoma I80 cells (Miyazaki,

1983;Kim et al., 1980). Protein-bound polysaccharides from hot water.extract of the

cultured mycelia of GL was found to suppressed tumor growth of sarcoma 180

grafted in A-strain mice (Kim et al., 1993;Kang et al., 1987;lto et al., 1977). The

ethanol precipitable Ilaction of aqueous extract ofGL significantly increased the life

span of Luwis lung carcinoma-implanted C57BU6 mice, when they were

administered alone or in combination with antitumor drugs or immunomodulator

(Furusawa et al., 1992). GL extract showed inhibition of the lung tumor incidence

induced by benzo (a) pyrene in newbom N:GP(S) mice (Yun et al.. 1995).

It is believed that the polysaccharides act as biological response modifiers in

host immune system mechanisms against cancer. Their antitumor effects are believed

not to function directly on the malignant cells but rather to inhibit the tumor by

promoting an increases defense system such as clrtotoxic T cell, natural kjller cell, and

lymphokine-activated killer cell activity. Wang et al (1997) demonstrated that the

antiproliferation effect of GL polysaccharide to human myeloid leukemia cell lines,

HL{0 an<I U937, in vitro culture system was mediated by cytokines, TNF-<4 IL-IB

and IFN-y, releases from human macrophages and TJymphocytes. The resembled

result of GL induced immune mediated antitumor effect was supporting by Liat et al

(1ee2) Copyright by Mahidol University

Prapatsoro Pakdee Literahle Review / 16

2.3.2.2 Immunomodulatory Activities

Stimulating the non-specific activities of macrophage by polysaccharide of

GL was a mitotic activity and e4pression of surface interleukin-2 receptors on murine

macrophage cell line TIB 7l by dosedependent pattern. Polysaccharide from GL

(PSG) augmented the phagocytic activity of TIB 71 cells against fluorescent latex

beads (Kim et at.,1997).PSG increased secretion of cytokines (ILl and TNF) when

the peritoneal macrophages of BALB/C mice cells were primed and triggered with

BCG and IFN-T (Kim e, a/., 1997). Increasing in the activity of IFN-y was observed in

BALB/C mice treated with extract of GL (Li et al., 1994).

GL polysaccharides increased the synthesis of nucleic acid and protein and

promote cell proliferation in murine Splenocytes (Xiao er dl., 7994). GL

polysaccharides, BN3A BN3B, and BNrC, increased IL-2 production of activated

mouse splenoc5rtes in vitro. They completely antagonized the mild inhibition of

murine mixed lymphocyte reaction induced , by cyclosporin d mitomycin C,

fluorouracil or cytarabine and partially antagonized the more severe inhibition

induced by hydroco{isone (Huang 1993;Lei et al., 1993;Ma et al.,l99l).

The aqueous e)dract of the fruit body ofGL was found to prevent remarkably

the experimenlal asthma in guinea pigs and contact dermatitis induced by picryl

chloride in mice. It also exhibited markedly a therapeutic effect against the lesion of

kidney and decreased the protein excretion into urine, in serum sickness nephritis

caused by immune complex in rat (Mari el a/., 1986).

The extraction fiom GL spore inhibited delayed-type hypersensitivity

induced by sheep red blood cells (SRBC), 2,4-dinitrochlorobenzene or allotypic

splenocytes in normal mice. It also inhibited the proliferation of murine splenocytesCopyright by Mahidol University

Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritioml Toxicology) / 17

or human tonsillar monofllclear cells induced by different mitogens in vitro. lt

suppressed the graft versus host reactior. in vivo artd lhe mixed lymphocyte reaction of

murine splenocytes in vitro (7hzng et al., 1994). And it inhibited in vitro mwine cell

proliferation induced by ConA or lipopolysaccharide (LPS) atd IL-2 production of

murine splenocytes stimulated by ConA (Xao el aL, 1993).

A novel immunomodulatory proteiq Ling Zhi-8 (LZ-8), was isolated fiom

the extract from GL mycetial and expressed mitogenic capacity toward mouse spleen

cells and human peripheral lymphocytes (PBL) in rzrro (Murasagi et al., l99l;

Frendscho et a/., 1993). The proliferative respons€ in PBL cultures was primarily due

to T-cells, but was monocye dependent. Stimulation of PBL with LZ-8 resulted in the

production of IL-2 and a corresponding upregulation of IL-2 receptor expression. It

markedly enhanced the expression of CDllb on the U937 cell line in a dose-

dependent fashion and increased the expression of CD2 on human T cell leukemia

cell line MOLT4 (Myasaka et a/., 1992). It induced cellular aggregate formation. The

aggegate formation correlated with a dramatic rise in ICAM-I expression and an

increased production of IFN-y, TNF-c, and IL- I p molecules associated with

regulation of ICAM-I expression (Frendscho et al.,t993;Miyaszkaet al.,1992).LZ-

8 also acted as a potent suppressor of bovine serum albumin-induced anaphylaxis in

CFW mice ir vivo (Mwamg;i et at.,1991).

2.3.2-3 Antiplatelet Aggregating Activity

The water-sotuble fraction ofGL suppressed platelet aggregation induced by

thrombin and the inhibitory substance was identified as adenosine. The content of

adenosine in this fungus was at least 40 mg/1000 g of dried fruit body preparation.


Pr4atsom Pakdee Litemhrre Review / l8

Inhibition of pldelet aggregption reduced the incidence of blood clots and stroke

(Shimizu el aL, t985).

2.3.24 Eepatoprotective Activity

The protein-bound polysaccharide fraction (M.W. 5000-20000 kDa) of GL

known to have p-1,3 bonds reduced serum AST, ALT, ALP, toral bilirubin levels, and

hydroryproline content in the liver of BDUs rats treated with CCI+ and thioacetamide

(Park et al., 1997). The tredment with the water extract from GL was effective in

reducing the liver damage induced by CCI+ GL prevent the increase of serum lactic

dehydrogenase (LDtf), glutamic oxaloacetic transminase (GOT) and glutamic pynrvic

transminase (GTP) levels caused by CCl+ in rats (Lin et al., 7995). Moreover

combined administration of GL offact and glutathione was more effective than either

one alone in limiting liver damage from CCI+ in rats @yun and Kim, 1987). Alcohol

extracts ofGL aDtagonized the elevation ofserum GPT and the accumulation of lipids

in the liver induced by, injection of CCI+. The extracts inhibited the infiltration of

lipids in the liver caused by dl-ethionine and decreased the mortality rate of mice

given high doses of digitoxin and indomethacin (Liu et al., 1979).

A few purified triterpenoids compounds from cultured mycelium of GL,

ganodosterong ganoderic acid S, ganoderic acid \ and trideacethylganoderic acid T

protect against D-galactosamine-induced hepatic injuries in rats cells (Frnrya et al.,

le87)

2.3,2.5 Antioxidant Activity

GL contains certain substances which are powerful antioxidants and capable

of scavenging superoxide radical and hydroxyl radical. Superoxide radical and

hydroxy radical production was suppressed by the extracts from GL (Chen andCopyright by Mahidol University

Fac. ofGrad. Studies, Mahidol Univ- M.Sc. (Food and Nutitional Toxicology) / 19

7-hang, 1987;Lin et al., 1995). The inhibition of GL in injection form on the formation

of hydroryl radical in the HzOz/Fe3*-EDTA,/vitamin C system was clearly observed.

GL in injection form had a scavenglng effbct on hydroxyl radicals with dose-response

behavior. GL also had significant scavenging effect on superoxide radrcal in vitro and

enhanced tie action of rabbit plasma in scavenging of hydroxyl radical (Jifeng et al.,

l98s).

2.3.2.6 Antihistaminic Activation

The methanol extract ofGL has an inhibitory action on histamine release from

rat mast cells. From the physiologically active fraction of the extract, two histamine

release inhibitory triterpefles, ganoderic acids C and D, were considered as active

components of anti-histamine releasing factor (Kohda et al.,1985).

The chloroform extract from GL markedly inhibited histamine release from rat

peritoneal mast cells induced by compound 48/80 and A-23187. One of the effective

constituents obtained from the ct oroform extract of GLcultured broth is oleic acid

(Tasaka ef a/., 1988).

2.3.2.7 Cardiovascular Effect

The water extract from GL mycelia had definite effect on cardiovascular

system srch as reduction of blood pressure, renal nerve activity, and heart rate in

anesthetized as well as conscious animals. The hypotensive action of the extract was

secondary to the primary effect of the exlract on the central nervous systerq which

suppressed the sympathetic outflow pee and Rhee, 1990).

The culture fluid of GL showed no efect on the normal electrocardiogram;

however, it antagonized the electrocardiographic changes (T-wave exchanges)

induced by pituitrin in rabbits and was noted to be inhibitory to isolated fiog heartsCopyright by Mahidol University

Prapatsorn Pakdee LiteBture Review / 20

(Chen and Zhang 1987,Ding 1987). GL generally improves the tolerance of rats to

hypoxia, particularly that of the myocardium. It decreased the orygen conzumption of

the body. lt was shown to increase the arterial blood flow of isolated hearts in rats. GL

led to a greater difference between the arterial and venous partial oxygen pressure in

hypoxic rabbits. In hypoxic gurnea pigs, it maintained the myocardial ATP and

glucose at a relatively higher level. These findings indicated that GL possessed

beneficial properties to the myocardial metabolism of hypoxic animal (Chen" 1987).

GL increased the capillary circulation and the number of capillaries in rats under

microscopic observation (Huang 1993).

Sweral highly orygenated triterpenes from TOYo methanol extracts were

identified to be mild inhibitors of angiotensin converting enzyme (ACE). From this

extract ten lanostane triterpenes, ganoderal d ganoderrols A and B and ganoderic

acids K, Y, F, E B, D and S were isolated and characterized. All of these compounds

except ganoderal A aad ganoderols A showed inhibitory elfect to ACE, and ganoderic

acid F had the highest inhibitory effect (Olafsson et al., 1997;Mongiwa et al., 1986).

None of these triterpenes, however, were as active as the positive control captopril

(Shrao et al., 1994).

2.3.2.8 Hypolipidemic Activity

The compounds isolated from GL namely, ganoderic acid B, ganoderic acid

C, which have oxygenated groups on both the 7- and l5-positions and \{I, which has

no functional group in the side chain and has both 7-oxo and lScr-hy&oxy groups on

the same skeleton, showed potent inhibitory effect on cholesterol biosynthesis from

24,25-dihydrolanosterol. The target site of inhibition was suggested as lanosterol 14cr-

methyl demethylase (Shiao e t a 1., 7994;Komoda e t a 1., I 989)Copyright by Mahidol University

Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 2l

2.3.2.9 Hypoglycemic Activity

The water extract of the mushrooms of GL showed significant hypoglycemic

activity on intraperitoneal administration to normal mice and inhibited of the plasma

glucose level in glucose-loaded rats. Three isotated glucans named ganoderans A" B,

and C were the active principles in mediaed hypoglycemic effect (Tomoda el a/.,

1986; Hikino et al-, 1985; ffikino e/ a/., 1989). The potencies of these hypoglycemic

effects were not paralleled with those of the antitumor etrect Qlikino and Mizuno,

1988) Intraperitoneal administration of ganoderans A and B to alloxan-

hyperglycemic mice reduced plasma glucose level (Tomoda e/ a/., 1986).

Ganoderan B contains a backbone and side chain involving D-glucopyranosyl

p-1-->3 and p-l-+6 linkages, and that ganoderan C has a backbone and side chains

involving D-glucopyranosl p-1+3 and p-1-+6 linkages and D-galactopyranosyl cr-

1-+6 linkages. 1-+6Jinked side chains are attached to the l-+3Jioked backbone in

ganoderans B and C (Tomoda et a1.,1986) Sigure 3 and 4).

*D'cl'"tIt6

s_s-clrlII

Ii

,-D-qq!It6

€ t)-f - D-O.r- (!+r)-r-D-GlcP-(l-

Figure 3. Possible stnrctrral fragments of the polysaccharide moiety in ganoderan B.


Prapatsom Pakde€ Literahfe Re\,iew / 22

I

I6

,-o-GlcPI

I

L4 t -e -,--5,q.-nt-r-,-o{.-6*} [-"1-.-o-cerp-(r-],

Figure 4. Possible slructural fiagments ofthe polysaccharide moiety in ganoderan C.

Hikino (1989) found that Ganoderan B elevated the plasma insulin level in

both normal and glucose.loaded mice, enhanced the hepatic glucokinase,

phosphofructokinasg and glucose-6-phosphate dehydrogenase activities, decreased

the glucose.6-phosphate and glycogen sytrlhetase activity but did nor afect the

activities of hexokinase and glycogen phosphorylase. It also decreased the glycogen

content in the liver but had no inlluence on the total cholesterol and triglyceride levels

in the ptasma aod liver- .

2.3.2.10 Antimicrobial Activities

The antimicrobial activity of the aqueou extract from the carpophores of GL

was the most potert against Micrococcus luteus. The antimicrobial combinations of

Gl..with four antibiotics (ampici[irL cefazolirl oxytetracycline and chloramphenicol)

resulted in an additive effect in most instances. Synergism was combined with

cefazolin against Bacillus stbtilis aad Klebsiella orytoca (Yoon et al-, 7994).

The low molecular weight fraction of the aqueous extract GL basidiocarps

strongly inhibited cyopathic effect of human T lymphoblastoid cells induced by HIV-

9-o-q?III6

,-D-l, r


Fac. ofcrad. Studies, lv&hidol Univ. MSc. (Food and Nutritional Toxicology) / 23

1. High molectrlar weight fractions did not prevent any Hlv-induced cytopathic effect

at any concentration tested on this cell line (Kim et al-, 1997).

2.3.2.11 Radioprotective Activity

Continuous intraperitoneal injection ofwater extract from GL before or after

irradiation of 500 and 650 cGy of X-ray was found to improve the 30-day survival

fractions of ICR mice- The administrxion also enhanced the recoveries of the body

weights and increased the recovery of hemograms of irradiated mice from radiation

damage by injecting before or after radiation exposurg especially for the treatment of

500 cGy irradiation (Hx et al., 1990). Extract from GL had the enhancing effect

against thymus weight and leukocyte count and repairing activity to the damage of

subset T-cells, CD+ and CDs, in the spleen of y-ray-irradiated mice (Chen et al.,

1995q' Chen et al., 1995b).

2 3 2.12 Other Activities

GL significantly raised the 2,3-diphosphoglyceric acid levels in human

erythrocytes and improved the oxygen transfer function ofthe erythrocyes (Chen and

Zhang, 1987; lileng et al., 1985). D6-polysaccharide of GL hastened the

incorporation of 3H-leucine to plasma proteins, resrlting in an acceleration of plasma

synthesis in the Iiver (Chen and Zhang 1987). GL extract prolonged the sleeping time

induced by barbiturate and reduces body temperature in mice (Huang; 1993).

Adenosine isolated from GL inhibited central inhibitory redrled spontaneous motor

activity, prolonged the death time induced by caffeine and relaxed skeletal muscle in

mice (Kasahara and Hikinq 1987). The preparation conlaining GL extract and ,v-

amino-B-hydroxybutaric acid or cyproterone accelerated blood circulation and

activates hair follicles, and promotes hair growh (flasunr:ma, 1987; Miyamoto andCopyright by Mahidol University

Praparsom Pakde€ Literature Review / 24

Maenq 1987). Oral liquids for eyesight improvement contain GL extract, glutathione,

Polygonum muhiflorum extract, and ginseng extract. Clinical tests indicated that the

oral liquids markedly improved the eyesight of cataract and weak eyesight patient (Li,

lees).

2.3.3 Toxicity

The toxicity of GL is low. A potysaccharide fraction from hot water extract

was examined for acute and subacute toxicity. In the acute toxicity tests on mice, this

agent did not show any serious and tethal effects. The results showed that 5Wo lethal

doses were higher than 5000 mg/kg. The oral administration of this fraction (5000

mg/kg) to mice for 30 days showed that there was a not change in body weight,

hematological features and organ weight (Kim et al., 1986\. Moreover, tie oral LDso

for GL synrp was found to be 69.6 mVkg in mice, and 4 mVkg in rabbits (Huang,

19e3).

2.4 Nitrate and Nitrite in Foods.

Nitrate in the environment originates from many sourceq for examplg as a

product of nitrogen fixation by microorganisms and plants, as a decomposition

product of plant and s€wage wastes, from inorganic fertilizers used in agriculturg and

in foodstufs as a preservative in meat and fish Nitrite is available either as a natural

constituent after reduction of nitrate or as food additive in meats, fish" aad cheese.

Sodium and potassium nitrate and nitrile are used to preserve meat producls for tlre

purpose of retaining coloq improving flavor, and inhibiting growth and toxin

formation by Clostridium botulirum (Olajos and Coulson, 1987). The tofcological


Fac. ofcrad. Studieg lUahitlol Univ. Irv(Sc. @ood and Nutritional Toxicology) i 25

significance of nitrates lies in its easy conversion to nitrite by nitri$,ing bacteria that

may present in foodstufs, the saliva and in tie GI tracl (Cassens e/ d1.,1979).

NOI + NADPI{ fx*_-elffi NO2- + NADP* + HrO

Nitrate in bacteria Nitrite

Figure 5- The conversion of nitrate to nitrite by nitrifting bacteria.

2.5 Mutagmicity and Carcinogenicity of Nitrite

High doses of sodium nitrite induced reversed mutation in Salmonella

typhimtiun and caused chromosomal aberrations in culture Chinese hamster

fibroblast cells (Ishidate et al., 1984). Sodium nitrate did not catrse an increase in

single strurd breaks in cultured mouse cells but there was a dose-related increase in

gene mutations and chromosome aberrations at high concentrations, possibly due to

deaminarion of bases (Kodama et ol., 1976). Cbromosome aberrations were

significantly increased by nitrate in culture hamster cells (Tzuda et al., 1976) and,

increase in 6-TG mutanls were seen in V 79 hamster cells in vilro @udayova, 1985).

Eryosure of newbom hamster cells iz vifro to sodium nitrite at concentrations

of 2100 or 4200 mg NOz / I for 24 h resulted in anarploidy, chromosomal aberrations

and cell transfonnations (Tzuda and Katq 1977). It was also shown that the

embryonic cell @ltured of the fetus obtained from pregnant Slrrian hamsters given

sodium nitrate at doses of 125, 25O or 500 mg/kg body weight by gavage on days I 1

and 12 of pregnancy showed a dosodependent increase in micronuclei and in 8

azaquairne and ouabain-resistant mutants. Cell transformation was also observed iz

vitro and implicafion of transformed cells led to tumor dwelopment (Inui et al.,

1979\. Copyright by Mahidol University

Prryatsom Paldee Literah[e Review / 26

Negative results were found in a host-mediated assay against Salmonella

typhimuriun at a dose level of 150 mg sodium nitrite/kg body weight (Couch and

Friedman, 1975) and in a mouse micronucleus t€st- However, administration of

sodium oitrite in drinking water at a mncentration of l25O m$ to non-pregnant or to

pregnant fats (on days 5-18 gestation) induced chromosomal aberrations in the bone

marrow of the adr t Ooth preg@nt and non-pregnant) and in the embryonic liver. The

ratio of trumber of metaphases with aberrations in treated and cortrol admals was

higher for embryonic liver compared to a&rlt bone marrow. The higher incidence

might result from the higher numbers of milotic cells in embryonic tissues @l Naahas

et al.,1984). However, Wistar rats of both sexes were given sodium nitrite in distilled

water d concentration ofO to 3000 ml on 5 dayVweek for more than 100 weeks.

Papilomas of the forestomach were seen in 8/45 treated animals (the group receiving

sodium nitrite 3 I drinking water; total dose, 63/kg) compared \ith 2/91 controls

(Mirvis er a/., 1980).

Males Wistar rats given sodium nitrite in diet at concentrations of 0, 800, or

16@ mg/kg body weight for 90 weeks I out of 8 rats in the lower dose group

developed a benign liver tumor and 5 out of8 rds in t}e higher dose group developed

liver tumor, three classified as benign and two as malignant. The tumors were derived

from parenchymal or hemangioendothelial cells. However, the nitrite-containing dias

were found to contain NDMA (N-nitrosodimethylamine), NPYR (N-

nitrosopyrollidine) and the authors concluded that these preformed nitrosamines were

probably the principle cause ofthe tumors (Aoyagi et al., 1980).


Fac. ofGrad Snrdies, Ivfahidol Univ. lv{.Sc. (Food and Nutritional Toxicology) / 27

2.6 AminopyretreNitrite Mod€l for Mutagenicity Inhibition

l-Aminopyrene is a natural compound produced in human feces or in

anaerobic incubation of l-nitropyrene with fecal bacterial (tloward et al., 1983; El-

Bayoumy et al., 1983; Kinouchi et al., 1982). It was known to be mutagenic after

metabolic activation to strain TA98 and TAl00 but less mutagenic without activation.

The mutagenicity of this compound with metabolic activation was decreased on

tredment with nitrite u&ereas the activity was increased metabolic activation

(McCann et al., 1975\. The numbers of His* revertant colonies oa TA98 ,without

metabolic activation mix were 870 co1onieV0.022 pmole. The results indicaled that

this .compound was transformed into poteffial direct action mutagens on treatment

with nitrite. Kato et al. (1991) also demonstrated that mutagenicity of l-aminopyrene

could be potentiated by nitrite. In their experiment, l-aminop5rrene (0.2 mmole)

ifferacted with sodium nitrite (0.8 mmole) in 5 ml acetonitriie-water (l:l v/v), the

mixture was adjusted to pH 3 and incubated d 3TC for 4 h. Then 0.8 mole

ammonium sulfamate was added. The mi*ure was extracted with 5 ml ethyl acetate.

The mixture was analyzed with HPLC and found to mntain l-nitropyrene and

unidentified nitro-compounds. The mutagenicity of l-aminopyrene was one-tenth that

of its nfuo-introduced product, l -nitropyrenq in the Ames test (Ames er al., 1973a).

Howwer, in the living systenL l-nitroprene is converted back to l-aminopyrene by

microsomal enzyme @l-Bayoumy et al., 1983).


Prapalsorn Pakdee

s\XI DA

N#rrrr l..rltoDyrcEc

l*.#d!*d;*6>l.dEo6rtr l..&rlryira.

DNA rddxt (C{ Grrdae)

Figure 6. Suggesed pathway for activation of I -nitropyrene.

Heflich et aL (1985) demonstrated that the pathway for activation of l-

nitropyrene proceeds as illustrated in Figure 6. The critical first step, a ratelimiting

step, was enzymatic reduction by nitroreductase to l-nitrosopyrene. This was

followed by a srbsequent reduction to the corresponding hydrorylamine, a species

capable of nndergoing acid-catalyz-ed decomposition to leld a covalent adduct at

position 8 of guanine.

The metabolism of l-aminopyrene was studied in animals. It was given orally

to rabbits and the mrresponding .l/-arylformamides was isolaled from the urine

together with the corresponding N-arylacetamides (Tatsumi er a/., 1989). Such

metabolic conversion of the arylamines was also observed in guinea pigs and rats,

Quantitative experiments showed that only minor amounts of the N-arylformamides

and iy'-arylacetamides were excreted in the urine or feces ofrats and rabbits given the

arylamines. Liver cytosols from several mammalian species exhibited a significant ,V-

formylating activity toward the arylamines in the presence of 1f-formyl-L-kynurenine

and lf-acetylating activity in the presence of acetf{oA.

Literahlre Rs/iew / 28


Fac. ofcrad. Studies, Mahftlol Univ. M.Sc. (Food and Nutritional Toxicology) / 29

2.1 Tlne Solmonella Mutagenic Assay (Ames Test)

The simplicity, sensitivity, and acqlacy of this method for screening large

numbers of environmental sources of potential carcinogens has resulted in its current

use in over I,000 government, industrial, and academic laboratories t}roughout the

world- The potential ofthis method for use as a bioassay for the development of safe,

useful chemicals raised many questions about ttre Edent to which this kind of

approach should be used in a program aimed at cancer prevention. McCann and Ames

(1977) discussed several aspects ofthe experimental basis for their current as$ssment

of the value ofthe test as a useful predictive tool:

l. The predictive value of the lest as an indicator of carcinogenic potential,

including both the strengths and weaknesses of the test at this stage in its

development.

2. Current applications of the test method to problems that were not

approachable using conventional animal test methods.

3. Some of the environmental chemicals that have already been pinpointed as

potential carcinogens by the test and the curr€rit status ofcarcinogenicity tests of these

chemicals in animals.

4. The evidence that the correlation between carcinogenicity and

mutagenicity in the Salmonella test re{lected more than a useful mincidence and fitted

into a compelling collection of evidence supporting a central role for somatic

mutation in the initiation of human cancer.

't"he fulmonella test wali first validated in a shrdy of 300 chemicalq most of

which were known carcinogens (McCann et ol., 1975; McCann and Ames5 1977,

McCann and Ames, 19?6). It was subsequentty vatidating in studies by the tmperialCopyright by Mahidol University

Prapatsom Pakdee Literah[E Review / 30

Chemical Industries (Purchase et al., 1976), the National Cancer Center Research

Institute in Tokyo (Sugimura et al-, 1976), aod the Inlemational Agency for Research

on Cancer (Bartsch et al., l98O). Nearly 900/o of the carcinogens tested were

mutagenic in these studies. However, Ames and McCann (1981) estimated the

correlation to be about 83%o. All the validations show that the test fails to detect a few

classes of carcinogens such as polychlorinated pesticides (Ames and McCaruU 1981;

Rinkin and l-egator, 1979; Rinkin and Legator, 1981). Prior to the initial development

of the Salmonella./microsome assay there were several studies that ernployed bacterial

systems to detect mutagenic agents @emerec et al., l95l; Lyer and Szybalski, 1959)

However, one ofthe problems with these earlier approaches was the use of screening

techniques that did not employ bacterial strains designed to detect broad range of

mutagenic mechanism. Thereforg Ames (1971) are developed a set of Salmonello

llphimurium strains that are permeable to a wide range of chemicals and also are

partially deficient in DNA repair.

2.7.1 The Salmonella Tester Strains

The reverse mutation system of Salmonella typhimurium uses the genetically

well{efined histidine requiring mutants developed by Ames and his colleagues

(Ames et al.,l973b\.The klmonella histidine reverse mutation is based on the use of

several selected Salmonella tlphirmznnrz strains that revert from histidine dependence

(auxotroph) to hisidine independence (prototroph) . Ames et al. (1973b) collected and

characienzd a large number of Salmonella typhimnriazr strains containing mutations

in different gene of the histidine operon (Table 5). The newly standard tester strains

contain other mutations that gredly increase their ability to detect mutagens such as:


Fac. ofcrad" Studieq lvhhidol l-hiv. fvLS& (Food ed Nfiitioal To<iolo$/) / 3l

rft mutation which causes patial loss of tb lipopolysaccharide barrier tlat

cod tlrc swfrce of the bacterh atrd increases permeability to Iarge rmlecules that do

not peretrde the rormal cell wall (Aw et al.,l973a).

- nur B mrtation is a detection of a gere coding for tbe DNA excision repair

system, resulting in increasd sensitivity in d*ecting rumy mutagens (Ames e/ a/.,

1973a; AM et al., 1973b),

- R-&c'tor plasmid GrKM 101) The strains containing the phsmid show

greatly enharced rcsponse to chemical showtr to be mdagenic ard also give clear

positive r€sponse to ch€mical described as weak, borderline or nonmut4g€ns with the

onSinal set of tester srains (McCann et al., 1975; Mortelmas aud socker, 1979).

Furthermore, MacPh (1973a 1973b) @lied that pKM 101 contains genes products

associated with error-prore repah, which may be responsible for the enhanccd

sensitivity seen in these strains.

Genot5pes of tlr,, fulnorclla typhimurium saains used for rnrtagenesis

te$ing are showed in table 3. Tbe sAndard tester strains: TA97, TA98, TA100 and

TAl02 contain tbe R-&ctor parc,m strains (McCenrrr et a1.,1975;I*vA 1982).


hapatsom hkdee Literature [Gview / 32

Table 3. Genot5pes ofthe Salmonella typhimzrizm strains used for mutagerrcsis testing

Histidine mutation

hisD66l0hisD3052

HisDl242

LPS Repan R-fictorHisc,l6 hisG428

TA9O

lTAeTI

TAIlO

TA98

TA1535

rrAesI

TA1978

TA94

TA1534

TA196/.

TM@l

TA1535

lTAl00I

TAl975

TA92

TAt950

TA24IO

TA1530

TA263l

[rAl02]

th

th

rE

+

+

+

Lgal

LSal

rh

AuwB

Ni:urB

+

LuwB

LuwB

LuwB

LuvrB

+

-R

+R

-R

+R

-R

+R

-R

+R

+R

Tester strains in brackets are rmnrmended for general mrtagenesis testing.

All sraios werc originally derived from Salmonella typhimurium LT2. Wild-type

genes are idicated by a +. The deletbn (A) through zvr B also ircludes the nitrate

reductase (cftl) ard biotin (bio) gerrs- The AgaI *rains ard the rhluw B strains have

a single deletion tkorgh gal chl bio uw B. The ,re repair - srains have a mutation in

gal E.R: pKM 101. Tbe testef, strain TA1536, included in the original tester set

(Anes et al., 1973a), and all other strains containing the histidine mutation his C207

werc discontirnred dre to the lack of specificity on few mutagens reversion and tester


Fac. of Grad. Studieg Mahidol Univ. ,, I,:"r . i M.Sc. (Food and Nutritional Todcolory) / 33

' -L t;i" lir'r"

strains TA97 replaces TA1537 and TA2637. Genotypes of these discontinued strains

and ofother derivatives ofhis C3076 were listed by Ames et al.

Therefore these standard tester strains are recommended for mutagenesis

testing. TA98 was derived from TA1538 by which plasmid pKM 101 was introduced.

lt can detect mutagens tlrat cause frameshift mutation due to its DNA sequence

(-CGCGCGCG-), which can be rwerted to histidine independence by a variety of

mutagens affected addition or deletion of the base pairs (Isono and Youmq 1974).

TA100 mntaining R-factor plasmid derivative of TA1535 can detect mutagens that

cause base'pair substihrtions. The others Salmonella strains related to these 4 strains

containing different characteristics in terms of DNA-repair capacity, cell permeability

and presence of plasmid pKM101 are also available and have been described

(Mc,Cann et a1., 197 5 ; I*it et al., 1982'1.

Ilowever, some mut4gens afect orily one strain of frameshift mutation strain

(TA1538 or TA98) or base-pair nrbstitution strains (TA1535 or TAl00), thus

imparting a degree of mutagen specificity to the assay. But, many or even most

mutagens can affect both types ofstrains at the different effective dose for each strain.

Mutagen specificity, thereforg is frequently associated with quantitative rather than

qualitative response @unkel e/ a/., 1984).

2.7-2 Evaluation Criteria for Ames Assay

The Ames assay .used

to waluate the mutagenicity of test article is

semiquantitative. The criterion used to determine positive effects was inherently

subjective and based primarily on a historical database. The positive results in the

present study were based on following criteria according to the Yahagi's

recommendation If the solvent control value was within the normal range a test

r lttrl)Jt


Prapatsom Hkde€ Literature Re!'iew / 34

article that produced a positive dose-response relationship over two corcentrations,

with the highes increase was not less than twice the solvent mntrol was considered

mutagenic (Yahagi et al., 1975).

The criteria used to determine positive effects were inherently subjective and

based primarily on the information shown in Table 5. Most data sets should be

evaluated using the following criteria @rusic( 1982).

a. Strains TAl535, TA1537 and TAl538. If the solvent control value is

within the typicat ratrge for the laboratory, a test article that produces a positive dose

response over thrce mncenlrations, witi the highest increase equal to three times the

solvent control valug is considered mutagenic.

b. Strains TA98 and TAl00. If the solvent control value is within the normal

range for the laboratory, a test article that pmduces a positive dose response over

three concertrations, with the highes increase equal to tw'ice the solvent cortrol

valug is mnsidered mutagenic. Occasionally a doubling is not necessary for TAl00 if

a clear dose-relaled pattem is observed over several concentrations.

c. Pattern Because TAt535 and TA100 are derived from the same parental

strain (Ga6), and because TAl538 and TA98 are derived from the same parental

strain @3052), to some extent thee are a built-in redundancy in the microbial assay.

In general, the two strains ofa set respond tothe same mutagen, and such a pattern is

sougtrt. Generally, if a strain responds to a mut€en in non-activation tests, it should

do so in activation tests.

d. Reproducibility. If a test article produces a response in single tests that

cannot be reproduced in addilional runq the initial positive test data lose significance.


Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food ad Nutsitional Toxicolory) / 35

The preceding criteria are not absolute and other extenuation factors may

e ter into a final evaluation decision However, these r:riteria could be applied to the

majority of situations and are presented to aid those individuals not familiar with this

procedure. As the database is inoeased, the criteria for evaluation could be more

firmly established. It must be emphasized that modifrcations of the procedure

involving preinobation conditions are neoessary for evaluation of specific chemicals

or classes of chemicals.

2.7.3 Method used for Detecting Mutagens

There are three methods that have been used for testing mutagenicity of

chemicals. These methods are spot test, plafe incorporation test and preincubation

method.

2.7.3.1 Spot test.

The spot test is the simplest way to test compounds for mutagenicity and is

useful for the initial rapid screening of large numbers of compounds. This test is

primarily a qualitative test and has distinct limitations. It can be used only for tesing

chemicals that are dimrsible in the agar. ft is be confirmed by demonstrating a dose-

response relationship using the standard plate incorporation test.

2.7.3.2 Plate incorporation tcst.

The test is the standard method that has been used for the mutagenicity of

chemicals. The test consists of combining the test compound and the bacterial tester

strain in soft agar, which is poured onto a minimal agar platg after incubation at 3/C

for 48 [ revertart colonieq are counted (Ames er al., 1973t, Ames ef al., 1973b;

Ames et al., 1975a). For initial screening chemicals were tested in concentrations over

a threeJog dose range. A positive or questionable result should be confirmed byCopyright by Mahidol University


demonstrating a dose response relationship using a narrower range of concentrations.

In a modification of t}le plate incorporation procedurg a preincubation step precedes

addition of the top agar. This modification is better for some compounds and appears

to be at least as good for other compounds lested (Ames et al., 1973a; Ames et al.,

1973b).

2.7.3.3 Preincubation method

The significant finding of preincubation assay was of equal or greater

sensitivity than the standard plate incorporation assay, when the mutagenic activity of

aflatoxin 81, benzoding benzo[a]pyrene, and methyl methanesulfonate has been

determined by both plate incorporation and preincubation procedure. Some mutagens,

such as dimethyl and diethylnitrosamine are poorly deteaed in the standard plate

incorporation assay a modification of standard procedures. The fact that the test

componnds and bacteria were incubated 20-30 minutes at 3fC at the higher

concentrations before adding into top agar provided more sersitivity of mutagenic

daection. The preincubation assay was first describing by Yahagi et al. in whrch

carcinogenic azo dyes were found to be mutagenic {Pival et al., 7979; Yahagr et al.,

1975).

The preincubation modification can be used routinely or when inconclusive

results are obtained in tlre standard plate incorporuion assay. This assay required an

e*ra step and therefore involves more work than the standard test but many

laboratories have been used to detect mutagenicity of l0 carcinogenic nitrosamines

(Yahagi et al., 1977) and several carcinogeaic alkaloids (Yamanaka et al., 1979). It

was used in screening assays have also been recommended @e Serres and Shelby,

1e7e). Copyright by Mahidol University

Fac. of Grad. Shrdieg Mahidol Univ. MSc. (Food and Nutritional Toxicotogy) / 37

2.73.4 Positive control (diagnostic mutagem)

In each experimen! positive mutagenesis controls were used for diagnostic

mutagem to confirm the reversion properties and specificity of each strain. The

characteristic reversion pattems of the standard stains to some diagnostic nrutagens

are described (Maron and Ames, 1983).

2.7.3.5 Antimutagenicity Tcst Usirg Ames Test

Considerable evidence indicated a strong .association between the

carcinogenicity and mutagenicity of chernicals (McCarm and Ames, 19761' Kawachr et

al., 1979). Since the antimutagenic activity has also been correlated with

anticarcinogenic activity ofvarious compoundq it should be possible to use mutagen-

testing procedure or Ames test for screening various compounds for antimutagenic

and hence for potential anticarcinogenic efects (Reddy et a1.,1983; Rahirrtula er a/.,

1977; Speier et al., 1978; Wattenberg, l9il7).

2.7.3.6 Cfitefii of Artimutagenic Activity

The antimutageric effects eryressed as inhibition (%):

Percentage ofinhibirion : (a-b/(a-c)x 100

where a: number of histidine revertants induced by the positive mutagen (product of

AP-nirfte reaction); b : number of histidine revertants induced by the positive

mutagen in the presence of sample; c : number of revertants oF the negative control

(spontaneous reversion).

Interpretation of data: compounds were classified as

based on the o/o iohibifion of the mutagenicity of tesed

qualitatively ranked according the following scheme:

positive antimutagen

mutagen. Dala were


PrqAsorn Pakdee Literatue Review / 38

Y" inhibition

>60 Yo

6040%

40-20Yo

204Yo

Ranking for arrtimutagenicity

Strongly active

Active

Weakly aclive

Not active

Positive antimutagens should be dose-responsive or positive dose-response

relationship (Calomme et al., 1995).

2.E Somatic Mutation and Recombioation Test (SMART)

The somaiic mutation and recombination test (SMART\ of Drosophila is a

rapid and sensitive test which detects mosaic spots produced as cellular clones on the

wings during tle fly's development as a consequence of several genetic mechanisms:

point mutatioq deletioq mitotic recombination and chromosome non-disjunction.

This eukaryote ofers the advantages of having a short generation timq needing very

inexpensive culhre media and allowing the breeding of large numbers of animals

using simple facilities. In additiol, it is well established that Drosophila possesses a

yersatile system for the metabolism of xenobiotics (Baars, 1980; Hallstrom e, ar.,

1984). The SMART provides a suitable substitute or at least a complementary rir viuo

method to mammalian in viw inve*igation. Drosophila has detoxification-activating

systems in many respects closely resembling the corresponding sysems in mammals,

which makes it possible to extrapolate data to mammals @aars, 1980; Hallstrom el

al., 1984; Zijlstra et al., 1984). The use of SMART assays is based on the featoent of

larvae, and besides the number of mutated spots appearing in the adult flies,Copyright by Mahidol University

Fac. ofGrad. Studieg Mahitlol Univ. lvlSc. (Food and Nutritioml Toxicolory) / 39

indicating the frequency of genetic eveots, the size of the spots indicates the time of

action during embryogenesis. The SMART is also able to d€tecf mutagenicity of

unstable mutagens (Graf et al., 1984), pesticides (Torres et a1.,7992), food colorants

(Tripathy et al., 1995) and m-administration of some agents with mutagens

(Abraharq 1994;Negishie/a/., 1994;NegishieraI., 1989;AbralramandGra{, 1996).

2.8.1 Principle of the Somatic Assays:

Drosophila melanogaster as a dipteran insect develops through successive

developmental stages of differenr duration. Drosophila undergoes complete

metamorphosis:(duration I day *. the optimal crrlture temperature of 25"C), l$ larval

instar (Ll, lday), 2d larval instar (L2, lday),3d larval instar (L3, 2days), and

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

to 40 days) (Ransorq 1982; Ashburner and Wriglu, 1987). During embryogenesis

primarily larval tissues (cuticle gut, fat body, nervous systefiL etc.) arc formed, and

during the larval period these tissues orlarge and finally form the body ofa large L3

larva ready for pupation. The adult structures (wings, legs, eyeg etc.) are formed in

the pupal stage from the so-called imaginal discs. These develop during larval life as

proliferating entities derived from groups of cells set apart during embryogenesis.

Such discs grow during the larval period by cell proliferation. The cells of the

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

syncytiurn, which happen to migrate to a given region of egg cortex. After cytoplasm

and membranes have surrounded these nuclei, the corresponding cells become

developmentally segregated from the reighboring 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: ProliferativeCopyright by Mahidol University

Prapatsom Pakdee Literatrre Review / 40

growth starts in the first instar and continues tl[oughout the larval period. Cell

proliferation is logarithmic in all the prenrmptive adult cuticular cells, although the

number ofcells per clone deviates fiom 2"d. On average, cells divide every 8.5 h and

growth is complete after 9-10 cell divisions. After pupatioq mitoses are still

detectablg but somatic crossing over initiated at this time results in single marked

cells. Twenty-four hours later, mitosis ceases altogether and visible cell differentiation

begins. During differemiation about 5q000 cells give rise to single identifiable

cutierlar processes organized in the B?ica1 adult pattern.

During disc growth in the larval stage if a wing imaginal disc cell is

genetically altered into a rutant fornr, a group of mutant cetls will result from clonal

6expansion during disc growth. After pupaion, in the course of metamorphosis ofthe

imaginal disc into an adult wrng the rmrtant phenot)?e will become expressed. The

mutant clone will be recopizable as a group of phenotypically ahered wing blade

structure (called a "spot"), e.g. showing multiple hairs instead of the single hair

forrned by each wild-type wing cell.

2.8.2 Genetic Basis of the Elfects Detected

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

destitrd 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

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

expressed autonomoqsly in wing cells. The srposed cells ofthe larval wing imaginal

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

chromosome 3. Multiple wing hairs (mwh\ are at position 0.0 cM and flare (/y') at

39.0 clrd, while the centromere is located at 47.7 cM (Lindsley and Grell, 1968;Copyright by Mahidol University

Fac. ofGrad. Studies, Mahidol Univ. MSc. (Food ad Nutitioul Toxicology) / 4l

Garcia-bellido and Dapenq 1974). The appeararce of multiple wing hairs is a

homozygous viable recessive mrtation producing multiple trichomes per cell instead

of the normally unique trichome. The second marker, flare Utf), u" a zygottc

recessive lethal but homorygous cells in the wing imaginal disc srrvive and lead to

wing blade cells with short, rlickened and misshapen triclromes. In stock cultures,

thefll alleteshave to be balancdover inverted chromosomes srch as TMI or TM3

because it is a recessive zygotic lethal. In all the erperimental series analyzd the

occrrrence of the various types of spots was as follows: most frequent were single

spots expressing the rm* phenotypg less frequent twin spots with both a tmvh and a

/l ub-clone, and quite rare single spots with the/y' phenotype.

There e>rist several mechanisms that lead to genetically marked clones (see

Figure 7). An important possibility is a mitotic recombination event between two non-

sister clromatids. Twin spots are e:<pected if recombination occurs between Jlf and

the centromere (Becker, 1966). A recombination went between mvh afi Jtt' rrlrry

result in a mvi single spot. If both types of recombination events (one between //and the centromere and a second between mwh olrrd ftf\ uke place within the same

cell, an// singte spot may result.

A "twin spot" is an indicative of a recombinational event. Nondisjunctional

or otler loses of the cbromosomes carrying the wild type allele represents another

mechanism that may lead to single spots. tvttotic recombination ia the chromosome

section benveen the centromere (spindle fiber arachment site) and the marker /y'leads to two dalglrter cells, one homozygous for nmft, the other homozygous forl1.


Prapatsom Pakdee

t C.ll 2C.lE E \suingQk Es

Lilerature R.eview / 42

zcells E'lslirlg Clon61 Cell

- mwll +^,t&t + ,----1

-

: /, il,.m*h +-\.,,. n.7X..-- ---?;{ .-..- \ nY{t\ +o

----+ r----mr

L .,4 ,4,

{, ,4 ,4IJNUnspd

",!r'/'oorrnal

'r:r"/"

- rNh +^mwh -

--?

-

-- 7; -n'nr/,. -"v,* n a,,'\-- -*{- \ d'kr' +o

--J ----ic-aOeHioo

",lri"*slTEle

, spoa

ltli4lt.t /,

singlespot

ent +

6wh r --'--)

---- <J

---, ---o > _{

--------------- ) -/'mrtr

* n a,,'\-,-mrr .- \ m*tt +o' >m---r.Foht rr{J(atir,

I. mkt +^

t$t,r.+---P.!

* &a--. \:l "*tt

*,-.,

- -=r-...---; r----lr----);----r,rN.rrdk*fdio{t

,ii*l II I srrEEspot

oo o.d fa0 id

'r"'i'"*E"lg|e

, sod*li*l,1JJ

Figure T.Genetic schemes illustrating various ways of spot formation in the somatic

mutation and recombination test wirh ilre wing cell markers multiple wing hairs

fnwh) and flare (/tl). Twin spots are obtained by recombinarion proximal to the JF

marker (b), while more distal recombination produces muh single spots only (d).

Deficiencies (cl point mutations (e) and nondisjunction evenls (f) give rise to tm)h

singls spotq or in analogous ways to fll siqgle qpots (not illustrated) (Graf et al.,

1e84)

Clonal e4pansion to these two cells will be recognizable on the wing blade

from the two nn:lticellular adjacent clones, one exhibiting the mwh phenotype

(multiple hairs) the aher thefll phenotype (misshape hairs).


Fac. of Grad. Studies, Mahidol Univ. MSc. (Fmd and Nutsitional Toxicolog,) / 43

On the other hand the origin of "single spots', showing either the raw& or the

/y' phenotypq cannot be clearly determined. Multiple wing hairs single spots may

result from a recombinational eveat occr:rring in the chromosome segment between

tlre two marker genes. But also a gene rnutal.lron or deletion of the tmvht gene will

result in a mwh single spot. Afil single spot may either result fiom a gene mutation

or a deletion of the Jtl gene, or from a rare double recombination with one'

recombinational event to the Ie& and the other event to the right of the/l locus

(Wurgler e/ a/., 1991).

2,83Approach of SMART

Three crosses of flies carrying the rnarker mth and fil on the left arm of

ckomosome 3 were generally sa up:

l. Standard uoss:Jf nn OLD IM3, ri f sep blk e' Ser virgin females

mated to mwh males. This is the reciprocal cross ofthe standard cross used previously

(Moraga and Graf 1989; Van Schaik aad Grat 1991).

2. High bioactivation QIB) cross: OR& ltl/IM3 females crossed with ORR;

mth males. This is the reciprocal cross of the one described by Frotich and Wurgler

(1989). A number ofpromutagens showed increased genotoxicity when the HB cross

was used compared to standard cross (Frolich and Wurgler, 1989; Frolich and

Wurgler, 1990; Frolich and Wurgler, 1990). However, the HB cross presents a

number of difficulties and disadvantages (Frolich and Wurgleq 1989; Frolich and

Wurgler, I99l). These are: (l) The presence of an irregular whorling in the pattern of

wing hairs making spot classification difficulq especially for inexperienced scorers,

(2) an undesirably high variation in resufts from repeated experiments, (3) the low egg


Praparsorn Pakdee Literattre Review / 44

production of the females used and the detay in development of the larvae of HB

cross.

3. knproved HB cross: ORR Jtl /TM3 females crossed with mwh males

(GraF and van Schaih 1992)- The main advantage of the improved IIB cross is to

combine tie high bioactivation mpscity with the ease of scoring the wings using the

same criteria as for the standard cross. The hybrid larvae of the improved IIB cross

show P450-dependent activation capacity equal to or even slightly higher than those

of the original HB cross. In additiorl the I}IB cross is more sensitive than the standard

cross in evalualing the genotoxicity ofpromutagens (Grafand van Schailq 1992).

Figure 8. Marker mutations of wing surface to show clone of cuticle secreted by cells

homozygous for multiple wing hairs, a) small single spotq b) flare on wing veirl c)

twin spots, d) large single spots.

I i,, ., 1---t-ry' r'L2--J_=

'f,r'<

'drl's

ffilit


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food md Nutritional Toxicology) / 45

2-8.4 Standard Mutagens for Mutagenicity of SMART

Urethane is generally used as positive standard toxicaots in evaluation

genotoxicity of the unknown compounds in SMART (Abraham and Grat 1996).

These chemical required metabolic activation to express their mutagenic activity

(Frolich and Wurgler, 1990).

2.8.4.1 Urethue (ethyl carbamate)

Urethane (NH2COOCH2CH3), also known as ethyl carbamatg is the ethyl

ester of carbamic acid (NHzCOOID. Urdhane may occur as a colorless, odorless

crystal or a whitg granular powder. It is slightly soluble ifl olive oil and soluble in

water, ethane, ether, glycerol chlorofonn, and Atryl ether, In the 1940s, urethane was

used as a hyprotic in man at doses of I g/persorlday and as an anesthetic for

laboratory animals. In 1943, it was discovered that uethane had a carcinogenic effect

in animals. It is regarded as an initiator, but also as a cocarcinogen and specifically as

a promoter of radiation-induced cancer @erenblum, 1982). Since 1948 it has been

lnown that urethane is mut4genic in Drosophila melantogaster. Today, humans are

exposed to urethane in their food and mainly alcoholic beverages.

oHC{H -o{-**322

(a)

ollqc-C}}N-N11

(b)

oHp-{H-O

olt

{-NH2

(c)

Figure 9. (a) Urethane (ethyl carbamate); (b) Vinyl carbamate; (c) Vinyl carbamate

epoxide. Copyright by Mahidol University

Prapalsom Pakdee Literatue Review / 46

2"8.42 Metabolic Activation and Detoxification of Urethane

Urethane was found to induced point mutatiorl gene conversioq

intrachromosomal recombination, chromosomal aberrations and sister chromatid

exchanges in yeast, plant sysems and mammalian cells (Schlatter and Lut4 1990).

Urethane exerts its carcinogenic effect following bioactivation to vinyl carbamate and

then to vinyl carbamate epoxide, which forms RNA and DNd adducts and initiates

tumorigenesis (Dahl et al., 1978; Leithauser et al., l99O). The activation of urethane

is important in exerting its carcinogenic efect. The two-step oxidation of urethane to

the active vinyl carbamate epoxide is catalyzed primarily by cytochrome P-450

subtype 2El (Grengerich et al., l99l). The metabolism of urethane is shown in

Figure 10 urethane is metabolized by two different pathways- The major pathway,

accounts in rodents for over 90o/o, is the hydrolysis of urethane by microsomal

esterase and amidases to ethanol, ammonia and carbon dioxide (Mirvis[ 1968; Park

et al., 1993). This major pathway is probably one for detoxification.

The minor pathway involves the oxidation of urethane via cytocbrome P-450

subtype 2El (CYP2E1) to 2-hydroxyethyl carbamate, to N-hydroxyethyl carbamate

and to vinyl carbamatg which is in turn converted by epoxidation to the putative

ultimate carcinogen vinyl carbamate epoxide (Guengerich and Kirq 1991; Miller and

Miller, 1983; Guengerich et al., 1991). Vinyl Carbamate epoxide is a major strong

ultimate reactive electrophilic, mutagenic and carcinogenic metabolite ofurethane and

vinyl carbamate in mouse (Park er al., 1993). Generation of the electrophilic vinyl

carbamate epoxide leads to the formation of RNA and DNA adducts and the initiation

oftumorigenesis (Leithauser er al., 1990)


Fac. ofGrad. Studies, Mahidol Univ.

ACTT\rATION

IU-SC. Cood and Nutritional Toxicology) / 47

INACTI\.ATION

/ ---fu.> .!c<l.ra +cor +NrlI Asid-a,

S j.-gt=?;*i,-o.--.,i*r\ccr!€{I['---\

I I --.*1..,-*..*o. I grx *=+) 3| -o-," *. I t qc<q-€+rxolr| ' I -t-----EJig- I

',,ji."* ---==#-+ n<,*cq*N*

L*"-_

l"'" "* +, oai.a e-d!.aorc

..aEing..qour&

-;s+:ffi> --\/<+cq+Nrt+3 oatcr!&.ia8 o.d?o.d

+ C!r.drd. ?

o!..-"."-*sl /)----- cs -------? *ufr,-..+\{,4\

t/\aroE-crl), o\.Eo<(,9!+s!fr.coor{*6*

qqr-.o{.'q"o

I

I

+D!{A..'B-.68 t

Illifiodfdr-.t'r5t

Figure 14. Known and probable activation and inactivation pathways of metabolism

of urethane (ethyl carbamate), vinyl carbamate and vinyl carbamate epoxide. (a)

Mouse liver microsomes + ethyl carlmmate or vinyl carbamate + adenosinel l*lf-ethenoadenosire. (b) Human liver microsomal cytochrome P450 ml. (c) Vinyl

carbamate epoxide + adenosine* l Jf-ethenoadenosine. GSH : gtutathione @artq

1ee3).



2.8.43 Mutagenicity of Urethane

Many studies were published concerning the mutagenicity of urethane in a

wide range of org;anisms (Field and Laqg 1988; Kada and Ishidate, 1980). In tests

with eukaryotic cells, positive and negative findings were about equal in frequency. It

seems that positive results are obtained only under conditions of appropriate

metabolic activation- Urethane was genotoxic in the somatic mutation and

recombination testin Drowphila melanogaster (number and shape of wing hairs after

treatment of larvae), in a standard strain and in a strain in which genetic control of

cytochrome P-450-dependent enzyme systems were altered (constitutively increased

P-450 enzame activities) (Frolich and Wurgler, 1990; Frolich and Wurgler, 1988).

The effects were dose-dependera and the modified strain was more sensitive to

urethane by about one order of magnitude than the standard strain. This further

suggests that the P-45O enz5rme system is involved in the activation of urethane. More

than l0 publications are available with quantitative data on DNA-adduct formation by

urethane. Sweral authors proposed a metabolic pathway, which leads to the formation

of vinyl carbamatg and, after epoxidatiorq to DNA and RNA adducl (Irilhauser e,

al., 799O; Miller and Miller, 1983; Ribovich et al., 7982). Since 7{2-oxoahyl)-

guanine is also formed by vinyl chloride (Liab et al., 1981). In bacterial test systems

the results were mainly negative. An explanation for this may be that in the standard

Salmonella/microsome assay, using ratJiver S-9, there was inzuIficient oxidation of

urethane to vinyl carbamate (the first step in the metabolic activation) to give positive

results. The fact that vinyl carbamate gave positive results in the Ames test @ahl et

a/., 1980) strongly supports this hypothesis. In additiorq there is a vast literature on

urethane carcinogenicity (Mirvictr" 1968; IARC, 1974; National cancer Institute,Copyright by Mahidol University

Fac. of Grad. Studies, Ivfahfulol Univ. IU.Sc. (Food and Nutritional Toxicology) / 49

1978; National cancer Institutq 1979-80) and urethane is a pluripotent carcinogen

with respect to tumor induction in different species, orBans, and the stages of

development of the animals (Zimmerli and Schlater, l99l).

2.8.4.4 Modilication the Mutagenicity of Urethane

A number of compounds have been found to be good inducers of cytochrome

P-450 zubtype llEl (CyPzEl) including ethanol, which can increase the metabolism

of urethane or decrease it depending upon the condition of exposure (Kwata et al.,

l99l; Yamamoto et al., 1990). Acute administration of high doses of ethanol may

postpone the metabolism of urethang possibly by blocking metabolizing enzymeq

including the group of cl,tochrome P450 (Waddell el al., 1987- Yamanoto et al.,

1988). Chronic administration of ethanol, in contrast to the amte situation, may lead

to induction of metabolizing enzymes systems such as P-450 (Lieber et al., 1987) and

thus modulated the carcinogenicity of urethane. Mirvish (1968) reported that

degradation ofurethane was inhibited up to 90 % by blocking esterase activity, which

indicated that ethanol may be formed in near equimolar amounts to the administered

urethane dose. It rernains to be shown whether the ethanol thus formed can modulated

the further metabolism of urethane. Kurat? et al. (l99l) demonstrated that acetone

was a v€ry potent, aorte inhibitor of the m rno metabolism of urethane when

metabolically derived from 2-propanol. Conversely, pretreatment using acetone for 24

and 48 hours before urethane, administration accelerated the clearance of urethane,

indicating that enzyme metabolizing uret}ane was induced by acetone.

Urethane is also metabolized by esterase to yield ethanol, carbondioxide and

ammonia-.Carlson (1994) shown that the cytochrome P-450 inducers, Phenobarbital,

p-naphtholflavonq esterase inhibitor, and paraoxoq were without effect (to theCopyright by Mahidol University

Pr4atsom Eakdee Literature Review / 50

conversion of (carbonyl-raC) ureth,ane to raCO2) while the CYP2EI inhibitor,

diethyldithiocarbamatg decreased rnetabolism to about 3olo of control. Ethanol

administered acutely inhibited urethane metabolism. Pyriding shown previously to

enhance this metabolism in microsomral preparaliong greally inhibited il in vivo.

Kemper et al. (1995) investilgated the role ofglutathione in protection against

vinyl carbamate epoxide-mediated adduct formation, and the irwolvement of

glutathione'S-transferase in detoxification of vinyl carbamate epoxide. They reported

that glutalhione inhibited formation ofethenoadenosing a measure ofvinyl carbamate

epoxide toxicity, in a concentration<lependent manner at concentration ranging from

I to 8 mM. This efect was significanitly enhanced by addition of rat liver glutathionc

S-transferase- In addition, pretreetment of mice with lYo dietary butylated

hydroxyanisole (BHA) caused paralld increases in cyosolic gtutathione-S-transferase

activity and cytosolic enhancement ,of detoxification of vinyl carbamate epoxide by

glutathione. The major conclusions of the study were (1) vinyl carbamate epoxide

could be detoxified by spontaneous conjugation with glutathione, (2) conjugation of

vinyl carbamate epoxide with gfutathione muld be catalyzed by glutathione-S-

transferasq (3) pretretment with BIIA protected against binding of active urethane

metabolites lz rzTo atd in vivo, and (.4) the protective effect of BHA against urethane

metabolite was mediated by incnnses in glutathion+S-transferase activity and

glutathione concentration. De Flora et aL (1986\ reported that N-acefylcysteine

(NAC), a precursor of intracellular glutathiong induced a significantly increase in

oxidizing glutathione reductase activity in rat liver preparations and counteracted the

mutagenicity of direct acting co,mpound (such as epichlorohydrin, hydrogen

peroxide), as a result of its re,rfucing and scavenging properties. At highCopyright by Mahidol University

Fac. ofGrad- Studies, Mahirlol Univ. I\{.Sc. @ood and Nufiitional Toxicologv) / 5l

concentrations, NAC completely inhibited the mutagenicity of procarcinogeos such as

cyclophosphamide, cigarette smoke condensate and aflatoxin Br by birding their

electrophilic metabolites. In contrast, deaeasiqg NAC conceftrations stimulated their

metabolic activatioq especially when liver preparations from enzyme-induced rat

were used. In addition, when adminirstrated with ttrc diet, NAC markedly inhibited the

induction of lung tumors in mice by uretlrane (De Flora et d1.,7986, De Flora et al.,

1986). Abraham et al. (1998) investigated the change in glutathione-S-transferase

dctivity in relation to the observed in vivo anligenotoxicity offresh vegetables, spiceg

tea and coffee. This experiment sholved that treatment with urethane alone resulted in

inhibition of glutathione-S-transferase activity. In additiorq only cofee could

moderately increase glutathione-S-transferase activity, while extracts of vegetables,

spices and tea did not exert any signifrcant effixt on glutathione-S-transferase level.

However, mmbination of uretbane witl extracts of vegetables, spices and coffee

attenuated the inhibitory efects observed with urethane alone in the mouse bone

marrow micronucleus test. They concluded that there was no indication of a firm

association between the observed pr:otection against genotoxicity and glutathione-S-

transferase activity (Abraham et aL, 1998).

A dose-dependent increase in the genotoxic activity ofuretbane was observed

in SMART @rolich and Wurgler, 1990). The frequency of induction of mutations in

the modification strain with increased P-450 enzyme activities was increased by about

one order of magniarde compared vdth tie standard strain. The frequencies of spots

per wirg in high bioactivation cross were higher than those of standard cross (Frolich

and Wurgler, 1990). This might resrlt from the constitutive expression of the enzymes

required for the transformation of ur,:thane into ultimate genotoxic metabolites.Copyright by Mahidol University

Prapdscn hkd€e Lit€rairre Review / 52

STAI]EMENT OF TIIESIS

Many carcinogens and mutirgens are found in food, whereas otber dietary

corryorcnt may help to preveff tk, development of cancer. Ganoderma lucidum ts

herb tlrat wildly use as a remedy for promotion of heahh and longevity in China 66

other Asian courrbs. It is reported to be effective in the treamem of rrrany diseases

and has antinrmor activity. Nitrite isr used as a preservative in many foods ard it can

convert sonre food ooostituerts to te direct mutag@ during tbe corditbn similar tor

gastric digestion And tbere wrc no information on co-administratbn and

pretreahert of GL with the genotoxin in Thailand- It is, therefore, of iderest to

elucidate the modulating effect of GL on the mutagenicity of sorne mutagens using

short-term tests.


Fac. ofGrad. SMies, I{ahktol t-hiv. N{.Sc. @ood rd Ntlritifrral TodcolQ$/) i 53

E)(PERIMMNT OBTECTIVES

General Objective

To detemrine tbe antimutagerricity of Ganoderma lucidun (GL)

Special objectives

l. In vitro *udy using Ames test

1.1 To determioe the nrdifying efect of GL incorporced imo nitrite

mixture a, the @inning ofthe reaction on the forrnation of mutagenic products.

1.2 To determine the effrt of GL on the rmrtageniciry of aminopyrene-

niEite rwtion product after 4 h inculbation.

2, 1z vivo sudy using somatic m$ation ard recombimrion test

2. I To deennine the modulating effect of GL simultarrcously administered

on urethane induced wing spot formatbn of Drosophila melanogaster.

2.2 To determine v/hether GL prctreatment to parent flies could coutrteract

the effect ofurethane in wing spot naufation in tlre progeny flies.


PrOasar Pakdee lviaterials and Nd*ods / 54

CEI.,{PTER ITI

MATERIA]LS AI\D METIIODS

3.1 Chemicah

Aminopyrene (Aldrich, St. kruiq U.S.A) was used to interact with nitrite in

ackl solrrion to goduce a standafil direct mrfagen of the Ames test. D-Bioti&

ammonium snrlfamate (NHrSO4lllIl) and Urethane (tlRE) were purchased tom

Sigma Chemical Conpa.ny (St. Iruis, Missouri, U.S.A.). L-Histidine

monohydrochbride, sodium chloride (NaCl), hydrocblorite acid (HCl), magnesium

sulfite heptahydrate (MgSO+7HrO), citrb acid mnohydrate GR (C}IO7.H2O),

potassium chloride (KCl) and di-sodium hydrogen phosphate (Na2lIPOa) were

supplied by E. Merck (OarmsaaL {iermany). D (+)-Glucose mooohydrde, crystal

violet indicator, di-sodium emrrroniurn hydrogen phosptrae tetratrydrate GR

(NaIrllIaHPO+aHO) and rnorpbline were bought from Fluka AG (BtrclL

Switzerlaod). Bacto agar was purchsed from Difco Laboratory (Detroit, Michigan,

U.S.A"). Oxiod nutriem hothNo.2 was supplied by Oxiod Ltd", (Basingstroke, Harts,

Englard). Sodium di-hydrogen phosprbde (NaH2PO+2H2O) was firnished by May &

Baker Ltd., (DegenharU Englaod). Arryicillin sodium was furdshd by Vesm

pharmaceuical Ltd., (Baogkok, Thailand). Sodium hydroxide (NaOH), di-porassium

hydrogen phosphate mhydrous pure (KzHPOc), sodium nirite (Nal.IOz) werc

purchased from BDH Chemicals Lt<L, (Poole, England). Glycerol was bougla from

Farmitalia Carlo Erba S.p.A Gum Arabic powder was purchased from BDHCopyright by Mahidol University

Fac. OfC-irad" Strdies, lvlahidol l-hiv. MSc.(Food and Nurftimal Todcolo$/y 55

Chemical Ltd., @oolq England). Chloral hydrate was supplied by Srichsnd United

Dispensary Co., Ltd., (Thailad). Other cbmicals were of lirboratory grade.

3.2 Sample prtparation

Steps in pr€paration are shown in Figr:re 11. Dri€d Ganoderma hrcidum (l0O

g) purchased from Arurryig muskoom ceder in Nakonpatom was blended to powder

and add distilled warcr (1000 mI). The solution was boiled with continuous stining for

lQ min; then, it was filtered- This hot water exffict was hought to dryness using a

vacuum e\rapoiator. The rcsidue was dissolved in steriledistilled water to ottain a

final concemaion of I g/ml and it was kept refrigerated.

Dry Reishi (100 g)

I

YBlend to powder

I

Boiled in IOOO ml distilled tte, with continuous stirring

Extract

I

YEvaporae to dryness

(t6 e)

IY

Dissolved in 16 mlsterile distilled water

Residue

IDiscard

Figure l1. Prcpardion of bot waer extac,- of Ganoderma lucidum


napasom hkdee Mateials aod tviettrcds / 56

33 Eryerinental D€siS

Tlp experiment was designed (Figu.e 12) to elucidate wtrther the offact

from Ganoderma lucidun (GL) was in tbe sornatic mutation aod

recombination test (SMART). Tbs extract was added to fly medium to ensrne thet all

flies would co rume it equally, Mixing a strong positive mrtagen urethane and the

extract ir tbe fly medium also performed amimutagenesis. Bringing rp the lrvae in

tle medium coraining tle exhact from GL for 3 day and then in the medium

containiry urethae (a500 ppm) udil they became aduh flies uras aired to elucidate

wbetbr d is ercract could mduhte on uretbane genotoxkity in vivo,

Sfurce the e)dract my be consumed simultaneously with some other food

componeffs especially ninite sah which is one of tlrc hrportant &ctor of sorrach

cancer in some are& This sak is very powerful in converting trotrgerbtoxic

compounds to be genotoxfo ones (Ieamworapotrg, 1990) as well as converting idirect

mtrag€n to direct mragen in tbe conditbn resemble to gasric pH during fuod

digestion Gansaaaanpai et al., 1996). Threfoi it was worth to elucidate such

heahh beneft of the eruract on inhibiting dirwt rutagenesis.

AlthouSh SMART is a good short-t€rm test for mfiagens garcrally required

metabolic activatbr" On tbe other han4 for a direct muagen that may increase tbe

risk of somach cancer, the me*abolic activation may destroy tle coryomd and gives

rise to a negative rcsporse. Therebre, in atr attempt to elucidate the heahh berefit of

consurytion of th ocract on risk of direct mutagen, AmeJSahnorclla asmy wittout

the metabolic activaion system was selected-


Fac. Of Gad. Studies, Mahidol L.hiv. M.Sc.(Food and l.tufitioml Todoologr/ 57

4b.37 "CpH 3-3.4

+ Nitrite

4b,37"CpH 3-3.4

Figure 12. Overall eryedme*al design

3.4 AmcsTest

3.4.1 The Br.tcrid Tester Strrin

Salmorulla typkxttriun tester ;tuairB usd in this study was histidine.

depeode.rt strains (His) TA98 and TAlfi), which werc capable of detecting

fiamcstrift msation asd tase-pair substitdiotr, respoctively. Dr. !/anoee Kusamaq

Natbml Carcer Insihfe, Ministry of Public HeahlU kindty p,oviM botr $raim. The

tester strains werc mpnb,lated as suggested by Maron and Aoes (1993). overdgrt

cultues of bacteria imculated trom frozen stoc& culture in oxoid nutried broth No.2

Weter extnct of G'anodemt lrciikan (GLl

otoboGI


naasorn Eakdee Nfuterials and lvde$rcds / 58

at 3TC were used for mutageresis asay see in Appendk. Cultmes were kept in

refrigerator rmtil use ard were used within 24 h"

3.4,2 Nutrient Agar

3.4.Ll Pleparation of minirne[ agrr pfuts

Minimal agar cotraining 1.5% Bacto-Difco agar was autochvd and then it

was mixed with 2% sterile glucose and Vogel-Bomer medium E (Appendix). About

30 ml of molten agar was poured on to the sterile petri dish. It was left mtil solidified

ald stored at 3fC in the incubator.

3.4.?-2 Prcpration of top agar

Top agar codainhg 0.6% Bacio-Difco agar and 0,5% sodiurn chloride was

autoclaved and was s{ored d room terryerdure. Before use, the agar was melted and

l0oZ ofa sterile sohrtircn of 0.5 mM hi$idirc and biotin was added to the mohen top

agar and then it was mnintrined at 45"C in tk wger bath"

3.a.23 Prcparation of Standard Dircc{ Mutagen

l-Amimpyrene treded with nitrite in acid solution was used as a positive

confinol because it bas been sbown to give direct-mutagenicity (Kangsadalampai e/ a/.,

1995; 1996). In brie{, approprhte volume (10 pl for testing on Salmaaella

tltphinurium TA98 and 40 pl for TAl00) l-mnnopyrere (0.0375 mgfun) in a tube

fitted with a plasic stopper was mixed with 0.71{.74 ml of 0.2 N hydrochloric acid

(sufficient to acidifi the reaction mixture to pH 3.0-3.5) and 0.25 lrl of 2M soclium

nitrite to otnain tb final vohrme of I00O pL Th fmnt concentration of nitite was

0.5M. Tk reaction tube was shaken at 37C for 4 h and then placing this tube in an

ice bath to sop the reaction- In order to decoqose the residual nitrite, 0.25 ml of 2M


Fac. Of Crad SMieq Mahidol ttrriv. I\4ft.(Fmd and Nutriticnal Tofmlog/y 59

ammonium sulfamate was added to the reaction mixture; then it was allowed to

for l0 min in an ice bath before mutagenicity assay.

Aminopyrene + Sodium nitrite

pH3-3.4 I 4hours

stop reaction ln lce { minadd 2M ammoniumsulfamate

I ",:TJil"*".I

Mutagenic products

Iigure 13 Preparaion of stardard direct rnutagen

3.4.2"4 Mutagenicity Study of GL

The concentraions of GL extract wef,e 0.125 dml,0.25 ghnl,0.5 g/ml and I

g/ml Orc-tenth ml of each corrcentration was added to the test tube cortaining 0.65

ml of 0.2N HCI ad 0.25 ml distilled water. The r€action tube was incubated d 37"C

for 4 h. thn placing this tube in an ice bath for I min to sop reaction. Then 0.25 rrl

of 2M amrnonium sulfrrnate was added to the reaction mixture. The reaction tube was

immersed in an ice bath for 10 min. The reaction mixture (100 pl) was mixed with 0.1

ml DMSO, 0.5 nl phosphae butrer (pH 7.$, and 100 pl of fresh ovemight culture of

tester strain and ircubated at 3fC in a shaking water bath for 20 min. After

incubatiorU 2 rrl of mohen soft agar (afc) contain 0.05 mM each of L-histidine and

D-biotin was added mixed gently, and poured onto a minimal glucose agar plate, The


PrapAscrn Pakdee Materials &d Methods / 60

histidine rcvertant colorfes were count€d after incubation d. 1fC for 48 b" Each

concentration was assayed in duplicated and done twice.

Positive and negative contols were ircluded in each test. The negative control

was DMSO. The positive control was prepared as described in 3.4.2.3

3.4.2"5 Mutagenicity of the r'.rtract Treated with Nitrite

To determine tbe mutagenicity of nitrite treated with GL ofiact thd were

gepared to be 0.125 gnts o.25 dnl" o.S dml ad I g/ml Sample solution (0.1 ml)

was added to tlrc test tube containing 0.65 ml 0.2N HCI and 0.25 mI 2M sodium

nitrite to obtain the final vohrme of 1000 pL Tb stoppered tube was incubaled at 37"

C for 4 tr Then, it was hmersed in an ice bah for 1 min to stop reaction, 0.25 ml 2M

ammonium sulfrmate was added to the reaction mixture, and the reaction tube was

left in an ix bath for anotber l0 min before mutagenicity assay.

3.425 Efiect of the Extract ftom GL m the Stmdard Mutagm

GL e:aract sohrtion (0.1 ml) was trarsferred imo a sferile plastic stoppered tube

containing 0.1 ml of AP-nitrite mixnre (preparcd as described m 3.4.2.3),0.5 ml of

phosphate buffer, pH 7.4 and 0.1 nrl of overnight bacterid culture (Figure 14). The

reaction tube was mixed gently aod incubated in a shaking water bath at 3fC for 2O

minutes. Then 2 ml of moften top agar corfaining 0.05 mM each of L-histidine and D-

biotin were adde4 mixed gentty, aod poured onto a minirnal glucose agar plate. The

Hist revetant colonies were scored after incubation at 37"C for 48 h. Killing efiect of

each corryourd on tester strains was determircd by the gp\ilth inhibition of ttre

backgroud lawn under a light microscope.


Fac. Of &ad- Studieg Mahidol t-Lriv. M.Sc.(Fmd and Nsitionat TGimlo$/y 6 I

Percent inhibition of the mutagenicity was calculated as suggested by

Calomme et al. (1995),

Percentage of inhibition : (a-b/(a-c)x 100

where a: number of histidine revertants irduced by the positive mutagen (product of

AP-nitrite reaction); b : number of histidine revertants induced by the positive

mutagen in the presence of sample; c: number of revertants of tie negaive control

(spontaneous reversion).

Inhibition more rhrn 60Yo, 604V/o, 44-2Uyo ard, ZO-U/o wer€ chssifid as

shongly active, active, weakly active and no effect, respectively. Whereas, increase of

mutagenicity 0-20yo, 204tr/o, 4O-6V/o and rnore than 60%o were classified as no

effect, weak enhancement, enhancement, strong enhancement, respectively.

AP-Ni.ritEkodr"t+ El !t'c Hstvphtuuritm

fr q*l| HH

I "#ffiH,

AmesTest :reverrard

- l;:Y-:'""@

Muta StreiB + *'fr:fr"His-

Figure 14 steps in studying the efrect of GL exract on the rmragenicity of 4 hours

incubation product of arninopyrene-nitrite rnodel.


Prapason hkdee

3.5 Somotic Mutation and Recombination Test

ldaterials and lv{etnds / 62

3.5.f l)rcsophila medium prtparation

Drosophila rredium was modified from the formula of Robert (1986). Sugar

(0.20 g), agar (0.028 g), yeast (0.10 g) and corn flour (0.25 g) were mixed ard boild

in a 15 ml test tube comaining 2 ml water or test solution ulltil sticky. Propbnic acid

(0.01 rrl) was added rul a preservative. lben" it was Ieft for l0 min and covered with a

cotton plug.

3.5.2 Mutagenicity Assay

The mutagenesis assay was carried out according to the method described by

Graf et al. (1984, 1986). Virgin females of the DDT-resistant Oregon R (R) strain

Drosophila melanogaster were allowed to mate with males of mwh/mwh strain to

produced trms Merozygous (mwh+/+Jtl') hrvae of iryroved high bioactivation

cross Q[IB). The three-day old larrrae (72 h) were collected washed with water, ard

transferr€d (with th€ help of a fine artist's brush) to glass tubes with medium

cortaining GL extract or distilled water as negative contr]ol Each GL extract sotrtion

was tested for its mutagenicity by mixing GL oaract solrtrion with Drosophila

medium. Final concertrdions of GL extract were 10,000, 100,000 and 1,000,000

ppn Both control and tredd larvae were allowed to ftod or tb€ culture medium at

25+1"C for 48 h urtil pupation. After metamorpbosis, the survival flies were collected

from tle tubes between days Ll*-l2 after egg laying aod stored in 707o ethanol Only

wings were collected as suggested by Graf and van Schaik (1992) ad their wings

were nrounted on microscope slides (described below); thus they were examined for

the mutant spots. Copyright by Mahidol University

Fac Of Grad. Studieg Maltidol {hiv.

3.53 Antimutagenicity Assay

Urethane (4500 ppm) was used as a positive conrol by mixing it with the fly

medium. The final concentrdions of GL extract in the fly medium were 10,000,

100,000 ad 1,000,000 ppm- Threeday old larrrae were trapsferred to test tube

cotrtaining Drosophila medium and test coryound (Figure l5), For each trreahnert,

the test concentrdion of urethane (given eitber alone or in mmbination with GL

extract solution) was selected as suggested by Graf and Wurgler (1986), Graf et al.

(1989) ad Abraham (1994), Expedment was prec€ded as descriH ln3.5.2.

IH

,#ffi-r)

Vireh ORR (fcoalc)+

ocrrt (male)

a€}+r&$'LirlA(

to frcshAcr*Ur"

25t10Cfu 72h

GL.dzeaor

GL crd..r.n+I'RB

Mutagenicity and

Flgure 15. Mutagenicity teSing ad amimutagenicity testing

Antimutagenicity Testing

Stffiifl-;qh-'tI


Prapasmn hkdee N{at€rials and lvtethods / 64

3.5.4 Eftect of GL extrac{ Pr€tr€atment to Parent flies on Urethane

Mutagenicity

Virgin females of the DDT-rcsistant Oregon R @) srain were allowed to mate

with males of mwUmuth strain They were tftmsfened to the medium containing GL

offact (10,000, 10q000, 1,000,000 ppm) and allowed to lay eggs. Iarvae were fed on

GL. esract for 3 days; then tlrcy were transfemed to test tubes comaining Drosophila

medium ard urettnne (a500 ppm) (Figure 16). Both cornrol ard GL e)ffact treated

larvae were treded with uretbarb il.25+lo0 for 48 h until pupation Experiment was

preceded as described m3,5.2.

ffi,ffi*\firgan ORR (fetnde)

+mrh {male}

E,A.r) € t cI.\E$t(-_:__:__J 25+1oCTo frestr

rncdium + GLlor 72

Effect of GL Pretreatmenton Mutagenicity

of URE in Drosophila melanogader

t$

,€ wng prepilation 5 day

+ - -$r r;frf1e25 + 10C**itr

lfgure 16. Pretreatrrrcnt

Drosophila me larn gaster.

study of GL extract on mutagenicity of urethane


Fac. OfCrad" Strdes, lvtahidol l-kriv. MSc.(Food and Nuritimal Toximlory)/ 65

3.55 Wing Prepantion and Micmscopic Analysis.

The flies in 70olo ethanol were washed with distilled water and placed in a drop

of Faure's solution (30 g gum arabic, 20 ml glycerol, 50 g chloral hydrate and 50 ml

distilled water) as suggested by Graf e/ a/. (1984). Wings were separatd from the

body with a fine paintbrush ttren they were lined up on a clean slide. They were

allowed to spread orr. A droplet of Faure's sohrtion was dropped on tbe slide and a

cover slip was put on A permanent prepar*ion was obtained by sealing th cover slip

with nail polish Next, the wings were aoalyzed under a compound microscope at

400X magnification The position ofthe spots was noted according to the sector ofthe

wing (see Figue lA. Ditrerent t),pes of sporc narnely, single spots showing either the

multiple wing hans (mwh) or the flare (/r) pherctype, and twin spots showing

adjacent mrh ad fll areas werc recorded separately. The size of each spot was

determined by counting tlrc number of wing cells (hahs) exhibiting the mutant

phenotype. The spots werc coutrtd as two spots if thy were separated by tlree or

more wiri+type cell rows. Muhiple wing bairs Qnwh) werc classified when a wing

cell coatained thee or more hairs insead of one hair per cell as in wide.type (see

Figure 18). Flare w'mg hah exhibited a quite variable eryression, ranging from

pointed stnrtened, and thickened hairs to arnorphic, sometimes balloonlike

extrusions of melanolic chitimus material (see Figure 18).


Prapdsont Rak&e Ndaterials and N4edrds / 66

trIgure l7..Normal balf mesothorax showing the regions A-E of the wing surfrce

scored for spots according to Graf et al. (1984).

figure 18. Trichornes on the wiry blade, a) norma[ b) deviate trichornes not counted

x mwh or ff , c) configurations irdicative of mwh, d) t,,pical manife*ation ofll

(Grag 1984).

c' ,1, Jd ''*

/#,M rb J, $ r,l

ralodd*@i

" lfio il4.ttt

t!rl

n //7

" / rd{r..lt


Fac- Of Crrad SMieg tvlahidol t[tiv. lvlSc.@md and f.{ilriticul To:ricologr/ 67

3.5.6 Data Evaluation ard Statistical Analysis

35.6. I Mutagenicity test

The wing spots dda were evaluated by the statisical procedure described by

Frei and Wurgler (1983). Briefly, induction frequencies of wing spots of color

tredrneft groups were comparcd with those of negative cotrrol group (waer). The

spots were grouped accordirgly to th following 3 types: (l) small single slrots of 1 or

2 cells in size Q) large single spots of 3 or more cellq and (3) twin spots, The

estination of Wot frequeocies and confidence limits of the estimated mutation

frequency were performed with significance level of o : p : 0.05. A multiple-

decision procedure was used to decide whether a result was positive, weakly positive,

inconclusive or negative according to Frei and Wurgler (1988). Staistical

considerations "nd calculation step by step are sbown in Appendix.

3.5.6.2 Antimutagenicity test

Antimutagenicity of GL extract was estimated as suggested by Negishi ef a/.

(1994). Relative spot induction frequencies of co-a&ninistration of each concentration

of GL extract aod a Sadard mutagen were conryared with those of a shndard

mutagen group. Relative spot indnction frequercies were calculated fiom the number

ofirduced spot per wing subtracted by the value ofthe control group. For exanple, in

Table 11, l00p/o of relative spot induction ftequency of uretiane of small siryle spots

conespords to 10.35-0.47=r.88 (number of irduced spot per witg), aad 89.47o/o

relative spot induction @uency of co-administration of GL extract (10 mglml) with

rnethane (4500 ppm) corresponds to 9.31-O.47=8.84.


Pragomhkdee R€$lts / 68

CIIAPTERTV

RESI]LTS

41 Mutagcnicity of Nitrite Trcated Enract fmm Ganodcrna lacidum

Water extract from GL was not on both S. typhimwium suahts

TA98 ard TA100. However, after being interacted with nitrite (pH 3.0-3.5) for 4 h at

3/C, the efract becare capable of causing base pair and fameshift mutation toward

both tester strai$ (Taile a) suggesting that there were some compouods could interact

with nitrite to form direct mutagenic products. Tb mutagenicity irdex calculated from

tb rumber of revertants per plate diviled by a correspordeff background revealed

th"t the products were good in inducing both tlpes ofmutation.

42 Antimutageoicity of Extract frtom Gotodenu lucidmtin Ames Test

The extract from GL inhihrited the mutagenicity of product of the Ap-nitrite

model in a dose-dependent nnrner on TAIO0. On S. tphimtrium TA lOO, the

mnfiagericity was inhibited (46%) W tb lowesr testing moutr (1,000 pglplate) and

the inhibition continued umil a partial kitling etrect was detected at 8000 1r1tplatl.

Surprisingty, it was derected on TA98 that tbe hwest testiog amormt (l,tl00 pglplate)

of the el<tract ircreased the mutagedcity ofthe AP-nitrite rpdel h:t wben the amount

was irrcreased to be 2000 or 40{D pglphte the mutagenicity was decreased 19 ad 35

%, respectively (Table 5). It is shown thar each idex (MI) of anCopyright by Mahidol University

Fac" Of Grad- Stdies, Mahidol t-Itriv. fvlsc.(Food ad l.hflriticnl Toximlogr) / 69

individual incubation mixture is less than tbat of the calculating derived from the

slllltrnrttion of the MIs of the AP-niffie model and tle one of nitrite treated extract

from GL. An exception was fudicated only that from the addition of 1ffi0 pglplate of

exuact to tbe AP-nitrite model which rlro*.6 6 higrrer actual MI.


Res{ts / 70

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Prapats$n Pakdee Results / 72

4J Survival of Isnrae and Adult trIics in Somatic Mutation and Recombination

Test

Table 6 shows the nunrber of aduh flies tom larra€ treated with ttre extract

from GL alone or with the mixture of uretiane (a500 ppm) ad varying amount of the

extract (10,000 - 1,000,000 ppm). Paccnts fly fiom the extract treated groups were

between 77yo d 90% while those of larrrae teated with the mixture were between

4f/o and ayo. lt $as 83yo survilal of the control group and 45% srvirral of uethane

Eeated grorp. The daa $ggest thar the addition of GL extract did not iryrove the

survival of flies from hrv.ae treated with URE

Table 6. Number of survival trans-heterozygous (nwh+/+ltl) aduh flies fed on

offact from Gazoderma lrcidtm and urethane

URE (IDor) Concenfiation of Number of Number ofGL (ppm) lantae

survirral

Per€ed ofsurvival

Nunber of

ro,"rd wings

0

0

0

0

4500

4500

4500

4500

10,000

100,000

I,000,000

0

lo,mo

100,000

1,000,000

70

70

57

5470

2@

100

100

100

59

49

83

90

8l

77

45

64

59

49

55

58

55

52

79

54

55

51


Fac. OfGrad Studies, Ivrahidol l_hiv. fvlSc.(Food and Nrlritional Toximlogz) / 73

Tabl,e 7. Nnnrber of survival trans-heterozygois (mwh+/+/F) adult flies from larvae

prefed with the npdiwn comaining the extract fiom GL for 3 days before continue

bringing up on oormal medium containing urethane

Extract &omGL IJRE No. of No, offly Percerr offly No. ofroundpretreamem fupm) Gpm) larv.ae' survival survival wrtrgs

0

10,000'

100,000'

1,000,000'

0

10,000

100,000

1,000,000

0

0

4500b

4500'

4500"

4500"

150

150

I50

2N

70

75

73

23

100

96

90

76

47

50

49

t2

72

65

58

68

58

53

55

J.'

70

76

70

loo

"larva were bnought ,p in rrcdium comahrng@transferred to rcw ft,estr mediumb

larva werc fed on uethane rmtil they becarrc adut flies.

" larva wer,e bro'rght up in medium containing tbe extract from GL br 3 days and were

transftrred to new fiesh nredium containing URE


Prapasrrn Pakdee R€sulB / 74

Percents of flies derived from larvae preheated with tbe ocract ftom GL

(10,000 - 1,000,000 ppm) for three day and fed on urerhane (4500 ppm) containing

medfurm were between 12-50olo while that of the positive comol group was 47%

(Table 7). This yalue was similar to those obtained with 10,fiD ppm and 100,000 ppm

ofthe extract.

.t 4 Effects of Gotodenu lrcidm Coadministratirm with Urcthane on Induced

Wing Spots

The data p€setred in Table 8 sbw the frequencies of difierert categories of

wing spots induced by tkee concemdions (10,000, 100,fi)0 and 1,000,000 ppm) of

the extract from GL. It is shown tbat the e><tract did not furcrease the ftequeocy of

mutafi spots (p<0.05). Frequencies of total spots iduc€d b,, the extract fiom GL are

within the range of 0.47 to 0.50 with no toxic effect while spontaneons fiequency is

0.54.

The reduction of wing spots induc€d by urethane was observed when the

e)firact fiom GL was co-admini$ered with urethane to the hry,ae. Th extract (10,000,

100,000 md 1,0fi),0fi) ppm in the medhrm) rducd the number of mall single spots,

large single spots, twio spots, and total spots comparcd with tbose of urethme treated

group. The rclaive induction Aequerrcies of total spots formation were reduced to be

78Yo,670/o arrd,52yo, respectiveh ofthe LJRE alone. It is rrcted that even at the very

high conceirffibn ofGL the mutagenicity of URE was still sipifcantly different fiom

that ofthe spontaneous control.


Fac. Of Grad- Studies, Mahidol L-hiv. MSc.(Food aod }tritiqul Todcolos/) / 75

4.5 Efrects of X'eeding Gonoenna lrcifum to Ncrborne Larvae for Three Days

on Wirg Spot Induced by Urtthane

No significam ditrerence (p<0.05) benreen wing spots induction (0.18) of

control group aod ttnse (0.24 to 0.25) ofthe extract (10,000, 100,000 and 1,000,000

ppn) pretreammt groups (Table 9) confirms that GL contain no mutagerl It is noted

that ttrc highest concemrdion of tbe ocract (1,000,000 ppm) was toxic to the hrvae;

thus, only thirty-th€e flies were available for ana$nis. The resub suggestd that the

exract tom GL mighf have some influence on the developnemal period of the larv"ae

possibly vh growth hormone.

Urethane in&rced both small single spots ad targs single spots. Less mrmbers of

twin spots were also induced. The preaeaments of tie e)<tract from GL (10,@0 or

l0O,(D0 ppm) to tbe hrwe bebre the administration of r:rcthane also reduce tbe

fiequency of mrtaf spots. The relafive indrrction of small single and total

spot formatbn the groups ttat prefed with 1Q000 or 1@,fi)0 ppm. of the extract were

reducd in tbe sarne rranner of the co-.administration study. Only tbe relative

@uencfus of twin spots of tbe pretremem sudy tha tk exract form GL did not

inhibit but irrcrease with tbe highest dose. Considering on the results obtained from

pretreatmetrt it was fornd that the reductions of mrtail spots werc le$s ttEn those of

the study on co-administraion feeding of URE with tbe extract from GL.


Resrlts / 76

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M.Sc.@ood and Nutritioal Toxicologr) / T7

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Proasan hkdoe Discrssim / 78

CIIAPTERV

DISCT'SSION

The human benefit on con$nrytion of an unconventioml extract from

Ganodenna lucidun was ewaluated using an in vitro tes oatrEly, Ames test ard, an in

vivo sbrt term t€st n4'nely, somatb rnutation and recombiodion test (SMART). For

Ames test, tre*ed with o<cess of sodium nitrite in acid sohrtion (pH 3)

for 4 h was used as a positive rnsagen and an exanrple of mutagen occurred drning

the gastric digeSioo- This reaction product was revealed its direct mutagencity toward

te*ers Salmorglla lyphinrurhm TA98, eod TAf00 (Kato et aI., l99l ad

Kangsadalampai et aL, l99,q. Since tbe product of the model does not require

metabolic acthation bebre eryr,essing its mrtagenicitl,; it allow us to dcermine the

couteractitrg activity of the saryle on tb mutagen rhat rnay cause mrtatiotr to the

stomach cell This oodel is currentty used at the Institute of Nutritbn in order to find

o$ some adimutagen agairst mutrgen formed in the gastric liked pH solution

In additbq tfu in vitro information obtain ftom Ames test might be

considq€d inzufficiem; therefore, tbe iesult sbuld be confirmed in a living sysen

Thus tle somatic ,mrtation ad recombioation test was ernployed. It is well

established tfut Drosophilo possess€s a versatile system for the meubolic of

xenobiotics. Drosophila has daoxification-activcing systems in many respects

closely resembling the corresponding systems ia mnmmelg urtich makes it possible

to extreohe the data to r .rammals, In SMART urethane was used as a positive


Fac. Of Crrad Sttdeg Mahidol t-Lriv. MSc.(Food and l.hllitiolal Todcolory) / 79

sandard mutagen since it required netabolic activation resemble to rnany known

dietary carcinogen

5.1 Mutagenicity of Eot Water Extrac{ from Ganodenaa lucidtatt

Using fulmonella typhimurium tq*er straiN TA 9g alld TA 100, it is

demonstrded that the bot water er0act of GL was not ge@toxic in the absence of

metabolic activation (ir vifro study) as well as on the sormtk c*lLs of Drosophila (in

viuo sudy), However, when the experioeft was cooducted to erylore wh€tler thextract would bave a chance to hteract wittr nitrite ard formed direct mrtagen in the

acidic soh*bn; it uras brd that tb product after nitrite trempnt was m$agenic in

Ames test.

The nurtagenic precursors itr GL rnay be sorre &ee amino acids. It was found

tiat sevedeen free amiro acids were detecte4 with glutamic acid aspartic acil, atrd

alanine were the high (qrrc, 2'4oiLtc,as,l99l;L€f..' 1986; Kawamata, l9g7; Chuog,

1986; Nagaraji 1987; Tseng, 198a). Primary nmines (6.9., amino acids) were

described as potential alkylatrng agents after nitrosation (G@ar et. al., 1996). T\ey

revaled the mutagedc activity of glycine upon nitrosation on Ames tester straiis

TA98, T4100, TA102, ad TA1O4. The nature and the nrechanism of genetic lesion

inducd by the uhimate genotoxicart arising from tbe nitrosation of glycine were not

fully uderstood. However, in our experimemal conditions ttrese alkylating activities

were not $udied aDd orthet mechanisms coull be involved in the genetb lesion

induced by nitrosated extract from GL.


Prapasrn hkdee Dsurssion / 80

Another possibility that directly acting mffagens may be produced in the

reaction mixture of the er.tract ant nitrite is proposed- Sirrce mycelium of GL

contafurcd various bw rnolecular weight components including amino acids. Kuzn et

al. (1994b, 199..k) ad Johansson et al. (1995b) also suggested rbd unideftified

mutagenic compourds (with properties similr to those of some known heterocyclic

amines) were fr"nd in non-meat pmducts and "guaoidino" conpomds (for instance

arginine) may be tleh precursors. They further suggestd that new, unidentified

mutagenic aromatic amines were shown to be produced bV dry heaing of single

amino acids or their binary combinations (thrcodre and arginine) produced high

mutagenic activity itr combination, ad l-mthyl-guanidine produced mutagenic

activity wkn heaed alone or in mixtures with amino acids (Knize et al., 1994c). The

GL saryle of tb preserfi shrdy was dried in a hot-air oven during manufrcturing and

before e*raction; tlrerefore, sorne indirect mutagens ready to interact with nitrite

might !s formed and resuhed as the cause ofdirect mutagenicity on the tester straiDs.

It is suggested ttrd tbe ilemification of suspected precursor in GL exract which

produc.ed direct mrtagen after nitrite tre*metr is warrmt to elucidate the risk/benefit

of consumer in drinking ofthe extract in the presence of nitrite cornaining food items.

Resutts of tie preseot st@ dernonstrated tht the extract Aom GL was not

genotoxic on the somatic cells of Drosophila t*s $rain. This confrms ttrat none of

the coryonems of extract form GL was mutagenic both in the direct mutagenicity

assay as well as in tlre iz vivo assay.


Fac. Of Crad Studies, Mahidol [_hiv. MSc.(Fmd and Nurftionl Todcolog/) / 8l

5.2 Antimutagenicity of Eot Water Extmct from GZ Study fu Am€s t€st

Direct mutagenicity of the product occurred after interactirg aminopyrene with

nitrite in acid solution pH 3-3.4 was reduced in tie presence ofthe extract from GL. It

was reported by Kato et al. (1991) tl,ut tbe prrodud was mainry nitropyrene and

Hensel ard Meier (1999) also reported that the mutagenicity of nitropyene was

hhibited (2o-5u/o) by :rrybglucao- The suggested rh2t the mode of actbn a direct

interaction of the potyrners with trp cells protected the testing organisms from the

attack of mutagea Mizutro (1994 revealed thzt aoongr polymer components in GL,

many polysaccharides and tbeir protein<orrylexes were extractd by hot wder and it

was shown rhat the cacinostatic substance in GL was a polysacchaide, beta (l-3)-D-

glucan" Ahhowh xyloglucan ad beta ( t -3lD-gfucan may be differem in sructure

but their similarity in chemical property rray be the same ard thus the effect on

antimutagerdcity found fiom this study may belong to tlre sarne mechanism.

studying on antimutagenicity of oaract fiom GL gave rise a question that a a

lcw dose (1000 pglplate) of tbe extract in'eas€d mutagenicity of nitrite treaed

aminopyrene in strain TA98 but the mutagenicity decreased with the higher doses.

siDr€ GL is a source sf tarmin (Mizutro, 1997) which is a polypherc! tb possible

mechanism of this two edged sit,ation of GL rray be the same as discussed in the

sudy of Pignatelli et al. (19t2) who srudied antimutagenicity oftea They propose.d

that c-nitroso derivartives might form fiom tlrc reaction between tea porypheml and

nitrite when low concentrdion of the extract fiom tea was studied during tbe initial

step. Ttrc rewly formed coryourd f,tber reacted with the nitrosating species to

generate a more powerfirl nitrosatmg agent- However, trrc posturated nrcchanism

implied that vn.n the ass:ly system containing inffeased armum of a catalytica yCopyright by Mahidol University

Pr@sorn Pakdee Disc1lssion / 82

active phenolic corpound it led to irrcreasing concentrdion of C-nitroso internrediate

and reduce the corrcentraion of the nfuomting species. This nrechani$n \^/suld

explain why a large excess ofphenolic compound could inhibit nitrosation.

5.3 Study in Somatic Mutation and Recombination Test

The protective effect of the extract from GL on genotoxicify itrduced by LIRE

was shown whes it vras administraled to tbe larvae cor-administration with urethane in

a dose rcspome manrrcr. tlRE is a well-known getptoxin tlat is metabolically

activared by the cytochrome P-450 enzyme system (Scblatter and Lutz, 1990) to be

viayl cartamarc epoxide (Park et al., 1993) ard is detoxified via conjugating with

glutathione (Kemper et al., 195). The dda obtained with the irproved high bio-

activatbn cross in th€ prcsent study showed a hiCher genotoxicity of urethane than

tbose published with the standard cross by Abraharn and Graf (1996). Therefore, GL

e$ract might be inhibit the catalytic activities ofcytochrome P-450 system ofphase I

or irduction of glutathione-,S-traosferase activity or increasing amount of glutathione

of phase II detoxiE ing in Drosophila seemed to be the explanation of this

phenorrenon.

G. hrcidun mrgtr reduce tie genotoxbity of tlRE by its Aee radbal scavenging

actiWy. Brcnnan and Schiestl (1998) showed tlat free radical species were produced

in Sacclurornyces cerevisiae following exposure to urethane. Treating with ttrc water

extract of GL, a marked decrease in the CClr-induced toxicity in rd liver was

observed GnL 199, ad tut (1999) also frrmd tbat Aactbns of GL had

antioxidative effect agains pyrogallol induc€d erythrocyte membrane oxidaion aod

Fe (Il)-ascorbic acid induced lipid peroxidarion Kim and Kim (1999) working on anCopyright by Mahidol University

Fac. O'fCrad. Stndies, Mahidol thiv. fvlsc(Food and f.lfiitianal To:ricologr) / 83

in vitro using isolated DNA demonstrated that the hot-wder ercract of GL shows

protection aginst hydroxyl radical-induced DNA damage. Ttrey also found that the

water-soluble polysaccharide isolated from the fiuit body of GL was as effective as

the hot-water exract in protecting against hydrcxyl radical-induced DNA strad

breaks, indicating that the polysaccharide conpound is associated with the protective

properties.

It was noted thaf prcEeatment resuhd in a bsser reduction of somatic mrsation

and have on inhibition but increase the number of rwin spots. Therefore, it should be

aware that no protection on uretbane mutagenesis in tbe preteatment study since twin

spot is the indicator of unusual recombination of DNA occurring during mitosis. This

infers to consunrers wlro rely on drinking such herbal extract tlrd it cannot give them

a long lasting protection against all types of mutagenic conryouds, especially the one

that causes somatic recombination.


Prapatsrn Pakdee Conclusim / 84

CHAPTERVI

CONCLUSION

The €xtract from Goodenna fuci&un (GL) is rct neither dir€ct nor indirect

mrtagen wbn assayed in tle backward mttratim of Ames fulnowlla mutag€ohity

assay in tbe absemce of activding system and in the wing spot somatic ,Intdlm and

rcoombinati(m test using Drosophila nplerogaster. Although tb erdraot was

mdag€nfo towar:dtr,thSalmowlla typbindriun sEains TA98 and TAl00 after behg

teated f,,ith rinite, it was aho an dimutagen of the prodwt derived fiom

ambpl,rene-nitrite rcacRrn. Co-adminiscaion of GL to larrae of improved high

bioactir.tion cross redrrced th fiequency of total spots iduced by urethaoc. All

coDc€ffidb6 of GL deoeCIed tbe Aeqrmcies of wing spots iduced by urethae. Tbe

autimragqlicity efrect of GL my imrohrc tbe inhibftion of the caalyti: activities of

phase I or iductioos of phase tr detoxifying in Drosophila as well as its fioe redical

sca\renging actntity. prercamnt of the edract to re$ibome larrae resrlted in a bsser

reductbn of somatic mutdion suggesting that tbe corulnner should consme tbe

extract along with any possible mutagen in order to cormeract the toxicity-


Fac. of Grad Studies, Mahidol Univ. ltdSc. (Food ad Nutsitional Toxicology) / 85

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Salmonella. Mutat Res 1977;48: l?l-9.Copyright by Mahidol University

Fac. ofGrad. Studies, lv{ahidol Uni\,. M.ft. (Food and Nutritional Toxicology) / I I I

Yamamoto T, Pierce WM, llurst HE, Chen D, Waddell WJ. Ethyl carbamate

metabolism: rr vlvo inhibitors and ,r, vitro errzyfiatic systems. Drug Metab

Dispos 1990; 18. 276-80.

Yamamoto T, Pierce WM Hurst HE, Chen DI, Waddell WJ. Inhibition of the

metabolism of urethane by ethanol. Drug Metab Dispos 1988; 16: 355-g.

Yamanaka [I, Nagao lvl Sugimura T, Furuya T, Shiria d Matsushima T.

Mutagenicity of pyrrolizidine alkoloids in the Salmonellalmammalian-

microsome test. Mutat Res 1979; 68 271-6.

Yoon Sy, Eo SK Kim YS, L@ CK, Han SS. Antimicrobial activity of Gotderma

l'ucidum extract along and in combination with some antibiotics. Arch

Pharmacol Res 99a;17(6\. a38-42

Yun TK Kim S[ Lee YS. Trial of a new mdium-term using benzo(a)pyrene

induced lung tumor in newborn mice. Anticancer Res 1995; 15: 839-46.

Zhang L, Wang fL Wang L, Xiao P. Immunological effect oF Ganoderma lacidum

spore extract in vivo and in vitro. Zhongguo Mianyixue Zazhi 1994; lle):

t69-72.

Zhu M, Chang Q, Wong LIt Chong FS, Li CR. Triterpene antioxidants from

Ganoderma lucidum. Phyotherapy Res 1999; t3(6): 529-31.

Zijlstra JA, Vogel EW, Breimer DD. Strain-differences and inducibility of

microsomal oxidative enzymes in Drosophila melanogaster flies. Chem Biol

Interact 1984; 48: 317-38.

Zimmerli B, Schlatter J. Ethyl carbamate: analytical methodology, occrnrence,

formatioq biological activity and risk assessment. Mutat Res l99l; 259 325-

50. Copyright by Mahidol University

Prapatsom Pakdee Bibliography /ll2

rn:n,dic(Afla, tranifilr. ino.n:laaiid:ltitfnna-ude. firrf,n9.rfi I aq.rmflir 1 fl::

d til nf;u y* e:r"nt z ai ; 25 40.

arna inrlqn, rfia #ufrfio{#ot. fianduda LING ZHI (Ganodetma hcittum). fiufi

!.4^ : ",nt.rlt J Rl-r,??flufl'tuat: vtun i.11. fliun: 2540


Fac. of Grad. Studies, Malfdol Univ. M.Sc. (Food and Nulritional Toxicolosi) / 113

APPENDIX

AMES TEST

MANIPUI.ATION OF TEE TESTING STRAINS

l.Preparation of Stmk Solution &nd Media

1.1 Vogel-Bonner medium E stock salt solution (VB salt)

Ingredient 1 liter

Distilled I{zO 670 ml

Magnesium sulfate (MgSOr.THzO) l0 S

Citric acid monohydrate 100 g

Potassium phosphate, dibasic (anhydrous) 500 g

2 \ter

1,340 ml

20g

200 g

1,000 g

(KzHPO+)

Sodium ammonium phosphate

(NaN}I{HPO4.4H2O)

175 g 3s0 g

Add salts in the order indicated to water and allowed each salt to dissolve

completely before adding the next. Filter the solutions and then autoclave at I 2 I 'C for

20 min.


Prapatsorn Pakdee

1.2 Minimal glucose agar plate

Appendix / 114

Ingredient

Bacto agar

Distilled Hzo

VB salts

40oZ glucose

1.4 Top agar

Ingredient

Bacto agar

Sodium chloride (NaCl)

Distilled tDO

300 rnl

4-5 g

280 ml

' 6ml

15 ml

350 nrl

5 ?5 0

330 ml

7ml

17.5 ml

Add agar to distilled water 'n a glass bottle. Autoclave at l2l"C for 20 min.

when the solution has cooled slightly, add sterile vB salts and sterile 40plo glucose_

Mix and pour 30 mI into each sterile petri plate. Mnimal glucose agar plates were kept

in incubator at 3/C before using.

1.3 Oxoid nutrient broth No.2

Dssolve 2.5 g of nutrient broth No.2 in 100 rnl distilled HrO. Transfer 12 ml of

nutrient broth for each flask (covered with sterile gauze). Autoclave at l2l"c for 20

min.

200 rnl

r.2 g

1.0 g

200 rnl

300 ml

1.8 g

1.5 g

300 rnl

Dissolve ingredients in water. Store in a glass bottle. Autoclave for 20 min at

121'C urd then add 20 rrl and 30 ml of 0.5 mM histidine HCI-0.5 mM biotin for 200

ml and 300 ml oftop agar respectively


Fac. ofGrad. Snrdies, Mahidol Univ. M.Sc. (Food and Nutdtional Toxicologl ) / l15

I.5 0.I M Lhistidine ECt stock

Ingredient 100 rnl

L-histidineHCl 2.O96 g

DistilledH0 100 ml

Dssolve 2.O96 g of L-histidine HCL (MW 209.63) in 100 nrl distilled water.

Autoclave at I2l'C for 20 min

1.6 lmM L-histidine ECL stock

Ingredient 100 ml

0.1 M L-histidine HCL I rnl

Distilled Hzo 99 rnl

I rnl of 0.1 M L-histidine HCI in 99 nrl of distilled water. Autoclave at l2loC

for 20 min.

1.7 lmM Biotin stock

lngredient

Biotin

Distilled HzO

Ingredieot

I mML-histidineHCl

l mM biotin

Dssolve biotin (MW 244.3) in distifled water. Warm it until dissolve completely.

Autoclave at 121'C for 20 min.

1.8 0.5 mM L-histidine ECI-0.5 mM biotin

100 nil

24.43 mg

10O ml

200 rnl

I00 ml

100 ml

Mix and autoclave at 121"C for 2O mnCopyright by Mahidol University

Prapatsom Pakde€ Appendl\ / 116

1.9 NaPO.r-KCI bulfer

Ingredient

0.5 MNaPOTpH 7.4

l MKCI

Dstilled HzO

330 ml

100 nrl

16.5 nrt

213.5 nt

Mix and autoclave at 12l'C for 20 min.

1.10 1.0 M KCt

Ingredient

Potassium chloride

DistilledHzo

1,000 rnl

74.56 g

1,000 rnl

Mix and autoclave at l2l 'C for 20 min.

1.1I 8mg/ml Ampicillin solution

Ingredient

Ampicillin (sodium)

0.02 N NaOH

Dissolved and store at 0'C.

1.12 0.lo/o Crystal violet

Ingredient

Dstilled IIzO

Crystal violet

10 ml

800 mg

10 rnl

l0 ml

10 rnl

l0 mg

Store at 0'C in glass bottle with screw cap.


Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food and Nulritional Toxicologr) / I 17

2. Procedure for Reisolation and Growing Culture.

Tester strains, TA98 and TAl00 are grown in Oxoid nutrient broth No.2 and

incubated ovemight n a 3TC shaking water bath. The grovrth period should not

exceed 16 hours. These cultures are reisolation by streaking on minimal glucose agar

plated which the surfaces were spread with 0. I ml of 8 mg/ml ampicillin, 0.3 ml of 0. I

M histidine HCI and 0-l nrl of lmM biotin. These plates are incubated at 3/C for 48

hours. After incubation, the 5 single colonies per strain TA98 and TA100 are picked

up and growth in Oxoid nutrient broth No.2 overnight at 3?'C in shaking water bath.

Each culture is confirmed genotypes of the strains and kept the cultures as the source

of bacteria for mutagenicity testing. For each 1.0 ml of culture, add 0.09 ml of

spectrophotometric grade DMSO. Combine the culture and DMSO in a sterile tube

and distribute 400p1 of the culture aseptically into sterile cryotube (lr{unc). The tubes

should be filled nearly full and then transfer to a -80"C freezer.

3. Confirming Genotype of Tester Strains

The broth cultures of TA98 and TA100 are used to confirm genotypes in the

following ways.

3.1 Histidine requirement

The his character of the strains is confirmed by demonstrating the histidine

requirement for growth on the minimal glucose agar plates enriched with histidine and

biotin.

Procedure:

plate a no histidine and biotinCopyright by Mahidol University

Prapatsom Pakde€

plate b

plate c

plate d

Appendix / 118

0.1 ml of lmM biotin

0.3 ml ofO.1 MHis-HCl

0.3 ml of 0.1 M His-HCl + 0.1 ml of I mM biotin

Four minimal glucose agar plates are required for each tester strains. Each of

the plates is applied on the surface with 0.1 ml of I mM biotin, 0.3 nrl of 0.1 M His-

HCI, 0.3 nrl of 0.I M ffis-HCl plus 0.1 ml of I mM biotin and no application (plate b,

g d a respectively). Made a single streak of each strain across these plates. Five

strains could be tested on the same plate. Incubated at 3/C for 48 h. The growing of

bacteria on histidine plus biotin plate is the result ofhistidine requirement.

3.2 R Factor

The R-factor strains (TA97, TA98, TA100 and TAl02) should be tested

routinely for the presence of the ampicillin resistance factor because the plasmid is

somewhat unstable and can be lost from the bacteria.

Procedure: For each tester straiJl add 0.3 ml of fresh overnight culture to a

tube containing 0.1 ml of 0.1 M histidine.HCl followed by adding 2.0 nrl of molten top

agar containing 0.5 mM histidine-HCl and 0.5 mM biotin. Mixed and poured on a

minimal agar plate. Rotated the plate to distribute the mixtures and allowed several

minutes for agar to become firm. R-factor and rfa mutation (see the next section) are

performed in the same plate by dividing the plate into 2 areas, one for R-factor and the

other for rfa mutation. For Ri.factor, commercial ampicillin disc or filter paper disc

containing 8 mg/nrl arnpicilin is applied on the surface of the agar by using sterile

forceps. The disc is pressed lightly to embed in the overlay. The plates are incubated at


Fac. ofGrad. Studics. Mahidol Univ. M.Sc. (Food and Nutridonal Toxicolory) / 119

37'C for 24 hours. The absence of the clear zones of inhibition around the disc

indicates resistance to ampicillin.

3.3 rfa mutation

Strains having the deep rough (rfa) character should be tested for crystal violet

sensivity.

Procedure: Pipetted 0.17o solution of crystal violet to the sterile filter paper

disc (l/4 inch) and transferred the disc to plates, seed with bacteria (the procedure is

similar to R-factor). Incubated at 3?"C for 48 h. The clear zone appeared around the

disc indicated the presence of the rfa mutation the permitted crystal violet to enter and

kill bacteria,

4. Spontaneous Reyersion

Spontaneous reversion of the tester strains to histidine independence is

measured routinely in mutagenicity experiments and is expressed as the number of

spontaneous revertants per plate. The revertant colonies are clearly visible in a uniform

background lawn of auxotrophic bacteria. Each tester strain reverts spontaneously at a

frequenry that is characteristic of the strain. Nevertheless, there is variability in the

number of spontaneous revertants from one experiment to another and from one plate

to another, and it is advisable to include at least 2-3 spont.rneous mutation control

plates for each strain in a mutagenicity assay.

Procedure: 0.1 nrl of sterile water is added to capped culture tube. Add 0.5 ml

of NaPOr-KCl buffer pH 7.4,0.1 rnl of fresh overnight culture of TA98 or TA100,

followed by 2-0 ml of molten top agar. Mixed and then poured on minimal glucose 'Copyright by Mahidol University

Prapatsorn Pakdce Appcndis / l2t)

agar plate- Rotated plates and left it to become hardens_ Incubated at 3/C for 48 h

and the his* revertants colonies were counted.

5. The Response to Standard Carcinogen

- Standard mutagens or positive mutagens are used routinely in mutagenicity

experiments to confirm the reversion property and specificity of each strain. The

standard mutagen, which used in this experiment, is nitrosoaminoprene. Tester strain,

which highly response to positive mutagens be collected.

Procedure: 1.157 mg of aminopyrene (l,tfW 217 21.) was dissolved in 0.075 nil of

0.2N HCl, 0.01 ml of the supernatant was pipetted to capped tube. Add 0.74 ml of

0.2N HCl, 0.25 nd of 2M NaNO:. The final concentration of aminopyrene was

6.1706x10{ M and the final concentration of nitrite was 0,5 M. Mixed and shaken in

water bath at 37C for 4 hours. Placed the tube in an ice bath and added 0.25 nrl of 2M

NH2SO3NFL. Standed for l0 min. Pipetted 50 pl and 100.trt sf thg mixture to each

capped culture hrbe for test the stock culture TA98 and rAl00. Then evaluated their

mutagenicity as described in spontaneous reversion. The characteristic of the stock

culture for TA98 and TA100 as the source ofbacteria for mutagenicity is.

a) contained R-factor (pKM l0l)) and rfa mutation.

b) His* requirement.

c) Low spontaneous reversion.

d) Highly response to standard carcinogen.

After the characteristic ofthe culture was tested, the mutagenicity test was

started. Copyright by Mahidol University

Fac. of Grad- Snrdieg Mahidol Uloiv.

SMARTTEST

Preparation of Stendarrd Cultur,e Medium

Ir{.Sc. (Food and }.{rldtional Toxicolory) / 1 2 I

Ingrcdient

1. Comflour

2. Sugar

3. Yeast

4. Agar

5. Proprionic acid

6. Water

125 C

100 C

50c

14c

5ml

1000 ml

Steps of prcparations of standard medium for Drosophila mehnoguter stocls.

l. Boil and blend sugar, agar, yeast ard com flour in 1000 ml water until sticky.

2. Add propionic acid.

3. Fill each 125 ml-erlonneyer flask with 50 rnl of the medium.

4. Close offttre flask with a phrg (rnade of gauze and cotton cover with aluminum

foil).

5. Sterile the flasks in an autochve microbial contamination t}at can harm tlre flies.


Prapatsom Pakdec Appendix / 122

Statistical Consideration (Frei, lgSg)

In experiments designed to assess the mutagenicity of a chemical, most often a

treatment series were compard with a control series. one might like to decide

whether the compound used in the treatment should be considered as mutagenic or

non-mutagenic. The fonmrlation of2 alternative hypotheses allowed one to distinguish

among the possibilities ofa positive, inconclusive, or negative result ofan experiment

(Sefu,1e81).

In the null hypothesis one assumes that there was no difference in the mutation

frequency between control and treated series. Rejection ofthe null hypothesis indicated

that the treatment resulted in a statisticalty increased mutation frequency. The

alternative hypothesis postulated a priory that the treatment results in an increased

mutation frequency compared to the spontaneous frequency.

The alternative hypothesis was rejected if the mutation frequency 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 were considered inconclusive, as

one could not acc8pt at the same time the 2 mutually o<clusive hypotheses. In the

practical application of the decision proced.rg one defines a specific altemative

hypothesis requiring the mutation frequenry in the treated series 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 shoutd mean that the treatment was

weakly mutagenic, but led to a mutation frequency which was significantly lower than

/r, times the control frequency.Copyright by Mahidol University

Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food ard Nutritional Toxicolog) / 123

Testing against the null hypothesis (Ho) at the level cr and against the alternative

a hypothesis (I{n) at the level p led to the error probabilities for each of the possible

diagnoses. positive, weakly but positive, negativg or inconclusive. The following four

decisions were possible; l) accept both hypotheses; these 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.

Calculation step by step

Estimation of spot frequencies and confidence limits of m"

Particularly in the case that both hypotheseq I{o as well as H1, 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 Aequency. The estimated value was

ru : (nr/ n) \(n / n) Nft

where N and N, represented the respective sample sizes in control and treatment

series, n" and r\ the respective numbers of mutations found, and n the total of

mutations in both series together. Exact lower and upper confidence limits pr and p"

for the proportion no/n on one hand, as well as er and q" for the proportion n/n on the

other hand, may be determined according to Sachs (SactL 1982; SactL l9g4). He gave

an easy method to calculate these values using an F-distribution table. To determined

Qr and po one-sidedly at the level cq and qu and pr also one-sidedly at the level p. InCopyright by Mahidol University

Prapatsom Pakdee Appendix / 124

this way and in agreernent with the foregoing section, a confidence limit mr > I led to

rejection of FI", while a confidence limit m" < m led to rejection of H1.

In the first step, F-distribution according to Sachs (1982, 1984) were used to

determine the value F,r,,z at the level ct = 0.05, where the degrees of freedom (v1, v2)

were given by the equations

vl:2(n-nt+ l) and{z:2*

In the second step, the F-value so obtained was used to calculate the lower

confidence limit (q1) for the proportion of spots in the experimental series

qr : r\ / [nt+ (n-q + I F'1"2]

This gave a lower confidence limit for the frequency of spots per wing in the

control, which was equal to

fr,r : Qrn/Nr

This was the following complementarity, namely that the lower confidence limit

for the number of spots in the experimental series (q1n) plus the upper confidence limit

for the number of spots in the experiment (g.n) was equal to the total number of spbts

(n) found in experimental and control series together, i.e.,

P"rr (l-qr) n

This gave an upper limit for the frequency of spots per wing for the control,

which is

"fo, : q,n/N.

The lower confidence limit mr of the multiple rn was 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.,Copyright by Mahidol University

a

Fac, ofGrad. srrr.ircs l!{.ahidol Unir'. M.Sc. (Food and Nxtritional ro)dcolog}) / 125

mt : Ltt - qr n/Nt

"f". " p,n/N"

Only in the case that nrl, the lower confidence limit of me, was larger than L0

would reject E{r. Since this was not the case, He remain accepted,

In the same way, the lower confidence limit of the spot frequency may be

determined in the control Jt r that w'ill gre "L " and the upper con-fidence limit of the

spot 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 Syo confidence limit m.

for the multiple m..

Again, the F-distritrution according to Sachs (1984) was used and determined

the value F,r, uz at the level 0 : 0.05, where the degrees of freedom (r..2) wcre this

time given by the equations

vl : 2(n-+ + 1) and v2 :2 4

The F-value so obtained was used to calculate the lower confidence limit (pl)

for the proportion of spots in the control

Pr= ru/ [n + (n-n" + l; R,,"ri

This gave a lower confidence limit lor the frequenry ol spots per wing in the

control which equal to

/l r :Prn/N"

Agairl There was complementarity, in tlut the lower confidence linrit for the

number of spots in the control (pln) plus the upper confidence limit for the number of

spots in the experiment (q.n) was equal to lhe total number ofspots (n), so that

q,n - (1-pr) nCopyright by Mahidol University

1

I

,t

Prrpfitsorn Pilkdcc Appcndir / 126

This gave afl upper limit lor the frequency of spots per wing for this series,

r.vhich is

,f,,, = q,n/N,

The upper confiderce limit n1r of the nultiple n\ can be determined as the ratio

between the upper confidence limit for the fiequency in the treated series and the low.er

confidence limit for the &equency in the control, i.e.,

m, :.,ilg! : funll'Y!

fr' PrnA{"

Ha was rejected if nr,, the upper confiderce linrit of nr", was less than m (m:2

for the total of all spots and for the small single spots, aod rn:5 for the large single

spots as well as for the rw-in spots), Substihrtion of m. by m1 or m, in the above

formulas provided the respective exact upper and lower confidence limits for the

frequencies estimated.


Fac. OfGrad. Studies' Mahidol Univ. M.Sc. (Food and Nukitiunl Toxicology) /

NAME

DATEOX'BIRTH

PLACE OF BIRTII

INSTITUTIONS ATTE,TTDED

FOSITION&OFFICE

BIOGRAPHY

Miss Prapatsom Pakdee

29 laauary 1974

Nonthatnri, Thailand

lvlahidol University, 1991-1994

Bachelor of Nursing Science.

Mahidol University, 1 9G2001

Master of Science (Food and Nutritional

Toxicology)

1995- 1996 Siriraj Hospital

Position: Registered Nurse

1 97-Present Vipawadee Hospitat

Position: Registered Nurse (part-time)

\


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Copyright by Mahidol Universitymulinet11.li.mahidol.ac.th/e-thesis/scan/3936164.pdf · g,anode&ma ltjcidum (fk) ruwt on niirosated prodi,'ct of amit{opyrene.niiriie MODEL IN AMES