EFFECTS OF ADRENERGICDRUGS AND MONOSODIUM GLUTAMATE ONTHE DEVELOPMENT OFSEX-DEPENDENTCYTOCHROME P450 SYSTEi-l
IN MALERAT LIVER
by
Cida Zhao, B.Sc. (Med i c i n e )
Athesis submittedto the School of GraduateStudies in partialfulfilmentof the requirements
for the de greeof Masterof Science
In t erd i s cip lina r y Programme in Toxicology MemorialUniversityof Newfoundland
June, 199 4
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ABSTRACT
The effects of adre ne rg i c drug s on the de v elop men t of male-spec ifi c cytochrome P450 in th e male rat liv er were stud i ed. Pups, cast rated onDay 0, we r e given (icv) a- antag on i s t or a-ag on i s t wit h or wi tho ut testosterone propiona te (sc.), end assayed on Day 65. Oth e r pups were admini s teredwith (ic v ) adrenerg icac;onists or antagonist s on DayI)&3 of lif e , castrat edon Day 55,and assayed onDa y70.
Neither a-ant agonist nor B-agonist defem inized the EHDM activit y inneonat a ll y ca str ated rats . Inthe ratscastrated onDa y55 ,a- anta g oni s t and aI-antagonistsuperdefemini zed the EMOM act ivity . a- antagoni st inc reasedthe total amount of P450 but ai-antagonistdidnot. In addition, theVmaxof EMDM in O-antagonist-treated animals was increased; the KIn was unchanged. Further,J3-antagon i s tstimulatedthe activitiesof 160-, 2a-, and 60-testosterone hydroxylases in the superdefeminized rats.
antagonist, given
Howeve r, both B-antagonist and QI- simultaneously, sup p r e s s e d th e superdefeminizationof EMDM activity.
The effe c t of mo noso d ium glutamate (MSG) on the prepubertaldevelopmentof male-specific cytochrome P450 in rat live r were also stud ied. Pups were treated on Day1-9 with2 or 4 mg/qMSGtecj ,and onDay35the EMDMacti viti e s
in 2 and 4 mg/gMSG-treatedgroups were the same as control va l u e s. However,MSG stimulated, dose-dependently, theEMDM activ ity on Day 65. In addi t ion, Vmaxof EMDM waselevated in MSG-treatedgroup; theKInwas unchanged. Final ly, 01mg/ gMSG stimu lated the activi tyof 6B- but no t 16a/2a-testosterone hydroxy] <loses.
ACKNOWLEDGMENTS
I wish to express my spec i a l tha n ks to Dr. Bruce B.
Virgo, my supe rvisor , for his ini tial dir ec tion in selecting a re sear ch are a , and his as s i s t a nc e and careful guidance throug hout the re searchpro jectand duringpre parat ionof the ma nu s c ript. I amalso gra tef u l tothe me mbers of my thesis committee, Drs.C. Lo omi sand D. Bi eger, for theiradvi c e.
In addition, financialsupportfromSchool of Pharmacy,
Memorial Univ8r s ity of Newfoundland is gratefully
ac kn owl e dg e d.
FinallyI aminde b t e dtomy pa re n tswho have give n memy lif e, educ a tion, an dmore than I cansay . I love thembo th dearly. My thanksarealsodu e tomy brot he r andsis t e r s for th e i r supportand un derstanding.
ABBREVIA T ION
SHTP, S-Hydroxyptophan ACTH,Ad renocor t ico tro pic Hormone AF P, a-Fe t opr o te i n
AMN, Amygd a l o i d Nucleus ARCN, Ar cua t eNu cl eu s
cAMP, Cycl ic Adenos ine 3',S '-Monopho s p hate CLO,Cl oni d ine
CNS, CentralNervou sSystem DAG,Diacy l g l y c e r o l
EMDM,Ethyl mor ph ineDemethylas e G-6-P , Gl ucose-6 -Phos ph ate
G-6-P-DH, Glu c ose - 6-P hosp h ate Dl!hyd r og e na s e GH, GrowthHo rmone
GHRH, Growt hHor mo ne - Releasi ngHo r mone rev, In tra c r ani a l Inj ec ti on IP)' In o sitol- l , 4, S-t r isp hos p h ate ISO, Is opro te r enolol
MPOA,Med ia l Preop ti c Are a mRNA,Messe ng e r Ribonuc l ei c Acid MSG, Sodium Glutama t e
NADP, a-Nicotin a mi de Adenine Dinucle o t i de Phosphat e NADPI a-Nicotina mi d e Aden i ne Dinuc leotide Phospha t e NADPH, Reduc e d Ni c o t ina mi de Aden ineDinucl eo t ide Pho spha t e
p-NPH, p-Ni tr o phe n o l HydrOlc:.ylase PAS, Perma n e n t Anovulator y-S ter i li t y PCPA, p-Ch lorophenylalan ine PH, Phen to lam ine PAY, Phenylephrine PRA, Prazos in PRO, Propranolol ec , Subcutaneous Injection SDN-POA, Sexually DimorphicNucleus SHS, ShaftSynapses
5S , Somatosta t in T4' Thyroxone
TP,'restos t.eccne Propio nate VMN, Ventromedial Nucleus WY, WY 27127
TABLE OF CONTENTS
Page ABSTRACT ••••.•. .•• •. •••.• .•.•..•.••. - .. ... .. ...• •ii i ,lI.CKNOWLEDGEMENTS•••••• •. .••. .•• ••• • • •••••.••• •••••.••. .•
ABBREVI ATION..•• ••• ••. . ..•••.. •.••.•.••• •••• .•••• •...•.• vi LIST OF TABLESANDFIGURES.• •• •.••.••••• •.•.••• •• •••••• •xiv 1. 0INTRODUCTION••• • •••••••••••••••.• •• •••. .•• ••• •• ••• . .
1.1 HEPATIC CYTOCHROME AND BRAINSEXUAL
DIFFERENTIATION ••.•••••• •••• ••• •••••••••••.•. .•••
1.1. 1Sexual Di f f e r en c e sof HepaticCytochrome P450Oxidations.•.. .. . . . ..•••. • •.. .•.. ....•• 1.1.2Sexual Differencesof Cytochro meP450and
NeonatalAndrogen•. ••••.•••. ...•• •..••• • 1.1.3SexDifferences ofHepaticCytochrome
Oxidationsand the SecretoryPa t t e rn of GrowthHor mone •. • • . •••• •••... •• • .•• •.•. . 8 1.1.4 Hyp o t h a l ami c Regulationof GHSecretion.... . 11 1.1.5Sexual Differentia tionof th eBrain ••• • •. • • • 12 1.1 .5.1Defem1nizationana Masculinization•• . 12 1.1 .5.2MorphologicalChangesof Brainand
NeonatalSex Hormones... ... .... . ... 14 11.5.3sax Steroids During the Neo n a t a l
Period . . ... ...•• •.. ••. . • •.... • .... 18 1.1.5.3.1And r ogenand Est rogen.•••••. 18 1.1.5.3. 2a-Fetopro tein(AFP)••••• •••• 20 1.1.S. 4 Hep at.Lc Sex-DependentCytochrome
P450•••• ••••. •••. • ••..•••••• ••••••••• 21 1.1.6CatecholaminergiCSystemin the
Defeminhationof the Brain... .... ... 22
Page 1.1.6.1 Neona t alcatecho1aminesand
behaviour. •...•... .... .•.. .... ... 22 1.1.6.2Neonatalcatecho1aminesand
re pr od u cti va endocrine function. . . ... 23 1.1 .6 . 3 Neonatal catecholamines and
SDN-PDA .. . ..•••.•• • • ••• .•. ..••.•.. • .. 24 1. 1.6.4
ce
tachotemrneeand androgen duringthe neonatal period.. .•..••..•• •.•..• 25 1.2 GH and Development ofMa l e-Sp e ci fi c Cytochromes
P450..•. ..•. •...•••.•.••. .•• •. . • .•• • ••.•••• 26 1.2.1Developmental Express ionsofMale-Specific
CytochromesP450 •.•.. ...•. ... . ....•. 26 1.2 .2GH andMale-SpecificCytochromesP450 ...•.•. 28 2.0 MATERIALS ANDMETHODS.... ... .•• •• •.• • .•..•. • . •• . .• .• 30 2.1GENERAL MATERIALSANDMETHODS•..••.••...•. • •.. •. • 31 2.1.1 Chemicalsand Biochemicals ••.... . .• ...•....• 31 2.1.1.1Ch emic a l s for animal inj ection..• •.•• 31 2.1.1.2Chemicalsforthe enzyme assay. .... .. 31 2.1. 1.3Chemicals for buffers..•.. .•.. ... 32 2.1.2Animals•. • . ••. •• •••.•..• • •....•.• .• ••.•.••.• 33 2. 1. 3Intraventricular Injection.• ...•.•.•.... .. 33 2. 1.4Castration.• •••••••• ••• ••••• .••••.•• .•••••.• 34 2.1.4.1 Castrationof the neonate... .••..•. 34 2.1.4 .2Castration of adult rats•..•.• •• • •••• 35 2.1.5SubcutaneousInjectionsnuringth e Neonatal
Period... ... ... ... ... 36 2.1.6Prenarationof Microsomes.•.•• ..•.• ••• ••• •.• 36
Pagp 2.1.7Me asu r e mentof Micr osomal Prot ei n . . . 37 2.1.8 EnzymeAssays •..•.. .. . ....•• . ... 38
2.1.8 . 1 Measurement of ethylmor ph ine demethy 1ase(EMDM )activity in hepaticmic r o s o mes.... ...••.. 38 2.1.8.2 Measu remen t ofp-nitraphenol
hydroxylase(p-NPH j act ivityin hepaticmicrosomes.. . ... .••••... ...•. 40 2.1.8.3Ouantitationof cytochrome P450 43 2.1.8.4 Measuremen t of NADPHcy to ch r ome c
reductase. ... .••. .•••.•. .•..•• .. 44 2.1.8.5 Kinetics of et hylmorphine
demethylase.• • •.•••...• •••.•.... 45 2.1.8.6Measurementof 2a-, 16a-,and 63-
tes tos t eronehy d r o xy l a ses 46 2.2ANIMALPROCEDURES .••.•. ..•. ... ..•.•••••• ••. .... 49
2.2 .1Roleof Adrenergic Recaptorsinthe Neonata lDefeminizationofHe p atic Drug Metabol ismin Male Rats.... ... .... .... . 49 2.2.1.1Drug metabolismin male rats
castratedne on a t al l y and given
a-,
a- ad r en e r g i c agonistsor antagonistsan dtestosteronepropionate ne on a t al ly.. ... 49 2.2.1.2 Drug metabolis min malera t sgiven
a-or 8-ad renergicagonis tsor antagonists neonatally.•.. . • ..•.. . • •. 49 2.2.1.3Drug metabolisminma l e ra t s gi v e n
propranolol tog e th erwi th other adrenergicagents ne o na t a lly... .... 50 2.2.2MonosodiumGlutamate(MSG) and the
Prepubertaland PubertalDevelopmentof Hepatic Cytochrome P450 System in Male Rats... ... ....•.. ••.•• .. .• .•• . ••. . .... .. 51
Page 2.2. 2.1 Dru gmetabo lismin35-d a y-0 1d rats
treatedwi thMSG duringtheneonatal
period... .. . ... 51
2.2.2.2Drug metabolism in 65-day-oldrats givenMSG during the neonatalperiod . . 52 2.3STATISTICALANALySES .. . . ... ... . . .... . . ... 52 3.0RESULTS... .. ... ... . . . ... . . . ... . . . ... . ... . . . ... 53
3.1THE HEPATICCYTOCHRmtFP450 SYSTEM:EFFECT OF ADRENERGICDRUGADMINISTRATEDNEONATALLy... .. 54 3.1.1Effect of Phentolami neand Is o p r otere no l
onDe femi nizationin NeonatalCastrates Defe minized withExoge n ousAnd rog en ••• ...•.. 54 3.1.1.1 Hepatic ethylmorphine demethylase
(EMDM)activity... .. .. . ... . .... 54 3.1.1.2 aepetac p-n i trophenol hydroxylase
(p-NPH) acti v ity... ... 56 3.1.1.3Hepatic cytochrome cred uctas e
activ i tyandcytochromeP450 amount .. 57 3.1 .2Effectof Neonatal AdrenergicAgents on
Def e mini z ed CytochromeP450 System. .. 59 3.1.2.1Hepa ti c ethylmor phi ne demethy lase
activ ity••••..••••••••.• . • • •••••• ••. 59 3.1.2.2 Hepat i cp-n i trophenolhyd rox yl ase
activity•••.••• ••••••••••• •••• .•.•••• 63 3.1.2.3 Hepaticcy t o chrome cre d u c t a s e
activity 63
3.1.2.4He p ati c cytochromeP450 amoun t .•. .... 64 3. 1.2 .5Kinetics of hepa t icEMOMin PRO-
induced"s up e r-d efemini z a t i o n "••• •.•. 66 3.1.2.6He p a t i c 68- , 16a-, and zc-
testosteronehydroxylasesin PRO- induced"s u p e r-de f e min i z at i o n ".... . .. 69
Page 3.1.3Effects of NeonatalAd r e n e r g icDrugs in
PRO- TreatedNeonates. • .. .. .. .. ...••• . •• 71 3.1.3 .1 He pa ti c ethylmorphi n ede meth ylase
activ ity . ... . .. . ... . ... .. .. 71 3.1. 3.2Hepaticcytochrome c reductase
acti vi ty and the amo u n t of
cytochrome P450•• • 74
3.1.3 .3He paticp-nitroph e na lhydro xylas e activi ty.. .•.• .•. . . . ...•• • . •.... . • 74 3.2THEHE PATI C CYTOCKROMEP450 SYSTEM; EFFECTS OF
MONOS ODIUM GLUTAMATE (MSG)ADMIN I STRATED
NEONATALLy.. . .... . . .. . . .... ...•. • "6 3.2.1MSG Effecr.u in Mal es.\ssa y edonDay 35 . 76 3.2.2MSG Effectsin Males As s ayedonDa y65 80
3.2.2.1 He pa t i c EMDM activ ityand p-NPH activity... ...• .... .•... .•• • .•.• •.• 80 3.2 .2.2Cytochromec reductase ac ti vi ty and
total amount of cytochrome P45 0.. .. 80 3.2.2.3 Ki n e t i c s ofhepa t i c EMDM. 80 3.2.2. 4 Hepat i c 63-, 16a- , and2a-
te s t o s t eron ehydrcxy La s e s ....•.••.• .. 83 4.0DI SC USS I ON . ••...••• .• .•. ••.• •... ... ... .. . .. •. . .•• 87
4.1ADRENERGI C RECEPTORS AND THEDEFE MINIZATION OFEMOM.• •.• • • •...•• • . .•....• ..•.•.. .. • • .... . .
aa
4.1.1 Ad rener g i cRe c ep t o r s andCNS
Def emi n i z at ion . .. .. .. ... . . ... ... 89 4.1. 2Mec h an is m of theActionof Adr en erg i c
Rec e p t orsin Oefeminiza tion..• •.•• •.. .. . . 96 4.1.3 Ki n e t i c s of EMDM and l6a- , 2a-,and 613-
Testoste ron eHy drox yLas ea in PRO-i n duc e d Supe rd e feminization.• •• •.• •. . •• • •• • • .. ••....104
Page 4. 2NEONATA L MONOSODIUMGLUTAMATEANDPREPUBERTAL
DEVEL.OPMENTOFMALESPECIF ICCYTOCHROMESP450 ... . 106 4.2 . 1 Prepu be rt alDeve lopmen t ..•. . • . • .••••• . ••. . •. 10 7 4.2.2 Pubertal Deve l o p ment ••..• •• •••••••••• ••••• •• I I I RE FE RENCES •••• • • •. ••• ••••••• ••• ••• • ••••••••••••••• •• •••• 115
Table
LISTOFTABLES AND FIGURES
Pa g e 2-1 Hepati cEMDM acti vi ty of intact andcast rat e d
maleratsun der diff e r e n t incub a tio n time ... . ... . 41 2-2 Hepatic p-NPH activi tyof male ratsunder
differentamountsof microsomeprot ei n&various in c ub a ti o n time.. .. . . .. . . . .. ... .... . . .• •. 42 3-1 Effec t of adrenergicagent son hepaticEMDMand
p-NPHactivitiesin neonatalcastrateddefeminized wi th elCogenous and r o g e n ... . .. .• •....• •... 55 3-2 Effectof ad r en e r g i c agents on hepaticcytochrome
c reductaseactivityan d cytoc hromeP450 amoun t in neonatal castrated defeminized withexogenous androgen ~. ... ... .... .... . . . .. ...•..• 58 3-3 Effect of ne on a t al adrenergicagents on hepatic
EMDM and p-NPHactivitiesin defemini zedmale rats... ....••••••.••••••••• •.•••.•.. .•.• ..••... 60 3-4 Effect of neonatal adrenergicagentson hepatic
cy t o c h r ome cre duc t a s e activityand cytochrome P450 amountin defeminizedmale rats... .. . . ... . 65 3-5 Kineticsof hepaticEMDM inpro p r ano l o l - s upe r-
defeminizedrats..••. .•• • •• •. .••... . .•. .•. .•... 68 3-6 Hepatic 68-, 16a-,&2a - t e stos t e r o n e hydro xylases
in propranolol-inducedsuperdefeminizatio n.• .. ... 70 3-7 Effect of neonatal adrenergicagents on hep a t i c
EMDM and p-NPH ac tivi t i e s
rn
propranolol-super- de f emi niz e d rat s ....••••...•. •... . 72 3-8 Effect of neonatal adrenergicagents on hepaticcytoch romec reductaseactivityand cytochrome P450 amountin prcpranolol -superdefeminized rats... •.•.•• •.. . .. . .•. ... .. ...•. . . ..•• .• 75 3-9 Effect of ne onatalMSGonhepaticEMDM and p-NPH
ac t i v i t i e s at Day 35•...••.•.••• •.••••• •. ••.••• .• 77 3-10 Effectof neona talMSG on hepaticcytochr ome c
reductaseactivityand cytochromeP450amountat Da y 35...•.. ... •.•.•.• •.••• ••..• • •••• .•• ...•..•.. 79
t
Page 3-11 Effect of neonatal MSG on hepatic EMDM and p-NPH
activitiesat Day 65••.. ..•.•..• .... ...•....••• 81 3-12 Effect of neonatal MSG an he pa tic cy t o c h r ome c
reductase act ivityand cytochrome P450 amount at Day35••.• ... ..•••.• •••••• ••.• •..•• •.•..•••• 82 3-13 Kinetics of EMDMin65-Day-oldmale rats treated
neona t a lly withMSG.•..• •..•..••...•. .••••.•.. .... 84 3-14 Hepatic 66- , l6a-, &2a.-testos terone hydrox y l a s e s
in65-Day-old ma l e rats treated neonatallywith MSG.. . . .. ... ... •.• •••.••.• •••.•••• • . •• • ••.••.•• •• 86
Figure
Cyto c h r omeP4 5 0 catalytic cycle
1.0 INTRODUCTION
Cy tochromes P450 are a superfamily of heme proteins. These cytcch rcmesare foundma inl y in the liver,but they also occurin othe r ti ssu e s,such as lunq, kidney , intestine,skin, testis, placenta , and adrenal (Si p e s and Gandollfi, 1991).
Theseproteinsare partsof a systemth a t oxidizessteroids, vitamins, fa t t y acids and many x.e no b i oti c s such as dr ugs, pe s t i ci d e s. The cytochro mesP450 are the terminaloxidaseand subst ratebinding enzy mes in some 30 different monooxygenase reactions that include aliphatic hydroxylatio n, aromatic hy d r oxy l a t i on , de a min a t i on , and N-hydroxylation , etc.(Sipes and Gandollfi, 1991).
Th e cytochrome P450 catal y ti c cycle is presented in Figure1: the xenobiot ic combineswiththe oxidizedform of cytochrome P45 0 (Fe].) to form a substrate-cytochrome P450 complex. This complex accepts an electron from NADPH via NADPH-cytochrome creductase, whichre du c e s the iron in the cytochromeP450 heme to theFe!' state. The reduced complex then combines with one molecule of ox ygen, which accepts another electronfromNADP. This molecu laroxy ge n rad ica l is hi g h l y reactiveand unstable an d oneatomof itis introduced into the substrate ,wh ilethe other is reduced to water. Then the oxygenated substrate dissociates, regenerating the oxidizedform of cy t o c h r o meP450.
Dor
UH
a
.y'
P.45 C(F,")"<
(RH ) P~450(Fc~ 'J)J+ (RH)P-450(Fc'J+)
HO H~I y c :
(P.H)P· 450(Fe3+)(HO:!-) (RH)P-45 0(Fe:l+)
\ f a,
(RH)P .4 50(Fc")(0,'-) (RH)P.4 50(Fc")(O,)
e -~
Figure1. CytochromeP450CatalyticCycle (modifiedfrom:RobertAN(1980). "Metabolismof toxic substances."In: Casarett andOoull'sToxicology,the Basic ScienceoCPo isons,2nded. editedbyDoullJ,KlaassenCD, andAmdurMO,MacmillanPublishingCo.,Inc.New York]
The act: i v i t yof cytochromeP4S0medi at e d re a c tio nsca n be in fl uenc ed by factors such as diet (And e r son and Ka p pas, 1991l, hormones (Lu n d et al.,1991 l, peptid e s (Ur b an et al. , 1990), chemicals(Bo ck et aI., 1990 ) , se x (Kato, 197 H. and age (Wa xman et al., 1985). Alterations in the acti v i ty of cy toc h rome P450 will influe n c e the normal physiologi ca l metabolismandthe metabolismof drugs and other xenobiotic s ; which mayinterferewithth e normal physiological func tion sin the body and ind uce pathogene s i s . Therefo r e , studi e s on cyt ochrome P450 are important in toxi co l o gy. This thesis is an attempt to exami ne the rol e of ne o nata l cldren erg i c rec epto r s in the centra l nervou s system in thede femi n iz a tion of hep atic male-specific cytocbromes P450, and therole Clf qrow t hhormo n e (GH ) inth epre pub ertaldevelopmentof hepatic cytochrom eP450 in malera t s.
1.1 HEPATICCYTOCHROMEANDBRAIN SEXUALDIFFERENTI ATIO N
1.1. 1 Suu a l Diff erencesof He patic CytochromeP450 Oxi dation s
Ox i da t ionof anumbe r of xenobi oti c s an dst e r o i ds 1nthe adult rat liver is sex specific . For ex a mp le , female rat s givenhexoba r b i t al sleep longer than male s (Holck et a1.,
1937)and sim i larresults had also been shown by otherauthors (Quinn et;al.,195 8 ). Microsomes from the li verof adult male rats oxidize ethylmorphine and benzo(a)py rene more rapidly thandothose fromth e females (Chu n g et a1., 1975 : Davieset al . , 1969; Gurtoo and Parker, 197 6 ; Hietanen, 1974), and althoughmfcz-cscmea from the livers of ad ultmale rats ha ve 10%- 30 %more cytochrome P450 than those f!"omfemales (Chung, 1977;Colby, 1980),thisdi f f e r ence does not account fo r the di f f e r e n c e s inth e ratesof metabolismof thesexe no b i o t i c s (Kamatakiet al. , 1980;xecc , 1974).Moreover, other studies (Kato and Kamataki , 1982: Kamata kiet a1., 1982; MacGeochet al., 1984; Morgan et al., 1985) showed that isoforms of hep a ti c cytochromes P450were sexu a lly di f f ere n t i a t e d in adult ra t s and that the ex istence of thesesex-specific isoforms, su chas cytochrome P450 2Cn , 2C12 , or 3Al/2, leads to the sexualdifferences in th e su bstr a te me t a bolis m.
1.1 .2 Sexu al Dif fe rence s of Cytochrollle P450 and Neonatal Androgen
Th e s e sex u al lydiff erent ac t i vi t ie s of cytoc hromeP450- ca ta l yzedoxidations are the res u l t of neonatal and pubertal exposureof thera t to androgen. Castration of male rats in the neonatalperioddecreases ethy l morphi neN-demethylation ,
propoxy c o umarin O- deprop y l a tion and benzo (a )p yre n e hy d ro xyl a tio n to the fema l e level (Sh imada et al., 1987);
neonata l administration of cas':;ra ted lllale pups with te s tosteron e inc rease d the activit iesof the enzyme s bu t not up to the lev elsof th e normaladult mal e s. However, others found that theac ti vityof ethyl mo r ph i n e demethy l a s e(EMDM)in neonatal ca s t r a t ed male rat s , trea t e d ne.>natally and in adulthood with testo sterone, was the same as that in in t a ct males (Re ye s and Virgo, 1988). The male isoforms of cytochromesP450 in adult male rats cast ra ted ne on a t a lly are bar e ly det e c table while the fema l e-s p e c ifi c isoform sappeared at a le v e l comp a r a b l e to those found in ad u l t fe males (Kamatakiet aI.,1983,1984; Kato and Kamatak i. ,1982;Waxman et aI., 1985; Jan s s o n et aI., 1985al. Moreover, the administrllt ion to these animalsof neona taland postpuberta l testostero ne caus e d the appearance of male-typecytoc: h ro mes P450 andthe disappeara nce of the felllale types (Sh i mad a et al.,1987). It appearsth a t the sexuallydi fferentcyt oc h ro me P450 enzymes ar e the results of neonatal and post-pUberta l exp os u r e of the ra t to androgen.
Sexual differentiation of cytochrome P450 enzyme is bel ieved to resul t from two dist inct androg e n-d e pe ndent processes: i) defemi niza ti on, whichsuppressesfe ma l e
characteristics , permitting malecharacte risticstode ve l o p ,
an d during the neonatal pe r i od, and ii )
masculinization, which enhances the ma l e characteristics in adul t h o od(Re y e s&Virgo,1988). Furthermore,de feminization is thought to be a ne ces s a ry prerequi si te for the masculinization (MacLusky and Naftolin, 1981 ). Neona tal castration complete l y and ir r ever sib ly de p resses the ec 'trvtttes of ethylmo r phine deme thylas e inadul tmal es , but theen zymeactivi ti escan be pe r mane n tl yincr ea sed , albe it no t to the le v e l s in intact ad ul t males , i f these an imal s ar e gi v e ntestoste ro ne immedi ately afte rcast ra t ion(Virgo, 1991 ). Adul t castration, howeve r ,decre ase s theenz ymeactivitiesto the def e mi n iz e d lev el s , whi ch can be mas c ulinized by testosterone. Thus, the s e datasho wtha t neonatal an droge n permanen tl y defeminizes the enz yme system, and puberta l androgen mas cu lin i z e s the defe minized en zyme s but not permane ntl y . Howe v er , neo nata l exposu r e of the ra t to andro gen is not an esse n ti a l pr e requis i t e for the se xua l differenti at i on of drug met a bolism. BothShi ma da (1987) an d Vi rgo(1991) ha ve reported that mas c ulin iz ation ca n occu r in the ab s ence of defemi ni za t ion. For exa mp le, neo n ata l ca s trati on comp l e t e ly supp res sed theactiv i tiesofthe hepati c enzymesinad u ltmale rats ,bu t the le vels of eth y l morp h ine N- de methy l a tion (Shim a.da et al., 1987:Vir go, 1991),
hydroxylation (Shi mada et al., 19 8 7)
propoxycoumarin O-depropylation and ben actajpy ren e increased to the levelsof int a ct adult malerats bype ripu b e r t a l testosterone treat ment. These data suggest that neonata l androgen is not necessar yfor the eff e c tsof pubertalandroge nthoughit can per mane ntly defeminiz e the enz yme system. Howe ver, older adult male rats (Day 75-80) castrated neon ata ll y do not respondtotestosterone(Virgo, 1991) ,end fema l erats alsodo no t re sp ond to either neona ta l or adu l t testosteron e; the reasonsforthe se phen ome n a are not yet known.
1.1.3 Sex Differ enCl;Isof Hep a t ic CytochromeOxid a t i on s and theSecretoryPat ternof Growt h Hormo ne
1t wa s pos tu lated origina llythatthe femal epi tuitary secre t esaho r mon e,"f e minotr o pi n", that femi niz e s the female cy t ochrome P450 syste m (Gusta fsson et al., 197 7), and that
"f emi no t r o p in" relea s e is blo c ked in the ma l e by a hypo thalamichormone, "fe minost atin" (Gusta fssonetal.,197 6 ; Gustafsson et al., 1977). By investigating differe nt pitu i t aryhormones ,it was foundthat onlygrowth hormonehas manifes t effectsonthesex-dep end e nt cyto chromesP450 inthe rat li v e r (Gus taf ssonet a1.,1983). Moreove r,growthhormone wa s found to playthe ro leof "femi n otropin". Fo r examp le,
exogenous GH feminhes the activities of cytochrome P45 0 sy ste m of intact, castrated, and hypophysectomized males (Kramer et al., 1975; Mod e et al., 19B1). Age nts suchas somatostati n (Virgo, 19B5) that block GH-rel e a se wi l l masculin ize the cytochrome P450 syste mof ca s t r a ted males whileagen t s suchas clo nidi ne (Virgo, 19 8 5 ) that stImutet,oGH rele a se will fe mi nize the cytochrome f'450 systemof intact males. All these obse rv ationssuggestthatGH playsthe role of"fe mi notrop in" .
Howev e r , GH exi sts 1nbothmalesand females. If it is the fac t o r that femi ni zesthe cytoc hro me P4S0 system, the r e sho u l d be some diffe r e nces in GHbet ween the males and the fema l es. The curren t data indi cates tha t the secretory patte r n s of GH arese xua llydiff erent. Mal e rats ex h i bitvery lownadi r leve l s of plasmaGHwith ma r ke d peaks every3-4h.
Female ra t s , in contra s t, ha ve a highe r basal GHlevel an d more irregu lar pea ks of smaller amplitude (J a n s s on at al., 19B5b ). It appears that it is th is sexual dimorphism tha t determin e s the activiti esof hepat i c cytoc h ro me P4 S0med iated oxi d a tions . Cytoch ro meP450 2ellis a mal e-speci fic isoform andex i s t s inhi g h le vel s in in t actadult malesbutlow le ve ls in in tact femal e s . Cytoc hrom e 2C12, aov e v e r , is a fem a l e- sp ec i fi<:isoform and appe a rs ina highconcentrationinintact
female adults but low in intact adult males (Mode et aI., 1989; waxmen et al., 1985). Hypophysectomy decreases the levels of bothis o f o r ms in adult rats. However, intermittent administration of GH, mimick ingthe male secretorypattern, causedcompletemasculinizationof the male specificP4S0
2en
in hyp o p hy s ec t omiz e d males ; when GH was administered continuously, mimicking the fema l e secreto ry pa t t e r n , the femalesp e c if i cP4S0 2C12 wasin duc edin the hypophysectomized females (Modeet al. , 1989; Legrav erend etal., 1992 ). The s e data sugges t tha t some isoforms of se x-d e p e nd e n t P4S0 ar e related tothe sexua l dimorphism ofGH sec r e t ory pattern.
Thissexualdimorphism ofGH secretion is th e result of exposureofmale rats to androgenduring the neonatal period and adulthood (Jansson and Frohman, 1987). Ne on a t a l cast ra tion caus e s a markedsup p ressioninbo t h the IDaqnitude and the duration of GH secretion , and higher ba seline GH le v e ls than that in eheei-opereued controls , ie. fe llli n i n e chara c t e ristics of GH secret ion. Al though ne on a t al te stos t e r o ne replacemen t therapyre stored high amp litudeGH pul s e s , they occurred mo re fre qu e n tly and the bas e line GH levels were ma r kedly hig h e r th an tho s e in in t a ct male...acs . However, neonatall y ca s tra ted rats given testosterone neonatally andalso du r i ng adulthood disp layed ano r malGH
pat t ern compa rabl e to that of intact males (J a n s so n and Fro hman . 1987). Th e r efo r e . neonatalandrogen ca n permanently defeminize the secreto ry patte r n of GH, and androgen in adulthood ca n masc u li nize the defeminized GH secre tio n.
Howeve r , a mas culine GH pattern was also seen in the neon ata lly ca s t rat e d malerats giventest os te rone only du ring adul thood(J a n s son and Frohman, 198 7) . Thu s , defem i ni zation is not an essenti al prerequisi te for masculin iz at i on. concurrently, neonatalca s t r a t ion complet elysupp resse sEMDM act ivi t ies in adult males, and neonatal te sto s t eron e re pl a c e me n t partiallyrestores the activity ;butbothneonatal and adultte s t o s t e r o n eco mp l e t e l y restores the enzyme activity (Virgo, 1991). Furt her,peripubertaltestosteronecan induc e an inta ct adult level of enayme act iv i ty in adul t males castrated neonatally (Vi r go , 19 9 1 )• Thus, th e sexual differentiationof thisenzyme systemis parallelto that of GHsecretorypattern.
1.1.4 HypothalamicRegulationof GH Secretion
GH secre t ionis under the cont rol of the hypothalamus . The hypothalamus produces GH-r el e a sin g hormone(GHRH) which stimulates the synthesisandre l e a s e of GH,and somatos t atin (55)whi ch in h i bits th e synthesisand releaseof the ho rmone
(Jansson at a1., 1985b). It has been reported (Painson et al., 1991 )that the secretionof GHRH shows episodic pulses, and that androgen ca n increase the amplitude of the GHRH pulses. Male rats also displaya periodic55 secretionwith peaks at the timeof thetr ou g h of GHRHsecretion, andGHhas apo s i ti v efe e d b a c k on 55secretion(Painsonet a1., 1991 );in contrast, femaleratsshowa conti nuoussecretory patternof 55 and the levelsare higher. These data suggestthat theGH secretorypattern1s regula tedby GHRHand55. As for sexual differences of the release of bothGHRHand SS, some evidence, which will be pr e s en te d in the followingsection, show th a t they might be determined by the sexual differentiati on of the brain.
1.1. 5 Sexual Differentiationof the Brain
1,1.5.1 Def emi n i z a tion and Masculinization
Se xua l differentiationofthebrain resultsfrom exposure to androgen during a sh or t period of neur aldevelopmentwhen ne r vou s tissue is still sufficiently plastic to respond permane ntlyand irreversib l ytogo n a dalho r mon e s . In rats, it actually consists of two process es, the permanent defeminization and th e reversiblema s c u l i n izati on (Ma c Lus ky and Naftolin, 1981).
The short critical period when androgen irreversibly defeminizes the brain is marked by a suppression of female characteristics and an enhancement of the potential of the central nervous system (eNS) to re s po n d to androgen in adulthood. Thisprocessis "de f emi nizati o n" and only depends on the hormonal environment of the perinatal period. At puberty, the higher levels of androgen masculinize the defeminized brain. This process, termed "ma s c u lin i z a tion", activates masculine characteristics but is not permanent.
Adult castration of male rats can inhibit the male type of drug metabolism, butth e drug metabolism can resume tono r mal after injection of testosterone propionate (TP) to the castrated males (Shimada et al.,1987; Virgo, 1991).
The criticalperiodforan d r oge n to permanently organi2:e the brain in rats is from just prior to birth to 10 days postpartum, ie, the perinatal period (MacLusky and Naftolin, 1981; McEwen,1983). Ho we v e r , for the P450 system,the limit is 5 to 6 days postpartum. I f exposure of the brain to androgen does not occur, the brain will innately develop female characteristics and the neural control of endocrine fun c t i ons and sexual steroid-relatedcvecctu oee P450 system wouldbe affected. The secreeceypattern of gonadotropins is sexually different, with a cyclic pattern in female rats but
an acyclic or tonic patter nin males. This sexualdimorphism re s ultsfr om the sexual diffe re ntiationofthebra in. If the brains of female rats ar e defeminized with neonatal te s t os t e r one treatment , the female rat s will show an anov ul a tor y syndr omedue to the occurrence of an acycl i c gonadotropin secre tion. Adult male ra t s cast r a t ed between birthand3 day s of life and tra nsp l a nt ed wit h ovarian and va gi na ltissuecan di sp lay va g i n alcycles , ovu lation (Ha r ris, 1964 ) and cyc lic gonadotropinsecretion in adulthood(Gorski, 1966) . Sim ila rl y, ne on a t al ca s t ratio n ca u ses a compl ete l y suppr e s sion of mal e- specific cytochr o me s P4S0 in adultmete rats but indu c estheex pr ess i onoffemale- spe cifi ccytochrome s P4S0 (Janssonet aI., 1985a).
1.1.5.2 Morpholoqic al Changes ofBrai n and Neonatal Se:K Hormon e s
It is we ll kno wntha t there are sexual di f ferences in bra in st ru cture that ar e sex steroid- d e pen de nt. These difference s incl ud e such aspects as sizeof nuclei in neurona l groups, numberof neurons(Go r s k i et aI.,1978; Breedlove and Ar no l d, 1980; Gur ne yandKonishi, 1980;Jordanet aI.,1982) , volumeof neu rons (Gorski et al.,1980), extent ofdendr i ti c extension andbra n chi ng (Meyer et al., 1973; Ayoubet a1.,
1982), number of dendritic spines (Greeno ughet al., 1977;
Ayoub et a1., 19 8 2 ) , and morphology (Gilldn er, 1982) and lo c ali z a ti on ofsy n a pses(Mats umotoand Arai, 1981).
In1966, Pfaff first discovered the existenceof sexual differe nc e s in the size ofnerve cell nuclei in rat brains. Late r , RaismanandField (1973) ,Dyer(198 4 ) , Greenoughet al. (1977) , Nishi zu ka and Arai (1981), and Matsumoto and Arai (19 81 ) foundsexua l dimorphisms in the patternof neuronal con nectionsinra t br ains . All th ese differe nces pr o ved tobe depe nd e n t on exposure of the eNS to and rog en during the perinata l pe r iod. For examp le , an inject io nof te s t o s ter on e into fema le rats of day 5 caused a marked inc r ease in the numbe r of shaf t syn ap ses (SHS ) in the medial amyg da loid nucleus (AMN). Th e inc i d ence of SHSinandroge nizedfemales was al most thesame as that in no rmal males. In contrast, neon a tal or ch idectomyre sulted in a sig n ifici.lOt de crease in the number of SHS to a le ve l compa rab l e to tha t of nor maI females (Nis h izuka and Arai , 1981). In ano the r experiment, treatmentwith te stoste r one onday5 tofemal e rats incr eased the mean nu mbers of sh aft and sp ine syna pses in the hypo t halami c ar cu ate nucleus (ARCN)i mcr ecver, the sy na ptic or g an i z ationoftheARCN inthe an d rog e n izedadul t femaleswas qui t ehomo l ogous to that observe d in the nor mal adult males
(Ma t sumotoan dAra i ,19 81 ). The seand othe rstu di e s indi cate thatthepresence of sex ste roidsduringthe per i nata lper i od Ny peraane n tl y dif fe r e ntia t e the developing pro cess of the brainand ir r e ve r sib lyorgani z ethe brain stru c tu r e.
The medial pre op ti c area (HPOA) of th ehypothalamus is cle arl yimportant in thecon t r olofgonadotrop inrelease an d sexualbeha v iour (P res loc k andMcCa n n, 1987). Also, th i sar e a is ge nerally accep t ed as a major sit e of ne onata l hormone actio n duringne ural deve lopme n t. Within the MPOAis anarea uf dar kl y st a i nin g neurons named the se xu a ll y dimorphi c nuc Ieua(SON-POA) (Gor skiet a1.,1980) .Thefunctionofth is nu cle u sissti ll notclear andmos tauthor s suppo s e tha t i tis involved in mas cul i ne reproducti v e be haviour (Arendash and Gorski,1982 ; Arend a sh and Gorski, 1983: Gors k i, 1991) .
The deve l opment of theSDN-POAstart s duri n g la tefetal life and ext.ends throughou t the first ten day sofpos t na t a l life (Jacobsonand Gorski,198 1 ;Ja co bsonet aI.,1980 ) . This period correspondsto thatof brainsexualdiffe rentiation . Thede vel opmentof SON-POAne u rons is dependent upon androqen.
Castrationof the 1-day- old male rat significan tly re du c e s SON- POA volumeinadultho od(Go rsk i etaI., 1978;Jac ob s o n et a1., 1981),but immed i ate reim p l an t a t ionof a testisor
injec t i o n of tes tos te ron e afte r neonatalca st rat i o n re e t .cce s SDN-POA valump.to normal (Ja cobsonet al. , 198 1) . Further, exogenous tes t o s teronegi ven postnata lly to the ge n eticfemal e sign ificant ly inc reases the vo l ume of SDN-POA in adulthood (Gor sk i et al. , 197 8;Jaco bsonetal.,1981). The r efore , the SON- POA, at lea s t in terms of its vo l u me, is clearly influencedbytheperinatalhormone environ me n t.
The structures of thl.'! ventromedial nu cle us (VMN)and the arcuateaccreue (ARN)arealsosexuallydif f ere nt in sy naptic pattern and nuclearvolumes of neuron s (Mc Ewenet al., 1982;
Toran- AllerandCD, 1980). The cell bodies of these nucle i contain GHRH; ind e e d , theARC nucleus is the major siteof GRRRinthebrain. Fu r t h e rmo r e, thes e morp ho l o gi c differ e n ce s in the rat are determinedbythe ne onatal steroid env i r o nmen t.
Recently, Chowen an d colleagues (1993) reported that neonatal and adult testoste r one had eff ects on the or g a n i z a tio n and activation of GHRR and 5S neurons in the hypothalamus. Neonatalte s t o s ter one incre ase d thenumbe rof GHRR neurons 1n adu l thood and adultte s t osterone stimulated GHRHmRNAle v e l s; further, infemal erat s ,adult testostero ne stimulated GHRRmRNA le v el s ,andthe magnitudp.of theinc rease was hi gh e r in the animalsexposed to neonatal testosterone
than tho s e not ex po s e d tone o natalsex st ero ids. Incont r as t, the numbe r of55 neuronswasnot effec ted byte stosteron e,but both neonatal testosterone and adu l t testosterone signif icantlyincreased 55 mRNA level s (Chowe n et al., 19 93 ).
1.1.5.3 Sex Ste r o i ds Duringthe Neonatal Period
1.1.5.3 .1 Androgenand Estr og e n
I t is known that the serumco n ce n t r a t i on ofte sto ste r o n e is significan tly higher in male rats than in female ones du ringthepe r i n ata l period(Pang et; al. ,1979). Androgencan permanentlydefeminize the eN5 duringthe critical periodof neural development. It has been suggestedth a t androgen is converted,vi aar oma t iz a t i on , to estradiol,wh ichis commonl y acceptedas theprecise molecu la rspecies of gonadalhormone that promotesthedefeminizationof thebr a i n (Dohle r, 19 86 ; Toran- Allerand, 19 8 6; Gorski, 1991). Administ r a tion wi th entaeserceene ca n block testosterone-induced (Doughty and McDon ald ,1974) or est rogen - i nd ucedde feminization(Doughtyet al., 1975). Furthermore, aromatizingenzyme in h i b i to r s can also inhib itthe defemin izingeffectsof both endogenous and exogenous testosterone (Booth, 19 77). As fo r th e br a i n- controlledhepaticcytoc hromeP450 system, ne o natal
test o sterone or estrog en def eminized se x-depend ent drug met aboliSIII, and ecoeet.e s e in hibitor prev en t ed the defemi nh atio n of he p a tic drug metabolism ind uc e d by sex steroids (Re y e s and Virgo, 1988). These data suggest th a t androge n is converted to est radiol todefe min ize the brain.
Furthe r,thefetal andneo n a tal ra t braincontains ar oma ti z in g enzymeswhichca nconve rt aromat i za b l e andro g entoes t rad i ol - 17D(Ge orgeandofede,1982 ;MacLuskyet al .,198 5 ; Lep har-t. et al., 19 92) , and estrog e n re c ep to r level s in the ma la and female ratbrainin c r eas e markedlyduringth eperina t alperiod (Mc Ewen et aL, 1982). Thes e evtde nces suppor t the possibilit y of andro g-en- a r omat h a tion andthe inv o lvementof estrad iol in the defe minizi ng proces s . There fore, it is assumed that and roge n en ters thecytosol of neuron and is co n vert e d toestra d iolby aromat a s e;est radio l then interacts wit h speci fic rece p to rsandul t i mat e ly the neu ro na lge norneto altermRNAac ti vitywh ich in tur n st i mulatesor inhib its the prOduc t i o n of some product, thereby modi f yi ng neuronal fu n ctio n(DOh ler, 1986 ; Toran-A lle rand, 1986;Gorski, 19 9 1 ) .
As forthe.eff e c t s ofse xster oid sonnerv e ce lls,it has been demon s tra t e d tha t estrogenor androgen can inc r e ase the rate and ext e n t of neuriticoutgro wth inneu ron s cont a in i n g est r o gen rec eptors (To ran -Allera n d , 198 0). Th e gro wt h and
deve lopmen t of axo ns anddend r ite s in tur n enha n ce synapse formatio nand neur o na l survival, and inc reas esne uronnumb er.
Sinc e differe nt circu l atin g le vels of sex st ero i ds exi t between se xes during the per i nata lperiod, numberofsynapses , the spat ia ldist r ibu t i on of syn a ps e s, neuronnumber, neuro nal vo l umeand neu ralcircuits wi ll sexuall ydifferentiate. This hypothesi s is cur r e n t ly in vo lve d in explaini ng the sexual morphogene ticmechanisms ofth e ster oidaleffect sduring the neura ldeve lopment(McEwen et al.,1982; To r an-A llerand,198 6 ; Gorski, 1991).
1.1.5 . 3 .2 a-FBtoprotein(Arp )
Neon a t al fema le s haveestrogen-richplasma, and yet the brains of female ratsdo not becomedefeminized. Th i s may be duetothe presenceof pLa a maa-fetoprotein.
AFP is a maj or fetalpl asma proteinwh i c h is present in most developing vertebrates. Inrats, this protein occursin hi g h concentration during the first 3 weeksof postnatal life and decreases li n e a r ly from its very hi gh concentration to trace levels at weaning (Raynaud et al. , 1971). AlthoughAFP ca n bind a varietyof hormones and otherbiolog icallyactive ligands in other species, rat and mouseAFP alone binds
@strcg@ns with a high affinity and pro t@cts th@ de v e IcpLnq brain from exces s ive exposure to estroqens. Sinc eAFP does notbi nd androgens, testosteroneis freeto ente r the rodent brai n . Thu s, felllale brainsdo not becomedefeminized.
1.1.5.4 Hepatic Sex-Dependent Cyt ochromeP450
Bas ed on th eabo ve , i t can be ccnciuee e tha t es t r ad i ol binds estrogen recept ors in the cytoso l to modula te the express ionofDNA and to organize thede ve l op menta l courseof th e brain s . Si.r\ce the sec r e t o ry patt ern of gr owt hhormone in adul t rats re sults from th e sex ua l diffe renti a t i on of the br ai n, and the secretorypatternreg u la testhe expre s s i on of sex-dep end ent cyto c h rome P450, we can in fer that sexual diffe renc es inthe activi ti e sof the cytoch rome P4 50 system aretheresult of sexual differ e n ti a tion of thebr a in . Reyes and Virgo (1988) showedthat va:r:ious sex-s tero ids whi c h can def emini ze gonado t ropi n sec r et i on also defeminized drug metabolis m, and that ar oraa t a s einhibi torwh ichcanblock the defemini zat i on of gona do tropin secreti on prevent ed the de feminization of hepa ti c cytochrome P450. Therefore, i t suggeststhatneo n a t a l androgen defeminize shepat ic cyt o c hrome P4.50 possibly viath e sexu aldifferent iationof the brain .
1.1 .6 Catecho 1ami ne r gicSy s tem in th eDefe min iza t10n of the Brai n
1.1.6.1 Ne ona t a l ca tec ho l ami ne s andbe hav i ou r
In addition to sex steroids, monoamine neurotransmitters are believed to be involved in the sexual differentiation of at least some aspects of CNS functions (D6hler, 1991). Thus, changingthe amounts of these monoaminergic transmitters in CNS during the critical period of sexual'd if f ere nt i a t i o n of the brain would alter the steroid-dependent expression of sexuallyor nonsexually-related behaviours in adultrats. It was repcz-xed that re s e r p i n e , which depletes norepinephrine from nerve terminals by inhibi t ing uptake int o storage vesicles , couldpreventthe defeminizing effectsof neonatal testosterone in inducing anovulatorysyndromes in the females (Gorski, 1973). Raum et a1. (1990) reported that prenatal exposureof malerat fetuses to cocaine,which could increase synapticconcentrations of norepinephrine, inhibit~dsexually relatedbehaviuurs in adult males. Reduction of seroton inby p-ch1orophenylalanine (PCPA) enhanced masculine sexual behaviour inma l e rats and potentiatedthe defeminizingeffect of exogenouste s t oste r one in females; in contrast, increasing serotonin by S-hydroxyptophan(SHTP)inhibitedmasculine
behaviou r induc ed byexogenous testos ter one in female rats (Wilsonet al., 1986). Neon atal admin i s trat i on ' of clonidine resultedin a sign if i cant re d u ctionin theca pa citytoexpress female lordos i sbehav iourinfema l e adulthoo d (Jarzabet;al., 1987). Neo na t a l dopamineor no r a d r e na line was foundto reduce the ope.... fieldact iv i ty infemaleadul t ra ts (Gonz a le z and Lere t, 199 2b ) . All the above evidence suggests tha t se xu a l differentiation of sexualbe ha v i o uris related to the neone tcI catecholami nergi csystem in the brain.
1.1.6.2 Neonatal catecholaminee and reproduct i v e en d o c r i n e function
The sec retory pattern of the gonadotrop in s is, as mentioned previous ly , sexual ly different, and thi s sexual dimorphismof gonadotropinsecretion resul t s fromth e sexual differentiation of the brain. I f the female bra in is de f e mi n i z e d with neonatal testosterone trea t me n t, the female rat will become anovulatorydue to the presence of acycli c gonadotropin secretion (Booth, 19 79; Dt:lrner, 1981; Goy and McEwen ,1980 ). Besides sex steroids, neurotransmitters during the neonatal period also have effects on the sexual different iation of gonadotropin secretion. A series of experiments (Jarzab and Dahler, 1984; Jar z ab et al., 19 86,
1987, 1989a,b,c) demonstrated tha t postnatal treatment of female rat s with the a-adrenergic antagonists prazosin or yohimbine, or with the nicotine receptor antagonist mecamylamine, significantly increas ed the release of LH by gonadal steroids in adulthood;and that postnataltreatmentof female rats with the a-adrenergicagonist isoprenaline, the B2-adrenergic agonistsalbutamol or B-adrene rgic antagonist alprenolol reduced the release re s p o n s e of LH to gonadal steroids in adulthood. Undoubtedly, these data indicate neurotra ns mitters engage in the sexual diff e r e n t i a t i on of gonadotropin secretion.
1.1.6. 3 Neonatal catecho1aminesandSDN- POA
SDN-POA is tho ught to be related to sexual behaviour (Ar endashan dGorski, 19 8 2 ; Arendashand Gorski,198 3 ; Gorski, 19 91 ) andsexua llydifferentiated (Gorski et al., 1980), th e effect of neonatal ca tec ho lamineson the anatomy of SDN-POA ha v e also been investigated. It was reported(Handa et al., 1986) that prenatal p-chlorop he nlami ne, an in h i bi t o r of sero toninbiosynt hesis, in cre a sed the volume of the SDN-POAin female neona tescompared to that of control males. Studies (Jarzabet al.,1989b,1990a,b ,c)show that salbutamol , a 132- receptoragonist, gi v en neonatal ly,increased SDN-POAvolume
in both fe mal e andmale ra t s but the effectwa s particu l a r ly manifes t in males; furthermore , the Ql- rec ep tor agonist clo nidi n e augment the stimulatory effector tes toat erc ne on SDN-POA differentiat ion in fe ma l e rats . Thesedatasuggest thatal t e r a ti o n ofcat e c ho l ami nele v e l sin theneo n a ta l brains wo uld mo d i f yth e sexual differenti a ti on oftheCNS.
1.1.6.4 Catecholaminesandandr og en during theneonatal peri o d
Thereis ev idenceshowingthat catechalaminesmodify the defemin izing effect of ne o n ata l tes t oste r one. Po stna ta l seroton indelayed thepe r mane nt anovulat ory- ster lli ty (PAS )- inducing effect of postnatal testosterone (Shirama et al., 1975), and PCPA was found to preventandrogen-induced PAS in fe ma l e rats(Re z ni ko v etaI., 1979). Tyramine ,which releases no r a d r e n a l i ne from nerve terminals, as well as the Ql/a2 receptor blockers phenoxy benzamine and phento lamine can inhibi t thedefemi nizingaction of testosteroneon ovulatory cycllcityin femalerats (Raum and Swerdloff, 1981). Although nuclear ac cumulat ion of estradiol derived from the ar o ma t i z a tion of testosterone isinhlbited by hypothalami c13- recept orstimulati onin the neona tal female rat (Ra um et aL,, 1984),the workof Cani ck et al. (19 8 7 )showed neonatal
administration of the B-agoni.st,isoproterenol, had no effect on aromataseactivityin maleor femal:apups (Cani ck et al., 198 7 ).
In spite of that the mechanisms of neurotransmitte r effectson defeminization is still not known;the fact of the involvementof these catecholamines in the de reeunraat rcn of the brain cannot be denied. Therefore, due to the fact that sexual differences of hepatic cytochrome P450 resul t indirectly fromthebr a i n sexualdifferentiation and that sex differentiated characteristh.:s can be influenced by adrenergic agonists/antagonists,we intend to study whetherthe neonatal adrenergic system is involved in the androgen-induced defeminization of hepat iccytochrome P450 in male rats.
1.2 GH and Development of Male-SpecificCytochromesP450
1.2.1 Developmental Ezpressionsof Male-specificCyt o c h r ome s P450
Sex -dependen t cvcocnrceee P450 of rat liver display developmental changes (Waxman et; aI., 1985; Imaoka et aI., 1991). Fo r example, during theneo natalperiod, the amount of cytochrome r4502Cl1is undetectablein both sexe.,, but in
male ratsthe amount of thisiso f o rm beginstori s e at 4 weeks of age and reaches its highes t leve l at puber ty. Thefemales show an al mo s t undetectab lelevelsof the isoenzymefromth e neonatal period to puberty (Wa xma n et al. , 1985; Imaaka et al.,1991) . Anothe r male-spec ifi cisoformof cytochromeP450, 3Al/2, also und e r g oes developmental changes, but i t mainly shows a developmentaldecline in thefe ma l e s (Waxman et a1 . , 19 8 5 ; Imaoka et a L., 1991 ). In the females, the amoun t of this is oe n z yme is high during th e neonatal period but decreases with age. Themales, however,keep a high level of P450 3A1/2 fromtheneo n a ta l periodto puhe rty (Wa x ma n et al., 1985; Imaokaet a L,, 1991 ) . The activitiesof zu- and l6a- testosteronehyd r oxy l a s e s are lowin newborn rats, bu t rise at around 4 weeks of age and then reach an adult high level at puberty; since 2a- and 16a-testos terone hydroxylations are selective forcytochrome
len
which is barelydetec tablein female rats from th e neonatal periodtoma t u r i t y , the fe mal e s display low activiti es of zu- and 16a-testosterone hydroxylases all the time. en-reeeoeteronehydroxylation, which is ca t a l y z e d by cy t o ch r o me P450 3Al /2 , shows a high activity in newb o rn female but dec reases its activitywith age. In ma l e s the act ivity of 6B-hydroxylase rises at two weeks of age, reaches the highestat 4 weeks of aqe , then it decreasesbut arisesagain at 6 week s of ag e (Waxman et al ., 1985; Imaokaet al.,19 91).1.2.2 GH andMale- Sp e cificCytochromea P450
Ethy l morp hinedemethylase(EMDM), a sex-specific he p a t i c monooxygenase which exists in both se xes but shows a much higher activi tyin adultmales, isneonatallydefeminizedby androgen(Reyes and Virgo , 1988;Vir g o , 19 9 1). The activity of thi s enzyme in malerats exh i bi ts developmentalchanges (Gramet al., 1969). Th e activi t yisunde tec tableinnewbo r n ra t s,begi n s to rise at 4wee ksofage andre a chad ultlevels at pubert y. Howev er , the femalesdi spl aya lowactivityof th e en zy meall th e ti me.
The activi t iesof£MOM(Krameret aI. , 1975;Vir go , 1983;
Vo c ke n t, mz and Virg o,1985)andmale-specificcy t oc hro lil8sP450 su ch as 2C11 (Janeczkoet aI.,19 9 0;Legra v e r en detaI.,1992) and3Al/2(Waxman et al.,1990)in adult rats areregulatedby GR. However, there are no rep orts regarding the fa c t ors res po n s ible for the developmental expre s s i o n of malespecif ic isofonnsduring theprepub ert al period. Plas ma GH immediately afte r bir th appears at very high levels, and then declines sh a r p l y toan almost unde t ect ab l e le v el; it the n begins ta ri s e at 4weeksofage, and reachesadu l t leve l s atpub ert y (Khor ram etaI., 1983; Rieuto rt, 1974; Strass er ani.! Mialhe, 197 5 ) .
Althou':jh the male patter nof GH secretionis organized during the neona ta l pe riod {J ans s o n and frohman, 19 B? } , it becomesmanifest only afte r pub e rty (Gabr i e l et al., 19 9 2 ) . Duringthepre pube rt a l period,thereis no apparentlysexual diff e r enc e in the secreto r y patternof GH, the prepuber tal expres s i on s of ma l e - s p e c i fi c P450 see ms no t rel a t e d to the secretorypattern of the hormon e . Therefore, wealsointend to inv est igate whether GH pl a ys a role in the pre pu b er t a l expre s sion of thema le-sp ecific P45 0 .
"
2 .0 MATERIALS
AND
METHODS
2.1 GENERALMATERIALSAND METHODS
2.1 . 1 ChUlicalsandBio che.ai c als
2. 1 .1.1 Chemica ls foranima l inj @ction
DL-isoprot@renol'HCI,DL-propra nol ol ' HCI, cl o nidln e -HCI.
phenyl ephrLn esHCI , phentolam ine'HCI • prazosin'HCI, te s t o s t e r o ne prop ionate , and pr op yle n e glycolwere purchased fromSi gma ChemicalComp a ny ; WY271 27is thepr oduc t of W}·gth Ltd•• Philadelphia, PA, USA; sod i um glutamate (MSG) is from G.E. Barbour Inc., Sus s e x , N.B.; and sodium chloride purchased from Fishe rSc i en t i fi c Company, Nepean , On to
2. 1. 1. 2 Chemicals for the enzyme aS8ay
Glucose-6-phosphate dehydrogenas e (G-6-P -DH), B- nicotinami de adenin e dinucleotide pho sp h a t €! (NADP),gluco s e- 6- phosphate (G-6-P ) . oxid ize d cyt oc hrome c and reduced B- nicotinamide adentee dinucleo t i d e phosphate were pu rc h as ed from Si gma ChemicalCompany ; semic ar baz ide hydrochloride an d 4-oi tropheno l werethepr odu c t s ofAIde~chChe micalCompany.
Inc., Milwa ukee, WI S, USA; magn e sium chloride, po t a s s i u m cyanide, sucrose and sodium hydr OXi d e werepurchasedfrom
MallinckrodtCanada Inc, Pointe-Claire ,Quebec;zinc sulfate, hydrochl or i c acid and sodium dithionite were from Fi she r Scientific, Fair Lawn, NJ, USA; ethylmo rph inechlorohydrate and perch l or ic acid were the produ cts of SDH Chemicals, Canada; l6a-hydro x y t e s tos t e r o n e (4-androst e n-16al, 1613- hydroxytestosterone (4-androsten -l613), l1a-hydroxy- testosterone (4-andros t en-11a) , 7a-hydroxytestosterone (4- and r os t en - 7a ) , 2a-hydroxytestos terone (4-and r o s t e n -2a ), 1113- hydroxytestosterone(4-androsten-l1B),6f3-hydroxy-testoster.:> ne (4-androsten-6B)andhy d r o x y testoste rone(4-and r o st e n -l 7J3-0 L- 3-0 NE ) were purchased fr om Steralo ids, Inc., Wilt on , N.H.; aceticacidglacialwa sfr om BaxterCorpo r a t i on, Toron to,Ont;
and L-ascorbic acid was purc hased from Uni te d States Bio ch emi c a l Corpor at ion, Clev e land, Ohio, USA; glycerol, ace t on e , me t h an ol and dich loromethanewere the pr odu c t s of Fishe r Scientific Company; BIC-HADPro tei n Assa y Dy e Re a g e n t Concen trate and bovine serum albu min ar e from BIO-RAD Laborator ies, Richmond, CA, USA.
2. 1.1. 3 Chemicalsfor buffen
Buf fers wer eprepar ed accor d ingto themethodsdescribed by Gomod (1955).
Trizma base (Tr is [hyd ro xyme t hy l )ami no-met hane) is the prod u ct of Sigma Ch emi c al Company; po 't aas Ium pho s p ha t e monobasic, potassium phc. sp hate dibasic and potassium chloride we r e purcha sed from Mallinc k rodtCanadaInc.
2.1 .2 Animals
Pregnant rats (Wis t a r), havi ng succ ess fu lly raised at ::'east one previouslitter, we r e purchasedfromCharlesRiver Can ad a (St. Constant, Que.) and were kept in solid-bo t tom plastic cages on chemical- free hardwood bedding. All ra t s were kept at 2l0Cund er 12h of light per day and ha d fre e access to tap waterand Agt.;ay PROLABchow(man u f a c t u re d by AgwayInc.,NewYo rk,USA). Newborn and adu ltcats were us e d in all expe r i lllen t s .
2.1. 3 In t raventricul a r Inj ection
On Day 0 and 3 of birth, ne who r n lIla l e rats were an e s t he ti z ed hypothermically. The pup was wrapped in a slightlydamppieceof gauze bandage andplaced in ice for15 minutes to in d u c e hypot he rm icanesthe sia (Pfeiffer, 19 3 6 ). The pup wasthen remov e d fromice, put inahold e r, and the hea d placedin a horizon t al position withthe skullside up.
A midsa gittal inci si onwasmade throughtheski n fr oma line joining the eyes toa line joining the ears, and the bregma was exposed . ;.. 271-gaugene e dl ewa s inse r tedvert i c ally in to the third ventr icle (coordinates: 0.5mm anteri or to the :)r e g ma; 1.5 mmla t e r a l to th e midsagi ttalfissure ) to a depth 2. 5 mm ventral tothe skull surface . The va lid i t y of these coordi nateswas co n fi rme d by in j e c ti ng dye (Methylene Blue- Certified , Sigma Chemical Company) and morphologi cal exam ination of th e brain after fixation in bu ffered 10 % formalin. Tenp.l of vetuc ae or dr ug solution was in j e c t e d intothe ven t ri cle ove r a periodof 3 minutes and the needle wasmaint aine din positionfo r a further 5 minutestoprevent backflow from the puncturewo und . The inc i s i onwas closed by sealingthe skintoth e skull wi t h cya noacrylateglue.
After the injection, thepup was lef t un d i sturbed and war meduntilitsbo dy returned to room temperat u re (i e. pink andactiveagain). They were thenwarmed at250Cfor at least twohou rsbeforebeing returnedtothe dam.
2.1.4 Castration
2.1.4 .1 Castration of the neonate
The procedure us e d was a modification of Pf ei f f e r's
method (Pfeiffer , 1936). Within12hou r s of bi r th , thepup wa san esthetize d hypothermic a llyby pl a c i ngit in ice fo r 15 min. The pup·....as then placed, ver.tralsldeup,on a surg ica l table. The area of incisi o n was wiped wi th a pie c e of Kimwipe1soaked in9S%ethan ol. Aninci s ion(2mml was made through the skinan t e ri o r to the urogenital papilla ata 4SJ_ angle to the left (of the pup) by use of very fine iris scissors . Thesk inwas thenfreedfromsub cu t aneousti ssues wit h a fine probe . Asecond inch ionwasmadehor izontally thro u g h the musculatureat the body wallof tile init ialcut.
The rigllt testi s was then extrudedby slightlypressing tile abdomen and excised witha pair of scissors. Tile lefttesti s was exposed by care full yprobing throughthe viscera and als o exc ised. Thewoundon the body wa llwas cl os ed by ali g n ing th e cut edgesof the musculature with sma ll forcepsand then sealingi twith cyanoa c r y l a t e glue. The opening on the skin wasthen closedbyo-..erlapping the cut edgesofthesk i n and sealing it withcy a no a c r y late glue. After the surge ry. the pupwas left undisturbed, warmed and returned to thedam.
2. 1 . 4 . 2 Castrationofadultrats
The rat was lightly anesthetizedwith ether andplac ed, ve n t r a l side up,on a surgic altable. Th.e limbs werefixed to
theta b l e . The haironth e scrotumwasremove d with a pairof eurqt ce,, sc i s s ors andth e skinofth e scro t um was wiped with 75%ethanol. Anin cis i o n was made throughthe mid lineof the scrotumfollowedby a second incisionthroughthe tunica of one testis. The testis was then exposed, the testicular artery and veinwerelig ate d, andthe testis wasexcised. The remainingtestiswasremo v e d similarly. The scrotal incision wa scl o s e d with3 to4sutures.
2.1.5 Subcutaneous Injections Duringthe Neonatal Period
OnDay 0to10,chemicals were injected under thedorso - lateral skin of thepup immed i ate l y posteri o r to the front limb.
2.1.6 Preparation of Microsome
The method us e d toprepare hepat icmicrosomewa s basedon tha t re portedby Virgo(1991) .
Fo o d was remo vedfro mtherat twelve hou rsbefore i twas ki lle d in orderto decreaseglycogen in the live r . The ra t wa s decapitated witha guilloti ne and exsanguinated. The abdome n was opened with apair of surgical scissorsand the
int a c t liver was ex cised as quickly as possib l e. The liver was then put int oa beakerofice- cold SOJlIMTri sHClbuffer pH7.4, contain i ng15 0mMKCL Aftertheliv e rwas ri ns e d. it was tak enout. wip e d dry witha pie c e or Klmwipe' to remove blood an d we i g hed. The trve r was then put int o a bea ker co n t ai nin g 3volumes of ice-coldSOmMTrisHCl bufferpit7. 4 with l50mmKel. homogenized with a Brin kmann Polytron at setting of 4 in a 4°Ccold room. The homogenate was then centrifuged at 9000'9fo r 20 min utes at 4°C . The result an t superna t a ntwas thencentr if uge d at10 5 , 000 'gat 4°Cfo r 60 minu t es. The super natantwaspou redof f and asi mil arvolume of fr e sh SO mMTris HCI buffer pH 7.4 was adde d. The microsomeswer e resuspendedwiththehomoge ni z e r atse t ti ng of 3. The su s pensionwascen t r i f uge don ceagainat lOS, 000'9for 60 mi nutesat 4°C. The supern atan t was dLscarded and the pelletwas homog eni zedin a volum.:!ofSOlaMTrisHCI bufferpH 7.4 equ al tohalfthevolumeof9,0 0 0 'gsupe r na ta nt originally containingthe lIlicro some s. Glycerine (20\ ofthe volume of th e suspens ion)was addedand the micro s omes were stor ed at -80°C untiluse.
2.1.7 Measurementof Micros omal Pr o t e in
Con cent r a ti on ofmi cro so mal prote i nwa s dete rmi ned
according to the Bradford's method (1976) using the Bio-Rad Protein Assay kit:
Diluted microsomal protein(1:3000 and I:6000)(0. 8ml) andQ. 8ml of standards (bovine serum albumin21.J.gjml, 31.J.gjml, 4pgjml, 6pgjml and 8].1gjmlj were placed in clean, dry test tubes, and O.8ml distilledwater was placedin "blank" test tube. Dye Reagent Concentrate O.2ml was then added to the te s t tubes. All test tubes were shaken without causingexcess foaming. After 5 minutes, the absorbance of the blank standards and sampleswere measured at 595 nm, The value of protein concentration wascalcu~atedfrom the standard curve.
2. 1.8 En zyme Assays
2.1.8.1 Measurement ofet h y l mo rphi n e demethylase (EMDM) activ ityin hepaticmicrosome
The activlty of EMDM was measured in vitro from the amount of formalinproduced from the methyl group released from the substrate (Nash, 1953). The incubation medium contained the following in a total volume of 3 ml: 0.1 mM NADPH,1 mM NADP, 3.3mMglucose 6-phosphate,2 Units glucose 6-phosphate dehydrogenase, 1.0 mM semicarbazide, 24 mM MgCI2'
3.
50 mM Tris Hel bu ff e r (pH 7.4), 6 rng microsomal protein, and 6.7 mM ethylmorphine. After 5 min of p r e Lncubat Lon of the mix t u re , the proteinwas added to the incubationnu a ture and there a c t i o n wa s allowed to proceedfor 20 min at 37JCunder air. The react i onwasthe n stoppedafter20 min by adding2ml 20%ZnSOtand 2 ml saturated Ba(OH )2to the incubation mix.
Af t e r theadditionof 3 mI of distilled water, there s u l t ant suspensionwas centrifuged at3500 rpm for 15 minutes. Fiv e ml of thesupernatant was removedto anothertube containing 2 rol double strength Nash Reagent (150 9 anunoniumacetate, 2 ml acetylacetone, 2 mlglacial acetic: acid and dilutedto 500 ml with distilled water), and th en he a t ed at 60 ~C for 30 minu t e s . The absor bance wa s then measuredimmediatelyat413 Concentrationof formalinintheoriginal incubationmiK was calculatedby:formalin(nmo l /ml)'"(ABS - 0.0069) / 0.0006 . Thisequation was obtained byusin gkn o wn concentrationsof formalinin the Nash reaction(Nas h, 19 5 3).
Theconcentration of MgCl1usedin the incubation was 24 mM, which wa s determined with prelimi na ry experiments to selectan appropriate MgCl1concentrat ion. We used 1.2 , 12, 24, 48 and 96 roMMgCl1, and foun d that the amountof product was highest at 24 and 48 mMMg C12• Si n c e there was no apparentdifferenceinth e amount of product between24 and48 mM MgCl 2used, we decidedto use 24 roM MgC12fo r the reaction.
The incubationtimewa s 20min. In cu b a t i on of the mix to-:5, 10, 20 and )0 mi nutes, re s p e c t ively showed th a t the specificac tiv ity ofethy lmorphinede me thy l a se wasthe sallie at 5, 10 and 20 minute incubationand lower at 30 mi n u tes (see Tab l e2-1) ; there fo re , 20min was chos e n.
2. 1.8.2 Measurementof p-nitrophenolhydro zylase (p-NPH) act ivityin hepa t icmicrosome
The activityof p-nitr ophenolhydroxylase was measured in vitra accordi ng to the methodof Koop (1 986 ). The incubation medium contained the followingin a to t al vo l ume of 1 ml:0.1 mMp-n itrophenol solution, 0.5 mg mi cro s omalpr o te in, 1mg NADPH and 100 mHpho s ph ate buffer pH 6.8 . After 5 min t)f preinc ubat i on,NArJPH wasadd e d to start thereaction and water was added to the refe r en c e. The react i on was allowed to proce e d for 6minat 37°C under air and stoppedbyaddi ng 0.5 1111 0.6Nperchloric acid. This mixture was then cen t rifuged for 15 min at 3500 rplll. At la s t, 0.1 ml of 10 NNaOM was added to 1 ml of the centrifuged supernatant , and the absorbance of this solutio n was meas ur e d at 546 om. The concentrationof th e product, ni t r o c atec hol , was calculated using amilli mole extinctioncoefficient of 9.53m?f1c m·1. The amoun tof the product was shownin preliminaryexperimentsto
'I'able2-1. HepaticEMD1'l activityof intactand castrated male ratsWiderdifferent incubationtime.
IncubationTime (minute)
[ Formalin Product 1 (nrnoI/ri,l) Int.r/
10 20 30
65 13 3 268 327
12 22 46 66
Table 2-2. Hepatic p-NPH activity of male ra ts under different amountsof microsome protein&various in c ub a t i on time.
(nitrocatechol J
(Microsome 1 (nmol/ mi x .)
(mg/pe rmix.)
---- ---
4 mi n.
•
mi n.s
mi n.0.1 0.44 1.0 4 2.20
0.25 1. 35 2.03 3.13
0.5 2.28 2.68 4.3 7
La 3.33 4.7 3 5.82
be pvoport Lc n a.l to both the time of in c uba t i o n end the concentretionof:nicrosomalpr o t e i n (see Table2-2).
2.1.8 .3 Qu ant i t a t i o n of cytochromeP450
The concentration was determined by the method of Omura
&Sato (1964).
A fewmi l lig r a ms of sodium dithionite we r e addedto the 2.5ml mt croeomes suspend e din 50 mM TrisHel buffer(p H 7.4) (2 mg/ml) in a cu v e t; to red uc e the cytochrome P450 . The absorbanceof the suspe ns io nwas then measured from400-5 0 0mn yieldinga baseline. The suspensionwas then bubbled with ca r bo n monox idefor30 secondsandit s absorbance was measured from 400-500 nmbasedontheobta inedbaseline. Twodifferent values of absorba nce we r e selected to calculate, using a coeffi ent E as 91 mM-1cm-l , the concent ra tion of cytochro me P45 0. The total P4S0 nmol/mgofthe microsomeis givenby the formul a :
(ABSm-ABSm )1<1000/91/mg MP.
Inthe above formula , .ABSm'was the absorbanceat 450 nm and'ABSao' the absorbanceat49 0nrn.
2.1.8. 4 Measurement of NADPHeyt.ochromecreduc tase
The activity ~f th is en zyme was determi ne d by the proc e du reofPhil li p sand Langdon (1962)using19.6CfQ·'mM·1as th e extinc ti on coe ff ic i entfor reducedcytoc h rom.;!:cand1001llM phosphate buff e r for the reactio n s.
Cne ml of50l1Mcy t o c h r ome c re a g e n t (1.86mgox i d iz e d cytochromecin1.a mlof10 0mMphosph a t ebu f fe r,pH 7.6) and 1 ml ofI roMKe N'(1.96mg inIO. Oml 100roMphos phatebuff er, pH 7.6), 90 pg microsome (lOOlJ,g mic r o s omal protein in 1.0ml of 100RIMphosphatebuffer,pH 7.6)weread d edtobo t h reference andsampleccveee. Th e n ,0.1ml of100roMphosphatebufferpH 7.6 buffer was added to the re fer ence cu v e t , and the
absorba nce of the mixture measured, wi th
spectrophotollleter under the function of time drive mode at 550om, to obtain a baseline. The reference cuvet was then replacedwith thesampl e cuvet. NADPH40.9J1Mdi s solve d in 10 0 1I1 of 100111Mphosphate buffer , pH 7.6, was added to th e sample cuvet; the absorba nc e of th e mixture wa s measured immediatel y. Two va luesof th e absorbancewerese l e c ted to calculate the actiVi tybasedon thefo r mu l a:
(ABS1-ABS1)"'3"'1 000 /19.6 /ti me /mgMP.
In theabove formula, 'ABS:' and - ABS:' were two values of the absorbance, '19.6' was the coefficent, '3 ' was the volu me of the mixt u r e , '1000' was us ed when millimole was conv e r t ed to nano mole,andthe -t i me' wastheperiod between the two ebso rbancesselec ted.
2.1.8.5 Kine tics of et h ylmo rp hinedemethy lase
The method to measure the kineti csof EMDM is ide n tical totha t for th e measuremen t of EHOMac ti vi tyexcep t thatthe concen t ra t i o n of ethy lm o r phin e varied, containing of 0.42, 0.8 4, 1.68, 3.35 or 6.7 roMet hylmorp h i ne. The valuesofKm andVmax ar eob tai n e dbas edonthe followi ng calculation:
ABS =b[E M] +a Km=b/a Vrnax=l /a
In th eab ov e formula s , 'ABS ' was the valueof absorbance,
•[EM] ' was the concentra t i on of ethylmorphine . Formula ABS=b [ EM]+a wa s obtain e d by line a r re g re s sion analy s i s; therefore, "b' wa s th e slop e of the cu rve, and 'a ' was the intercept of th ecurv e with the or di nate .
