ri to

MECHANISMOF OCHRATOXINA-STI MULATED LIPI DPEROXI01\'1'ION

by

@Ra be ea Fahmy omar , B.Sc•• Honours

AthesissUbmi t t e d totheSchool ofGrad u a t e Studies in partial fUlfillmentof the

requirementsfor thedegreeof Master ofScience

Tox i col og y

Me mo rial Universityof Newfoundla nd St .Joh n's,Ne....tound l l: nd

Hay, 1990

Na tionalUbrafy 01Canada

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Canada

11 ABS'i'RACT

OChrat oxin A (OTA) is a myco toxi n and a nephrotoxi c carcinogen. The me ch a n i sJI by whic h i t st imul ate s lipid perox idation inv estig a t e d in a rec on stitu ted syste m con sis tin g of phos pholipi dvesicles(liposomes). flavop r otein NADPH-cytochr ome P-4 5 0 reducta s e, EDT A , Fe) 'ions,andNADPH .

Lipidperoxidation,measuredeither as meLcnd La Idehyde formed or by oxyge n uptake. wa s greatl y stimula ted in the presence of OTA. Omission of EDTA lowered the ext e nt of lipid pe roxi da tio n but did not eliminate it. Flu oromet ri c and spectrophotometric st ud i e sdemo nstr a t e d the fannationof a 1:1 Fe)'-OTAcompl e x. The rate of reduct ion of Fe)' to FeZ'wa s grea t ly enhanc ed in the presenc e of OTAan d there was a fu r th er increase in the rate wh e n EDTAwas also include d.

Cytocr.r o me P-4S0 (a nenzyme normall y presen t inmicro s ome s)

";"" 0;found to effect i vely replace EDTAin the reconst i tuted

• stemand it s role in microsomal lipid peroxidat ionwas al so implicated sug ge s t i ng th at this hemoprotein could play an impo rtant rol e inOTA-st imu lated lipi d peroxidationin....Y..!..Yg.

ESR studies showe d th a t the FeJ··OTA compl ex prod uc ed hydroxyl rad icals in the pre s en c e of NADPH and NADPH- cyt oc h r o me P-450reductase . The lac kofany diminuti on of li p idpero xid ationby catalase and severa l hyd r o xyl radical scav e ngers suggest&: thathy d ro xy l radical product ion by the FeJ~-OTAcomplexlIlaynot be a significant facto r in th e lip i d

iii

peroxidation in Yit.r.Q. but these results do not preclud e hydroxyl radicalsproduced by the Fe3+-OTAcomj-Lex from playing an importantrole in the toxicity of OTA.

str uctu r e-act iv ity relationship studies indicated that the pres en ce ofa free carboxyl gr oup and chl o r i ne atom as we llas the L-Phe mo i ety on OTAcont ri b u t edsi g nific a nt ly to th estimula to r y effecton lipidperox ldation. Earl ierstudie s had shown an ebectu t.erequirementfor a free phenolic hydroxyl grouponOT A.

My resul ts indica t e that OTA stimulates lipid peroxldation by compLexir.q with Fe³+ and fa cil it a ting i t", reduct ion . Su bs e que nt to oxygen binding , an ir o n-oxyg en co mp lex of undeterm inednatureinitia tes li p i d peroxidation.

The extent of OTA-dependent lipid peroxidation in vivo and its roleintheto xi c ityof OTAremain tobe determi ned .

iv

To Hy Parents

1\CRNOWL E DGEHENTS

I would like to thank Dr . A. D. Ra him t u la for his supervisionand his valuableguidance throu ghout thi swork. I thank Dr.C. Sharpe for his kind supp or-t; , I am grateful to Dr. B. Hasinofffor devoting a considerableamoun t oftime and effort towards the tSR studies. Ialso thankth e members of my supervisory commi1:.tee, Dr.E. Barnsl e y and Or.F. Shahidi for theirconstructivesuggestions. A spec ial than k s toMs . Marie Codne r for technica l assistance and helping to make bench work a more enjoyable experie n ce. I th an k Dr . Phil Davi s for demonstrating the operation of GC and giving me access to it. I am also grateful to th e members of Biochemistry Depar tment and itshe:d Dr. K. Ke ou g h for their generosity. I am indebted to theKidneyFounda tionof Canada for generous fina nc ialsupport.

I wish to extend specialth<.l!lk s to myparents forth e i r kind blessings.

TAB LE OP CONTENT S

PAGE ABSTRACT•.•• .••••• . •• • ••••.•• • • .• •• •.••• •.• . ... ...•... . i i ACKNOWLEDGEMENTS .• •• • • .. ••• .••• • .•.. • . ••• .• • • •.••.••..• LIST OFTABLES ••• • • •. •. . •••. • ..••.. .. ••••••••... •.• • • LIST OFFIGURES •••• •.• .•• • •••.•••..•. ••..• ... .. .. . . . xi LISTOFABBREVI ATI ONS . ... ... . . . .... . . . ... . . ... xiv

1. INTRODUCTION.•••..•..• •. .... . .... . . .. ... .... .... 1 1.1 Ochr a toxi n A .• ..•...•• • • • • •••...•.... .. .. 1 1.1.1 Occurre nceand huma n exp o s u r e ••• .. • 1 1.1. 2 Toxic ity..• ••• ••• ..• •...•... ... . 1.1.3 Ca r ci noge nici ty and muta ge nicity. . . 10 1.1. 4 Absorpt i on and metabolism. . ... . .... 11 1.2 Li pid pero xidation .••. •. . •.• •••••• • • . . •• . . . .. 12 1.2. 1 Defini t i on .. . .. . . ... . ... .. . .. 12 1.2 . 2 Measurement•••• .•• • •• •.•• . ..•. • . • . . 15 1.2 . 3 En zymat i clip idpe r ox i d ati on

sys te ms •..•••. . .••...•••..••••... 24 1. 2.4 Phys i olog i c a l sources of iron... 25 1.2. 5 Ini t i ati o n . ...•••.• .• • ..• •.. • •. • .... 26 1. 2.6 Cellul artoxi city •.•.. . ... . ... .•. 28

vii

1.3 Objective of thesis .••••••..•• • •. • • •••••.. 31

2. BIOSYNTHESIS OFOCHRATOXIN A•••• ••• • • ••••••• ••••• J2 2.1 Organism•• ••.•..••.•.•.••.• •• .•••... ..•. ..• • 32 2. 2 Culture.. . .. •...•..•..••••••..• . .•.. .•. .• •• 33 Extraction •••.••.•• ••.••••••••.•••••• •.•... 35 Purification... . . . .. . . ..• . . ••• • .. . .. •..••• 36 2.5 Crystallization ..•.. . • . .••.• .••••..••••.. •• 37 Determination of purity.••.... •. .•..•••••• . 38

MATERH.LSAND METHODS.•• • • • •• • • •••• •• ••••• • • • •• . • • 46

3. I Jl:AT ERIA LS •••••• •• ••• ••• ••••• • • ••••••••••.• . 46 3.1.1 Chemicals . . • . .•.• • ••. . . ...•. 46 3.2 METHODS ••• ... ....•.. . •...• ...•.•• •.• •••.••. 47 3.:2.1 Preparationof microsomcs •.• • •• .• •. 47 3.2.2 N'ADPH-cytochrome P- 450 rectuct ece

(Fp) .•.. ••. .••.. ••.• .• .••••.•••••••

3.:2. :2. 1 Fp purification ••. •• .• .... 3.2 .2.2 Fp assay .••.. .••••••••..•• 50 J.:2.3 CytochromeP-45 0 ••••.••••..•. •.• • • . 53

3.2.3.1 cytoch romeP-450

purificatio n•. • •.•.• •.•••. 53

3.2.3 .2 Cytochrome P-450 assay 55

3.2.4 Prepara tionof rnicrosomes from protopo rphyrinIX-treatedrats••. .• 58

viii

3.2.5 sy nt hesisofOchratoxinA

ana l ogu e s •••• • •••.. . . ..•..•..•• •... 59 3.2. 5.1 Ocratoxino{•• • ••• •• ••• • ••• 59

3.2.5.2 OchratoxinB 60

3.2.5.3 OchratoxinC•• •• •.••• ••••• 60 J.2.5. 4 Replaceme nt of L-Phemoiety

in OTA by diffe re ntamino acids •.. .. •••. ••• ••.• ••... 61 3.2.6 Lipidextractionand preparation of

phosp holipidvesicles••...••....••• 62 3.2.7 Lip i d peroxidationassays

.... ....

64 3.2.1 Spectrophotometric measurements

..

65 3.2.9 Reductio nof Fe]oI-to Fe2+

...

65 3.2.10 Oxygenuptake studies

...

66 3.2.11 Fat ty acid anal ysis inmrcrosoeos 6' 3. 2.12 ESRstudi es

.. .. . .. . .. ... ... . ...

sa

4. RESiJLTS.• ••.••• • •.••.••••• • •••.•.•.•• • •• •••• . .. .•.69 4.1 Roleofvariouscompon en tsin lipid

pe r ox i da t i o n •.•... • • . •• . • . •• • • . •• • • • • •.•... 4.:2 Effect offr e e radica l sc a v e ng e r s on 1 ipid

perox i dati o n .. .•.•. ... . ... .. ... .... .. 79 4.3 Effect of varyi ng the conc e ntrat ionof

individua l componentson l ipid

peroxida tio n•. • ••. ••.• • . •... ..• • •• •••• .•. • . 79

4.3.1 varying the EDTA concentration •....79 4.3.2 Va ry i ng Fp concentration ••••.•• • •.• 82 4.3.3 varying the pH•••• • •• ••• • ••• •.• •••. 82 4.4 rrwo.tvenenc of cytoch romeP-450inlip i d

peroxidation..•••.•• ••. .•.•• •• •••••• • •. ..•.Ci7 4.4.1 Effectof varying cytochrome P-450

co n ce n t r a ti o n •...••. •.•....•••.. .•.87 4.4.2 Effect ofinh i bi ti ng cytochrome

P-450 •••••••••••• ••.•••• . • • . ••• •.• • 90 4. 5 Role of OTA and EDTA in the reduction of Fe3+

toFe2~•• •• •• • • • •• •••• • ••• •••• ••••••••• •• •••90 4.6 Formation of Fe3+-OTAcomplex•• •.•••.•.•. .• 95 4.7 De te cti o n of hydroxyl radical formation by ESR,

and effectof various components on free radical formation •.••••• ••••.•• .• ••• ••• •• •• 10 5 4.7.1 EffectofFp •• ••••• •.•• • • •• ••••••• 105 4.7.2 Effect of ethanol•••. • ••••••• •• .•• lo a 4.7.3 Ef f e c 't:ofOTA, Fe3+and catalase .• 111 4.8 structure-activ ityrelationship studies ... 111

5. DISCUSSION•.•.•.•... ...•.• .•••.. . . .•..•...•••.•.• 124

6. CONCLUSION •••• •• • • ••••• .• ••.•.••••.•••• • • •.•.•. 137

7. BIBLIOGRAPHY•. • ••.••• • •• • •.••.•••• ••• •••. .••• • •. 139 7.1 Li st 0'.references•••••. • ••• •• •••• • ••••••• 139

A. APPENDIX •• . ••••••• • ••• •••••• ••.•• ••• • •••• •. • •157

LISTOJ!'TABLES

PAGE

Table1. The Rrvalue s ofochratoxi r.A ... . . .•... 39 Table2. Effe c t ofvar ious agentson

OTA-st imu lated lipidperoxida tion .• ... 78 Ta b le Al. Th e R(valuesof OTAan dit s

analogues... ... . .... 158

Figure1.

F'igur e2.

Figure 3.

Figure 4.

Figure5.

Fi g u r e 6.

Figure7. Fig u r e8.

Figure9.

Figure10.

xi

LIST OF FIGURES

PAGE

Chemi ca l structure of

ochratoxi nA.. .. ..•. ... . .••.••. •. .•.. Fa c t o r s in fl u e nci ng the

occurrence ofmyc o t oxins inhuman food and ani mal

feed .

The major polyunsaturated fatty acids inrat liver

microsomes. •...•... ... ... . 12 Simplifiedreactions of the

process of LrpId pe r ox i da t io n. 16 Scheme repre sent ingthe

fo rmati on of malonc'lialdehyde

duri n g lip i d peroxid a tio n. 17 The complexformedbe t wee n

th i obarb itu r i c acid (TBA)

and malondialdehyde (MDA). ... . . ... . 20 Th eHPLC profil eof ochra to xi n A... 40 The UV spectrum of och ratoxinA... .. . 42 Th e fluoresce ncespectrum of

..:..:h r a to xi n A. ...• •. .... ... .. . ... 411 Gelelectroph o reticanalysis

of the purifi ednative NADPH-

,•.. 51 Figure 11.

Figure 12.

Figure 13 .

Figure14.

Figure 15.

Figure 16.

Figure17 .

Figure18.

Figure19 .

Figure20.

xii

cytochromeP-4 5 0red ucta se. 50S-ge l ele c t r oph o r et i c analysis ofthe purif iedcy to chromeP-450.••• . 56 Effe c t of va r ious componen tsof the reconsti tutedsyste mon OTA- stimulatedMDA formation. •.. • .•.• • ••• 70 Ef f e ct of OTA concentration on MDAformat i o n. •.• •••.•.•• •.••• • •• . •.• 72 Ef fe ·::t of various components on OTA-stimula t e d oxygen uptake.... . .•.• 74 Ef fe ct of OTA concentrationon oxygen uptake. ••••••••• •..••••• • • ••• • 76 Ef f e c tof EDTAconcentrationon OTA-st i mulatedlipid peroxidation.... 81) Effect of Fp concent rationon OTA- stimulated lipidperoxidation. .• • .. •• 83 Effectof pHon OTA-stimulated lipid peroxidation. ••.•. . ..••••• •.•.• Ej Eff e ct of cytoch rome P-450

conce ntra tionon oTA-stimulate.d lipid pero x i d ati on ... ..•• ..••...•. 88 Fattyacid analysisof microsomal lipids extractedfrom control and coba l t pro t o po r p h y ri n IX

tr e a ted rats . ••.•.• • .• ••••• ••••.••••• 91

Figure 21.

Fi gure 22 .

fi g u r e 23 .

Figure 24.

xi ii

Effectof inhibiting cytochro me P-450 onOTA stimulated lip i d

perox idat i on. ..•. . •••. .•• ••• • •••••••. 93 Effectof variouscompone n t son fe rr icre duc t ion . .•.•.•... . .. .. . . 96 A: Spectrum of t"'e Fe³'-OTA

complex for me d in methan o l. B: Spec t r u mof the FeH- OTA

complex in Trisbuf fer. .. .••. •.•.• • • • 98 Tit ra t i o n ofOTA by FeC13.•••••••••• ••100 Figure25 . Quenching ofOTAfluo re s ce nce

byFe³+. • •••••• • •••• ••••••• • • •••• ••• •10::

Fi gures26- 28. ESRspectraunder di f f e r e n t

Figu r e Fig u r e 30.

Fig ur e 31.

Fig u r e 32.

Figure33.

Fi g u re 34.

experimental cond i t ions.••• . . .•• • • •.. 106,1 0 9 , 1 12 str uc tu r e ofOTAanalo gues•.... ••••..116 Ef fe ct of vario u sochr ato x i ns

on MDA formation ••••••..• .. ... ... .. ••118 Eff e c t of vari ous och ra tox ins

on iron reduction•••. ••• .• • •• . •••.•• . 120 Effectof var iousOTA an a l o gu e s on MDAformation••• •••••.• •.. .• •••.••122 Aut o xidationof ferrouschel ates.•• ••129 Sch e me rep re s e n t i ng the over a ll suggestedmec hani smofOTA-

stimulatedlipid peroxid ation••••• ••• 133

Figure Al.

xdv

HPLCprofiles of OTA and its

analogues.••••••• •••• •. .•••••.•....•.• • 159

LIST OF ABBRBVIATIONS

BPS bath ophe n.lnt h r o l i ne di s ul f on i c acid BHA butylatedhydroxyanisole BHT butylatedhydroxytoluene

CHAPS (3-((cholamidopropyl)-dimethylarnmonioJ -l - pr-apaneau Lfonatiaj

DMPO

m-r

ESR Fp

FMN

GIu-Oo:

MDA DTA

00 DB

DC zro-ecc PB

Ser-Oo:

SOD

5,5-dimethy l-l-pyrrolIne-u-cxtcre dithiothreitol

electronspi nresonance

the flavoproteinNADPHcytochrome P-450 reductase

flavinmononucleotide gl ut amic acid ochratoxin0:

maLo ndLeLd e j-jde ochra t ox inA oc hratoxin0:

ochratoxinB ochra t o x in C prolineochratoxin0:

sodium phenoba rbital serineochratoxina superoxidedismutase

TBA TCA

xvi

2-thiobarbituricacid trichloroaceticacid.

CHAPTER 1 INTRODUCTI ON

1.1 OCHRATOXINA

1.1 .1 occurr e nceand human exposur e

Och r atoxin A (OTA) is a myc ot oxin pr oduc e d by Asp erg illu s ~, Pen i ci ll i um vi ridicatum and oth e r fung a l spe c i es. It was dis cover ed in 1965byDeScott (1) as th e tox ic met a b o l i t e ina culture medium of Asper gil l u s oc hraceusWilh,and chemically ch a r acteri zedby Van der Merwe et a1. (2) and SteynandHolzapfel (3,4) . Its che mi ca l stru c t u r econ s i s ts of a s- c mcrc-e-nycrcxy-a,4- di hy d ro -3-me t hylLeoc oumaz-Lnmoiety li nked by an amide bond toL-/J-pheny 1alani ne(Fig. 1). The toxigeni c mol ds known to prod uce the tox (5) are the followi ng species :

Aspergi llus~ Peni cilliumviri d i c at um A. ost ianus P. cyc 1 0 c ium

~ ~ A. petrakii P. pa l i ta ns A scle r o tj o ru m P. purcures c ens.

A. sul p h nrell s variabile

The occurrence ofOTAin food and feed is widespread.

Figure1. cneetceastructureof ochratoxinA.

eoea f1 ~

©- CH,- ~H- ~- C ~~~H,

C'

It ispres ent as a centamina ntin pla nt pr oduct s, es pec i all y cerea ls, be ansandpeanuts (5). OTAis alsofeund in meat s, dried fishand nu ts (6) aswe l l as inthekid ne y, liver, and bl ood ofslaughte redpi gs(7). Ina rec e nt analy sis of 1200 blee dsa mpl e s ebt ainedfrom pigs sla u g hter e d inWe ste r n Canad a, Mar quardt et al. (8) fou ndthat 76 \ had det ec ta ble level s of OTA,11.3% had OTAle vel s > 10ng/ro1with the highe s t bein g 229ng/ml. Scottet al. (9) de t e ct edOTI\in conc entratio n s of up to27 /-I9/mlin 18 out of29 samplesof he ated gra i n fr om aasxaccne wenfarms (Ca na d a ),wh ereasthe hi ghe s t obs e rv edconcen trationef resi dues inan im a l produ c t s (baconfr om pigs)is 0.067 /.lg / ml (10 ) . Th e pot enti al for huma n expo s u re existsbecaus e ofthedire c t consumptio n of con taminat edcere alsand the consumptio nof ani malsthat retain OTAinthe irtis s uesaf t erbeing fed contam i nated feed (Fi g. 2).

Ochratoxin A is sus pe ctedofbeingthe ma in etielog ic<l1 agent respons ibl e forBalka nendemi c nephro pat hy (BEN) and as s ocia t eduri nary tr acttumors , di se a s es whic haffec t mUltiple memb e rs of famil i esresiding in particul ar areasof Bulgaria, Roma niaand Yugos l avia (11 ). BENandporcine neph r opathy (adise a s ewithmorph olog i c a l andclin ica l sympt oms similar tothose of BEN) ar e found inar eas Whe re home-growncereal s arecontami na ted wit h OTA (12,13).

Ga ltiereta L, (14) have shownthat aTApers ists long e r in pig sthanin othe r spec ies ,whichsu g gests tha t pro bl e msof

Figure 2. Factorsin f l u e ncing the occurrence of mycotoxins in human food and animal feed (175).

Insect,Dire&

IJoent <uma g!

EmlironmenUl mreracnens Orougnt&

reenerature

~

'WIYCOTOXINS^{ESIO UAL}

Food,,, )

l1umanconsump tion

~

^______ ADsOrpt IOn an lm.1l$

Fe~'"

,n'IS"" / ~

u@taDOllsm

&Sec retion

~

OTA residues inthe humanfoodchain may be greater vhe n pork rather thanot h e rme a t s is consu med .

1.1 .2 Toxici t y

Oc hratoxin-Ais todc toIilanytest ani ma l s incl ud i ng ch icke n s.dogs,duckl in gs, mic e, rat s, hens, sheep,swi ne and rainbow tr o ut (15, 16 ) . The toxic effectof OTAis di s pl ay e d initially on thenepllron,an dthe pr oxima l tubule is thepr lrrar y target site (6). Later, theglomeruli as ....ell as the interstitiamay be involved. The chan gesof renal funct ion in OTA-exposedrats and pig sare chara cterizedbyan increase in po l yuria , glu cosu r i a.

p r-ete Ln u r-Ia an d bl o od ur e a nitroqen,and a decreasein uri ne osmola r i ty,qlomerul a rfil t r at i o n rat e (Gr R) and ':"nulin r-Leara nce (17) . Th e changes inre na l st ruc t u r e inpiq ~ are ch a ra ct e r iz ed bydegenerationof th,pro x i maltubul es , lead i ng totu b u la r at ro py accompan iedbyint e r s t iti al fibro sis. Later, hyal inizati onot the qlolllerulimay occur (26,27). In ta t sora l doses of OTAle d toreduced plasma fibrinogen, factors II,VII and X andth romb o c yt e and megakaryocy tecounts (28). In chickens lympho c yt openi a developedateverydose levelte sted (29), and co n ce nt ra t io n s ot ser um immunoglobulins (Ig A, IgGand IgM) were reducedto 57-66\ of norneL values in th e toxin-expos ed groups PO} . Inmice , myelotoxic itywaf;observed reSUltin g

in bone marrow hypocellularity, decreased maz-r-cw pluripotent stem cells, and granulocyte-macrophageprogenitors (31).

The treatment of pregnant mice and rats Lp. with OTA resulted in increased prenatal mortality, decreased fetal weight, and various fetal malformations (32). In chickens fed diets containing 0, 1, 5 and10 mg O'l'A/kg over a four- week period, the rate of growth and relative weight of the bursa of Fabricius were depressed, and the relative weights of theli v e r , kidney, pancreas and various sections of the gastrointestinaltract were increased, but there was no effect on the heart and spleen (33). The transportof p-aminohippurate (PAH) and tetraethyl ammonium (TEA) by renal slices was also found to be inhibited by OTA (18).

Kane et a L, (19) observed a particularly good correlation between the increase in urinary excretion and a decrease in renal activities of l-glut:amyl transferase (r- GT), alkaline phosphatase (ALP) and Leuc Lne aminopeptidase (LAP) within a week of the oral administrationof 145 JIg OTA/kg body weight/day forl~weeks. An inhibition of gluconeogenesis was alsoobserved in kidney slices from rats which had been fed with='mg/kg body weight for 2 days (20), and renal phosphoenol pyruvate carboxykinase (PEPCK) was selectively lowered by 50%. Suzuki et aL, (21) observed a 60% decrease in hepatic glycogenlevels and a concomitant increase in serum glucose and blood and liver lactate levels in the rat after daily administration of OTA (5 mg/kg) for]

da y s . Admin ist r a ti o n of OTA to miceinhibited protei n synth e s i s (22). The doq r e e of inhib ition of protein syn th e sis 5hr afte r adminis t r a tion of 11119'OTA/kqwa s 26\

inliver , 68\ in kidneyand 75\in spleen. Ph e nylala nine (100 mg/kql injected toge t h er wi th OTA(10 Dig/kg) pre ve nted the in"ibitionof protein synth e sis In all of theseorgans . OTA is tho uqht.toinh i b i t protei n syntheolis throug h co mpe titi o n with phenylala nin e in the reaction catal yz e d by phenylala nyl t-RNAsyn t heta se.

l.!:l...Y..llstudiessho we d that OTA inhi b itedliver mitochondr i alre s p irat ionprimaril yby alteringthemembrane permeability (23,24). Re c e nt l y,Ra himtulaand associates (25) reporte d tha tOTAdi s r u p t ed microsoma l ca lc i um ho me osta s i s by imp a i rme nt ofthe endoplas mic reti c u l u m me mbr a ne, probably viaenhancedlipidpe r oxid a t ion.

The L050 veIue c of OTAin variousspe c iesare listed below(materialsafetydcca sh e et (MSOS), siqrnaChemical Co•• 1989):

~J§ Bouteof Administration IJl>Q

Rat Oral 20.0 mg/Kg

Rat lop. 12.6 mg/Kg

Rat r,v. 12. 7 mg/Kq

Mouse Ora l 46. 0 rng/Kg

Mou s e iop. 22. 0 mg/Kg

Mous e Lv . 25.7 mg/Kg

Dog Ora l &.2 mg/ Kg

Piq Oral 1.0 mg/Kg

Chicken Or al 3.3 mg/Kq

10 1.1.3 Ca rcinogenicityand ...ut a g e ni ci ty

Bendeleat eL, (3 4 ) found thllt40 ppm OTA fed to (C57BL/6J xC3H) Flmice for up to24 months inducedrenal neoplasms in males only. Femalemice had a slightincrease in hepatocel lul arneoplasms. Ea rlier, Kanesawa andSuzuki (35) had observed an increasedincidenceof both hepaticand renal tumorsin male DDY mice fed 40ppmQTA. In a recent study conducted bythe U.S . NationalToxicol ogyProgram (NTP) (186 ), OTA in cornoil was administeredby gavage to group of male and femaleF344/N ratsfor upto 2 years.

There was clear evidenceat:anincreasedincidenceof uncommon tubular cel l adenomas and carc i no ma s of the kidney in both sexes. In addition, female ratshad .vninc r e a s e d inc ide n ce and mUltipl icityof fibrcadenomas of themammary gla nd. Other non-neoplasticrenal chang esobse rve d included tubular cellhy p erp l asi a,tub ulil r cellproliferation, cytop lasmicalterations, karyomegaly,and degenerationof the renal tubular epithelium.

eTAdid no t produce geneticor related effects in a varietyof shor t term tests (36,37) . From the sameNTP stUdy, OTA was not mu t a g e ni c infour strains of~ typhimurium(TA97, TA 98,TA100, or TA 1535 ) whente s t e d bothwithandwithout exogenous metabolicactivatio n. Also, OTAdid not significan t ly inc r e a s e the numberof ch r omosoma l aberrationsin cult ured chinesehamsterovary cells. aTA

11

induced sister chromat idexc henqee inthe presence, but not inthe ab s e n c e , of me ta b o licactiva tio n (18 6 ). OTA has also been shown to ca usesing le-strandbreaks in DNAisolated fro,.,li versand kidneysofratsthat ha d been fedthe eq u i val e n t of 4 ppm OTA for12 weeks (38).

J\.bsor ption an c1 metabolism

The primarysite of OTA absorptionis though t to be in th e sma l l intestine . When OTA was injectedinto theLumen of the stomach,smal lintestine, caecum,or colonof male Wista r rats, thehighestab s o rpt i on wasin th eprox imal jej u n u m(39). Inmice,when OTA was givenorally,the site ofhighest abso rptionwas the duodenum (40) . In th e latter study, immunohistochemical stainingre v e al e d that the highest concentration of eTA was in the intestine,with decreasi ng le v els in the kidn ey and liver (40).

Many environmen t a l carci r o g e n s an d to x i n s re q uire ox id a tiv e metabolism,"lost oftenby the cytochromeP-450- dependent mon o x y g e n a s e system, in order toexert theirtoxi c or carcinogeniceffects (41). eTA iskn o wn to be metabolizedbyra t livermi crosomesto (4 S )-4-h yd rox y-e-rA an d (4R)-4 -hydrox y-OTA(42), while wi t h rabbit liver microsomesan ad ditio nal meta bo l i te, 10-hydroxy-eTA, is formed (43). Invivo, OTA metabolitesdetected in the urine of ra t s includeochr atoxina and 4-hydro xy -oTA(4 4 ). In

12

rats intubatedwithlabelled OTA(3Hin Phs),thr eeother metaboliteshave been detectedinlive r extracts,butthese havenot been identified(:38).

1.2 LIPID PEROXID ATION

1.2.1 Definition

In euk a ryotes , membrane flu i d i t y is mai nta ine dby the Lncorpor-at.Lc nof pol yu n s a t u r a t e dtat t y acid (PUFA) ch a i ns intomembranelipids. Most of these PUFA chains occuron the 2-C pos ition of the glycerolmoiety of phospholip ids , particu larlyphosphatidyl ch o line and phosphatidyl eth a nol ami n e, al tho ugh some al s o occur in neutrallipids . In the membranes of ratliver microsornes, themostabundant PUFAs (e x p r e s s€:d as percentage of total fattyacids) are shown in rig.3. The presenceofan adjacentdouble bond weakens theca r b on-a l l y lic hydrogenbonds . These ally!ic hydrogens, especially th os e onthe carbo n atom betwee n doublebonds,can be abstractedby re acti v especies containing one or moreunpairedelectrons (f r e e ra d i c a l s).

The lip i d radical thus formed will then re a c t withmo lecula r oxygen, and the ensuingchainreactionresu lts in the breakdownof the PUFA. This reaction se qu e nc e is known as lipid. pe r oxidation. Li pid peroxidationpropaga te s bycol- lision of a radical moleculewith a non-radi cal mol e c ul e and

13

Figure3. The major polyunsaturated fatty acids in ratliver microsom e s (176).

14

;vv=v=vvvyCOOH

v=v=v=v=v=v=v\

_eOOH

17% ara ch ido ni cacid

lOXlino l ei c aci d

5% dccos.a be xaenoteac i d

5%lino l en i c acid

15

term i nates wh e n tworadical s col lideeachother. The reacti o n s oflipidperox ida tionmay be classif i edinto thre e lIai n~tep s: 1) ini tia tion,2) prop a g ationand 3) terJllina t i on (Fig . 4) .

1.2. 2 Keasu rem e n t

The pro duct s of l ipidpero x idat i o n includ e l ipid epo xi d es, hyd r-o p e z-cxLdea,ep o xy alcoh o l s,andth e sho rt- chaincompound s suc h asmalondialdehyde (MDA), ethane, pentane, and 4-hydroxyalkenals (45-48). Lipidperoxidation ha s beenmeasu r·",d byth e detecti o n of conj u gate d die ne s forme d du r ingthe earlyphas e ofthepero x i dat i o n react i on sequence (49-51). and le s s c01lUDonl y by mea sureme n t ofli p i d hydrop e roxides (52,53 ). Themostcommonproc edu r esare based on th e measu rementof theproductsof 1 ipid hydro peroxid e br e a kdo wn suc hasMCA (Fig. 5). Thi s is the Dlost wide lyused methodbecause of itssimp licit y and sen sitiv i ty. Th i s SUbstanc e (MCA ) hasbe e n commonl y dete cted by thethi ob arbi tur ic acid (TBAI re action (54-57) . In addition , l ip i d pero x i da t i o n has been as sayedrecently by theevo l u t i onof short-chainalkane s (e t h a ne and pentane) both.1.n...YiY..Qandin...Y.i.U.2 (58-61).

In my stUdi e s , differentparameters were measured as indicesof lipid pe roxi dat ionto study the roleofOTAin stim ula t i n gl ipid peroxida t i o n. The cl a s si c TBA reac tionto

16

Figure 4. Simpl ifiedreactions of the pr ocess of lipid pe r-ox Ida 't Lon(I77).

17

Lipid peroxidation

(1 ) (2) (3)

LH + R' ~ L' + RH

L' + 02 - - - . . LOO' LH +

LOO'~

LOOH + L' L + L' - - - - 7 LL LOO' +

LOO'~

LOOL + 02 LOO' + L' - - - - 7 LOOL

R' ,. afre eradi c al

tH lipid unde rgoinglipidpe r oxidaticn I.' • lipid radical

tOO'• lipid pe rnxy radical

Initiation Prapagation

Termination

18

Figure 5. Sc hemedemons tra t i ng the forma tionof lIlalondialdehyde(MOA) during lipidperoxidation induced by ferrous-oxygencomplexesor by hydroxyl ra d i c a ls (134).

·19

.7::~·\:~".~· .j· .

^110'

~O_lM

.

"It-QUCH,

Figure 6. The complex formed between thiobarbituric acid (TBA) and malondialdehyde (MDA). The complex can be measured at 535 nm as an index of lipid peroxidation.

:S'(7

^{0 H}

OH TeA

2l

+

o

H 0

i l l C-j-\

HI

^{H H}

MOA

TBA- MOA Complel

22

measureMDAasanMDA-TBAadduct (Fig . 6) was the first in d e x to be meas ured (as desc rib ed in the Methods sec t io n).

It has been reported that MDA i tselfis react iveand fo r m!;

Schiffbaseswith aminogroups of various biomolecules, evq, amino acids andproteins, nucleic acidsand amino sugars (6 2-65 ) , and suchreactions wo ul d de creasetheMDAav ai l able forrea ctionwith TBA. Onthe ot h er hand, theac idi c conditi onsused durin g'.t ..a TBA reacti onmayhydrolysethe Sc h iff bases and maximizeMDA measurement. Recently, Pompella et a1. (6 6) inve s tig atedwhether someof th e commonly used methodsto de t e c t lipidperoxidation.i.n...Y.iY..

correlatewitheachothe r. They tested and compared the followingmethod s: i) measurement of MDA formation: ii) dete ct ion of dieneccnjugation; iii) measurement of the los s of PUFA; and Ivj eecermnetacn of carbon yl fu nc tio n s formed inac y l residuesof membrane Phospholipds. Correlations among the values theyob t a i ne d withthe semethodsshowed high at.atiLs t.Lca L significance, indicating that the procedures measure lipid peroxidation in vivo with comparablereliab~lity . Analogous ly, the four methods also appea r-a.;toco r r e l a t e when appli e d toin vitrq microsomal lipid peroxidation, reaffirmingconfidence in these procedures.

Oxygen uptake was the second method to be used as a measure of lipidperoxidation (describedin the methods section). It is also an acceptable me t ho d but it is not as

23

wid e l y used as the MDA-TBAreaction . It isknovn that lipid peroxidat ion is accomp a n i edbyuptake or oxygen in the fo rm a tion of pero xyradicalsand insubsequentdec ompos ition reac t io ns. I tis worth llIe nt i oning that (f r om myre su lt5 ) the all ou nt ofo)(yg e n uptake is about60 fold the amount of KDAformed ove r thesameperiodot: ti llle. That is becaus e oxy g e nuptakerepre sl;mtsa measure forthe vn cte lipid per-oxidationprocessplusa direct reductionor oxygento sup e r oxi d e ani on s and "202' whil eMDA is only one of several componentsresul ting from th ebreakdownof the pe ro x idized lipid . In addi t io n , measur ement of HDA formationcannot account for perox id izedlipids th at have not yet been broken down , wherea s oxygen uptakedoes incl udethisamount .

It isgenera lly accept e d tha t ir on pl a ys an impo r tan t role inlipid per oxida tio n . Fe r rous ir o n is able to produ c e hyd r oxyl ra d i c al s via the Fe n ton react ion, andit ca nalso par t i c ip a t e in th eini t ia t i o n of lipi d peroxi da tion by int e r ac tingwith uns aturatedlipid (UI) under goi nglipi d pero xidat i on to fon.lipidra d ica l (L"). The ra teof reducti on of ferric tofe r r o u s iron isa usef ul me a sur eme n t (des c r i bed in the Method ssection ) Whi c hwa salso usedto con firmtherole of OTA in fac ilitatingferricred uct ion SUbsequentto forming a comp l e xwi th it.

24 1.2.3 Enzymatic lipidperoxidlltion sys t e ms

Enzymatically induced microsomal lipid peroxida tionwas first describedbyHochstein and Ernster in 1963, who demonstrated the requirement for nicotinamideadenine dinucleotidephosphate, reduced form (NADPH)and adenos ine 5'-diphosphate (ADP) and the enzymatic nature of the process (67). In a subsequent study they showed thene c e s s i ty for iron (68) whichha d been a contaminantof thei roriginal ADP solutions. Later, Pederson and Aust(69) characterized the enzymatic na t u r e of lipid peroxidation further by demonstrat ing th a t NADPH-cytochrome P-450 reductase (Fp ) was

the enzyme linking NADPH oxidationto the ADP-Fe3+-dependent peroxidation of microsomalmembranes(69). They also developed a reconstitutedlipid peroxidation system consisting of phospholipidvesicles (liposomes), purified Fp, ferric chelates and NADPH. In their reconstituted system , a second ferricchelate, EDTA- Fe^H (i nadd i t i o n to ADP-F e'+j wasalsorequired (69). Therefore, Aust et a1.

(69) suggest ed that there may be a microsomal component(sj that directly reduces ADP-Fe H (for which EDTA-Fe" can SUbstitute) inthe reconstituted l ipidperoxidatio nsystem.

In support of this,Ho c hs t e i n and Ernster (70) suggested that cytochromeP-450may be involved . Later, Aust et al.

(71) demonstratedthat when cytochrome P-450was incorporat edint ophospholipid vesicles, EDTA-Fel'was not

25 required.

1..2.4 Physiologioal sour cesof iron

Because of the important role of iron in promoting lipid peroxidation, i tis worth gi v inga brief description of it s physiologi cal sources. Iron is transportedin the plasma and extracellularfluidsby transferrin,a glyco proteinwith two Fe" binding si t e s per mo lecule. In t r a c e l l u l a r l y iron is storedin fe rritin(72). This is the major phy s i ol ogicalstore of iron. It is ala r ge proteinof 44000 0molecularweight whichcan store up to 450 0 mol of i ron pe r mol of protei n,alth oughit isusua l ly not sat u rate d (7 3 ) . Inaera tedaqueoussolu t i o ns iron existspredominantlyas re". At physiological pH values, theChe mi s t r y of ferric ion isma inl y that of hydrolysis to yield insoluble fe r r i c hydr o xide s and oxyhydr oxide s (74 ). Forthisreas o n , low molecularweight c-hnlaticz-s of iron are required to exchange iron inboth direc.t ionsbetween transferrin and ferritinand betweenfe rri t i n and in t r a c e llu l a r iron- c on t a i ningcompounds (75). Heme pr o tein s , mos t ly hemoglo binand myoglobin,ca n also serveas other sources of intracellulariron (76).

26 1.2.5 rnitiat i o n

Li pidperoxidation is some times amajormechanism of cellularinjury inorg a n ism ssUbjected to oxidati vestress (reviewe d77-79) . Surprisingly lit tl e is known, howe v e r, aboutth e chemistryof initiation of peroxidation in membra n e systemssuch as liposomes or microso mes. The natu r e of the free radicalspeciesultimately responsible forth e initiation ofir o n- d ep e n de nt lipidperoxidationhas beenthe subject; ofco ns i d era ble debate. Theprincipa l candidatessuggestedfor this roleare the hydroxylradical (OH·) andth e ferrousdioxyge ncomplex (perferry 1 ion). The hydroxyl radical is anext r e mel y reactivespecies, reacting very rapidl y withmost organicmolecules (8 0 ) . Hydroxyl radicals can be formed via the Fenton reactionas follows:

The su p eroxideradical (0;-) is producedat a numberof intr acel lul arsites (81-8 3 ) and H:O:ca n then be formed readi ly from theno n e n zymi c or superoxlde dismutase- catal yzeddismutation of

0;-

(83). Subs equently , chelated iron canyieldOWi.nth e Fentonreactionas above. An alternativehypothesisfor the ini tiatingspe c ie s is that fe r r ou s ion , in undergoing au toxida tion to ferricion , passesthroughan in t e rm e d i a t e (Fez'- --0 2+-- -~FeJ' O;-)

27

st a te (fer rousdioxygencomplex ). Uponthedi scov e ry of ADP-Fe)+-i n i t i a ted microso mal lipid peroxi da tio n , Hochst ein and Ern ste r (68 ) postulated the invo l v eme n t of an ADP-Fe3+-- -02 complex. Fur t he rmore, Aus t's groupha spu r s u e d the idea that an AOP- f errou s dioxygen comple xinitia tes lipid peroxida tio n (84.85). The con c e pt of a ferrousdi o xyg e n comple xhas beencriticized on thegro un d s thatthe complex is insuf f i c iently reactive to....ardsPUr A (86). Thehig hl y- react ive hydrox y l rad i c al canoft en be dete ctedin mic r os oma l or lipo so mal lipidperoxida tionsys t e ms (87-90). The hyd r o x y l ra d ical isknowntobe ca p a b l eof ini tiating lip i d pero xida tionby abst r ac ti ng a hydrog en atom from tatty acidside chains (91,92). Rz02-degradingenzymes or sca vengersot OH', however , ra r e ly inh i bi t ir o n-d e pend ent peroxidationinmicrosomal or liposomal systems(87-901. It hasbeen proposedthat th e terryl ion (se e below) is th e trueFen t o n re a g e n t ra ther th a n OK".andth i s wouldnot be availab le for scavengingbyconv e nt iona lOR scav engers (95,9 6 )•

re" ...

R20₂~.:.,FeH-OR~[Fe]~-O-+--).Fe2·-0) ---~FeZ....OH"

en' H· fe rry l io n

Su p e r-ox Lde radic al smayplayaminor rol eininitiating lipid per o xidation un-serconditio ns in which th ey act to re du ce Fe]·toFe l+ (97,98 ). Rec en tl y,Aust et al. (99 - 100)

2.

have proposedthatasp e cifi c Fe1+-Oz-Fe^h complex, or at least a 1:1ratio of Fez>to Fe^J', actsas an initiator of peroxidat ion in liposoma l andmicrosoma l systems, butsome dou b t s have been ra isedaboutthis complexas a specifi c initiator ofperaxidat io n. Attempts to isolate sucha complex have failed (99,1 01) . TheFeh/Fe^J+ratiosrequ i red formaximal stim u lat i on ofperoxictation have been reported tovaryfrom1: 1 to1: 7indiff erentexperiments (10 2) , perhaps suggestingthataspe ci fic stoichiometri c comple x is notreq u i red. It mus t beconc ludedthatth e ident i tyof the ini tiat in g species of lipidperoxi da t ionprodu cedby re r rcus iron is stillanope n questLon,

1.2.6 Cellul ar toxicity

Th e peroxictative breakdown ofPUFAhas been imp lica ted in the pathogenes is ofmany typesof inj u r y and especia lly in the hepaticdamage induced by seve ral toxic sub stances. Among these toxic substances are theha l oal ka ne s , carbon tet rachloride (10 3-105).trIcm o rcbrcnometnene (103 ,106) , chlorofo rm(107), 1,2 - di b romo eth a n e (10 8 ) and haloth a ne

(10 9). In addition, paracetamol (110 ), bromob en zene (111 )I

Lrc-i (112). bipyridyl compounds (113 ), allyl alcohol (114) and in some in s ta nc e s , ethano l (7 9 , 115 , 1 16 ) have been shown tostimulate lip i d peroxidat ion. Thepe rox i d a tion of PUFA withinbiological membrane s res ult s in a complex series of

29

bio c h e mica l. and bi ophys l c a l even ts whi ch lead to inact iva t ion of enzyma ti.c functionsin se ve r al subce l lular organelles (103,104 ,11 7 -11 9). These alteratio ns in clude chan gesin thephysicalpropertiesof the lipidbila yer, reactionsbetwe enacyl peroxy l radicals andmembrane pr otein s and formation of re ac t i ve products originating from the degradatio n of pero xidi z ed fattyacids (1 17-1 1 9 ).

Thestimulationof lipidperoxidationin either artificia l neab r-e n es of liposomesor in subcel lular organelles has beensho wnto increase membrane rig i dity (120,121). Such aloss of fl uidit y does not seem to be depe n dent upon an inc rease in the rat i o be tw ee n chol esterol andphospholipids (120 ),but is ratheran effect of the formation of cross-linkingbet ween acyl chains (122) andof the depletionof long chai nPUFA (120). In add i tio n toth e changes in flui d i t y , li pid pe r oxida t i on causes an increase intheionicperrneabil i-::.yan d affectsth e surfacepote nt ials of themembranes (118). In th e liver, themembranesof the mitochondriaand endoplasmi c reti c u l um conta in unsa t u rated fa tt y acids in hi gh pro porti o n and th ere fo r e arevul n erab l e to per oxid at ive att ac k . At the sa me timethe y cont ain enzymesof the electrontransportsyste ms whi chmake them capabl e of produci ng freerad ical species (103 ,1 04 ,11 8 , 11 9).

The consequences ferthecel l oflipidper oxid at i on reactio ns and pr oducts are many. Mic r oso ma l membra nes undergoing perox idat lon in vitro show fr ag me n ta tion and

30

tu r bid ity ch a nges. destruc t i on of cyt ochrome P-450 (~23), andlos s of la t enc y and activ i tyof gl uc o s e-6-pho s ph at e and UDP-glucuronyltransfer ase(123 -125 ). Theplasmamembrane ca~+-ATPaseis inactivated beca us eof oxi da tion of essential sulfhydrylgroups in theenzy me (1 2 6 ) , result i ngin defective control of cytosolic calciu m. Ribosomes be c ome deta c h ed fromthe endop lasmicretiCulumduring 1ip i d peroxidation (127). Inmitochondria ,peroxidationcauses membraneswelling , deteriorationof el ectron transpo r t,and or g a n ellely s i s

r

rae, 129 ). Lipid peroxi d atio n of lysosornes ca-raes lysis and enzyme re l ease (13 0, 13 1 ) . and th e er yt h r oc yt e plasmamembranerespond s ina similarmanner

(132).

Cephaloridi ne, a be ta - lactamantibi oticof the cephalosporintype,causes ren a l in jury in humans and in laboratory animals. Like OTA, itsma in toxic effect is considered to be onthe proximaltubu les (118,~79 ). Several biochemicalmech anis ms have be enproposed to explai n the nephr o t oxic effectsof cepha lorid i n e (18 2 ,18 0 ,~8 1). The mos t rec enthypothesis sugge s t s an involve ment of lipid perox i d ati on ini t i a t ed by reacti ve oxygen species (18 1 ,183j • Recentstud iesshowed that the for mat i on of ce p h a l or i di ne- Induce-rreact iv e oxyg e nspec i e sand peroxidat io nof renal cor tica l membran e lipidS was inhi bi t ed by rad i c a l scavengers andan tioxid a nt s (182,18 4) .

31 1. 3 Objectiveofthethesis

Lipid peroxida tionhas beenpro posed as th e mechani sm or toxicityof a wide andever-increasing range of compounds

(1J4,135). Rece ntly,Rahimtu l a et al. (133) re p o r t e d that the addition of OTA torat liver or kidney rnicrosomes, theadministrationof OTAto rats enhanced lipid peroxidation.

Themajorobjectivp.of my stud ieswas to investigate themechanismbywhich ochratoxin A stimulatedlipid perox i d a t icn. Fo r thispu rpo s e, I used a re c o n s t i t ute d system co ns i s t i ng of phospholipidvesicles (liposomes), the flavoprote in NADPH-cytochrome pw45 0 reduc t a s e , OTA,EDTA, Fe^J'and NADPH.

Therol e of purifiedcytochrome P-450 as a possible substitutein vivo tor EDTAwa s also examLne d inthe reconst itutedsystem.

UsingESRI atte mptedto charac t eri z e the freeradicals fanned inthe recons t i tut e d systemin the prese nc eOJ: OTA.

Finally, Iex a min e d sevez-a I OTA analogues in a study of structure-activity rela t i o nshi p s to characterize the components effectivelycontributing to th e stimUlatory effect of OTAonlip i dpero X'idati on.

32 CHl\ P TER 2 BIOS YNTHESI SOF OCHR1I.TOXIN A

OTAis av aila b le commerci a l lyfrom a few chemica l compan ies , but i tisve r yexpe ns ive (100mg cost about $ 2,000). I therefore co ns i d e r e d th e productio n of th et cxin in our la b o r at or y . Th e OTA produced wascharacterizedby thin lay e r chromatog raphy (TLC)I high pre s s u r e liquid chromatography (HPLC).uvab s or pt i on an d fluorescence spectroscopy,and wa s found to be ide ntical to that purchasedfrom Sigma ChemicalCo. The earl ypa r t of my research (lipid peroxida tion) wa s done with OTApurchased fro mSigma cbemLc ar Co. Inmost of the laterwork I used OTA producedin our laboratory.

organism

Asper g illusochraceusNRRL-3174, obtain edfrom 0:.-.S.W.

Peterso n, Nort he rnRegiona l Res e a r ch Center, U.s. Dept. at Agricul ture , Peoria , Illi no i s , was us ed tor OTAproduction.

Cu l t uresweremaint a i nedat SeConmodifiedczapekaq a r » (se esection2. 2) wi t h 20%sucrosesupp lemented wi t h0.7%

yeast extract.

33 2.2 Culture

Flask s (250 ml) contain i n g100 rnlof mod i f i edCz a pe k liqu idmedi umtlr with 4%sucros eand 2%yeastextract, were stop peredwithcotto n plug s andautoc lavedat 12 1°Cfo r 15 min (136). Media were inocu l a t edwith a spor e suspensionof A ochrace usand incuba t edat2SoCfor 12 day s wi t h o u t

agi tation. The initia l pH of the med i a wasab o u t 6.7, and the fina l pH was abo ut 6.4.

-Mod i f i edcza pe k agarmedium **Modifi e d Czapek liquid

~

(f or fun gus pres e rvat i o n ) (f or OTA production)

Sucrose 200 gm Sucrose 40 gm

NaND] 3 gm NaNO] 3 gm

K:HP04 1 gm KzHP0^{4 1 gm}

Mg SO, . 7H 2O 500 mg Mg SO•.7H2O SOD mg

KCl 50 0 mg KCl 500 mg

FeSO••7H1O 10 mg FeSO••7H2O 10 mg

Yeas t extr-=t 7 gm 'leas t ext r a c t 20 gm

(Ditca ) (OH e a)

Aga r 15 qm Di st .H2O 1 L

Dist. H2O 1 L

Sin c e OTA co n tai n s achlo rine atom, theeffec t of dif f ere nt c iconcentra t i ons inthe cul turemed ium onOTA

34

pro d u ctionwas investigated. Chlo r i de conc e nt r a tionsof0, 25, 100 , 250 and 500 mg/L were tried,and itwa s found that thema x i mu m toxin yield was achieved at 100mg / L ci.

Hence,th i s concentrationwas used in the regularmediumfor OTAproduction instea d of 500 mqjLthatwasused before. An added advantageof lower m" concentration is that itcanbe used topr od uce3&Cl-l a b elle d OTAof higher specific activity . othe rsnev- alsorecommended thata lower ci' concentrationbe used (137) .

Because OTA contains a phenylalanine (Phe) moiety , the effectof adding exoge nousPhe tothe regularmedium was investigated. I t was found that th e yiel d ofOTAinc r ea s e d by about 25% when Phe (1 0mM) was incl ud e d in th eculture medium.

Cultureswere incubated fo r different per iodsof time (from 3 days cpto 30day s priorto harvesting). The highest tox in produ ct ion wa s found to be between10and 12 days of incuba t io n,andso thi s time period was used for all further ex p e r i ments. It was also of interesttodete r mi ne thetoxi n concen t ra t io n in th e fun g a l myceliumandin the cul ture filt ra te. Twelvepointsevenpercent of the total toxinyieldwas found inthe culture filtrate which isin good agreementwithval uesin the l iteratu re

r

ao - i ae: . The remainingtoxin (87.3%) was in the mycelium.

35 2.3 Oc hra to xin11.ex trac t ion

Aftermeasurin g the final pH, the culture medium was acidifiedtopH2.0with.ronc,He l and then thoroug hl y homogen izedusinga pojytn-onhomogenizer (Brinkman Instruments) for 3-5min at30, 000 rpm tobreak a l lfunga l hyphae. To 1 Lof ho mog ena t e about 10 mlof saturated NaCl solutionwas added, and themi x t u r e was extracted with lLof chloroform. After vigorousshaking, th emixtur ewa s centrifugedat 2000 rpm for 5 min. Threeseparatelayers wereobtained :an upperaqueouslayer. a lower ch loroform la ye r and a fluffy la ye r in between containingcelldebris. Both th e chloroform la ye r and th e aqueous layer were otiroaat.oqr-aphedbyTLC (K5Fsilica gel , layer thickness 250 J.l, Whatman) andchr o ma t og r a ms wereexamined under UV. OTA purchasedfrom Sigma Chemical Co. wa s used as a standard. Thesolvents}'~~'IS used we re: benzene, methanol, acetic acid(95:5:5v/v) (solven t A)l benzene, acetic acid(8:1 v/v) (solventB)lbenzene, aceticacid (4:1 v/v) (solvent C). OTA was no t detected inthe extractedaqueous solution after a sample (50~l)had been chromatographed, and sothe aqueous layer was disca rded. Thechloroform layercont a i ned material chromatographical ly ide n tic al with OTA. The ch loroformlayer(about800 ml) was driedoveranhy d r ou s sodiumsulfate (30-4 0 gm) and then reducedun d e r vacuum to about25m!.

J6 2. 4 Och ra to xi n Apur ifica tion

Tothe reduced vo l ume of OT Ain chloroform, an equal vo l ume ofNaHC0 3 solu tio n (0.5M) was add e d (1 3 8 ) . Af t e r vigorous shakin gthe mixturewas centrifuged at 4000 rpm for 10 min. The ch loroformlayerwas extracted again with an equal volume of fres h bicarbonate solution. T·.18 combined bicarbonate ex t r act s and the resIc ua r chloroformla ye r were tested for OTA by TLC as previouslydescribe d. and OTA was not detectedin the chloroform layer. Theco mb i ne d bicarbonatelayers were acictHied cautiously topH2.0 with cone.Hel andthe nthe toxinwas re- e xtractedwith chloroform(3 x 70 mL), Again, thechloroformex t r a c t s and the residual bicarbona te la yer werete s t e d for OTA by TLC, and noto x i n was detected in the bicarbonate la ye r. The chloroformlayerthat conta': -d the OTA (about 200 ml)was driedover anhyd r ou s sodiumsulfa te(20-25gm) and the volume wasreduced to about 10eu.

Fo r furtherpurifica t ion,a silica gel co l umn was us e d (1 38). Silica gel 60 (mesh 230-400) was dried at noocfor 1hr, suspended in benzeneand packedin t o a '500 x 38 mm column. Some ofthe ben ze n e was allowed to drainto aid se ttlingof the silica gel. Whe n the silica gelha d set tled, ath in layer of anhydro ussodiumSUl fate wa s added to theto p ofth e co l umn. The benzene was thendrained just to theto p ofth e sod iumsulfa te la ye r . The reducedvolume

37

of OT'"inchlo ro fo rm wa s ap plie d to the column whic h....as then el ut e d'<li th thesolven t be n z e n e:a c et i c acid,94: 6 (v/v) at a flowrate of 1.5ml/min. Frac tionsof15 rol eachwe re collected . Progressof OTAthrough the column could be obs erved by sh i n ir.qa UV l ightatthecolumn. eTAwas elutedbetween fractions 31-5 4. onlytho s e fracti ons tha t showeda singlespotof aTA onTLCwere combined. Fung al pi qments and other contaminan ts of OTA stay ed behi ndwhi le OTAwaselutedas a separateband. The co l lec tedOTAwas pure (judged by TLCat this st e p), colourl essand had ablue fluoresce nceunder a UV lamp.

Ochrat.oxinA crystal l izat ion

From 6 L of cUltu remedium, 380 mg ofpur e OTA was obtained. The purified OTAwas dissolvedin hot benzene (ca 2S ml) andfiltered. The filtrate wasev aporat ed ina steam bathunt i lcrys talssta rted to form andth encoole dtoro om temperatu re (138). The crysta ls we r e col lectedby filtration, wash e dwit h coldbe n z e ne and dried (yie ld359 mg). Ac c o rd ingto Ne s heim (138) ,thecrys t alline OTA ccnt.e Lns I mol of be nze n eof cry stall iz a t i on, and soafte r sUbtractionof 1mol of benze neof crysta l lization,the net toxin yieldwasabout 302 mg.

3.

2. 6 Dete rminat i on of OTA pur ity

TLCon sili ca gel plates containingaUVfluorescent indicator was used asasimple, rapidan d reli abl emethod for checking the purityof thetoxin especiallyduringthe isolationprocedure. Theproduced toxi n gavea single spot on TLCwhich had thesame R, valueas commercialOTA inJ solvent syst emsAI BandC (Tabl e 1). HPL C was alsoused

to determine the purity of the produced OTA. The toxingave a sharp clean peak that came out inthe same position as that of standardOTAand bothpeakar e a percentageswe r e comparable (Fig. 7).

OTAwas further character i zedspectrophotometrica lly between400 and 200 nm, It showed 2ma i n peaks at 332and 214 nmidentica l toth e maxima for standa rdOTA(Fig . 8) . The molar absorption co e f f i c i e n t of OTA at 332 nm(6 3 3 0 cm' M' l) was used to calculate the OTAconcentrat io n(139). Th e prod ucedOTA wa s alsosc an n e dfluoro metr i c ally us i ng an excitati onwav ele ng t h of 340nmand scanning the fluore s c ence bet ween500 and 300 nm (Fig.9).

Usi ng theabove techniques , i twas clear thatthe producedtoxinwas comparabl e in purityand spe ctral characteristics to standardOTA purchasedfromSigma Chemical Co.

39 Ta b l e 1: Th e Rr valuesotochra t o x i n 1l..

Solvent system

Benzene,acetic acid (4:1,v/v) Be nz en e ,acetic acid (8:1, vJv)

Benze ne,ac et i c acid , methanol (95:5 : 5 , v/ v/ v)

SigmaOTAo

0.68

0.41

0.45

Is ol at e d OU,"

0.68

0.41

0.4 5

"J /11of 25 roMwerespo t t edon TLC plates wit hflu ores c e n t indica to r.

.0 Figure 7.The HPLC profileof CTA.

A. OTApreparation

B. Standard OTA from Sigma Chemical Co.

Bothsampleswereinjectedinmet h ano l (10 /.11 of a 2.5mMsolution) int o a Perkin Elmer Series 4 Liquid Chromatograph. The column used was Partisil 10 005-2. The solvent systemcons i s t e d of a mixtureof a) acetonitrile:me thanol (Soo : s oo, v/ v )65%and b) 5roMsodiu macetate:acetic ac id (5 0 0:14 , v/v) 35%. Theflow rate was1. 5ml /minand the photomet ricde t e c t ion was per f o r me d at 332 nm.

41

OT-Z:

A

QTA

: I' \

~I

ST-l :

eo

10.0 60 w

'"

Z

o

.. ~

g , C;,"'

s.e"",---~---c:_::_­

0- Uw 0-w

o

QTA

1\

I'

I I i

^I

i \

\

'

....

20 '0 60 so

ELUTIONTIME(mlnj

42

Fiqu re8. The t1Vsp e ct rum. of OTA.

The conti nu o us li ne re p r e s ent s standardOTA from sigmaChe mi c a l Co. (- ) , wh i l e thebr ok e n line repres ents myOTApreparation (- -). Both samples areat a concentration of 25 IJ-M OTA in ) ml met hano l . The instrument (PerkinElmer Lambda )6 spectrophotometer) settings were: wavelengthsc an 400-200nm,char tspeed 60 mm/min and scan speed 60nm/ min.

UJo.ro

o z C1i a:

a

U)

~ 0.40

43

OJ

WAVELENGTH (nm )

44 Figure 9.The fluo re s c e n cespectrumof OTA.

The continuousline representsstandardOTA from Sigma ChemicalCo. (- ) , while the broken line rep resentsmyOTApreparation(- -). Both samples are at a concentrationof 50~MOTAin) ml methanol. The instrument(Perkin Elmer LS-S spectrofluorimeter) settings were as fallows: Excitation wavelength340 nm,while emission was scannedfrom 300 to 500 nra, The Ex/Em slits were set at 5/3 nm, chart s peod 60 mm/min and scan speed 120 nm/min .

w

U

w

Z

u en w

0:

o ::>

-'

u,

45

'"

WAVE LENGTH

(n m)

46 CHA PT ER 3 MATERIALS AND MET HODS

3.1 Materi als

3.1.1 Chem icals

2I,5' -ADPag arose, ba t h ophe n a n t h r o l inedisu lfonicacid (BPS ) , butyla ted hy d r o x y a n i s ole (BHA), but y l a t e d hy d r o xyto l ue n e (BRT), catalase,Cha ps (J-(cho lamidopropyll- dimethyl ammonio]-l-propanesulfonate), cnctIc acid , cytochrome c, DMPO(5 , 5-dime t h y l-l- py r r o l in e - l -Ox i d e ), EDTA

(e t hy l e ne d i a mi ne tetra c e t ic acid-disodiumsalt),glycerol, Lubrol PX, NADPH, OTA, Renex 690, superoxidedismutase and 2-thiobarbituricac i d (TBA) werepu r c h a s e d from sigma ChemicalCo. (St. Louis,MO, U.S.A). Anhydrousferric chloride, ferric nitrateand fer rouschloride wereobtained fr omBDHCh emicals,Da rtmou t h, Nova Scotia, Ca n a d a . DEAE- Sephace l was purc h a sedfro m Pharmacia, Dorval, Queb ec, Canada. All ot herchem icals we r e ofthe highes t gr ade commerc i a ll yavailab l e.

47 3.2 Meth od s

3.2 .1 prep arationof microsomes

Male speeque- uev t eyrats (200-2 20g) were obta i nedfrom Charles Rive r Canada, La Prairie, Quebec and were allowed free access to standa rd lab orator y rat chowand wa t.e r . Untreated rats or rats pretreatedwith sodium phenobaz-l-Lt.a I

(PS) (O.lt PBindrink i n gwa t e r for 5 days) werefasted overnightpr i ortouse. Livermicrosomes wereisolatedby differentialcentrifu gat ion as described ear l i e r (1 4 0 ) . Livers were excLsed and pooled (6 livers'" ·100 gm wet weight) in ice-cold50mMTris-Hel buffe r (pH 7.4) containing1.0mM EDTA, l.lS%:xci, and 20 ~MBAT. The livers werechoppedinto pieces, homogenizedinJ parts (300 ml) of the above buffe r toon e part liver (wetwt) usinga Teflo n/gl asshomog enize r, and th en centrifuged at 10, 000xg for 15 min. Foll owi ng filtrationth rou g hch e esecloth, microsomeswereisolatedfrom th e 10,0 0 0xg eupe'rnatant, by cent rifugati o n at 110, 0 0 0 x g for 60 min. The microsomal pe lletswerere s u s p en de d in 10 0 mM sodium pyrophosp hate(pH 7.4 ) (about 200ml ) conta ini ng1.0mMEDTAand 20 /,LM BHT and againcentrifugedat110,000 x g for 60 mi n.The final microsomal pellets weresusp e nd ed in 10roMphosphate buffer (pH 7.4) (35ml ) containing0.25 M su c roseat apr o t e i n concentrationof 50 mg/ml and storedat-8 0 °C untilused.

..

Proteinwas de teB ined bythemet hod.of Lowry et al. (141 ).

Hicroll"o me sprepa r e d as describ edabo v evereus ed forthe purificationot theflavop r o te in NAOPH-cy toch r ome P-450 reduc t a se . When1l1crosOJ:le swer epreparedfor lipid ex t r a c t ion , BUTwas excl ude d fr o mthebuffe rsand the final mic r osoma l pelle ts we r esuspendedin10mH Tris-Helbuf f e r

(pH1.4) (16 ml fro m 2ra t s ) atapro t e in concentra ti o nat 40 mq/ ml.

3.2.2 NADPH- cytochrome P-4S 0 redu cta se (Fp l

3.2.2.1 Fp Purifi cati o n

The flavoproteinNAOPH-cytoch romeP-450redu c tase (Fp) wa s purified trolllive retcrc seee s is ol atedfr olllPB- pretre ate d rats essent ia lly asde s cri be d byHurra y Arelieset a1. forha ms t er microsollles(14 0) . Th e followi ng proced ure s ve z'e allperfo rmed at ..·C. Mi c r os omes (50mgpr o t ein/Ill) were diluted to10 mg protei n/Illwith100aMTri s-HeIbu ff er

(pH 7.7) co nt aining 1.0mHEDTA,1.0mHdithiothreitol (OTT).20 JJH BHT, 5JJM flavinmonon uc18ot i dQ(F"MN),and 30\

glycerol (b u ff e r A). IJl\ll\e d i a t e l ypri or to use,CHAPS(3- (c h ola mi do prop y l )-dimethy l a mmon i oj-l-p rop a n e sul f on a t e ) wa s diluted1:1(v/ v) ....it h bufferA. A solut i on of 10\ CHAPS

(inbuffer A) was add e d slowlydropwi s e wi t h stirri ngto a fi nalconcentra tion of1\ (..../v ;1.1ll"ogCHAPS/mg protein) and

"

the mixturewas stirred for 30 .1n. Then , a1.5\ solution of protaminesulfate was addeddropwiseto a final co nc e nt ra t i o n of 0.07\ (v/v).After stirring for an additional20 1II1n, the mixtu rewasce nt r i f ug e d at 110,000x q for 60 mi n. Thesu p erna t a n t wasremov e d and the re s u l t i ng gray-coloredr ti ght l y packed pelle t was resuspended in bufferA withthe aid of a Teflon/glass ho mog e n i ze r to a protei nconcentrationof50mg/ml. A 10\ (w'lv) solution of sodiumcholatein wate r was thenaddeddropwise with sti rri ngtoa fi nal dete rge n t: p ro teinratio of 3mg/mg. Ten minute s la t e r , a 20\: (v;v)solut i o nof Lubr ol PX(ethylene oxide condensateof fattyalcohols, Sigma Chemica l Co.,MO, USA)in water was added dro pwise to a final concentrationof O.S\ (w/v) and the mixt u r e was stirredfor an additional30 rai n . The detergent-treatedfractionwas again centrifuged at 110,000 x 9 for 60 lIlin and the reSUltingsu p e r na t a n t was app l i e d directlyto a 2',S'-ADPagarose column (2.Sx 4. 0 em)at a flowrateof 1 ml/min. The dti n i t y column had beenequilibrated previouslywith 100 11M phospha tebutter

(pH7.7) containing0.4\cho late ,0.1\lIMECTA, 0.1mMDT'l', 20/-1MBHT, 5/-111 FMN,and 20\ glycerol. Once loaded, the re s inwa s wa s he dwi t h IS colum nvol umes of equili b r a tion buffe r , 12 col umnVol umes of 100mMph o sphate buff er (pH 7.7) conta i ni ng1.0\ Cha p s, O.S\ Lubr olPX,0.1raMEDTA, 0.1 mI1DTT, 20 /-1MBHT, S /-1MFMN, and20\gl yc erol, andagain with IS columnvolumesofequ ilib ratio n bu ff e r.All washes

50

were performed at a flow rateofa1 rol /min. Fp was then el ute d from the affinity resin with a small volume (about 25 ml) of equ il i b r at i o n buffer to which 10mMNADp· had been added (flow rate= 0. 5 rol/min ). The peak Fp-containing fractionswere pooled and dialyzedtwi c e with 2 liters of 50 10M phosphate buffer(pH 7.4 )co n ta i n i n g 0.1roMOTT and20 ' gl yc e r ol for a total of 28 hr. The purified Fp was then st o re d in small al i qu ots (0.5ml each )at -BODe. The purifiedFp showed a single bandon sodium dodecyl sulfa t e - polyacry lamidegel electrophore sis (Fi g . 10).

3.2.2. 2 Fpassay

Theenzymewa s assayedas des cr ibedby Lake(142) using

cytochromecas the electr on accepto r. Bri e f ly , 1 mlof 0.125mMcytochromecsolution in 0.1M phosphate buffer, pH 7.0 , and 0.2ml of 15^roMKCN 1n water were pipetted into each of atwo matchedJ ml spe c+:ro photometer cuvettes . Te n microlitersof Fp were pipetterl into eachcuv e t t eand phosphate buffer was added to the testand referencecuve t t e contents to bringthe volumosup to2. 4 and 2.5ml, respective ly. Aftermi x i ng,the cuvett e s were placed in theircell compartmentsof thespe c t ro pho t o me t e r (thermostatt ed to22°C ) , and after ) minthe rea ct i onwas

Figure10.

51

Gel el e ct rop hores i s ana l y si sof thepurified na tiveNADPH-cyt och rollleP- 450reductase(Fp)

(O. l77Jlg protein , la ne s 2-4) alongwi th Phar1llacialowmolecularweight standard (lanes 1,5 ). The Fpwas ju dg e d tobe electrophor e t i callyhOllogeneous andits mol ec ul ar weig h twasdet ermi ne dto be 76000.

52

53

initiated by adding O. lml of 10mMNADPH to the test cu ve t t e only. 'rne contents were againmixed and the in c r e a s e in absorbance with timewas recorded at 55 0nm.

us ing anextinctioncoefficientfor the reduc e d cytoc h romec at 550 nmof 0.02 1em'l~M-l, the specific activityof the fla vopro te i n wasca l c u l a t e d to be18, 000 uni ts/ mg pr o tei n (142). Oneun i t of enzymeacti vityis. def ined as that amou nt whic h cataly zes the red uct i on of 1 nrnol cytochrome clmi n.

3.2.3 Cyt o c h r ome P-450

3.2. 3.1 Cytochrome P-4S0 purification

Cy t o c hrome P-450 was purified fromliver microsomes isol ate d fromPB-pretreatedrats as descri bed byGuengeric h (143) . Microsomeswere sus pe ndedto 2109pr o t ei n/m l in0.1 Mpotassiumphosphate buffer (pH7.25) co nt ai n i n g20%

gl ycerol , ImM EDTA, and ~O}.1M BHT. Sod i u m cholate (r ecr ys t allized from 50\ aqueousethanol) was added dr opwi s e (fro m a separatory funnel) to the stirring suspensi on ov e r 20 minto givea final conc e nt r a tio nof 0.6% (wt/ vo l).

Aft er st i r r i ng foran additional 30 min, the cla r if i e d sol u t io n was ce n t r ifug e d at 100,00 0x 9 for1 hr. An amou nt of th e supernatant equ iva l e n t to2,000 nmoles of cytochrome

54

P- 4 5 0wasappliedto an v-eutnc-cceyi agarosecolu mn (2.5 x 50em) pr e v i ou s ly equilibratedwith JOOrotof0.1M po tassi u mphosphate buffer (pH 7.25)con t a ining1rnMEOTA, 20% glycerol, and0.6 \ (wtjvol) sod i umche l at e at a flow rateC'f1 rnl/min. The cyt oc h r ome P-4 S0, a reddis hbrown prote in, was bound to the topone-th irdof thecolumn. For thefirst column,all stepswerecarriedoutat4°C and the sodiumchelate usedwaa recrystallized. Th ecolumnwas wa s h e d wi th600 rol of 0.1Mpo t a s s i u mph os p h a t e bUffer (pH 7.25) containing1rnMEDTA, 20% glycerol,an d0.42\ ('Nt/ v ol) so-Hum chelate. CytochromeP-4 S 0was el ut ed using about 1,500ml of0.1 Mpo t a s s i u m phosphatebuffer cont ainin g 1roM EDTA, 20 %glycerol, 0.33% (wt/ v o l ) sodiumcholate, and 0.06%

(wt/vol ) aenex 690(polyoxyethylene (10) nonyl phenol ether, ICI AmericasInc.,WA , USA). The elutedfractionswere monitored forcytochrome P-4 50 (A'II) ' The A'1Jpea k fractions werepooledand concentrated to about 50 rol using an Amicon ultr a f ilt r a t i o n apparatus and a PN-JOmembrane. The concentratedsol utionwas dialyzedagainst1 L of a 20%

glycerol-D.l%"roMEDTAsolution (a bout3 hr) and thenversus 1Lof10mMpotassium phosphatebuffer(pH 7.7) containing 0.1mMEDTA,20% glycerol,

o. n

(wt/v ol ) LUbrol p.x:: , and 0.2%

(wt /vo l ) sodiumchola te (not re c ry s t a l l i zed ) (abou t )hr)• The cytochromeP-450wa s furtherpurifiedby DEAE- cellulose chromatog rap hyat roo mte mpe ra t ur e (a bout 22°C). The dialyzedcytochromeP-4 5 0 solutionwasap p liedtoa 2.5

55

xSOemcolumn of Phannacia DEAE-'i e p h a c elprev i ously equili br a t ed with1 Lof 10mMpo t a s s iumphos ph ate bUf f e r (pH 7.7 )conta ining 0.1m1"IEDTA. 20t glycerol, 0.1\ (vt/vol ) Lubro l PX. and0.2\ (wt/ v o l ) sod i um cholate (not

recrysta llized). Theccauen waswashedwi t h700 ml of the eq uilib ra t ionbuffe rand then elu t e d wi th 2 L of the equfLf.br-acLcn buffer in which the concen t ra tion ofNael incr e a s e d line arlyto 0.25H. The last major Am peak con t a i ns thebulkof thecytochromeP-450. The peak fractions we r e pool e dand concentrate dto about 20ml with an Amiconultrafiltrat i onapparatus usinga PM-JOmembr an e. The concentrated sol ut io nwa s stirredwi t h Bic-Be a ds SM-2

IBia-Rad Labs;0.2gm/m9 pro t ei n) tor J hr toremov eexc e ss dete rqe ntand then fil t e r edthrou gh gl a ss wool. Finally , the enzyme pr e para t i o n was di alyze d over nightagains t 50 volu mes of 10mM'rete- eceea r e bu ffe r (pH 7. 41con t a ining 0.1 IIlH EOTA and 20' glycerol. The dialyzedenzyme"'as storedin alique t s (0.25 IlIl each) at- 80°C. 50s-gelelectrophores i s re v e aled th e pre s e nc e of a si ng lepro t e inba nd (Fi g.11).

3.2. 3.2 cyto c hrom eP-4S 0assay

CytochromeP-4 50wasas s a y e das de s c ri bedby Guenge ri ch (14 3 ) us ing an extinc tioncoe f f i cien t of91crn-^lli\M- l.

Brie fly, 6 mqmicrosom a l pro t ei n or 0.6mg pr o te in of the

Fiqure11.

"

8Ds- qel el e c tropbo r e ticana l ysi sof the pu rifi ed cytoc bromeP-UO (lanes3, 4and5;

0.25,0.5 and 1.0~q protein, re spe ctiv ely) alon gwithPharma cia lowmolecu l a rweight standa rds (la nes6,7) (fro mtop tobottom 9400 0, 6700 0, 4300 0, 3 0 00 0,20000and140 0 0 ) andmicrosome standard (la n es land 2: 2 1J.9 prote in). Thecytochrome P-450 molecular weight was de teIlllinedtobe54000.

57

_ ^...