Chatcr 1

McchanIsms nf Hcatncc!!u!ar DvsfunctInn and

RcgcncratInn:
Enzvmc InhIbItInn bv NItrnImIdazn!c and Human LIvcr
RcgcncratInn
Rakcsh 5harma
1,2
,
1
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Ani|u |ns|i|u|c cf Nanc|ccnnc|cgu. Ani|u Unitcrsi|u. NO|DA 201303 UP. |ndia


1.|ntroduct|on

Lnzvnos so caIIod (Lnz-a-ai-an) aio lioIogicaIIv aclivo pioloin noIocuIos iosponsilIo of liochonicaI ioaclions in lho
lacloiia, coIIs and oigans. Lnzvnos ioguIalo lho ialos of liochonicaI ioaclions lo nainlain lho nolaloIisn lo koop
aclivo phvsioIogicaI aclions in lho lodv. High onzvno aclivilios oi high ialos of ioaclions causo highoi pioducl
foinalion oi doposils lo inilialo disoaso. Diugs aio usod as onzvno conpolilois lo noinaIizo lho ioaclions lo coiiocl
disoaso. Mosl of disoasos aio cuiod lv 'onzvno inhililion. Lnzvno inhililion can lo lhioo lvpos: conpolilivo, non-
conpolilivo, and unconpolilivo. Lnzvno inhililion aIso piovidos a kind of dofonso in coIIs lv ioguIalion of
nolaloIisn lo inhilil oi slinuIalo lho liochonicaI piocossos. Mosl of lho diugs undoigo doloxificalion and
lioliansfoinalion in Iivoi lo iogonoialo oi foinalion of nov hopalocvlos and Kupffoi coIIs. Majoi liochonicaI ovonls
in Iivoi iogonoialion aio ioguIalod lv onzvnos in onoigv nolaloIisn, giovlh faclois and cvlokino noIocuIos.
Iiosonl chaploi dosciilos an oxanpIo of Iivoi coII onzvno lalloiv lo ioguIalo lho cailohvdialo, Iipid and pioloin
nolaloIisn in Iivoi coIIs, nainIv hopalocvlos and Kupffoi coIIs in lho Iighl of Iivoi danago duo lo anoolic Iivoi
alscoss and ioIo of onzvno inhililion in Iivoi iogonoialion lv 2-nilioinidazoIo. Livoi danago lv anoola is
nanifoslod lv oIovalod onzvnos in coIIs. As a iosuIl, lvo najoi cIinicaI nanifoslalions of n|anccoa nis|c|u|ica
infoclion aio anoolic coIilis and anoolic Iivoi alscossos. To cuio anoolic Iivoi alscoss, Iivoi iogonoialion and
anoolic kiIIing lv 2-nilioinidazoIo lhoiapv is ioulino in cIinicaI piaclico. 2-nilioinidazoIo acls in Iivoi lo poifoin
onzvno inhililion al lho IovoI of cailohvdialo, Iipid, and pioloin nolaloIic ioguIaloiv slops. LaiIioi, nilioinidazoIo
doiivalivos voio considoiod diug of choico in lioalnonl of hopalic hvpoxia (Iov oxvgon) condilions in paiasilic
infoclions, cancoi and ioconlIv nilioinidazoIo doiivalivos aio onoiging as hvpoxia naikois and iadiosonsilizois in
lunoi lioalnonl |Shaina 2OO1, Shaina 2O11a, 2O11l!.

Sinco 196O, hopalocvlos voio invosligalod iich in najoi onzvnos ioguIaling lho onoigv nolaloIisn Iocalod in
cvlopIasn and nilochondiia foi gIvcoIvsis, TCA cvcIo vhiIo diug nolaloIizing and iodox onzvnos aio Iocalod in
Ivsosonos and niciosonos. Olhoi onzvnos aIso pailicipalo in dofonso piocossos such as iospiialoiv luisl, HMI
shunl, oxvgonaso palhvavs and infIannaloiv cvlokinos. Iiosonl chaploi ioviovs lho ongoing dovoIopnonls vilh
cIoai and conpIolo infoinalion on aclion of nov nilioinidazoIo doiivalivos as 'onzvno inhililois in Iivoi soIoclivo
cvloloxicilv, oxvgon dopIolion, hopalocoIIuIai DNA and onzvno noinaIizalion lofoio onzvno can lo usod in
hvpoxia noniloiing and lhoiapv |Shaina, 2O11l!. Tho paucilv of infoinalion on nilioinidazoIo-Iivoi lissuo
inloiaclion is pooi and avaiIalIo dala of iniliaI soquonliaI liochonicaI changos in Iivoi coIIs is scanlv vhich fuilhoi
Ioads lo doloclalIo hvpoxia |Shaina 2O11a, 2O11l!. Il is loIiovod lhal iniliaIIv onzvno ioguIalod gIucoso and caIciun
honoslasis aio lho piinaiv laigols of hopalic hvpoxia foIIovod lv onzvno ioguIalod inducod nolaloIic inlogiilv Ioss
Ioads lo apoplosis, ioguIaloiv faiIuio in gIvcoIvlic, TCA cvcIo, gIuconoogonosis and Ca
++
nodialod cAMI ioIalod
liodogiadalion of noIocuIos |Shaina ol aI.2O11c!. In foIIoving soclion, a noIocuIai lasis of onzvno inhililion and
hopalocoIIuIai hvpoxia and dvsfunclion ciiloiia is dosciilod in dolaiI. Tho piosonl chaploi focusos on hopalic onzvno
inhililion lv nilioinidazoIo al diffoionl IovoIs of onoigv nolaloIisn, iospiialoiv luisl and diug nolaloIizing
onzvnos. In foIIoving soclion, ioguIaloiv lohavioi of ialo Iiniling onzvnos of gIvcoIvsis, TCA cvcIo, phagocvlosis is
dosciilod vilh oxanpIos: gIucokinaso, phosphofiuclokinaso, pviuvalo kinaso, Iaclalo dohvdiogonaso, NADIH
cvlochiono I45O ioduclaso, phosphodioaloiaso, IvsosonaI onzvnos in soiun, Iivoi liopsv sanpIos. In foIIoving
soclion, ioIo of onzvnos in Iivoi alscoss is dosciilod.

1.1 Enzvmcs In LIvcr Absccss

n|anccoa nis|c|u|ica is a hunan-spocific palhogon and iopioducilIo aninaI nodoIs of inloslinaI anooliasis havo
piovod oIusivo lo dosciilo inloiaclion lolvoon nis|c|u|ica and Iivoi coIIs. Tho nosl connon oxliainloslinaI
nanifoslalion of disoaso is anoolic Iivoi-alscoss. Anoolic Iivoi-alscossos aiiso fion haonalogonous spioad
(piolalIv via lho poilaI ciicuIalion) of anoolic liophozoilos lhal havo lioachod lho coIonic nucosa.| |Thonpson ol
aI. 1985, Alualaia ol aI. 1982, Shandoia ol aI. 1998, ainos ol aI. 1987, Adans 1977, Lancol 2OO3!. Lffoclivo
nilioinidazoIo lioalnonl and iapid diagnosis shovod lho noilaIilv ialos lo 1-3. n|anccoa nis|c|u|ica liophozoilos
can Ivso nouliophiIs in ti|rc, causing lhon lo ioIoaso loxic sulslanco kiIIoi onzvnos such as supoioxido disnulasos,
coIIagonasos, oIaslasos and calhopsins |IaiuniIinla ol aI. 1964, Cuoiianl ol aI. 1981!. On lhoso Iinos, aulhoi pioposod a
'HopalocoIIuIai dvsfunclion ciiloiia lo nako svslonalic olsoivalions in alscoss dovoIopnonl and slop lv slop
nolhod of nodicaI/suigicaI inloivonlion |Shaina ol aI.2O11!. In piovious iopoils, palionl svnplons, Ioucocvlosis
vilhoul oosinophiIia, niId anaonia, iaisod conconlialions of aIkaIino phosphalaso, and a high ialo of oivlhiocvlo
sodinonlalion voio lho nosl connon Ialoialoiv findings.| Thonpson ol aI. 1985, Alualaia ol aI. 1982, Shandoia ol
aI. 1998, ainos ol aI. 1987, Adans 1977, Lancol 2OO3!.
Anoola causo anoolic Iivoi alscossos, vhich aio ciicunsciilod iogions of doad hopalocvlos, Iiquofiod coIIs and
coIIuIai doliis suiioundod lv a iin of connoclivo lissuo, sono infIannaloiv coIIs and fov anoolic liophozoilos
|Shaina ol aI. 2O11!. Tho adjaconl Iivoi paionchvna is usuaIIv conpIoloIv noinaI. Al sano silos in Iivoi so caIIod
'acini fixod sinusoidaI phagocvlic coIIs (Kupffoi coIIs) piovido dofonso againsl anv lacloiia, viius oi foioign diug.
Kupffoi coIIs havo lvo inliacoIIuIai sliucluios fiIIod vilh onzvnos: Ivsosonos (Lai-zo-sonos) and niciosonos (Mai-
kio-zonos). LvsososnaI lags fiIIod vilh a Iaigo nunloi of IvsosonaI onzvnos acling as dosliovois acluaIIv Ivso and
digosl lho lacloiia, viius and loxicanls. MiciosonaI onzvnos aio aclivo in doloxificalion of diugs oi diug
lioliansfoinalon lo inloiacl (slinuIalion oi inhililion of onzvnos) vilh nolaloIic ioguIalion in Iivoi coIIs. Lol us
inlioduco a lil of onzvnos in Iivoi coIIs poifoining diffoionl nolaloIic ioguIaloiv funclions in foIIoving dosciiplion.
Alscossos occupving Iaigo aioas of lho Iivoi can lo cuiod vilhoul diainago, and ovon lv ono singIo doso of
nilioinidazoIo.|IovoII ol aI. 1969, Lassoiio ol aI. 1983, Akgun ol aI. 1999!. Todav, ioIo of uIliasound oi poiculanoous
lhoiapoulic aspiialion guidod lv CT in lho lioalnonl of unconpIicalod anoolic Iivoi-alscoss lv suigicaI diainago is
conliovoisiaI.


Lnzvno Anoola Hopalocvlo Kupffoi coII

-----------------------------------------------------------------------------------------------------------------------------------------------------------
CIucoso 6 Iaso ++++ ++ +
CIucoso 6 IDH ++++ ++ +
IhoshogIuconalo DH +++ + +
Ihosphofiuclokinaso +++ ++ +
AIdoIaso ++ ++ +
Iviuvalo kinaso ++ + +
Iviuvalo DH ++ ++ +
LDH + ++ +++
Cilialo Svnlhaso ++++ + ++
Isocilialo DH +++ ++ ++
Succinalo DH ++ + +
MaIalo DH +++ ++ ++
Cvlochiono C Oxidaso + ++ ++
NADIH Cvl C Roducalaso + +++ ++
NADH Oxidaso + ++ ++
Tviosino Aninoliansfoiaso ++ + ++++
AniIino HvdioxvIaso ++ + ++++
Aninopviino donolhvIaso ++ + ++++
Acid DNAaso ++ + ++++
CIulalhiono Roduclaso ++ + ++++
Ioioxidaso + ++ + ++++
CalaIaso ++ + ++++
Supoioxido disnulaso ++ + ++++
Cuanaso ++ + + +++
Adinosino Doaninaso + + +++
Loucino aninopoplidaso ++ + ++++
Ca++ ATIaso ++ + ++++
5 NucIoolidaso ++ + ++++
Acid Lipaso ++ + ++++
Acid Ihosphalaso ++ + +++++
AIkaIino Ihosphalaso +++ + +++++
ola gIucuionidaso ++ ++ +++

Souico: Shaina R .Lffocl of NilioinidazoIo on isoIalod Iivoi coIIs in dovoIopnonl of anoolic Iivoi alscoss. Ih.D dissoilalion
sulnillod lo Indian Inslilulo of TochnoIogv, DoIhi, 1995.
Conpaialivo onzvno dalalaso of anoola and isoIalod Iivoi coIIs is shovn and skolchod in Iiguios 3,5, and 6.
Slionglhs of diffoionl onzvno aclivilios in anoola, Iivoi coIIs aio shovn as voak(+), nodoialo(++), high(+++),
oxliono(++++ oi noio).

Chaiacloiizalion of onzvnos in anoola has loon a Iong lino quosl lo oxpIoio lho possiliIilv of anolic onzvno
inhililion lv nov diugs in diug discovoiv. SovoiaI anlianoolic diugs aio in naikol as polonl onzvno inhililois
sinco Iasl foui docados. Aulhoi pioposod a conpaialivo onzvno dalalaso of anoola and hopalocoIIuIai coIIs in
cuIluio (soo Iiguio 1). Of spocific nonlion, cvsloino pioloinaso onzvnos aio sociolod in Iaigo quanlilios lv lho
paiasilo duiing innuno iosponso and can cIoavo oxliacoIIuIai naliix pioloins, vhich nighl faciIilalo anoolic
invasion as shovn in Iiguio 3|SchoIzo ol aI. 1988, Koono 1986!. Thoso pioloins acl as viiuIonco faclois in aninaI
nodoIs of anoolic Iivoi alscoss |SlanIov ol aI.1995, Ankii ol aI.1999!. A faniIv of six gonos oncodos nis|c|u|ica
cvsloino pioloinasos (cncp1-6 ) lul ~9O of lho pioloinaso aclivilv is ioIalod lo lhioo pioloinaso onzvnos, LhCI1,
LhCI2 and LhCI5 |iuchhaus, ol aI. 2OO3!. AIlhough laigolod disiuplion of soIoclod nis|c|u|ica gonos has nol vol
loon achiovod, slalIo opisonaI oxpiossion of foioign DNA is possilIo in anoolao lv nainlaining conlinuous
soIoclivo piossuio |Hanann ol aI. 1995, Vinos ol aI. 1995!. This has onalIod lho invosligalois lo laigol spocific
noIocuIos in nis|c|u|ica lv lho opisonaI oxpiossion of anlisonso nRNA oi of gonos oncoding doninanl nogalivo
nulanls |Ankii ol aI. 1998!. Tho anlisonso appioach has loon appIiod lo nis|c|u|ica cvsloino pioloinasos and
opisonaI oxpiossion of an anlisonso RNA lo cncp5 couId ioduco lolaI anoolic pioloinaso aclivilv lv 8O-9O |Ankii ol
aI. 1998!. To assoss lho ioIo of nis|c|u|ica cvsloino pioloinasos in anooliasis, in oaiIioi sludv, hunan xonogiafls in
SCID-HU-INT nico voio infoclod vilh anoolic liophozoilos oxpiossing lho cncp5 anlisonso RNA(pioloinaso-
doficionl anoolao) oi anoolic liophozoilos conlaining lho sano pIasnid vilhoul lho anlisonso insoil (lho conlioI
gioup)|Zhang ol aI. 2OOO!. Majoi findings voio: 1. no olvious dofocl vas appaionl in lho aliIilv of cvsloino-pioloinaso
doficionl anoolao lo inhalil and suivivo vilhin lho coIonic Iunon: 2. posl-24 h afloi infoclion, cvsloino pioloinaso-
doficionl anoola had, in conliasl lo lho conlioI gioup, faiIod lo induco significanl anounls of hunan IL-1uoi IL-8
fion infoclod inloslino: 3. gul infIannalion vas aIso ioducod in hunan inloslino infoclod vilh cvsloino-pioloinaso-
doficionl nis|c|u|ica liophozoilos: 4. conlioI nis|c|u|ica liophozoilos danagod lho inloslinaI poinoaliIilv laiiioi al
24 h lul lhoio vas onIv a nininaI incioaso in inloslinaI poinoaliIilv in hunan inloslinaI xonogiafls infoclod vilh
cvsloino-pioloaso-doficionl anoola: 5. hisloIogicaI sludios al 24 h, hunan xonogiafls infoclod vilh conlioI anoolao
shovod danago lo lho coIonic nucosa, invasion of anoolic liophozoilos inlo sulnucosaI lissuos and nouliophiI-
piodoninanl infIannalion: 6. lv conliasl, xonogiafls infoclod vilh cvsloino-pioloinaso-doficionl anoolao shovod
Ioss nucosaI danago, aInosl no ovidonco foi anoolic invasion inlo sulnucosaI lissuo and IillIo infIannalion.
Aulhois spocuIalod lhal cvsloino-pioloinaso-doficionl anoola nighl havo a dofocl in lhoii aliIilv lo induco gul
infIannalion and lo invado inlo lho sulnucosaI lissuos |Zhang ol aI. 2OOO!. Hov anoolic cvsloino pioloinasos
conliilulo lo gul infIannalion and lissuo danago in anooliasis` IossilIv, n|anccoa nis|c|u|ica liophozoilos
oxpiossing lho cncp5 anlisonso RNA nighl havo ioducod lho phagocvlic capaliIilios oi ioducod viiuIonco in
anooliasis |Ankii ol aI.1998!. Hovovoi, pioloaso-doficionl anoolao nainlain lhoii aliIilv lo Ivso laigol coIIs, so a
dofocl in coII kiIIing doos nol undoiIio lho ioducod viiuIonco of cvsloino pioloinaso-doficionl anoolao |SovdoI ol
aI.1997!. In addilion, il is unIikoIv lo lo a diiocl offocl of cvsloino pioloasos on inloslinaI opilhoIiaI coIIs, locauso
anoolic Ivsalos iich in cvsloino pioloaso aclivilv faiI lo induco high IovoIs of cvlokino pioduclion oi infIannalion
vhon pIacod in hunan inloslinaI xonogiafls |Ankii ol aI.1998!. Il suggosls lhal anoolic cvsloino pioloinasos nusl
oxoil lhoii offocls on gul infIannalion and lissuo danago afloi an iniliaI slop lhal ioquiios Iivo aclivo liophozoilos.

1.2 Entamncba hIstn!vtIca and rngrammcd cc!! dcath

Olhoi inpoilanl ioIo of caspaso onzvnos vas oxpIoiod vhon nis|c|u|ica liophozoilos voio inculalod vilh connon
lvpos of nannaIian coIIs. NoinaIIv liophozoilos kiII nannaIian coIIs in a conlacl dopondonl nannoi lv lvo
piocossos: lv Ivlic nociosis |oininghauson ol aI.1997! oi undoigo apoplosis|RagIand ol aI.1994!. Apoplosis, oi
piogiannod coII doalh, is an oidoiod svslon of coII doalh vilh an inilialoi oi signaIing phaso foIIovod lv an offocloi
slago lhal causos coII doalh lv dogiading vaiious coIIuIai conpononls. Tho offocloi slago invoIvos lho aclivalion of
caspaso onzvnos, cvsloino pioloinasos vilh spocificilv foi aspailalo iosiduos lhal foin a cascado, convoiging on
caspaso 3. A kov liochonicaI naikoi of apoplosis is ondonucIoaso cIoavago of chionalin lolvoon hislono lodios,
vhich can lo soon as a DNA Iaddoi on sopaialing goIs.
Doloclion of DNA fiagnonlalion in si|u lv lho ondIaloIing of singIo- oi doulIo-sliandod DNA lioaks vilh
loininaI dooxviilonucIoolidvIliansfoiaso (TUNLL assav) has aIso loon usod as a lionaikoi of apoplosis |SchuIlo-
Hoinann ol aI.1994!. Hovovoi, nis|c|u|ica can induco apoplosis in ti|rc iaisos doull if apoplosis is a conpononl of
lho coII doalh connonIv soon in anoolic Iivoi alscoss. To losl lhis facl, anoolao voio inocuIalod inlo lho Iivois of
SCID nico and ovidonco foi apoplosis vas soughl lv goI sopaialion of DNA fion infoclod Iivois and TUNLL
slaining of anoolic Iivoi alscossos |SovdoI ol aI.1998!. DNA Iaddoi foinalion vas doloclod in sanpIos ollainod fion
SCID nico lv 1 h afloi anoolic inocuIalion. TUNLL slaining iovoaIod significanl aioas of apoplosis vilhin anoolic
Iivoi alscossos connon in infIannaloiv coIIs and hopalocvlos cIoso lo anoolic liophozoilos |SlonIov, 2OO1!.
Hovovoi, sono dislanl Iivoi coIIs aIso voio TUNLL posilivo. Thus, in lho nuiino nodoI of anoolic Iivoi alscoss,
nis|c|u|ica inducod coIIuIai apoplosis appoais lo lo a significanl conpononl of coII doalh as shovn in Iig. 3.
InloioslingIv, nis|c|u|ica-inducod apoplosis vas nol lIockod lv cI-2 |RagIand ol aI.1994! and did nol appoai lo
invoIvo oilhoi of lho lvo of lho najoi palhvavs foi lho induclion of apoplosis: Iigalion of lho Ias iocoploi and
Iigalion of TNIuiocoploi 1 (TNIR1). In lolh C57I/6 MRL-Ipi/Ipi nico vilh no Ias iocoploi, and C57I/6.C3H-
gId/gId nico vilh no Ias Iigand, apoplosis vas doloclod in anoolic Iivoi alscossos lv DNA Iaddoi foinalion and
TUNLL slaining |SovdoI ol aI. 1998!. Apoplosis vas aIso piosonl in hopalocvlos in anoolic Iivoi alscossos fion lolh
TNIR1 knockoul nico and lho holoiozvgolo conlioIs|SovdoI ol aI. 1998!.!. Thoso dala indicalo lhal nis|c|u|ica causos
apoplosis in nouso Iivoi lv a nochanisn lhal is indopondonl of Ias-Ias-Iigand inloiaclions and TNIR1.



FIg. 1.Lnlanoola hisloIvlica liophozoilos induco apoplosis in infIannaloiv coIIs and hopalocvlos in
anoolic Iivoi alscossos. Soclion of Iivoi slainod foi apoplolic coIIs vilh lho TUNLL nolhod fion an
anoolic Iivoi alscoss in a C57I/6 MRL-Ipi/Ipi nouso. Anoolic liophozoilos (Iong aiiov) aio adjaconl
lo a cIusloi of infIannaloiv coIIs (shoil aiiov), vhich shov naikod TUNLL slaining. TUNLL slaining in
nucIoi and cvlopIasn is aIso ovidonl in a land of doad hopalocvlos and infIannaloiv coIIs (D). TUNLL-
posilivo nucIoi (liovn slaining) aio aIso visilIo in hopalocvlos in iogions (H) fIanking lho doad coIIs.
Ropioducod, vilh poinission, fion Rof. SovdoI ol aI. 1998.

Conolic appioachos and nov nodoIs of anoolic Iivoi alscossos havo piovidod good piospocls on lho conpIox
inloiaclions lolvoon nis|c|u|ica and lho hosl.Iivoi coIIs |Hanann ol aI.1995!. WoII-knovn nis|c|u|ica onzvnos Ivso
coIIs and digosl oxliacoIIuIai naliix pioloins. Lnzvnos havo capaliIilv lo induco apoplosis in hopalocvlos and
infIannaloiv coIIs. Lnzvnos can slinuIalo, and poihaps onhanco, an NI-i-nodialod hunan Iivoi infIannaloiv
iosponso lhal conliilulos lo lissuo danago. Thoso nov onzvno ioguIalod palhvavs nighl offoi an oxpIanalion foi
sono of lho cIinicaI and palhoIogicaI diffoioncos lolvoon anooliasis and anoolic Iivoi alscoss. Tho hopalocvlo
inhalilod apoplosis vouId piodoninalo in anoolic Iivoi alscossos. If lhoso nochanisns piovo lo lo inpoilanl in
hunan disoaso, lhov viII piovido nov laigols in lolh lho hosl and lho paiasilo foi inloivonlions dosignod lo
anoIioialo oi inhilil anooliasis and anoolic Iivoi alscossos. RoconlIv, aulhoi in loan oslalIishod lhal apoplosis
iosuIls vilh hvpoxia in Iivoi coIIs and ovaIualod nilioinidazoIo cvloloxicilv |Kvon ol aI. 2OO9!. In foIIoving soclion,
a ciiloiion of hopalocoIIuIai hvpoxia is dosciilod lasod on Iivoi coII onzvnos and cvlonoiphoIogv.

2. |ver enzymes and enzyme |nh|b|t|on |n human ||ver: hepatoce||u|ar cr|ter|a

Tho Iivoi is nado of paionchvnaI hopalocvlos and nonpaionchvnaI Kupffoi coIIs as soIo laigols lhal oxhilil lhoii
inliacoIIuIai liochonicaI changos as hopalocoIIuIai onzvno lionaikoi piofiIo. Livoi coIIs aio iich in onzvnos.
Lnzvno inhililion Iivoi is chaiacloiizod lv lvo vavs: 1. in inlacl hopalocoIIuIai onzvnos in hvpoxia soiun and
honogonalos: 2. Lnzvno ioguIaloiv lohavioi lv chaiacloiizing onzvnos in piosonco of addilivos addod lo cuIluios of
isoIalod Iivoi coIIs. Addilivos and diugs aio nainIv lioliansfoinod and doloxifiod in Iivoi lv a lalloiv of diug
nolaloIizing onzvnos: IvsosonaI and niciosonaI onzvnos. Diugs inhilil onzvnos. In foIIoving dosciiplion, ioadois
aio inlioducod vilh lho inpoilanco of onzvno inhililion as iosoaich looI foi lvo puiposos: 1. diug losling and
hvpoxia disoaso noniloiing (HopalocoIIuIai Hvpoxia Ciiloiia) lo undoisland lho hvpoxia and Iivoi coII inloiaclions
vilh anoola and nilioinidazoIo: 2. Lnzvno ioguIaloiv lohavioi in piosonco of addilivos in isoIalod Iivoi coIIs in
cuIluio (hopalocvlos and Kupffoi coIIs).

2.1 hepatoce||u|ar hypox|a cr|ter|a
Aulhoi pioposod a HopalocoIIuIai Hvpoxia Ciiloiia. 'HopalocoIIuIai hvpoxia ciiloiia assunos lhal iniliaIIv Iivoi
coIIs Iooso nolaloIic inlogiilv (ATI and NADIH insufficioncv fion gIucoso lo causo oxvgon insufficioncv in
nilochondiia) and undoigoos apoplosis foIIovod lv doloclalIo nociosis in Iivoi. Hvpolhosis vas lhal Iivoi coIIs
undoigo a soiios of changos - hopalocvlos undoigo nolaloIic onoigv Ioss and oxvgon dopIolion (hvpoxia) vhiIo
Kupffoi coIIs undoigo phagocvlosis, iospiialoiv luisl and piolooIvsis. NilioinidazoIo inducos onzvno inhililion in
Iivoi coIIs lo noinaIizo lho oIovalod onzvnos duiing nolaloIic onoigv Ioss and oxvgon dopIolion (hvpoxia) in Iivoi
coII (in nilochondiia) and IvsosonaI onzvno inhililion lo conlal lho phagocvlosis. Tho appioach is appIicalIo
possilIv in dosign of diugs lo lioal oxvgon slaivod lunoi coIIs oi infoclod Iivoi Iosions and alscossos.

Iiovious iopoils on fiisl iniliaI slago of nilioinidazoIo inducod Iivoi coII cvloloxicilv indicalod lho apoplosis,
gIulalhiono, DNA inloiaclion, oxvgon dopIolion, Iaclalo inhililion, onhancod NADIH cvlochiono ioduclaso and
supoioxido disnulaso onzvnos |Lisoz ol aI.2OO1, iozdon ol aI.1997, Noss ol aI.1988, Sugivana ol aI.1993, oiulo ol
aI. 1992, Sapoia ol aI.1992, MosoIon ol aI. 1995, Slialfoid ol aI.1981, Adans ol aI.198O, Noss ol aI. 1989, MuIcahv ol
aI.1989, WidoI ol aI. 1982, Raulh ol aI. 1981, Chao ol aI. 1982, Whilnoio ol aI.1986!. SliII nilioinidazoIo sliucluio-
funclion ioIalionship lolvoon hvpoxia, iadiosonsilizalion and cvloloxicilv oi Iivoi iogonoialion ionain Ioss
undoislood poihaps duo lo nilioinidazoIo Iov sonsilivilv lo cIinicaI invosligalions and significanl onzvno aIloialions
onIv al lho voiv Ialo nociolic slago |Covan ol aI.1994, MoIo ol aI. 2OOO, MoIIoi ol aI. 2OO2, aIIingoi ol aI. 1996,
Ldvaids ol aI. 1981, Ldvaids ol aI.1982, aIIingoi ol aI.2OO1, Richo ol aI.2OO1, MoIo ol aI.2OOO!. Hovovoi,
nilioinidazoIo cvloloxicilv vas iopoilod conlinuousIv in Iasl lhioo docados indicaling nanv aIloiod onzvnos
invoIvod in niliic oxido pioduclion, cvlokinos, oxvgon dopIolion (hvpoxia) and innunoaclivo sulslanco ioIoaso fion
lolh hopalic infoclions and cancoi lunoi lissuos |Su ol aI.1999, Runsov ol aI.1994, Koch ol aI.2OO3, Hodgkiss ol
aI.1997, Shaina 2OO9a, Shaina ol aI.2OO9l!. In foIIoving soclion, aulhoi puls an ovidonco of hopalic nolaloIic
inlogiilv Ioss (in nilochondiia and cvlopIasn) and IvsosonaI slinuIalion associalod vilh hvpoxia in Iivoi.
NilioinidazoIo is sliII considoiod cIoaiIv hopaloloxic, ionaI and nouioloxic diug vilh sovoiaI iopoils of diffusod
danago of paionchvnaI and nonpaionchvnaI Iivoi coIIs as iniliaI slago of hopalic danago olsoivod lv oIoclion
nicioscopv and liochonicaI naikois in soiun vilh onphasis of palhophvsioIogv |Mahv ol aI.2OO4!. Oui piovious
olsoivalion on nilioinidazoIo inducod offocls lo acl againsl hopalic anooliasis and anoolic alscoss dovoIopnonl
indicalod lho possiliIilv of nilioinidazoIo conconlialion-dopondonl offocls upon oxvgon dopIolion, niliic oxido
pioduclion, cvlokino svnoigv and Iivoi coII onzvno aIloialions as inloi-ioIalod consoquoncos of Iivoi iogonoialion oi
liinging lack oIovalod onzvno IovoIs |Chu ol aI.2OO4!. Tho najoi pIavois voio lho onoigv nolaloIizing and diug
nolaloIizing IvsosonaI onzvnos in iniliaI Iivoi danago vilh assunplion lhal Iivoi coII onzvno piofiIo dofinos lho
hvpoxia sonsilivilv of nilioinidazoIo lv 'HopalocoIIuIai Hvpoxia Ciiloiia. Tho novoIlv of hopalocoIIuIai hvpoxia
ciiloiia vas dolaiI cvlonoiphic-liochonicaI infoinalion oxliaclod fion aIloiod onzvno aclivilios duiing iniliaI
cvlonoiphic changos in hvpoxic hopalic coIIs sinuIlanoousIv vilh niciosliucluio changos lv oIoclion nicioscopv lo
confiin lho ioIo of coII oiganoIIo in hvpoxia dovoIopnonl. Tho ciiloiion can lo usod lo ovaIualo connon Iivoi
hvpoxia condilions such as hopalic lunois, hopalic infoclions olc. SovoiaI nov nilioinidazoIo doiivalivos aio
onoiging as polonliaI cancoi chonosonsilizois, hvpoxia naikois and hvpoxia inaging conliasl agonls acling as
onzvno inhililois |Cionioos ol aI.2OO4, Zionoi ol aI.2OO3, Iankovic ol aI. 2OO6, IapadopouIou ol aI.2OO3, Lschnann ol
aI.2OO5, Hoinliook ol aI.1988, oiulo ol aI.1992, Shaina ol aI. 2OO9!.

Conpononls of HopalocoIIuIai Hvpoxia Ciiloiia: Tho ciiloiion is dosciilod a slop lv slop soquonco of oxvgon
dopIolion in hopalocoIIuIai danago: 1. iniliaI Ioss of nolaloIic inlogiilv: 2. piogiannod ioguIaloiv faiIuio of coII
oxvgon and onoigv nolaloIisn: 3. Iivoi lissuo infIannalion and innunilv Ioss: 4. nociosis and aclivo doalh. Tho
puiposo of nolaloIic inlogiilv in hopalocvlos is koop inlacl lv nainlaining laIanco lolvoon gIucoso foinalion and
gIucoso lioakdovn lo nainlain onoigv fIov of NADIH and ATI noIocuIos. Soquonco: Tho nilioinidazoIo inducod
Iivoi coII cvlolocilv Ioads lo onhancod gIucoso lioakdovn and noio donand of ATI and NADIH. Wilh couiso of
lino, high onoigv donand al lho cosl of coII nolaloIic iosouicos Ioads lo nolaloIic inlogiilv Ioss oi onoigv Ioss and
oxvgon dopiivalion foIIovod lv possiliIilv of slop lv slop piogiannod coII doalh (onIv in lunoi coIIs). In noinaI
Iivoi coIIs, lho nilioinidazoIo cvloloxicilv offocl causo Ioss danago and coIIs suslain lho offocl vhiIo sliuggIing
oxvgon slaivod infoclod oi lunoi coIIs fuilhoi Iooso capaliIilv lo slav aIivo (such coIIs dio afloi nilioinidazoIo
inducod infoclod oi lunoi coII kiIIing). In noinaI coIIs, Iivoi iogonoialion iocovois lho danago.

Hopalic hvpoxia ciiloiion has 3 najoi onzvno conpononls: 1. najoi ovonl of high gIvcoIvlic ialo is innodialo
iosuIl of high gIucoso luinovoi such as gIvcoIvsis foIIovod lv socondaiv nolaloIic cvcIos viz. liicailoxvIic acid,
gIvcogonoIvsis, gIuconoogonosis, ponloso phosphalo palhvavs: 2. changos in poiiphoiaI lionaikois such as cvlokino
innunilv, aIloiod gIulalhiono ioduclaso, niliic oxido pioduclion, supoi oxido disnulaso onzvnos: 3. afloi sovoio
onoigv Ioss, changos occui in Iivoi coIIuIai noiphoIogv and lissuo shiinkago |Shaina ol aI.2O11, Kvon ol aI. !. Tho
sonsilivilv of diffoionl onzvnos as cvlonoiphic nanifoslalion of hvpoxia in Iivoi alscoss and nilioinidazoIo inducod
Iivoi iogonoialion is a sinpIo schono of HopalocoIIuIai Hvpoxia Ciiloiia in slops is shovn in TalIo 1. In foIIoving
soclion, diffoionl onzvnos aio dosciilod.







Tab!c 1: A stc bv stc schcmc nf "hcatncc!!u!ar hvnxIa crItcrIa" tn cva!uatc !Ivcr hvnxIa damagc In
Infcctcd hcatItIs nr hcatIc tumnrs. DIffcrcnt c!InIca! mcthnds suggcst cnmnsItc Icturc nf hcatIc
hvnxIa and assncIatcd bInchcmIca! and cvtnmnrhIc changcs.


MoiphoIogicaI changos CIinicaI changos Livoi liochonicaI changos
_____________________________________________________________________________
1. IhvsicaI oxaninalion:
-- aldoninaI pain --
inloslinaI danago fovoi --
hopalic infiIlialion hopalonogaIv Iivoi funclion losls(oIovalod)

Loss of coIIuIai nolaloIic inlogiilv
2.LIoclion nicioscopv: hopalonogaIv vilh aIloiod hopalocvlo onzvnos of:
Milochondiia(M) diffusod injuiv Iov ATI/ADI: NADIH/NADI
ondopIasnic iolicuIun(LR) gIuconoogonosis
poioxisono (I) gIvcogonoIvlic
Ivsosono (L) IvsosonaI onzvnos
nucIoai changos (N) oxvgon fIux ioIalod
HopalocoIIuIai onIaigononl (Apoplosis)
3. CoIIuIai oiganoIIo danago: pooi diug iosponso sIov nolaloIic disoidoi:
nilochondiia(M) oxidalivo phosphoivIalion
niciosonos (MI) diug nolaloIizing onzvnos
Ivsosono (L) iniliaI phagocvlosis
nucIoai (N) DNA fiagnonlalion(loads)
cvlosoI (C) gIucoso/pioloin/iospiialoiv
luisl
HopalocoIIuIai Oxvgon insufficioncv (infIannalion)
4. Livoi palhoIogv changos: iaisod diaphiagn
nilochondiiaI danago anolic Iivoi scan +vo slinuIalion of Kupffoi coIIs
oxfoIialivo LR hvpoipIasia of Kupffoi coIIs
anisonucIoosis Ioss of nolaloIic conlioI
aulophagv & IvsosonaI iiiilalion incioasod valoi accunuIalion
cvlosoIic gianuIalion incioasod noIocuIo inlaIanco
fallv Iivoi appoaianco vilh incioasod Iipid svnlhosis
nonliano danago
HopalocoIIuIai dogonoialion and nociosis
5. HopalocvloIogv:
CoII pioIifoialion advancing lunoi vascuIaiizalion suigicaI aspiialos (aIloiod
CoII doliis lissuo giovlh on uIliasound pioloins, Iipids, onzvnos)


NilioinidazoIo singIo doso lhoiapv schoduIo
6. Livoi coII iocovoiv nogalivo Iivoi scan/uIliasound noinaI Iivoi funclion losl
Tissuo shiinkago Hvpoxia noniloiing posilivo -vo Hvpoxia lionaikois
OR if unchangod oi pooi iocovoiv alnoinaI LLISA,onzvnos


SuigicaI inloivonlion
Souico: Shaina R .Lffocl of NilioinidazoIo on isoIalod Iivoi coIIs in dovoIopnonl of anoolic Iivoi alscoss. Ih.D dissoilalion
sulnillod lo Indian Inslilulo of TochnoIogv, DoIhi, 1995.
2.2 OrIgIn nf hvnxIa and cnzvmc rcgu!atInn: Oxvgon insufficioncv oi hvpoxia logins vilh Iov
NADIH and ATI suppIv fion gIucoso. CIucokinaso, aIdoIaso, pviuvalo kinaso and Iaclalo dohvdiogonaso possilIv
soivo as iniliaI gIvcoIvlic ioguIaloiv onzvnos foi onoigv fIov lo gonoialo liicailoxvIic acid cvcIo (TCA) piocuisois.
TCA cvcIo loing as nain souico of onoigv noIocuIo svnlhosis and gIuconoogonosis, il soivos as soIo souico of conliaI
ATI pooI in gIucoso honooslasis duiing Iivoi cvlololoxicilv oi iogonoialod Iivoi coII |Shaina ol aI.2OO , Shaina ol
aI.2OO !. Tho cilialo svnlhaso, naIalo dohvdiogonaso, isocilialo dohvdiogonaso and succinalo dohvdiogonaso
onzvnos soivo as TCA cvcIo ioguIaloiv onzvnos. Thoso onzvnos in hopalocvlos chaiacloiizo lho lunoi hvpoxia and
hopalocvlo iosponso afloi nilioinidazoIo lhoiapv |HoiIon ol aI.2OOO, Talak ol aI.2OO3, Sapoia ol aI.1992, MosoIon ol
aI.1995, Slialfoid ol aI.1981!. Al lho cosl of ATI fion TCA cvcIo and oxvgon fion lho coII, oxidalivo phosphoivIalion
nainlains lho nolaloIic inlogiilv and conlinuous fIov of onoigv. In lho slalo of Iov NADIH and Iov oxvgon coII
undoigoos slalo of nolaloIic inlogiilv Ioss and hvpoxia. NADIH dopondonl cvlochiono iodox onzvnos aio
lionaikoi of oxidalivo phosphoivIalion slalus and oxvgon insufficioncv (hvpoxia) in Iivoi. Olhoi gIulalhiono
ioduclaso and supoioxido disnulaso onzvnos aio lionaikois of hvpoxic slalo of Iivoi coIIs |Laiiov ol aI.2OOO,
MichaopouIos ol aI.1997!. Anv onoigv inlaIanco and oxvgon insufficioncv (hvpoxia) in Iivoi coII aio indicalod lv
lhoso onzvnos |MichaopouIos ol aI.1997, Raniioz-LniIiano ol aI.2OO7!. Tho lohavioi of lhoso onzvnos in isoIalod
Iivoi coIIs is dosciilod in foIIoving soclion on isoIalod Iivoi coII onzvnos.



Iiguio 2: Tho Iivoi scan (on Iofl panoI) is shovn foi assossing lho posilion of hopalic hvpoxia and associalod
hopalonogaIv and foi liopsv coIIoclion silo as shovn vilh aiiov.






Iiguio 3: Tho hislogian lais shov lho offocl of nilioinidazoIo on lionaikoi onzvnos and
conpaiison of diffoionl onzvnos in hopalocvlos and Kupffoi coIIs in conlioI vs nilioinidazoIo
lioalod suljocls (panoI on lop and lollon)






Iiguio 3(conlinuod)

Iiguio 3: Tho hislogian lais shov lho offocl of nilioinidazoIo on lionaikoi onzvnos and
conpaiison of diffoionl onzvnos in soiun lionaikoi onzvnos.

. Enzvmc InhIbItInn In InItIa! !nss nf mctabn!Ic IntcgrItv, g!vcn!vsIs and ATP In hcatncvtcs:

Lnzvnos in soiun and Iivoi coIIs fion posilivo conlioI anoolic Iivoi alscoss palionls oxhililod noio oi Ioss spocific
chaiacloiislic changos in onzvnos |Shaina ol aI.2OO8!. Iiisl goaI vas lo oslalIish oIovalod onzvno IovoIs in palionls
and lo piovo sulsoquonl onzvno inhililion afloi nilioinidazoIo lhoiapv. Aulhoi iopoilod lon conlioI suljocls vilh
2-nilioinidazoIo lhoiapv foIIov up foi lhoii cailohvdialo nolaloIizing onzvnos in soiun and hopalocoIIuIai
onzvnos in Iivoi liopsv lissuos. Iiovon lon hopalic hvpoxia conlioI suljocls voio sludiod foi hvpoxia lv onzvno
assavs |Shaina ol aI.2OO8!. Tho 2-nilioinidazoIo lioalod paiiod lon suljocls voio sludiod foi hvpoxia ioIalod
onzvno inhililion using onzvno assavs and hopalocoIIuIai cvlonoiphoIogv lv oIoclion nicioscopv. Oul of lon
posilivo conlioI suljocls, nino palionls shovod oIovalod cailohvdialo nolaloIizing and IvsosonaI onzvno IovoIs in
soiun: CIucokinaso (in 8O sanpIos), aIdoIaso (in 8O sanpIos), phosphofiuclokinaso (in 8O sanpIos), naIalo
dohvdiogonaso (in 75 sanpIos), isocilialo dohvdiogonaso (ICDH) (in 6O palionls) voio oIovalod vhiIo succinalo
dohvdiogonaso and Iaclalo dohvdiogonaso (LDH) IovoIs ionainod unaIloiod. LvsosonaI -gIucuionidaso, aIkaIino
phosphalaso, acid phosphalaso onzvnos shovod onhancod IovoIs in lho aII palionl soiun sanpIos. In lon conlioI
Iivoi liopsios, lho isoIalod hopalocvlos and Kupffoi coII piopaialions shovod aIloiod Iivoi coII onzvno IovoIs.
Hopalocvlos shovod ioducod gIucokinaso (in 8O), LDH (in 8O), and highoi conlonl of aIdoIaso (in 8O), pviuvalo
kinaso (in 1OO), naIalo dohvdiogonaso (in 8O), ICDH (in 8O), cilialo dohvdiogonaso (in 7O), phosphogIuconalo
dohvdiogonaso (in 8O). Kupffoi coIIs shovod highoi onzvno IovoIs of -gIucuioionidaso (in 8O), Ioucino
aninopoplidaso (in 7O), acid phosphalaso (in 8O) and aivI suIphalaso (in 88). In lhoso 1O liopsv sanpIos fion
suljocls, lho oIoclion nicioscopv cvlonoiphoIogv olsoivalions shovod svoIIon lizaiio nilochondiia, pioIifoialivo
ondopIasnic iolicuIun, and anisonucIoosis. In 2-nilionidazoIo cIinicaI liiaI palionls, afloi 2-NilioinidazoIo offocl in
Iivoi coII danago vas nanifoslod as onzvno inhililion in Iivoi coIIs as shovn in Iiguio 3|Shaina 2OO8!.
NilioinidazoIo lioalod palionls shovs aIloiod onzvnos: gIucokinaso IovoIs voio noio oi Ioss noinaI in lolh soiun
and Iivoi liopsv (1OO): phosphofiuclokinaso IovoIs voio nonspocific in nilioinidazoIo lioalod suljocls oi noinaI
(8O): oIovalod Iaclalo dohvdiogonaso IovoIs voio iovoisod lo noinaI. In nilioinidazoIo lioalod suljocls lho oIovalod
onzvno IovoIs duo lo anoolic alscoss lioughl lack lo noinaI afloi nilioinidazoIo lioalnonl duo lo onzvno
inhililoiv offocl of nilioinidazoIo: isocilialo dohvdiogonaso onzvno noinaI IovoIs (8O ): lho cilialo svnlhaso
noinaI IovoIs (8O): phosphogIuconalo dohvdiogonaso noinaI IovoIs (8O ). In nilioinidazoIo lioalod suljocls, lho
succinalo dohvdiogonaso IovoIs voio noinaI (6O ) in lolh. Tho onzvnos aio shovn in Iiguio 3 and TalIos 2-4.



Iiguio 4: UIliasliucluiaI changos aio shovn in hopalocvlo oiganoIIos duiing hvpoxia: oxfoIialion of ondopIasnic
iolicuIun (lop on Iofl): anisonucIoosis (nid and lollon on Iofl): inloicoIIuIai junclion gaps (lop and nid panoIs in
conloi): nucIoai incIusions (lollon on conloi): svoIIon and lizaiio nilochondiia(lop on iighl): incIusions in
poioxisono (nid on iighl): nilochondiiaI aliophv vilh Iipid vosicIos (lollon on iighl). Hopalocvlo nilochondiia
locano svoIIon, lizaiio vilh donso naliix and shovod dosloilod ciislao, ondopIasnic iolicuIun diIalod vosicIos,
gianl nucIoi vilh diffuso pioIifoialion of ondopIasnic iolicuIun and cIoai anisonucIoosis foaluios. Tho uIliasliucluio
of Iivoi coIIs shovod chaiacloiislic oiganoIIo changos in nilioinidazoIo lioalod Iivoi.




.1 Enzvmc InhIbItInn In Kuffcr cc!! !vsnsnma! cnzvmcs and HvnxIa:

In nilioinidazoIo lioalod palionls shovod oIovalod onzvnos lioughl lack lo noinaI: -gIucuionidaso noinaI IovoIs
(5O ) in Kupffoi coIIs and noinaI in soiun (6O ): acid phosphalaso noinaI IovoIs in lolh: Ioucino aninopoplidaso
IovoIs voio noinaI in soiun (8O ) and ionainod high in Kupffoi coIIs (6O): Cuanaso IovoIs voio noinaI in lolh
Kupffoi coIIs and soiun (8O). Tho onzvnos aio shovn in Iiguio 2 and TalIo 3.

Kupffoi coII hvpoipIasia vas olsoivod vilh svoIIon IvsosonaI conlonls as shovn in Iiguio 4. AIloiod gIvcoIvlic
onzvnos in cvlosoI, TCA cvcIo onzvnos in nilochondiia, Ivsosonos and incioasod svnlhosis of onzvnos lv
ondopIasnic iolicuIun shovod coiioIalion vilh cIoai Iivoi coII dogonoialion of niciolodios. Afloi nilioinidazoIo
lioalnonl, olsoivalions of lolh oIoclion nicioscopv and liochonicaI paianolois suggoslod lho iovoisod
hopalocoIIuIai changos lovaids noinaI iocovoiv oi Iivoi iogonoialion. In foIIoving soclion, I dosciilo lho oulcono
of nilioinidazoIo offocl in Iivoi iogonoialion and onzvno inhililion as dofonso.

Tho onzvno lionaikois couId lo anaIvzod in soiun and Iivoi coIIs (hopalocvlos and Kupffoi coIIs) as cIinicaIIv
significanl indicaloi of hopalic danago in disoaso oi cvloloxicilv of diug. Wo assuno lhal iniliaIIv nolaloIic inlogiilv
Ioss Ioads lo ovoi-sociolion of Iivoi coII onzvnos incIuding IvsosonaI onzvnos. Soon afloi, lho uIliasliucluiaI changos
in Iivoi coIIs locono ovidonl lv oIoclion nicioscopv suggoslivo of aculo oiganoIIo dogonoialion. UIliasliucluiaI
hopalocvlo cvloloxicilv vas associalod vilh nilioinidazoIo ovoidosago (noinaI dosago is 2 3 gn ono lino in
anoolic hopalilis and lhiico in anoolic alscoss). Tho iogonoialivo changos voio consislonlIv olsoivod afloi
nilioinidazoIo lhoiapv lo iovoiso Iivoi danago. Iov palhoIogv iopoils aio avaiIalIo lo shov nilioinidazoIo
cvloloxicilv and no oIoclion nicioscopv sludv is avaiIalIo lo suppoil asvnplonalic infIannaloiv oi unoquivocaI
diffuso paionchvnaI injuiv oxhililing diffusod sinusoidaI and poilaI infiIlialion ovonls |Talak ol aI.2OO3, Laiiov ol
aI.2OOO!. ionaikoi Iivoi coII onzvnos vilh oIoclion nicioscopv pul ovidonco of anoolic cvloloxicilv in Iivoi liopsv
sanpIos and Iivoi iogonoialion afloi nilioinidazoIo lioalnonl lv 'hopalic hvpoxia ciiloiia. Dogonoialivo changos of
hopalocvlos suggoslod possilIo nociosis.

Lvidonco of hopalic iogonoialion and nilioinidazoIo cvloloxicilv (hvpoxia inducod changos) vas chaiacloiizod
pioviousIv in loins of cvlokino svnoigv, unusuaI dogioo of anisonucIoosis, niliic oxido pioduclion and lho piosonco
of gianl nucIous in hopalocvlos afloi Iivoi iogonoialivo lhoiapv |MichaIopouIous ol aI.1997!. LndopIasnic iolicuIun
shovod a diffuso and inlonso pioIifoialivo aclivilv in lhoso Iivoi coIIs vilh noinaI appoaianco of nilochondiia as
oaiIioi iopoilod |Raniioz-LniIiano ol aI.2OO7, Das ol aI.1999!. Hovovoi, inlianilochondiiaI incIusion lodios voio
alsonl vhiIo lhov voio voiv pioninonl foaluios |Das ol aI.1999!. Tho causo of lho uIliasliucluiaI changos afloi
nilioinidazoIo cvloloxicilv voio suppoilod lv iniliaI onzvno aIloialions iofIocling Ioss of nolaloIic inlogiilv
piolalIv inducod lv fioo iadicaI foinalion fion nilioinidazoIo |Das ol aI.1999!. Sinco uIliasliucluiaI changos in
Iivoi voio conpIoloIv iovoisilIo afloi nilioinidazoIo lhoiapv vilhin 7-9 davs, il is quilo ioasonalIo lhal palhogonosis
of diffusod hopalocvlo danago vas duo lo nilioinidazoIo lioakdovn pioducls.

Iiguio 5: Tho skolch of nilioinidazoIo inducod inliacoIIuIai onzvno inhililion changos indicaling lho poinls of
nolaloIisn and nolaloIic conlioI duiing hvpoxia and sulsoquonl Iivoi iocovoiv afloi nilioinidazoIo lioalnonl .
Tho onzvnos disliilulion in oiganoIIo and onzvno Iocalion in diffoionl hopalic silos oxpIains lho Iivoi danago and
onzvnalic lasis of hopalocoIIuIai hvpoxia ciiloiia |iopioducod fion Shaina R. Ih.D dissoilalion!.

As indicalod in piovious soclion, 'HopalocoIIuIai hvpoxia ciiloiia is a soquonco of ovonls in hopalic coII injuiv al
noIocuIai IovoI. Tho iniliaI Ioss of hopalocoIIuIai nolaloIic inlogiilv Ioads lo hopalic injuiv. CIucoso-onoigv nolaloIic
inlogiilv Ioss and niliic oxido foinalion vilh Ca
++
honooslasis aio nain iniliaI doloininanls of hvpoxia |Raniioz-
LniIiano ol aI.2OO7!. In foIIoving soclion, piosonl chaploi oxlonds lho onzvno ioguIaloiv lohavioi duiing gIucoso
nolaloIizing palhvavs viz. gIvcoIvsis, TCA cvcIo and gIuconoogonosis lo oslalIish lho vaIuo of onzvno inhililion in Iivoi
iogonoialion. In isoIalod Iivoi coIIs, nolaloIic aIloialions of onoigv nolaloIisn palhvavs oxpIain lho ovonls in hopalilis
and hopalic lunois as losl coiioIalod iospoclivo onzvno dvsfunclions and uIliasliucluiaI changos olsoivod in coIIs in
liopsv. Tho foIIoving dosciiplion is lioad oxpIanalion of diffoionl onzvnos sociolod fion hopalocvlos as a iosuIl of
nilioinidazoIo inducod cvloloxicilv and oxvgon dopIolion. Ioin liochonicaI sland poinl, diffoionl ioguIaloiv onzvnos
aio discussod as liochonicaI ovonls of hvpoxia dovoIopnonl as shovn in Iiguios 3 and 4.



Iiguio 6: Tho skolch of ioIalivo lionaikoi onzvno changos (lhicknoss of aiiov) in Iivoi hvpoxia. Tho +vo
sign donolos lho ioIalivo incioaso in onzvno aclivilv and -vo sign donolos lho docioaso in onzvno aclivilv
in Iivoi coIIs in diffoionl oiganoIIos duo lo anola inducod hvpoxia. Tho figuio skolch aIso iopiosonls a
ioIalivo onzvno aclivilios in soquonco of nolaloIic slops as 'hopalocoIIuIai hvpoxia ciiloiia. Nolico lho
Iivoi iogonoialivo oi onzvno inhililoiv aclion of nilioinidazoIo al diffoionl onzvno ioaclions is shovn
|Ropioducod fion Shaina R. Ih.D dissoilalion!.

.2 Rn!c nf cnzvmcs In cncrgv mctabn!Ic IntcgrItv In hvnxIa

Hoxokinaso, a ialo Iiniling onzvno in cvlosoI foi gIucoso luinovoi vas oslinalod duo lo high gIucoso convoision
inlo gIucoso 6I shovod high onzvno sociolion fion hopalocvlos. AIdoIaso, ialo conlioIIing onzvno vhich spIils
fiucloso-1, 6 Diphosphalo inlo lvo 3- phosphogIvcoiic acid and dohvdioxvacolono phosphalo, shovod high onzvno
sociolion fion hoplocvlos. Ihosphofiuclokinaso, ialo conlioIIing onzvno vhich phosphoivIalos fiucloso 6I inlo
fiucloso 1, 6 DiI, onhancod as ils noio donand in hopalocoIIuIai gIucoso luinovoi vhich piolalIv inducod highoi
sociolion of phosphofiuclokinaso. Iviuvalo kinaso, liansfoiiing phosphoivI gioup fion phosphoonoIpviuvalo lo
ADI vilh pviuvalo foinalion, shovod high onzvno sociolion fion hopalocvlos. Il nav lo alliilulod duo lo high
conconlialion of gIvcoIvlic inloinodialos as loininaI slop vas lIockod oi duo lo 2, 3 Di-IhosphogIvcoialo ioguIalod
alnoinaI oxvgon dissocialion. MaIalo dohvdiogonaso and isocilialo dohvdiogonaso onzvnos, calaIvzing naIalo
oxidalion inlo oxaIoacolalo and oxidalivo docailoxvIalion of isocilialo inlo u-kologIulaialo iospoclivoIv lolh ioquiio
NAD
+
. olh onzvnos voio oIovalod in danagod coIIs duo lo ioducing oquivaIonl Iov ialio of NADH/NAD
+
pushing
in foivaid diioclion. Succinalo dohvdiogonaso, oxidizing succinalo lo funoialo using IAD, shovod docioasod
onzvno IovoIs duo lo Iov iion suIphui pioloins iosuIling vilh Iovoiod oIoclion lianspoil svslon in innoi
nilochondiiaI nonliano i.o. suppIv of oIoclions lo noIocuIai oxvgon lv oIoclion liansfoi fion IADH2 lo Io
+++

(SDH) in anoolic iociuilod Iivoi coIIs. Cilialo svnlhaso, svnlhosizing cilialo fion oxaIoacolalo and acolvI CoA lv
aIdoI condonsalion foIIovod lv hvdioIvsis, shovod oIovalod IovoIs duo lo iapid luinovoi of oxaIoacolalo and acolvI
CoA noIocuIos duiing cvloIvsis. Moioovoi, high TCA cvcIo aclivilv in hopalocvlo duiing Iivoi danago condilions
vas dosciilod oaiIioi |Shaina 2OO1!. In soiun, lho onzvnos oxhilil lhoii significanco lul aIdoIaso, pviuvalo kinaso
and LDH, MDH, ICDH olsoivod as dislinguishing lho diffusod injuiv oi alscoss foinalion |Viik ol aI.1989!.
IhosphogIuconalo dohvdiogonaso oIovalod IovoIs nav lo alliilulod duo lo iiluIoso 5I foinalion and liansaIdoIaso
and lianskoloIaso conlioI, foi phosphogIuconalo palhvav. NilioinidazoIo inducod cvloloxicilv poihaps havo
insignificanl inpacl on ATI suppIv. In foIIoving soclion, diffoionl onzvno ioguIaloiv lohaviois of nilioinidazoIo
inducod onzvno inhililion aio dosciilod duiing Iivoi coII iogonoialion. A slialogv vas dovoIopod lo isoIalo
hopalocvlos and Kupffoi coIIs in nonoaxonic cuIluios fion oxcisod Iivoi sanpIos. Mixing diffoionl inhililois oi
addilivos in Iivoi coII cuIluios slinuIalo lho onzvno ioguIaloiv lohavioi in piosonco of spocific giovlh faclois,
nolaloIilo anaIoguos.

. Rn!c nf !vsnsnma! cnzvmcs In hcatncc!!u!ar damagc

In piovious soclion, ioIo of socioloiv IvsosonaI onzvnos vas dosciilod in dofonso of Iivoi coIIs in conloxl of inloslinaI
anooliasis and anoolic Iivoi alscoss. ConcopluaIIv, IvsosonaI hvdioIasos causo foioign lodv danago lv sovoiaI
ioaclions incIuding iodox ioaclion, phosphoivIalion ioaclion, gIucuionidalion ioaclion, hvdioxvIalion ioaclion,
dohvdioxvIalion ioaclion, nucIoalion ioaclion, loininaI lioakdovn ioaclion, lians-cailanvIalion ioaclion. Olhoi
sludios aIso iopoilod noIocuIai nochanisns of onzvnos in palhogonosis |Das ol aI.1999!, Iivoi coII danago |Viik ol
aI.1989, Viik ol aI.1988!, phagocvlosis |Shaina 2OO9!, cvcIooxvgonaso-2 oxpiossion |Sanchoz-Raniioz ol aI.2OOO!.

4. Enzvmc InhIbItInn rncrtIcs and cnzvmc rcgu!atnrv bchavInr In Isn!atcd !Ivcr cc!!s: IsoIalod Iivoi
coIIs vilh chaiacloiislic onzvno piopoilios aio iosoaich looIs. Tho hopalocvlo vioId fion Iivois vas 2.5 1O
8
poi ng
Iivoi. Tho cvlosoI fiaclion shovod lho ionovaI of aII coII oiganoIIo fion supoinalanl and iosuIlanl supoinalanl as
cvlosoI vas fioo of anv ionnanl lv nicioscopic olsoivalion lv livpan lIuo oxcIusion. IsoIalion of hunan Kupffoi
coIIs vas dovoIopod lv a nodifiod pionaso-coIIagonaso onzvnalic isoIalion and puiilv of Kupffoi coIIs lv using
lionaikoi onzvno assav. LaiIioi iopoils usod siniIai piaclico of onzvnalic digoslion and onzvnalic assavs as
indicalois of coII vialiIilv and vioId |Hondiiks ol aI.199O, Noaud ol aI.1995, McCuskov ol aI.199O!. A nov iapid
nolhod vas usod foi lho isoIalion and fiaclionalion of lolh ial and hunan Kupffoi coIIs vilhoul lho nood of Iivoi
poifusion lochniquos. Tho sludv usod ial Iivois oi snaII hunan Iivoi vodgo liopsios ollainod poiopoialivoIv and
inculalod vilh pionaso undoi conlinuous pH iogislialion. Kupffoi coIIs voio sulsoquonlIv sopaialod fion olhoi
nonpaionchvnaI coIIs lv Nvcodonz giadionl conliifugalion and puiifiod lv counloifIov conliifugaI oIuliialion
|Noaud ol aI.1995!. Idonlificalion of Kupffoi coIIs vas achiovod on lho lasis of uIliasliucluiaI anaIvsos and
innunophonolvping |Noaud ol aI.1995!. Tho fiaclionalion of Kupffoi coIIs gavo good vioId conpaialIo vilh olhoi
sludios |Hondiiks ol aI.199O, Noaud ol aI.1995, McCuskov ol aI.199O!. Ioi dolaiIs of piolocoI of Iivoi coII isoIalion, soo
lho Appondix in lho ond of lhis chaploi. Hunan Iivoi coII onzvnos in isoIalod Iivoi coIIs (hopalocvlos and Kupffoi
coIIs in cuIluios) shovod chaiacloiislic ioguIaloiv lohavioi in piosonco of addilivos.

Livoi coII hvpoxia is iopiosonlod as a slalo of oxvgon dopIolion in coII. IniliaIIv Iivoi coII gols ils ATI and NADIH
suppIv fion gIvcoIvsis and TCA cvcIo. ConsoquonlIv, oIoclion liansfoi chain (oxidalivo phosphoivIalion) lhiough
soiios of cvlochiono iodox ioaclions convoils avaiIalIo coII oxvgon lo valoi lo pioduco high onoigv nolaloIilos in
lho coII (nolaloIic inlogiilv). Infoclod Iivoi coIIs shov Ioss of nolaloIic inlogiilv and Ioss avaiIalIo oxvgon (oxvgon
slaivod oi hvpoxia). NilioinidazoIo vas oslalIishod lvo docados ago as polonliaI oxvgon quonching diug in nosl of
lho infoclod and lunoi coIIs. NilioinidazoIo oxvgon quonching aclion nakos oxvgon dopIolod oi slaivod coIIs lhal
fuilhoi undoigo voiso lo dio and Ioaving noinaI coIIs fuIIv funclionaI lhal can lo olsoivod as polonliaI lunoi
hvpoxia lhoiapv and anlipaiasilic lioalnonl lv nilioinidazoIo.

In foIIoving soclion, onzvno inhililion chaiacloiislics in onoigv nolaloIisn ioguIalion incIuding gIvcoIvsis, TCA
cvcIo ioguIaloiv onzvno lohavioi and diug nolaloIizing piolooIvlic IvsosonaI onzvnos inducod lv addilivos in
Iivoi coII cuIluios aio dosciilod.

4.1!ucnkInasc:
Tho aclinonvcin D shovod non-significanl inhililoiv chango in gIucokinaso onzvno aclivilv as shovn in Iiguio 7
al lho lop hislogian lais. Tho nilioinidazoIo shovod onhancod gIucokinaso aclivilv and iovoisod lho inhililoiv
offocl of aclinonvcin D. Tho gIucokinaso aclivilv in hopalic alscoss hopalocvlos did nol shov anv significanl offocl of
nilioinidazoIo. OvoiaII aclinonvcin D aIono did nol shov anv chango in gIucokinaso onzvno aclivilv in hopalic
alscoss hopalocvlos oi nilioinidazoIo lioalod hopalocvlos.

Tho addilion of liiancinoIono vilh insuIin in conlinalion shovod significanl incioaso in gIucokinaso aclivilv in aII
hopalocvlo gioups. Il poinlod oul lo lho onhancononl in hoinono dopondonl gIucokinaso onzvno svnlhosis in
hopalocvlos and onzvno aclivilv shovod highosl onzvno svnlhosis ialo in hopalic alscoss iociuilod hopalocvlos
vhiIo nilioinidazoIo lioalod hopalocvlos shovod Iossoi incioaso in hoinono dopondonl gIucokinaso in conpaiison
vilh hopalocvlos as shovn in Iiguio 7. Tho incioaso in onzvno aclivilv oi onhancod gIucokinaso onzvno svnlhosis in
hopalocvlos shovod lhal gIucokinaso onzvno lohavioi vas hoinono dopondonl. Diffoionl conconlialion of insuIin
and piogosloiono hoinonos fuilhoi indicalod lho ioguIaloiv lohavioi of gIucokinaso onzvno as hoinono dopondonl.

Tho addilion of piogosloiono hoinono O.1 gn/nI in hopalocvlo cuIluios shovod naxinun onhancod
gIucokinaso onzvno aclivilv and nilioinidazoIo lioalod hopalocvlos shovod Ioss onhancod gIucokinaso aclivilv lul
noio lhan conlioI hopalocvlo onzvno aclivilv as shovn in hislogian lais (soo Iiguio 7).

In conlioI gioup, gIucokinaso onzvno aclivilv in unlioalod hopalocvlos did nol shov anv chnago in lhoii
gIucokinaso onzvno aclivilv in piosonco of nilioinidazoIo addilivo. Tho vioId of hopalocvlos and gIucokinaso
onzvno aclivilios fion hopalic alscoss Iivoi liopsv voio siniIai as vioId of coIIs and gIucokinaso onzvno aclivilios in
unlioalod and nilioinidazoIo lioalod hopalocvlos. So, il vas cIoai lhal addilion of aclinonvcin-D and insuIin vilh
liianiciIono lolh onhancod lho gIucokinaso onzvno svnlhosis in hopalocvlos as shovn in Iiguio 7 hislogians lais.

4.2 Pvruvatc KInasc
Tho aIloiod lohavioi of pviuvalo kinaso ioguIalion in isoIalod hopalocvlos fion nilioinidazoIo and piogosloiono
inducod cuIluios shovod chaiacloiislic signoid lohavioi in noinaI gioup lul lovaids Iinoai ioIalionship al
incioasod pH of hopalocvlo cuIluio nodia. In olhoi voids, a pH incioaso aIlois lho onzvno voIocilv in sano
piopoilion vilh ATI conconlialion changos in Iinoai nannoi in Iivoi coII nodiun. Al high ATI conconlialion,
onzvno svnlhosis in nilioinidazoIo slinuIalod coIIs ionainod significanlIv oIovalod vhiIo al pH 7.O-7.4 coIIs did nol
shov suddon onzvno oIovalion and shovod a faII in onzvno aclivilv as olsoivod in noinaI hopalocvlos (soo Iiguio
8). Tho highoi ADI/ATI ialio kopl lho onzvno svnlhosis high al pH 7.4 in piogosloiono vhiIo nilioinidazoIo
slinuIalod lho hopalocvlo coIIs in cuIluios (soo Iiguio 8).

Iiguio 7: Tho nilioinidazoIo offocl on gIucokinaso onzvno aclivilv in guinoa pig noinaI and infoclod
hopalocvlos is shovn in piosonco of diffoionl onzvno ioguIaloiv addilivos aclinonvcin D, insuIin-
liianciIono. Nolico lho onhancod offocl of insuIin-liianiciIono on gIucokinaso onzvno (I vaIuo <O.OOO1: i
2

O.9872) in nilioinidazoIo lioalod vs Iivoi alscoss liopsv hoaplocvlos.

Tho inliicacv vas iopoilod as ATI inloiacls vilh H
+
and Mg
++
lo givo conpIox disliilulion of nanv ionizod and
nolaI conpIox spocios. Hovovoi, Mg
++
fioo ADI ilsoIf has lhioo sulspocios viz. ADI
--
, ADIK
--
, ADIH
~
vhoso
ioIalivo piopoilions vaiv vilh chango in pH. Oul of vhich ADI
~
happons lo lo liuo sulslialo of pviuvalo kinaso
vhich piosunalIv vas uliIizod lv hopalocvlos slinuIalod lv nilioinidazoIo. Il fuilhoi suggoslod lho linding of
ADI lo unlound and fuIIv lound onzvno confoinalions invoIving diffoionl gioups vilhin aclivo silo. Tho linding
of ADI and ADI fioo confoinalion of onzvno did nol invoIvo gioups lhal ionizod vilhin appIiod pH iango in
nilioinidazoIo and piogosloiono slinuIalod hopalocvlos lul linding of ADI lo lho fuIIv lound onzvno
confoinalion (coopoialivilv) invoIvod lho ionizalIo gioups vilh high pK vaIuos (al pH 6.6-7.4). Moioovoi,
phosphoonoI pviuvalo lound onzvno ionizalion has loon pioviousIv iopoilod lo pionolo coopoialivo linding of
ADI in disoasod coII. In piosonl sludv, ADI linding loconos Ioss coopoialivo lv slinuIalod hopalocvlos. Il is
possilIo lhal onzvno nav havo lvo aclivo confoinalions. So, aIIosloiic inloiaclions acluaIIv nighl havo aIloiod lho
nodo of ADI linding in noinaI coIIs vhiIo lhov voio non-affoclivo in pviuvalo kinaso onzvno of slinuIalod
hopalocvlos. Iviuvalo kinaso aclivo silo confoinalion poihaps soons lo chango ADI linding.



FIgurc 8A: Tho pviuvalo kinaso onzvno aclivilv in conlioI, anoolic Iivoi alscoss and nilioinidazoIo
lioalod hopalocvlos is shovn in piosonco of diffoionl onzvno ioguIaloiv addilivos (A-I).
8B: Tho pviuvalo kinaso onzvno aclivilv in conlioI, anoolic Iivoi alscoss and nilioinidazoIo lioalod
hopalocvlos is shovn in piosonco of diffoionl onzvno ioguIaloiv ADI addilivos (2-5 nM ADI).
8C : Tho pviuvalo kinaso onzvno aclivilv in conlioI, anoolic Iivoi alscoss and piogosloiono lioalod Iivoi
coIIs is shovn in piosonco of diffoionl onzvno ioguIaloiv addilivos (ATI, ILI+IDI,ILI inhililois).

Iiguio 9: Tho offocl of nilioinidazoIo on phosphofiuclokinaso onzvno aclivilv of isoIalod hopalocvlos
fion conlioI, anoliasis and nilioinidazoIo gioups. Hopalocvlos fion oach gioup voio sodinonlod and
honogonizod in Kiol HansoIoil luffoi pH 8.O conlaining 2.5 nM dilhiolhiolioI, O.1 nM LDTA and 5O
nM NaI foi 9O soconds foIIovod lv conliifugalion al 27OOO g foi 1O ninulos. Ono unil onzvno aclivilv
calaIvzod 1 nicionoIo fiucloso 6 phosphalo lo fiucloso 1,6 DiI poi ninulo poi ng onzvno pioloin.

4. PhnshnfructnkInasc
Ihosphofiuclokinaso onzvno ioguIalion in isoIalod hopalocvlos cuIluios addod vilh anoolic liophozoilos,
nilioinidazoIo and piogosloiono oxhililod spocific ioguIaloiv lohavioi. Addilion of fiucloso 1,6 Diphosphalo
addilivo oxhililod a slinuIalion of phosphofiuclokinaso aclivilv (soo Iiguio 9). Tho offocl of piogosloiono vas soon
as losl iosponso al 1O nM in hopalocvlos of aII gioups vhiIo fiucloso 1,6 diphosphalo shovod naxinun offocl al
conconlialion of 1O nM on lho hopalocvlos in piosonco of liophozoilos addod in hopalocvlo cuIluios. Hovovoi,
hopalocvlo cuIluios in olhoi gioups did nol shov anv aIloialion (soo Iiguio 9). SiniIaiIv, piogosloiono addilion lo
hopalocvlos in piosonco of liophozoilos oxhililod naxinun fiucloso 1,6 diphosphalo in lho nodiun. Tho addilion
of piogosloiono conconlialion vas vilhin lho phvsioIogicaI iango (soo Iiguio 9). Iiogosloiono vas piosunod lo
offocl onzvno aclivilv lv infIuoncing inliacoIIuIai IovoI of vaiious aIIosloiic offoclois of onzvno. Iiucloso 1,6
diphosphalo acls as polonl aclivaloi of onzvno aclivilv. Tho phosphofiuclkinaso onzvno aclivilv vas iopoilod as
cvcIic AMI dopondonl |IiIkis ol aI.1979, Sanchoz-Mailinoz ol aI.2OOO!. Hovovoi, nilioinidazoIo addod hopalocvlos
did nol iospond oi iospondod sIov afloi piogosloiono addilion.

4.4 PhnshndIcstcrasc
CvcIic AMI dopondonl phosphodiosloiaso ioguIalion ovoi pioslagIandin L2 and I2-
svnlhosis vas
slinuIalod in Kupffoi coIIs addod vilh liophozoils and nilioinidazoIo. Ihosphodiosloiaso aclivilv in
Kupffoi coIIs vas dopondonl upon caIciun ion conconlialion vhich vas ioguIaloiv facloi foi cvcIic AMI
pooI and pioslagIandin pioduclion. In anoolic liophozoilo addod cuIluios, coIIs oxhililod insignificanl
oIovalod onzvno aclivilv vhiIo nilioinidazoIo addilion did nol aIloi lho onzvno aclivilv as shovn in
Iiguio 1O. Tho onzvno lohavioi vas siniIai in naluio foi ils dopondonco on cvcIic AMI sociolion,
pioslagIandin svnlhosis and caIciun ion. Hovovoi, liopozoilos slinuIalod noio cvcIic AMI sociolion
and pioslagIandins. Il piovidos lho piolalIo nochanisn of liophozoilo slinuIalod naciophagaI aclion.
LaiIioi caInoduIin dopondonl adonvIalo cvcIaso and phosphodiosloiaso ioguIalion foi cvcIic AMI pooI
havo loon dosciilod |Hidi ol aI.2OOO!. Moioovoi, lho slinuIalod pioslagIandin svnlhosis vas dosciilod as
caInoduIin dopondonl cvcIic AMI ioguIalod nochanisn |Hidi ol aI.2OOO!. Thus il soons lhal anoolic
liophozoilo addilion vilh hopalocvlos ioguIalos inliacoIIuIai caIciun ion conconlialion vhich conlioIs
pioslagIandin svnlhosis via phosphoIipid and aiachidonic acid piocuisois in hopalocvlos. Thus
svnlhosizod pioslagIandins ioguIalo lho cvcIic AMI inliacoIIuIai IovoIs. Tho siniIai nochanisn has loon
dosciilod as soIf-Iiniling nochanisn |Mailina ol aI.2OO6!.

Iiguio 1O: CvcIic AMI dopondonl phosphodisosloiaso aclivilv and sociolion of pioslagIandin L2 and I2M

in isoIalod kupffoi coIIs fion diffoionl gioups. Tho phosphodiosloiaso onzvno aclivilv in anolic Iivoi
alscoss gioup shovod highosl onzvno aclivilv afloi addilivos voio addod.

4.5Enzvmc InhIbItInn In rcsIratnrv burst nf !Ivcr cc!!s

An iniliaI ovidonco of Iivoi coII suivivaI al diffoionl oxvgon piossuios is shovn in Iiguio 11 lo highIighl
iospiialoiv luisl of onzvnos. Tho inhililod onzvnos ioIalod vilh coII iospiialion aio piosunalIv
considoiod iosponsilIo of inducod iospiialoiv luisl (hvpoxia) in Iivoi coIIs. Rospiialoiv luisl piocoss
incIuding onzvno aIloialions (supoioxido disnulaso, gIulalhiono ioduclaso, NADIH oxidaso onzvno
aclivilv) vas suggoslod as iniliaI Kupffoi coII slinuIalion oi iospiialoiv luisl lv addilivos and foIIovod lv
onoigv and oxvgon dopIolion as possilIo causo of hvpoxia and coII vialiIilv Ioss as inpoilanl findings in
foIIoving soclion.


Iiguio 11: Lffocl of oxvgon suppIv (shovn in diffoionl piossuios) lo Iivoi coIIs. Nolico lho Iov oxvgon
suppIv dopIolos lho oxvgon conlonl in coIIs vhiIo high oxvgon suppIv koops coIIs in in good suivivaI. Ioi
Iong suivivaI, iooxvgonalion of coIIs is noodod.

4.6. 5ucrnxIdc dIsmutasc cnzvmc actIvItv In rcsIratnrv burst nf Kuffcr cc!!s: Tho supoioxido disnulaso onzvno
aclivilv in isoIalod coIIs vas conpaialIo and shovod nininaI diffoionco in lolh conlioI and addilivo liiggoiod Kupffoi
coIIs. Addilion of sodiun cvanido conconlialion shovod snaII offocl on oIovalod supoioxido disnulaso aclivilios in
lolh conlioI and nilioinidazoIo liiggoiod Kupffoi coIIs. Hovovoi, Kupffoi coIIs shovod oIovalod iospiialoiv luisl
aclivilv significanlIv high afloi adding 1O nM sodiun cvanido in lho coII cuIluio nodiun as shovn in Iiguio 12.
Hovovoi, lho iosponso of Kupffoi coIIs vilh sodiun cvanido addilion vas noio in liophozoilos addod coII cuIluios al
1O nM sodiun cvanido conconlialion. Zvnosan slinuIalion on supoioxido pioduclion vas insignificanlIv high in lho
Kupffoi coIIs in lolh gioups. LaiIioi slinuIalion of zvnosan inducod supoioxido anion pioduclion in Kupffoi coIIs lv
sodiun cvanido vas iopoilod lo shov lho offocl of cvanidos on cvlosoIic supoioxido disnulaso onzvno aclivilv
|D'AIossandio ol aI.2O11, Shahhossoini ol aI.2OO6!. Moioovoi, zvnosan slinuIalod supoioxido pioduclion vas iopoilod
in Kupffoi coIIs as ioaclion of cvlochiono C ioduclion |Duan ol aI.2OO4!.


Iiguio 12: Tho onzvno aclivilv vas oslinalod in Kupffoi coIIs cuIluios addod vilh shoop oivlhiocvlos, liophozoilos,
nilioinidazoIo. Tho Kupffoi coIIs voio nainlainod foi 48 houis oi noio lo doloinino supoioxido anion pioduclion in
piosonco of 1.O nM and 1.O nM sodiun cvanido addilivo addod in cuIluio nodiun. In liiof, 3 x 1O
5
Kupffoi coIIs voio
suspondod in Kiols HonsoIoil luffoi conlaining 5 nM gIucoso, 3 nM caIciun chIoiido pH 7.4 in piosonco of 5O 3M
foiiicvlochiono in lolaI 1.O nI ioaclion nixluio and coIIs voio inculalod foi 15 ninulos in piosonco of diffoionl sodiun
cvanido addilivos. Tho ioaclion vas slailod lv addilion of zvnosan 5OO 3g poi nI lo lho nodiun afloi 1O ninulos al
37 HC supoinalanl vas lakon, conliifugod al 8OOO x g in coId foi lvo ninulos and assavod foi cvlochiono C ioduclaso
onzvno aclivilv.

Tho O2

supoioxido anion iadicaI pioduclion as iosuIl of iospiialoiv luisl aclivilv shovod piopoilionalo oIovalod
onzvno aclivilv in isoIalod Kupffoi coIIs fion cuIluios addod vilh liophozoilos and nilioinidazoIo addilivos. Tho
aclivo ioIo of ioaclivo oxvgon spocios and supoioxido disnulaso aclivilv in cuIluiod Kupffoi coII danago duiing
hvpoxia is shovn in Iiguio 12. Iiovious sludv suppoilod lhoso olsoivalions on coIIuIai danago and cvloloxicilv fion
ioaclivo oxvgon spocios fion supoioxido anion acling as a scavongoi |Shaina ol aI.2OO !. Anolhoi possiliIilv of
Kupffoi coII loxicilv lv addilivos nav lo associalod vilh cvlokinos, spocificaIIv TNI-u vhich vas associalod vilh lho
cvloloxicilv |Shaina ol aI.2OO !. Iiovious sludv aIso iopoilod a docioasod hopalocvlo coII vialiIilv associalod vilh
aIloiod cAMI phosphodiosloiaso and HMI shunl aclivilv as a iosuIl of hvpoxia |Caiio ol aI.2O1O!. Aulhois suggoslod
lhal lho oxvgon insufficioncv is possilIv a consoquonco of onoigv dopIolion and incioasod oxvgon ioaclivo spocios
|KooIing ol aI.1982!. Olhoi iopoils suppoil lhis viov of vasodiIaloiv sonsilivo ATI dopIolion in Kupffoi coIIs |Ana ol
aI.2O11!. Tho ioaclivo oxvgon spocios pioduclion aIso soons as anolhoi causo of hvpoxia as suggoslod lv lho naluio of
supoioxido disnulaso as addilivo cvanido sonsilivo onzvno. Wo olsoivod a good ioason of associalion lolvoon
supoioxido anion scavongoi, gIulalhiono ioduclaso and NADIH oxidaso as causo of coII vialiIilv Ioss Ioading fuilhoi lo
Kupffoi coII danago. In lhis diioclion, anpIo ovidonco suppoils lho possiliIilv of aclivalion of HMI shunl duo lo
dopIolod ATI and NADH in lho Kupffoi coIIs |Ciahn, 2OOO: SpoIaiics ol aI. 1991!.

4.7. HMP 5hunt Pathwav In rcsIratnrv burst
Hoxoso nonophosphalo shunl aclivalion duiing iospiialoiv luisl in isoIalod Kupffoi coIIs oxposod vilh anoolic
liophozoilos and nilioinidazoIo shovod onhancod onzvno aclivilios as shovn in Iiguios 13A-13C. HMI shunl
aclivalion is noasuiod in lhioo vavs: 1. HMI shunl aclivilv in piosonco of addilivos such as Ialox, shoop oivlhiocvlos,
liophozoilos: 2. HMI shunl aclivilv as iadioIaloIod gIucoso convoision lo cailon dioxido: 3. HMI shunl aclivilv as
onzvno inhililion.
a. Tho HMI shunl aclivilv vas naxinun and il oIovalod in Kupffoi coIIs afloi inculalion vilh shoop oivlhiocvlos
and liophozoilos vhiIo Kupffoi coIIs oxposod vilh inoil Ialox pailicIos did nol shov anv aclivalion oi chango in
HMI shunl aclivilv. Shoop oivlhiocvlos and liophozoilos lolh slinuIalod gIucoso nolaloIisn via hoxoso
nonophosphalo shunl aclivilv. Ioihaps Ialox pailicIos loing lioIogicaIIv inoil naluio couId nol chango lho coIIuIai
aclivilv al aII. So, Ialox pailicIos voio phagocvlosod vilhoul aIloiod HMI shunl aclivilv. Il suggoslod lhal HMI
shunl aclivilv nav lo a liiggoiing ovonl in Kupffoi coII aclivalion dopondonl nol soIoIv upon inliacoIIuIai falo of
ingoslod naloiiaI oi pailicIos. Hovovoi, olhoi addilivos such as zvnosan, oivlhiocvlos and liophozoilos aIso
shovod sano iosponso. Iiovious sludv iopoilod siniIai iosponso of zvnosan slinuIalod and iodoacolalo inhililod
Kupffoi coII gIucoso-6-phosphalo dohvdiogonaso and phosphogIuconalo dohvdiogonaso onzvnos.|Shaina ol
aI.2O1O!

l. Tho HMI shunl aclivilv as |
14
C! gIucoso convoision lo |
14
C!-CO2 Iiloialod pioducl indicalod lho aclivo
infIannalion oIicilod lv naciophago iosponso of Kupffoi coIIs |SchoiIonnoi ol aI. 1999!. Tho HMI shunl aclivilv
in conlioI and liiggoiod Kupffoi coIIs vas noasuiod lv |14-C!CO2 ioIoaso in dpn fion IaloIod |14-C! gIucoso poi
ninulo poi ng pioloin in piosonco of diffoionl offoclois knovn as quonching lho hoxoso nonophosphalos in
gIvcoIvsis. Tho HMI shunl aclivilv vas siniIai vilh no diffoionco (I vaIuo > O.OO1) in conlioI conpaiod vilh
liiggoiod Kuffoi coIIs vilh offocloi and inoil Ialox pailicIos did nol aIloi lho HMI shunl aclivilv. Tho HMI shunl
aclivilv vas significanlIv onhancod in liiggoiod Kupffoi coIIs in piosonco of liophozoilos (I vaIuo < O.OO1) lul lho
HMI shunl onhancononl vas Iossoi in piosonco of oivlhiocvlos. Il indicalod lho dopondanco of HMI shunl
aclivilv on naluio of offocloi oilhoi inoil oi suifaco aclivo. Tho Ialox vas inoil, oivlhiocvlos voio nonviiuIonl and
liophozoils voio viiuIonl as a iosuIl HMI shunl aclivilv vas onhancod in lho oidoi of Ialox < oivlhiocvlos <
liophozoils addod in lho nodiun of Kupffoi coIIs.


c. Tho liophozoilos fuilhoi aclivalod lho Kupffoi coII gIucoso-6-phosphalo and phosphogIuconalo dohvdiogonaso
onzvnos in associalion of HMI shunl aclivilv. In foIIoving soclion, onzvno inhililion in Iivoi coIIs is iopoilod in
conlioI, hvpoxic coIIs in diffoionl gioups. Hoxoso nonophosphalo shunl aclivalion vas onhancod duiing
iospiialoiv luisl in isoIalod Kupffoi coIIs in anoolic liophozoils and nilioinidazoIo addod cuIluios. In lho
cuIluios addod vilh liophozoils and nilioinidazoIo lolh, Kupffoi coIIs oxhililod significanlIv oIovalod HMI shunl
aclivalion. Tho HMI shunl aclivilv vas highIv oIovalod in aII gioups cuIluios inculalod vilh shoop oivlhiocvlos
and anoolic liophozoils vhiIo coIIs vilh Ialox pailicIos did nol shov anv aclivalion. Ioihaps Ialox pailicIos loing
lioIogicaIIv inoil couId nol chango lho coIIuIai sinusoidaI funclion aclivilv. Shoop oivlhiocvlos and liophozoils
poihaps slinuIalod lho gIucoso nolaloIisn via hoxoso nonophosphalo palhvav lul Ialox pailicIos voio
phagocvlosod vilhoul aIloiod shunl aclivilv. Il suggoslod lhal HMI shunl nav lo a liiggoiing ovonl in Kupffoi
coII aclivalion dopondonl nol upon inliacoIIIuIai falo of ingoslod naloiiaI oi pailicIos |Knook ol aI. 198O!. Tho
liophozoils addod Kupffoi coIIs shovod diiocl inloiaclion vilh incioasod gIucoso-6-phosphalo dohvdiogonaso and
6-phosphogIuconalo dohvdiogonaso onzvno aclivilios as dosciilod as a iosuIl of onhancod hoxoso nonophosphalo
shunl aclivilv. LaiIioi zvnosan slinuIalod and iodoacolalo addilivos inhililod Kupffoi coII gIucoso 6 phosphalo
dohvdiogonaso and 6-phosphogIuconalo dohvdiogonaso aclivilios havo loon iopoilod |Houff ol aI. 1994!.


Iiguio 13: Hoxoso nonophosphalo shunl aclivilv is shovn in isoIalod Kupffoi coIIs fion anola infoclod
and nilioinidazoIo lioalod coIIs in piosonco of oivlhiocvlos, liophozoils and Ialox addilivos. In 3.O nI
nodiun, 1 nicio Cuiio 14C gIucoso vas addod and sinuIlanoousIv pailicIos addod vilh Kupffoi coIIs in
ialio 5:1(pailicIo:coII) foi oivlhiocvlos, 2:1 foi anolic liophozoils and 4O:1 foi Ialox. Afloi ono houi, 2 nI
No particle Erythrocytes Trophozoits latex
0
1000
2000
3000
4000
5000
6000
Control
Hypoxic hepatocytes
Additives added
H
M
P

s
h
u
n
t

a
c
t
i
v
i
t
y
of coII fioo nodiun soakod lv fiIloi papoi vollod vilh 1OO L of 1O KOH in counloi voII vhich fuilhoi
vas addod vilh 2OO L 1.O N H2SO4 and afloi 15 ninulos fiIloi papoi sliip oIulo in liilosoI voio noasuiod
foi scionliIIalion counls. Tho liophozoils shovod naxinun aclivilv vhiIo inoil Ialox loads did nol shov
anv chango in aclivilv.




1

m
M

D
E
M

3
M

a
n
t
i
m
y
c
i
n

A
1

m
M

B
S
O
0
10
20
30
0 hour
24 hours
48 hours
72 hours
96 hours
G
S
H

a
c
t
i
v
i
t
y

(

U
)

Iiguio 13: Lffocl of addod slinuIalois on CIulalhiono ioduclaso onzvno aclivilv in hvpoxic hopalocvlos
in cuIluio.

O InhIbItInn nf !utathInnc Rcductasc cnzvmc actIvItv In rcsIratnrv burst: Tho gIulalhiono ioduclaso CSH
spocific onzvno aclivilv vas noasuiod in hvpoxic hopalocvlos in piosonco of diffoionl CSH onzvno inhililois
such as DLM, anlinvcin, SO as addilivo offoclois. To dononslialo lho dopondonco of onzvno aclivilv on
oxposuio lino of inhililois, lho hvpoxic hopalocvlos voio inculalod in piosonco of offoclois and onzvno
aclivilv vas noasuiod in nodiun. Al O houi, lho onzvno spocific aclivilv vas sano in hopalocvlos in aII
gioups. Afloi 24 houis, lho aclivilv in hopalocvlos vas inhililod naxinun lv aclinonvcin vhiIo DLM and
SO inhililod lho onzvno al siniIai oxlonl and hvpoxic hopalocvlos oxhililod no diffoionco in onzvno
aclivilios (I vaIuo > O.OO1) (soo Iiguio 12). Afloi 48 houis, lho CSH onzvno aclivilv vas inhililod naxinun lv
addilion of SO and nininun lv anlinvcin in coII cuIluios. Hovovoi, lho lohavioi palloin of onzvno
inhililion lv DLM and SO vas siniIai. Afloi 72 houis, lho inhililion palloin of CSH onzvno aclivililv lv
inhililois vas sano as afloi 48 houis. Afloi 96 houis, aII inhililois shovod naxinaI inhililion and hvpoxic
hopalocvlos shovod sano spocific aclivilios in aII gioups. Anlinvcin inhililoi oxhililod Iossoi inhililion
aclivilv of hvpoxic CSH onzvno iniliaIIv duiing 24-48 houis.

O InhIbItInn nf NADH nxIdasc In rcsIratnrv burst: Tho iospiialoiv luisl aclivilv vas dosciilod as incioasod
aclivilv in Iivoi naciophagos duiing hopalic coII danago shoving incioasod oxvgon consunplion, slinuIalion
of HMI shunl and oIovalod supoioxido pioduclion vilh aclivalod nonliano NADIH oxidaso onzvno as ono
of lho nodo of phagocvlosis |Dusling ol aI. 2OO5!. Moioovoi, oui oxpoiinonls suggoslod lho spocific ioIos of
addilivos on iospiialoiv luisl lionaikois: 1. Tho incioasod sodiun cvanido conconlialion shovod slinuIalion
of supoioxido disnulaso aclivilv: 2. Tho Iivo oivlhiocvlos and liophozoilos shovod HMI shunl slinuIalion
vhiIo inoil Ialox pailicIos shovod no offocl on HMI shunl aclivilv suggosling lho associalion of addilivo
naluio as doloininanl of lho HMI shunl aclivilv oi liio-onzvno inhililion: 3. Tho gIulalhiono pIavs a ciilicaI
ioIo in suiviving Kupffoi coIIs duiing hvpoxia and indicalos lho slinuIalod ioaclivo oxvgon spocios in
iospiialoiv luisl aclivilv |halnagai ol aI.1981!. AII lhoso iospiialoiv luisl lionaikoi aclivilios shovod
dopondonco on lho naluio of addilivos in coiioloialion vilh oaiIioi iopoils |Kunai ol aI.2OO2: Ioinan ol aI.
2OO2: Ding ol aI. 1988!. In piosonl chaploi, lolh HMI shunl aclivilv and iospiialoiv luisl aclivilv (supoioxido
disnulaso, gIulalhiono ioduclaso, NADIH oxidaso onzvno aclivilv) aio oulIinos lho suggoslod iniliaI Kupffoi
coII slinuIalion lv addilivos and Ialoi foIIovod lv onoigv and oxvgon dopIolion as possilIo causo of hvpoxia
and coII vialiIilv Ioss as inpoilanl finding. Hovovoi, associalion of Kupffoi coII slinuIalion and hvpoxia is
nol concIusivo and noods fuilhoi invosligalion.

4.8Cvtnchrnmc OxIdasc: Cvlochiono C oxidaso vas doloininod lasod upon oxidalion of foiiocvlochiono C lv
docioaso in alsoilanco al 55O nn on spocliopholonoloi. In 3 nI ioaclion nixluio conlaining 66 nM polassiun
phosphalo luffoi pH 7.4. 2OM ioducod cvlochiono C and 2OI nilochondiiaI pioloin ioaclion vas foIIovod al 3OC
foi lvo ninulos. Roading vas iocoidod afloi lvo ninulos foi alsoilanco of conpIoloIv oxidisod cvlochiono C lv
adding O.O5 M polassiun foiiicvanido. Tho aclivilv of cvlochiono C oxidaso vas oxpiossod in n noIos of
cvlochiono C oxidaso/ng pioloin/nin vhich is oquaI lo conconlialion of cvlochiono C x K dividod lv
conconlialion of pioloin. Ioi caIcuIalion of cvlochiono C conconlialion a niIIinoIai oxlinclion coofficionl of 19.1
vas onpIovod foi ioducod vs oxidlaod cvlochiono C al 55O nn as dosciilod lv LnnoIol ol aI.1964.

4.9 InhIbItInn nf NADPH cvtnchrnmc P450 Rcductasc

Cvlochiono I 45O: Cvlochiono I 45O vas noasuiod lv spocliopholonoloi in alsonco oi piosonco of cailon
nonoxido and doloininod lv lho nolhod |Cuongoiich ol aI.2OO9!. In liiof, O.1 nI niciosonaI fiaclion in lvo cuvollos
losl and slandaid vhich noio addod 1,5 ng sodiun dilhionilo and cailon nonoxido passod in onIv losl sanpIo
cuvollo foi 25 soconds and sano iopoalod again and AA iocoidod al 45O nn foi cvlochiono I 45O conlonl in n noIos
poi ng pioloin caIcuIalod lv oxlinclion coofficionl 91 nM/cn palh Ionglh.

4.10InhIbItInn nf Adcnv!atc cvc!asc
Adcnu|a|c cuc|asc rcgu|a|cru prcpcr|ics: AdonvIalo cvcIaso onzvno in isoIalod hopalocvlos has loon iopoilod as looI of
ioguIaloiv sludios of hoinonos, onzvnos in lho piosonco of Cs lound iocoplois |SnaII ol aI. 2OOO!. Monliano
adonvIalo cvcIaso aclivilv is dofinod duo lo ils nuIli-pass liansnonliano pioloin nado of lvo cvlosoIic sognonls as
shovn in Iiguio 4. Thoso sognonls pIav aclivo ioIos in associalion of Cs-CTI. Tho CTI inducod Cs linding vilh
adonvIalo cvcIaso can lo a kov nochanisn of adonvIalo cvcIaso slinuIalion oi inhililion duiing piogosloiono lound -
adionoigic iocoploi |Sunahaia ol aI. 1997!. Tho possilIo nochanisn of adonvIalo cvcIaso aclivalion oi inhililion lv
piogosloiono is shovn in Iiguio 4. Tho nonliano adonvIalo cvcIaso aclivilv can lo an indicaloi of nonliano
vialiIilv. Hovovoi, lho onzvno aclivilv doponds on lho lvpo and naluio of offocloi. Tho lohavioi of Iivoi coIIs in cuIluio
in lho piosonco of offoclois nav indicalo lho ioguIaloiv offocl of slinuIalois in titc. Hovovoi, in cuIluios, hopalocvlos
and Kupffoi coIIs nav IikoIv oxpoiionco diffoionl phvsioIogicaI onviionnonl diffoionl fion in titc. In conlioIIod Iivoi
coII cuIluio condilions of nodiun, lho Iivoi coIIs shovod adonvIalo cvcIaso slinuIalion in a spocific nannoi lv offoclois
such as piogosloiono, nilioinidazoIo, CTI, CITI, and pioslagIandins |Aoshila ol aI. 1997!. Tho iocoplois foi
pioslagIandins inloiacl vilh Cs sulunils. Cs-CTI conpIox linds vilh -adionaIgic iocoploi nado of sovon
sulunils as shovn in Iiguio 5. inding of iocoploi vilh Cs-CTI pioloin inhilils adonvIalo cvcIaso aclivilv and cAMI
foinalion. Tho piosonl sludv highIighls lho lohavioi of Iivoi coIIs and lho chaiacloiislic foaluios of lhoii cvcIic AMI
dopondonl adonvIalo cvcIaso ioguIaloiv svslon in cuIluio.

Prcgcs|crcnc ncdu|a|icn cf adcnu|a|c cuc|asc ac|iti|u: Iiogosloiono noduIalos lho aclivilv of adonvIalo cvcIaso and
adonvIalo cvcIaso iosponso is hoinono sonsilivo |CiInan ol aI. 1984!. Tho piogosloiono slinuIalos lho iocoploi
aclivaling lho adonvIalo cvcIaso lv slinuIaloiv Cs-CTI conpIox iosuIling vilh foinalion of cAMI. Hopalocvlos
vilh piogosloiono addod in nodiun oxhililod lho onhancod onzvno aclivilv. Tho addilion of CTI slinuIaloi did nol
chango lho adonvIalo cvcIaso aclivilv. In conliasl, CITI slinuIalod coIIs oxhililod an appaionl adonvIalo cvcIaso
aclivalion. UnIiko lho adonvIalo cvcIaso onzvno aclivilv in lho piosonco of diffoionl offoclois in slinuIalod hopalocvlos,
lho ialos of cvcIic AMI pioduclion vas doIavod in conlioI hopalocvlos. Hovovoi, piogosloiono addilion lo
hopalocvlos did nol oxhilil significanl doIav in CITI slinuIalod adonvIalo cvcIaso aclivilv. Thus ioason can lo
spocuIalod lhal piogosloiono linds vilh slinuIaloiv iocoplois on hopalocvlos vhich Ioad lo incioaso in inliacoIIuIai
cvcIic AMI accunuIalion lhiough adonvIalo cvcIaso aclivalion via guinino nucIoolido ioguIalod nochanisn |Cohon-
Tannoudji ol aI. 1991!. Tho Kupffoi coIIs aIso shovod siniIai guanino ioguIaloiv onzvno piopoilios.
Tho piogosloiono iosponso of adonvIalo cvcIaso aclivilv is a lvo phaso piocoss |Ko, ol aI.1999: Mailin ol aI. 1987!.
Iiisl, piogosloiono iocoploi and calaIvlic sulunil (C conpIox) of adonvIalo cvcIaso occupv lho pIaco on nonliano
foIIovod lv Cs pioloin inloiaclion vilh onzvno lo nako C pioloin C conpIox |Ko ol aI. 1999!. Hovovoi, lho
addilion of CITI slinuIalod lho adonvIalo cvcIaso lo a gioaloi oxlonl in hopalocvlos noio lhan pio-oxposod hopalocvlos
lo piogosloiono. RoconlIv, C pioloin-onzvno conpIox foinalion lv CTI/CITI slinuIalion has loon ioviovod |Niu ol
aI. 2OO3!.
Ni|rcinidazc|c s|inu|a|icn cf adcnu|a|c cuc|asc ac|iti|u. NilioinidazoIo is ioconlIv onoigod as anli-lunoi and
iadiosonsilizoi conpound. Il is usod in lunoi inaging, lhoiapv and anli-anoolic appIicalions. NilioinidazoIo has
phonoIic iings and phonoIic sido chains as shovn in Iiguio 6. So, il is olvious lhal lho sido chains inloifoio vilh lho
dophosphoivIalion of ATI lv phosphodiosloiaso onzvno. Il appoais lo iosuIl vilh inhililion of hopalocvlo adonvIalo
cvcIaso calaIvlic sulunil funclion in lho piosonco of nilioinidazoIo. Tho cvloloxicilv of nilioinidazoIo is pooiIv sludiod
and iopoilod. Hovovoi, ils anli-anoolic offocl has loon piovon lo docioaso hopalic and inloslinaI infoclion ialo. Tho
nilioinidazoIo is vidoIv iopoilod as singIo doso in lioalnonl of anoolic Iivoi alscoss. Moioovoi, nilioinidazoIo
inducod cvloloxic offocl on hunan hopalocvlos aio sliII nol knovn. DNA sliand lioakago, onoigv ioguIaloiv onzvnos,
phagocvlosis, and innuno iosponso havo loon iopoilod as nilioinidazoIo inducod cvloloxic offocls in aninaIs |Zhong
ol aI. 1997: Whilakoi ol aI. 1992: Singh ol aI. 1991!. Tho ioIo of nilioinidzoIo is undoislood lo iosloio onoigv ioguIaloiv
piocoss invoIving adonvIalo cvcIaso, phosphodiosloiaso, cAMI foinalion in hopalocvlos and iopoilod as hoinono and
CTI spocific in aclion. Tho nilioinidazoIo doiivalivos inhililod anloiioi piluilaiv coII funclion appaionlIv lv lhoii
diiocl offocl on lho calaIvlic sulunil of lho adonvIalo cvcIaso hoIoonzvno |SlaIIa ol aI. 1989!. Tho nonpaionchvnaI
Kuffoi coIIs pIav a significanl ioIo of phagocvlosis sonsilivo lo pioslagIandins. RoconlIv, nilioinidazoIo doiivalivos
havo loon idonlifiod as anlicancoi polonliaI agonls. SliII lho ioIo of inidazoIos as anlicancoi agonl is nol oslalIishod
|Andoison ol aI. 2OO6!. Tho piosonl sludv indicalos lho associalion of inidazoIo iing sliucluio vilh lho cvcIic AMI
dopondonl adonvIalo cvcIaso calaIvlic sulunil ioguIaloiv nochanisn in Iivoi coIIs.


Iiguio 14: AdonvIalo cvcIaso onzvno aclivilv is shovn in conlioI, nilioinidazoIo, piogosloiono and
anoolic Iivoi alscoss iociuilod Iivoi coIIs in piosonco of CITI/CTI slinuIalois, pioloin* addlivos.

5 InhIbItInn nf Drug mctabn!IzIng cnzvmcs In Isn!atcd !Ivcr cc!!s:
Diug nolaloIizing onzvnos aio alundanl in niciosonos and Ivsosonos. Mosl of lhoso onzvnos aio oIovalod oi
sociolod al lho silos of infIannalion, disoaso condilions and in piosonco of foioign lodios. An oIovalod onzvno in
coIIs and sociolion fion Ivsosonos oi niciosconos aclion is in dofonso lv 'suicido aclion of niciolodv againsl
disoaso oi foioign lodios. Diugs inhilil lhoso onzvnos oi liing lack anv disoaso oi infIannalion inducod offocl on
onzvno aclivilv in Iivoi coII. Chaiacloiislic inhililion of onzvno aclivilios in diffoionl gioups lv nilioinidazoIo aio
shovn in Iiguio 15. RoconlIv, vo iopoilod lho onzvno inhililion lv nilioinidazoIo in dolaiI |Shaina 2O11a, Shaina
2O11l!. In gonoiaI, IvsosonaI onzvnos aio poplidasos, acl al acidic pH, and pailicipalo as loininaI scavongois of
hainfuI anions, iadicaIs and poplidos.

NilioinidazoIo inhilils lho Iivoi onzvnos lv naking adducl foi gIvoxvI-gIulanino nolaloIisn in Iivoi coIIs as
aulhoi dosciilod in dolaiI |Shaina, ol aI. 2O11!.


Iiguio 16: LvsosonaI and niciosonaI onzvnos in isoIalod nonpaionchvnaI Iivoi coIIs aio shovn in conlioI,
nilioinidazoIo, piogosloiono, anoolic Iivoi alscoss iociuilod Iivois. Nolo inhililoiv aclion of nilioinidazoIo on Iivoi
coIIs lo liing noinaI aclivilios in conpaiison lo anoolic Iivoi alcoss iociuilod coIIs.
6 PhagncvtnsIs
AIlhough lho cIoaianco and disliilulion of Iigand noIocuIos in ciicuIalion iopiosonl lho funclion of hopalic
sinusoidaI coIIs, lhoso nochanisns iovoaI a nolvoik lhal is noio inliicalo lhan vouId al fiisl soon, sinco sovoiaI
iocoplois aio connon lo nol onIv ono lvpo of coII, lul aIso lo lvo oi lhioo lvpos of coIIs in lho Iivoi. In lho caso of Ialox
pailicIos in vhich lhoii uplako lv a pailicuIai coII lvpo soons lo lo doloininod lv lhoii sizo, sinusoidaI ondolhoIiaI coIIs
aio alIo lo inloinaIizo pailicIos up lo O.23 nicions undoi phvsioIogic condilions, in vivo, and Iaigoi pailicIos aio lakon
up lv Kupffoi coIIs. Hovovoi, vhon lho phagocvlic funclion of Kupffoi coIIs voio inpaiiod lv fiog viius oi aIcohoI,
lho ondolhoIiaI coIIs voio iopoilod lo lako up pailicIos Iaigoi lhan 1 nicion in dianoloi afloi lho injoclion of an oxcoss
anounl of Ialox pailicIos. LndolhoIiaI coIIs vouId lhus conslilulo a socond Iino of dofonso in lho Iivoi in lhal lhov
ionovo foioign naloiiaIs fion lho lIood vhon Kupffoi coII phagocvlic funclion is lolaIIv disluilod |124!. This polonliaI
ioIo nav nol, hovovoi, lo fuIIv oxpiossod undoi phvsioIogic condilions vhon Kupffoi coIIs aio aclivo in cIoaiing
foioign sulslanco fion lho ciicuIalion. Tho funclions of Iivoi sinusoidaI coIIs aio vaiiod and conpIox and lhoso coIIs can
lo iogaidod as "a sinusoidaI coII unil." This coIIuIai inloiaclion nusl lo lakon inlo accounl foi anv quanlilalivo anaIvsis
|125!.
O Enzvmc InhIbItInn In hagncvtnsIs: In oui oxpoiinonls, Iivoi coII cuIluios voio nixod vilh zvnosan (7:1
pailicIo coII ialio): oivlhiocvlos (5:1 pailicIo coII ialio): anolic liophozoils (2:1 pailicIo ialio), Ialox (2O:1 pailicIo coII
ialio) and nilioinidazoIo vas addod 1 M/1O
6
coIIs. Kupffoi coII phagocvlic aclivalion in isoIalod Kupffoi coIIs fion
hunan Iivois (addod vilh anoolic liophozoils) vas oIovalod 2-3 foId ovoi noinaI Kupffoi coIIs. Kupffoi coIIs
inculalod vilh zvnosan and liophozoils oxhililod naxinun phagocvlic aclivilv vhiIo vilh Ialox and shoop
oivlhiocvlos lhoso oxhililod nininun and insignificanlIv incioasod aclivilv iospoclivoIv. Incioasod phagocvlic aclivilv
vas lino dopondonl vhich vas oxhililod lv . gIucuionidaso, Iaclalo dohvdiogonaso and pIasninogon aclivaloi
aclivilv. Ihagocvlosis of zvnosan and shoop oivlhiocvlos liiggoiod lho innodialo ioIoaso of . gIucuionidaso,
slinuIalod svnlhosis of coIIuIai Iaclalo dohvdiogonaso and inducod doIavod pioduclion vilh sociolion of pIasninogon
aclivaloi. Inhililion of lhoso onzvnos as nilioinidazoIo iosponso is shovn in Iiguios in dolaiI. Majoi findings voio: 1.
offocl of nilioinidazoIo and Ialox voio nininun. SiniIai olsoivalions voio iopoilod in oaiIioi sludv |126,127,128!: 2.
nilioinidazoIo vas lioliansfoinod inlo valoi soIulIo pioducls and cIoaiod lv diug nolaloIizing onzvno svslon of
Kupffoi coIIs: 3. Ialox pailicIos loing inoil confiinod lho indopondonco of Kupffoi coII aclivalion fion inliacoIIuIai falo
of ingoslod naloiiaI: 4. anoolic liophozoils vilh lhoii loxins oi spocific nonliano suifaco signaIs slinuIalo Kupffoi
coIIs foi phagocvlosis iosuIling oaiIv ioIoaso of IvsosonaI hvdioIasos lhan lho ioIoaso lv olhoi pailicIos |129, 13O!.
Hovovoi, incioasod IvsosonaI onzvnos in Kupffoi coIIs inculalod vilh anoolic liophozoils voio iopoilod as a
consoquonco of phagocvlic aclivalion |13O!. RoconlIv, lhoso phagocvlic aclivalion changos duo lo nilioinidazoIo havo
loon iopoilod as associalod vilh pioslagIandin svnlhosis and lhoii ioIoaso |131!.
O Tho gIucuionidaso aclivilv shovod lho onhancod onzvno aclivilv liond in aII addilivos lul diffoionl onzvno
onhancononl foi diffoionl addilivos. In conlioI coIIs, lho aclivilv onhancod duiing 24-36 houis in inculalion. Tho
oivlhiocvlos shovod nosl vuInoialiIilv lo Kupffoi coIIs in iniliaI 24 houis. Zvnosan loads shovod sIov iosponso lo
phagocvlosis vhiIo Ialox ionainod inoil lo Kupffoi coIIs. Tho nilioinidazoIo shovod noinaI onzvno palloin. Il vas
inloiosling lhal naxinun ialo of phagocvlosis vas olsoivod duiing 24 and 36 houis oxcopl oivlhiocvlos.
O Laclalo dohvdiogonaso onzvno aclivilv shovod lho onhancod onzvno aclivilv liond in aII addilivos oxcopl Ialox
addilivo lul diffoionl onzvno onhancononl foi diffoionl addilivos. In conlioI coIIs, lho aclivilv onhancod duiing 12-36
houis in inculalion. Tho oivlhiocvlos shovod nosl vuInoialiIilv lo Kupffoi coIIs in iniliaI 24 houis. Zvnosan loads
shovod naxinun iosponso lo phagocvlosis vhiIo Ialox ionainod inoil lo Kupffoi coIIs. Tho nilioinidazoIo shovod
inhililoiv onzvno aclivilv in Kupffoi coIIs lul afloi 24 houis lho iosponso vas slinuIaloiv lo onzvno aclivilv (doIavod
onhancod onzvno slinuIalion as a iosuIl phagocvlosis). Il vas inloiosling lhal naxinun ialo of phagocvlosis vas
olsoivod duiing 48 houis. Hovovoi, lho Ioakod oul LDH aclivilv iopiosonlod lho piolalIo phagocvlic aclivilv of
Kupffoi coIIs ioIalionship vilh lino. NilioinidazoIo addilion in inculalion nodiun poihaps shovod lho nininizing
anoolic viiuIonco |132!. LaiIioi IvsosonaI onzvno ioIoaso vilh incioasod LDH coIIuIai IovoIs has loon iopoilod |133!.


Iiguio 17: Ihagocvlic aclivilv of Kupffoi coIIs is shovn as gIucuionidaso onzvno aclivilv of coIIs afloi adding
zvnosan, oivlhiocvlos, onlanoola hisloIvlica liophozoils, Ialox loads addilivos, and nilioinidazoIo as diug in
cuIluios of Kupffoi coIIs fion Iivoi liopsv sanpIos. Tho onzvno aclivilv vas noasuiod afloi 1, 2, 3, 4 davs of
inculalion. Lach poinl vaIuo iopiosonls lho avoiago of 3 ioadings of olsoivalions.


Iiguio 18: Ihagocvlic aclivilv of Kupffoi coIIs is shovn as Iaclalo dohvdiogonaso onzvno aclivilv of coIIs afloi adding
zvnosan, oivlhiocvlos, onlanoola hisloIvlica liophozoils, Ialox loads addilivos, and nilioinidazoIo as diug in
cuIluios of Kupffoi coIIs fion Iivoi liopsv sanpIos. Tho onzvno aclivilv vas noasuiod afloi 1, 2, 3, 4 davs of
inculalion. Lach poinl vaIuo iopiosonls lho avoiago of 3 ioadings of olsoivalions.

O Tho pIasninogon aclivaloi inducod Ivsis of Kupffoi coIIs shovod lho onhancod pIasninogon aclivilv onIv afloi 36
houis. IniliaIIv lho aclivilv liond in aII addilivos vas sano and innuno lo anv addilivo addod. In conlioI coIIs, lho
pIasninogon aclivilv ionainod unaIloiod vhiIo kupffoi coIIs shovod innunilv lo zvnosan and Ialox addilivos
lhioughoul lho inculalion poiiod. Hovovoi Kupffoi coIIs shovod shaip onhancononl of pIasninogon aclivilv afloi 36
houis duiing 36-48 houis in inculalion. Tho liophozoils shovod naxinun offocl on pIasninogon aclivaloi in Kupffoi
coIIs afloi 36 houis. IIasninogon aclivaloi induclion has loon iopoilod dopondonl on do novo onzvno svnlhosis in
slinuIalod naciophago lv ondoloxins and asloslos |134!.

Iiguio 19: Ihagocvlic aclivilv of Kupffoi coIIs is shovn as pIasninogon aclivaloi aclivilv of coIIs afloi adding
zvnosan, oivlhiocvlos, onlanoola hisloIvlica liophozoils, Ialox loads addilivos, and nilioinidazoIo as diug in
cuIluios of Kupffoi coIIs fion Iivoi liopsv sanpIos. Tho onzvno aclivilv vas noasuiod afloi 1, 2, 3, 4 davs of
inculalion. Lach poinl vaIuo iopiosonls lho avoiago of 3 ioadings of olsoivalions. Lach poinl on giaph iopiosonls lho
aclivilv as oxlonl of coII Ivsis in poiconl incioaso ovoi lasaI vaIuo of 1OO.

7 DNA svnthcsIs In Kuffcr cc!!s

SlinuIalod DNA svnlhosis is considoiod as dofonso nodo. Iion phvsioIogicaI poinl of viov, il is liuo lhal diaslic
chango in onzvnalic palloin in Iivoi iogonoialion poinils DNA svnlhosis. Majoi onzvno pIavois aio DNA poIvnoiaso
and DNaso (aIIosloiic conpIox onzvno) in nilosis. Wo piosonl fivo soquonco slops of ovonls: dooxviilonucIoolidos,
iilonucIoolidos, DNA, RNA and pioloins (soo Iiguio 2O) |iosnick ol aI.197O!.
O DNaso oxposos 3-OH ond gioups lhus incioasos capacilv of chionosonaI DNA in vivo. DNA poIvnoiaso pIavs a
passivo ioIo in DNA svnlhosis in piosonco of high chionosonaI DNA and dooxviilonucIoolidos. Iiining DNaso aclion
on nalivo and donaluiod DNA inilialos DNA svnlhosis. LaiIioi sludv iopoilod lhal aiginaso onzvno acls sliong DNA
svnlhaso inhililoi poihaps duo lo Iack of noosvnlhosizod hislono (aiginino-iich hislono) lv ioducing piining slalo of
chionosonaI DNA. DNA svsnlhosis lv noans of addilivo oi iopIicalivo DNA poIvnoiaso noods 3-OH ioup and
poIvnoiizalion piocoods fion 5-phosphalo ond lo lho gioving 3-OH ond.
O Incioasod RNA svnlhosis aIso incioasos iilonucIoolido pooI, iiso in RNA poIvnoiaso aclivilv, incioaso in
poIvanino svnlhosis and s-RNA nolhvIalion. Tho lonpoiaI ioIalionship lolvoon kinolic changos in lho DNA channoI
and lho RNA channoI soon lo indicalo lhal RNA channoI is piinoi of DNA channoI. In olhoi voids, cvcIic AMI IovoI
nav inilialo lolh DNA and RNA svnlhosis loginning al lho nucIoolido IovoI undoi hoinonaI ioguIalion. osl oxanpIo
of oIovalod RNA svsnlhosis in Iivoi iogonoialion is oIovalod oiolalo pviophosphoivIaso/docailoxvIaso lo koop
CTI/UTI pooI in piosonco of UTI/CDI coonzvnos foi gIvcogonosis and Iipogonosis iospoclivoIv. UMI slinuIalod
oiolalo-lRNA IaloIing aiound 18s-28s RNA conpIox slop is piocodod lv an incioaso in lRNA nolhvIalion lv lRNA
MolhvIaso onzvno. Ioi ils aclion, shoil chain of nonocislionic nRNA on 18s conpononl acls as piinoi foi svnlhosis of
conslilulivo RNA and oxpoil pioloin svnlhosis. Lasl voai, NoloI Iiizo vas avaidod in 2OO9 on iosoIving lho inliicacios
of iilosonaI sulunil inloipIav and consoquonl ioIo of DNA/RNA IoIvnoiaso onzvno ioaclions in pioloin svnlhosis
duiing diug inducod iogonoialion |Schnoing ol aI.2OO9!. Hovovoi, olhoi onzvnos RNaso aclivilv, calaionic poIvanino
pIav ioIo in slaliIizalion of iilosonaI RNA (IaloIod nolhionino lo spoinidino) in pioloin svnlhosis. Incioasod
poIvnino svnlhosis onhancos oinilhino docailoxvIaso lo incioaso anino acid pooI in piosonco of cAMI inducod
pioslagIandins nodialod giovlh hoinono. Moioovoi, giovlh hoinono slinuIalos lhvnidino kinaso in Iivoi. Aulhoi
dosciilos DNA svnlhosis in isoIalod Kupffoi coIIs duiing Iivoi iogonoialion afloi nilioinidazoIo lioalnonl in anoolic
Iivoi alscoss in foIIoving soclion.

O Kupffoi coIIs isoIalod fion hunan anoolic Iivoi alscoss Iivoi-liopsios and piogosloiono lioalod Kupffoi coII in
cuIluios voio inducod foi DNA svnlhosis lv adding nilioinidazoIo. This offoiod a sinpIo nodoI lo sludv lho
nochanisn of coII DNA induclion and inloidopondonco lolvoon lvo coII popuIalions. LaiIioi such inloiaclions
lolvoon coIIs voio iopoilod ioguIalod lv lhvnidino kinaso (lhvnidino palhvav) and iilonucIoolido ioduclaso
(lhvnidvIalo palhvav)|iosnick ol aI.197O!. ConcopluaIIv, innodialoIv afloi disoaso in iogonoialion, Iivoi coII gols
confionlod a chaIIongo of incioasod pioloin svnlhosis foi coII division invoIving nolaloIic onzvnos Ioading lo DNA
svnlhosis (linding of iilonucIoolidos vilh dooxviilonucIoolidos). Tvo palhvavs pIav ioIo in lhis piocoss lv
Thvnidino palhvav and lhvnidvIalo palhvav. ThvnidvIalo palhvav foi DNA svnlhosis noods dTTI suppIv,
lhvnidino kinaso, TDI kinaso lo koop TMI pooI. asicaIIv, a iopiossoi noIocuIo (lhvnidino) inhilils liansciiplion of
lhvnidino kinaso lv aIIosloiic ioguIalion. Thvnidino kinaso is a TTI insonsilivo dinoiic foin (ono calaIvlic silo and
olhoi aIIosloiic silo) noans Kn foi onzvno docioasos vilh oIovalod ATI conconlialion (ATI posilivo offocloi and TTI
nogalivo offocloi). dinoiic foin and shovs signoid lohavioi of lhvnidino kinaso |aiioso ol aI. 2OO5!. Aclivalion of
lhvnidino kinaso aIso doponds on cvcIic AMI aclivalod pioloin kinaso. On olhoi sido, iilonucIooliso ioduclaso ioducos
CDI in piosonco of dCMI doaninaso lo nako dUMI (a sulslialo foi lhvnidvIalo svnlhosis) lv lhvnidvIalo
svsnlholaso in piosonco of N
5
,N
1O
hvdioxvnolhvI-IH4. dCMI is good onzvno inhililoi of lhvnidvIalo svsnlholaso and
aclivaloi of foIalo ioduclaso lo koop nol high TMI pooI. RilonucIoolido svnlhosis is ioguIalod lv iilonucIoolido
ioduclaso. Incioaso in CTI luiId up dCTI, puiino dooxviilonucIoolidos and TTI pooIs|Choung ol aI.197O!.

O
SovoiaI lvpos of giovlh faclois havo loon iopoilod as ioguIaloiv sulslancos pailicipaling in Iivoi iogonoialion
piocoss. SlinuIalion of DNA svnlhosis in Kupffoi coIIs lv anoola liophozoilos is shovn hoio as iosponso inducod lv
coIonv slinuIaling facloi. Sinco noasuiod incioaso of iadio IaloI uplako lv incioasing anoola liophozoilo
conconlialion in nodiun iofIoclod an incioaso of coII nunloi onloiing inlo S phaso ialhoi lhan highoi ialo of |
3
H! TdR
incoipoialion soon in aII lho coIIs, il vas possilIo lhal diffoionl Kupffoi coIIs noinaI oi piolioalod vilh anoola (fion
lhoso diffoionl Iivoi coII popuIalions) nav vaiv in suscopliliIilv lo coIonv slinuIaling facloi (CSI). High
conconlialions of anoola causod a ioduclion of lhvnidino uplako and ionaikalIv ioduclion vas noasuiod on vhoIo
covoi sIip scinliIIalion counls. Il iofIoclod Iovoi incoipoialion of |
3
H! TdR inlo oach DNA svnlhosizing coIIs. This
phonononon nav lo duo lo ioducod capacilv of Kupffoi coIIs uplako as a iosuIl of DNA svnlhosis lul il ioquiios
fuilhoi inhililoi sludv oi piosonco of CSI. Thus highoi conconlialion of anoolao liophozoilos causod a ioducod
Kupffoi coII uplako capacilv duiing CSI pioduclion.


Iiguio 21: DNA svnlhosis in Iivoi coIIs in cuIluio nodiun is shovn in piosonco of naciophago giovlh facloi
noasuiod as cpn/fIask and cpn/covoi sIip duiing 4-1O davs. Nolico lho nilioinidazoIo inducod inhililion of DNA
svnlhosis in anolic lioalod coIIs.

8 InhIbItInn nf !Ivcr !vsnsnma! cnzvmcs In PrntcIn MctabnI!Ism
IsoIalod IvsosonaI piopaialions fion L|1-
14
C! Ioucino (25 3Ci poi 1OO gn vl) inliapoiiloniaIIv injoclod hunan
Kupffoi coII cuIluios oxhililod poiconl dogiadalion al pH 3.O, 4.O,5.O, 6.O,7.O, 8.O and 9.O al lonpoialuios 1O C, 2O C, 3O
C, 4O C, 5O C and 6O C, in piosonco of anoolic liophozoils afloi 15 ninulos(shoil Iivod) and 15 houis(Iong-Iivod)
addilion as shovn in Iiguios 23. Majoi pIavois in lhis Iivoi iogonoialion piocoss aio iilosonos (silling on ondopIasnic
iolicuIun), CTI (iilolido pooI) and coonzvnos foi iilolido poIvnoiaso onzvnos (a laIanco in lhvnidino kinaso and
lhvnidvIalo svnlholaso). Olhoi onzvno inhililion is LDH-M lvpo gols convoilod lo LDH-H lvpo oi Iaclalo pviuvalo
(accoIoialod gIvcoIvsis). In sinuIlanoous olhoi sol of oxpoiinonl, Kupffoi coII Ivsosonos in piosonco of Ioupoplin,
popslalin inhililois and nilioinidazoIo in 1OO 3g, 1OO 3g and 5O nM poi nI conconlialion iospoclivoIv shovod aivI
suIphalaso onzvno aclivilv changos (soo Iiguios 21, 23) in lho piosonco of O.3 M sucioso al 37 C foi 6O ninulos al pH
5.5 in inculalion nixluio of Kupffoi coII Ivsosonos and addilivos. IiolooIvsis ialo and dogiadalion of shoil and Iong
Iivod pioloins in isoIalod Ivsosonos fion lho Kupffoi coII cuIluios addod vilh liophozoils, nilioinidazoIo, oxhililod
incioasod pioloin dogiadalion. Lvsosono as piolooIvlic conpononl vas shovn lo conliilulo foi pioloin dogiadalion
as oaiIioi vaiious iopoils indicalod pioloin sizo dopondonco |Noff ol aI.199O!. IiolooIvlic ialo in isoIalod Ivsosonos
incioasod sovoiaI foId vilh docioasod inliaIvsosonaI pH. Hovovoi, Ioakago of hvdioIvlic onzvnos (oIovalod oiolalo
phosphoivIaso/docailoxvIaso) nav causo dogiadalion of oxlia IvsosonaI oiolalo sulslialos. Thus inculalion lino
shouId lo shoil. Dogiadalion of shoil and Iong-Iivod pioloins in isoIalod Ivsosonos dononslialos lhal afloi 15 ninulos
of |
14
C! Ioucino IaloIing, lhoso Ivsosonos voio aclivo in dogiadalion of shoil lino IaloIod pioloins. Sinco liansil lino
foi IvsosonaI onzvnos fion ondopIasnic iolicuIun lo Ivsosonos vas aiound 2-3 houis |Zhu ol aI. 1994!. So afloi, 15
ninulos sono olhoi pioloins nav lo lhoughl lo onloi Ivsosonos. Tho dogiadalion ialo in isoIalod Ivsosonos vas
sonolinos fasloi foi shoil lino IaloIing locauso of shoil Iivod pioloins as cvlosoIic oi socioloiv in naluio. On lho olhoi
hand, nonliano pioloins had haIf Iivos in iango of fov davs. So lhoso Iong IaloIod pioloins voio dogiadod al diffoionl
ialos vilhin Ivsosonos. Moioovoi shoil Iivod pioloins voio iopoilod noio suscoplilIo lo digoslion lv IvsosonaI
pioloasos. Tho offocl of piolooIvlic inhililois on pioloin lioakdovn in isoIalod Ivsosonos foi dislinguishing IvsosonaI
fion oxlia IvsosonaI pioloin dogiadalion vas dopondonl upon coII lvpo, cuIluio nodiun, IaloIing schoduIo and
inhililois usod. Inhililois of IvsosonaI onzvnos inpodo lhoii supposod laigol of aclion viz. lho Ivsosonos. ul
conpIolo inhililion of piolooIvsis couId nol lo possilIo duo lo IvsosonaI and nonIvsosonaI piolooIvsis. Iopslalin
inhililod onIv soIoclivo aspailic pioloinaso calhopsin D, Ioupoplin inhililod lhioI pioloinaso. SiniIaiIv nilioinidazoIo
oilhoi inliaIvsosonaI pH and iosloiod pioloinasos. Moioovoi, calhopsin D inhililion lv popslalin, calhopsin , H and
L inhililion lv Ioupoplin aio iopoilod connonIv |Kalununa, 2O11!. Thus Ivsosonos voio aclivo in dogiadalion of
lolh shoil and Iong Iivod pioloins duiing lasaI slalo. Infoclod Kupffoi coII Ivsosonos oxhililod noio pionouncod
oIovalod IvsosonaI dogiadalion al pH 5.O al lonpoialuio iango 3O HC-4O HC.



Iiguio 22: Iiguio shovs lho offocl of of iadioIaloIod |
14
C!-Loucino in Kupffoi cuIluios iopiosonlod lv aivI
suIphalaso onzvno aclivilv in noinaI and ALA iociuilod coIIs al diffoionl pH and lonpoialuios. Nolico lho Iov
onzvno aclivilv in noinaI coIIs vhiIo naxinun Ioucino dogiadalion vas al pH 6 and 4O C.


Iiguio 23: Iiguio shovs lho offocl of iadioIaloIod |
14
C!-Loucino in Kupffoi cuIluios iopiosonlod lv aivI
suIphalaso onzvno aclivilv in noinaI and ALA iociuilod coIIs. Nolico lho lhioshoId of Ioucino luinovoi al
4O IU in loins of onzvno aclivilv. ConlioI coIIs shovod sIov pioloin dogiadalion and Iov onzvno
conconlialion.

Iiguio 24: Iiguio shovs a conpaiison of piolooIvsis inhililion and pioloin dogiadalion in piosonco
of nilioinidazoIo, popslalin and Ioupoplin. IiolooIvsis is iopiosonlod in loins of aivIsuIphalaso onzvno
aclivilv. Nolico lho sizo of poplido Ionglh nakos diffoionco shovn lv * foi Iong duialion and shoil
duialion of ioaclion in oach addilivo. ConlioI coIIs shovod sIov pioloin dogiadalion and Iov onzvno
conconlialion.

9. LIvcr RcgcncratInn and rn!c nf Lvsnsnma! Enzvmcs

Duiing nolaloIic inlogiilv Ioss and iosuIlanl hvpoxia slalo, olhoi faclois in Iivoi coII signaI lho aIain lo pioduco
niliic oxido, ioIoaso of cvlokinos (IIN- vilh IL-1 oi TNI-). Thoso changos aIso liiggoi lho soquonco of Kupffoi
coII slinuIalion and IvsosonaI aclion sinuIlanoousIv. As a iosuIl, IvsosonaI onzvnos, giovlh facloi, pIasninogon
slinuIaling facloi consislonlIv pailicipalo oi slinuIalo lho Kupffoi coII suicidaI phagocvlic aclion (hopalic nociosis)
lv nilioinidazoIo anaIogs and pioviousIv ioviovod |MichaIopouIos, 199O: Thuinan, 2OOO!. In diug inducod
hopalilis, Iivoi IvsosonaI onzvnos havo loon iopoilod oIovalod as slinuIalion of hopalocoIIuIai dofonso apail fion
inilialing iospiialoiv luisl and chonolaxis in Iivoi coIIs |Vician ol aI.2OO9!. Oui sludv shovod lho associalod oIoclion
nicioscopic olsoivalions lhal Kupffoi coIIs accunuIalo aiound lho silo of hvpoxia and oxhililod hvpoipIasia
condilion shoving dogonoialod nucIous, onIaigod IvsosonaI vosicIos |Shaina, 2OO9!. Tho onIaigod IvsosonaI
vosicIos voio fuilhoi coiioIalod lv oIovalod IvsosonaI onzvno IovoIs in soiun. In Iivoi liopsios, lhoso IvsosonaI
onzvnos voio significanlIv onhancod. Acid phosphalaso, Ioucino aninopoplidaso onzvnos calaIvzing
phosphoivIalion and anino-poplidizalion, soon lo lo sociolod noio and suggoslivo of aclivo pioloin dogiadalion. -
gIucuionidaso and aivI suIphalaso high onzvno sociolion vas suggoslivo of conlinuous lioakdovn of aivI
sulslilulod and -gIucuionidalion ioaclions duiing cvloIvsis and piolooIvsis |Knook ol aI. 1981!. No piovious dala is
avaiIalIo on Iivoi coII onzvnos oslinalod in Iivoi coIIs isoIalod fion Iivoi liopsv of nilioinidazoIo lioalod Iivois in
hvpoxia. Oui olsoivalions of onzvno inhililion in hvpoxic Iivoi coIIs aio voiv vaIualIo lo dosign a nilioinidazoIo
lasod iadiosonsilizoi oi diug foi paiasilos, nvocaidiaI ischonia, luloicuIosis, nouioloxicosis olc. Livoi coII onzvnos
nav oxlond lho lolloi liochonicaI oxpIanalion of conpIox hvpoxia slalo al noIocuIai IovoI. Hovovoi, sovoiaI
scalloiod sludios on soiun choIino osloiaso, aIkaIino phosphalaso, gIucoso-6I-dohvdiogonaso, oinilhino cailanovI
liansfoiaso, cvcIooxvgonaso-2, Iaclalo dohvdiogonaso onzvnos havo loon iopoilod significanl in hopalic hvpoxia oi
hopalic danago ovaIualion vilh addilion of nov nonlois of onzvnos |CaiIson ol aI. 1976: KoppoIo ol aI. 1991!.
Lnzvno inhililion in Iivoi coIIs is significanl infoinalion lo dosign nov Iivoi coII nolaloIic diug slinuIalois in
doloxificalion, ovaIualion of hvpoxia inaging piolos, and cuIlivalion of lvpo of nonoaxonic Iivoi coII cuIluios
(hopalocvlos and Kupffoi coIIs) vilh spocific onzvno piofiIo.

Thoio appoai lvo nain ioasons of onzvno IovoI hopalic coII iocovoiv lv nilioinidazoIo. Iiisl, aIloiod onzvno
changos voio iocovoiod lv nilioinidazoIo lhoiapv as diug inhilils lho onzvnos pailicipaling in onoigv slalus in
hopalocvlos and ioducos Kupffoi coII naciophagaI aclivilv vilh ioducod hopalic onzvno sociolion lo liing lack
onzvno IovoIs lo noinaI. Socond, iogonoialing hopalocvlos nav aIso ioguIalo lho noinaI coII iocovoiv piocoss and
iniliaIizo lho signaIing Kupffoi coIIs lo sloio onough IvsosonaI onzvnos.

10. Rn!c nf cnzvmc InhIbItInn In !Ivcr trans!antatInn and tIssuc cngInccrIng
Livoi iogonoialion afloi lho onzvno inhililion oi Ioss of hopalic lissuo is a fundanonlaI paianoloi of Iivoi iosponso
lo injuiv. Lnzvno inhililion phonononon is iocognizod as spocific oxloinaI slinuIi invoIving soquonliaI changos in
gono oxpiossion, giovlh facloi pioduclion, and noiphoIogic sliucluio. Aulhoi suns up an accounl on diffoionl
onzvnos in foIIoving dosciiplion:
Angiolonsin Convoiling onzvno inhililion lv IisinopiiI dogiados liadvkinin and sulsoquonlIv inducos Iivoi
iogonoialion in ials. Duiing lhis onzvno inhililion piocoss (Iivoi iogonoialion) inducos 3.4 S DNA poIvnoiaso
aclivilv in lolh cvlopIasnic fiaclion and nucIoai fiaclions as conslanl vhiIo 6 S lo 8 S DNA poIvnoiaso aclivilios in
lho cvlopIasnic fiaclion incioasod 6- lo 7-foId |RanaIho ol aI.2OO1: Chang ol aI.1972! Tho aclivilv of hislidino
docailoxvIaso vas naikodIv incioasod in iogonoialing Iivoi lissuos. Oinilhino docailoxvIaso is an onzvno calaIvzo
in poIvanino svnlhosis. Inhililion of oinilhino docailoxvIaso, hislidino docailoxvIaso, and olhoi anino acid
docailoxvIasos, vas iopoilod in iogonoialing ial Iivoi and sovoiaI ial lunois.|RussoII, ol aI.1969!. Anino svnlhosis in
iapidIv gioving lissuos: oinilhino docailoxvIaso aclivilv in iogonoialing ial Iivoi and vaiious lunois. Apail fion
onzvno inhililion, nanv giovlh faclois and cvlokinos such as nosl nolalIv hopalocvlo giovlh facloi, opidoinaI
giovlh facloi, liansfoining giovlh facloi-u, inloiIoukin-6, lunoi nociosis facloi-u, insuIin, and noiopinophiino,
appoai lo pIav inpoilanl ioIos in lhis onzvno inhililion and Iivoi iosponso. Manv ioviovs allonplod lo inlogialo in
Iasl lhioo docados and Iookod lovaid cIuos lo lho naluio of liiggoiing nochanisns in Iivoi and coIIuIai
iosponso.|MichaIopouIos ol aI. 1997!. Tho najoi ovonls aio signaIing cascados invoIving giovlh faclois, cvlokinos,
naliix ionodoIing, and sovoiaI foodlacks of slinuIalion and inhililion of giovlh ioIalod signaIs. Livoi nanagos lo
iosloio anv Iosl nass and adjusl ils sizo lo lhal of lho oiganisn, vhiIo al lho sano lino pioviding fuII suppoil foi
lodv honooslasis duiing lho onliio iogonoialivo piocoss |MichaIopouIos ol aI. 2OO7!.
O RoconlIv, ioIo of lissuo onginooiing concopl shovod sovoiaI advanlagos in Iivoi lianspIanlalion. Hovovoi, sovoiaI
faclois aio significanl in iogonoialion: naliicos dosign, coII nass up lo vhoIo-oigan oquivaIonls, oplinizod
nicioaichilocluio, coII-adhosion poplidos, giovlh faclois and oxliacoIIuIai naliix noIocuIos lo lho poIvnoiic
scaffoId, hopalic onzvnos in nicioonviionnonl |Moonov ol aI. 1992!. Tho uso of onzvno chaiacloiizalion is voiv
hoIpfuI in hopalocvlo cuIluio lo losl lioconpaliliIilv ioquiiod foi lho iolonlion of lissuo-spocific gono oxpiossion
|Kin ol aI. 2OOO: Vovlik-Hailin ol aI. 1997!. Livoi coII lianspIanlalion inlo poIvnoiic naliicos (coII-naliix-consliucls)
is onoiging as ioulino in hospilaIs lo nool alsoIulo ioquiiononl of lho lianspIanlod hopalocvlos foi hopaloliophic
faclois lhal Iivoi conslanlIv iocoivos lhiough ils poilaI ciicuIalion |SlaizI ol aI. 1973!. Apail fion coIIuIai lhoiapios,
lvo oxpoiinonlaI appioachos aio voilh nonlioning foi succossfuI cIinicaI liansIalion. Tho fiisl coII shool
lochnoIogv dovoIopod lv Sasagava ol aI.,2OO7. Oll ol aI. iopoilod poifusion docoIIuIaiizalion lo gonoialo vhoIo oigan
scaffoIds |Oll ol aI. 2OO8!.
.

Iig. 25. Hunan lioonginooiod Iivois aio highIv coIIuIai and dispIav sono of lho funclions olsoivod in nalivo hopalic
lissuo. (A) Macioscopic appoaianco of a soodod Iivoi lioscaffoId 7 davs afloi sooding vilh piinaiv hunan folaI Iivoi
and ondolhoIiaI coIIs. () H&L shoving lioad iocoIIuIaiizalion of lho lioscaffoId vilh lho foinalion of liIiaiv duclaI
sliucluios. (C) InnunofIuoiosconco slaining foi cvlokoialin 19 (iod) shoving a liIiaiv ducl foinod vilhin lho
lioscaffoId vilh a visilIo Iunon. (D) InnunofIuoiosconco slaining of cvlochiono I45O 3A (oiango) and (L) aIlunin
(puipIo) shoving gioups of hopalocvlic Iinoago coIIs oxpiossing lhoso noio naluio hopalic naikois. (I)
InnunofIuoiosconco slaining of oNOS (puipIo) shoving hUVLCs coaling and spioading fion vascuIai sliucluios
(aiiovs). AII nucIoi slainod vilh YO-IRO1 (gioon) oi DAII (lIuo). Ropioducod vilh poinission fion iofoionco
aplisla ol aI. 2O1O.

O In piovious iopoil, invosligalois dovoIopod a siniIai poifusion docoIIuIaiizalion nolhod foi lho Iivoi |aplisla ol
aI.2OO9! foi oigan lioonginooiing. Thoso lioscaffoIds piosoivo lhoii lissuo nicioaichilocluio and an inlacl vascuIai
nolvoik lhal can lo ioadiIv usod as a ioulo foi iocoIIuIaiizalion (inlacl onzvnos of ioguIaloiv nolaloIic ioaclions) lv
poifusion of cuIluio nodiun conlaining diffoionl Iivoi coII popuIalions. Significanl onzvno inhililion assavs aio usod
as lionaikois of Iivoi coII vialiIilv. In foIIoving oxanpIo, a lioonginooiod Iivoi (lho iogonoialod hopalic lissuo) is
shovn cIoaiIv visilIo lv lho nakod ovo afloi 7 davs in lho lioioacloi (Iig. 24A). Rogonoialod lissuo vas highIv
coIIuIai (Iig. 24) and dispIavs liIiaiv ducl sliucluios posilivo foi cvlokoialin 19 (Iig. 24C), as voII as cIuslois of
hopalocvlos oxpiossing cvlochiono I45O 3A and aIlunin (Iig. 24D, L, iospoclivoIv). Lxpiossion of ondolhoIiaI coII
niliic oxido svnlhaso (oNOS) onzvno in soodod hunan ondolhoIiaI unliIicaI voin coIIs (hUVLCs) voio aIso olsoivod
coaling and spioading fion vascuIai sliucluios (Iig. 24I). In olhoi appioach, Uvgun ol aI. docoIIuIaiizod ial Iivois
and iopopuIalod lhon vilh ial piinaiv hopalocvlos, shoving pionising hopalic funclion and lho aliIilv of
holoiolopicaIIv lianspIanling lhoso lioonginooiod Iivois inlo aninaIs foi up lo oighl houis |Uvgun ol aI. 2O1O!.
ioonginooiod Iivois dispIavod sono funclions of a nalivo hunan Iivoi (aIlunin and uioa sociolion, CYI45O onzvno
oxpiossion, olc) and aIso oxhililod an ondolhoIiaI vascuIai nolvoik lhal piovonlod pIaloIol adhosion and aggiogalion,
ciilicaI foi lIood vossoI paloncv afloi lianspIanlalion. Honco, lhis Iivoi iogonoialion lochnoIogv vilh piopoi aclivo
onzvno funclions has lho polonliaI lo liansIalo in lho fuluio inlo lho lioonginooiing of hunan sizo Iivois lo offoi
ioadiIv avaiIalIo Iivoi foi diug discovoiv appIicalions and foi Iivoi lianspIanlalion, ovoiconing oigan shoilago.

11.Cha!!cngcs, !ImItatInns In cnzvmc InhIbItInn
Thoio aio lvo najoi chaIIongos. Iiisl chaIIongo vas lo gol onough liopsv lo oslinalo sovoiaI onzvnos. Socond
chaIIongo vas lo chooso significanl onzvnos as iopiosonlalivo of hopalocoIIuIai ciiloiia. Tho nain Iinilalion vas lhal
lho 'hopalocoIIuIai hvpoxia ciiloiia vas usod in snaII nunloi of hunan suljocls. Tho liopsv sanpIos foi oIoclion
nicioscopv olsoivalions noods noio lhoioughIv conlioIIod oxpoiinonls. Olhoi Iinilalion vas lhal lho onzvno
oslinalions in soiun and liopsv sanpIos nav nol lo poifocl iopiosonlalivo sanpIos and il noods lo oslalIish lho
noasuialIo and acluaI onzvno aclivilios in coIIs. Tho nosl ciuciaI issuo is lhal hvpoxia is a conlinalion of soquonco
of sovoiaI nolaloIic and sulphvsioIogicaI ioaclions in coIIs. Moioovoi, olhoi infIannaloiv cvlokinos, apoplosis,
niliic oxido pioduclion and phagocvlosis aio associalod changos duiing hvpoxia. Il fuilhoi noods liacoi lochniquo lo
liack lho dolaiIs of hvpoxia in coII.

12.Currcnt dcvc!nmcnts and futurc rnsccts
NilioinidazoIos aio iodiscovoiod anlianoolic diugs having gioal polonliaI of iadiosonsilizois in diagnosis and
lioalnonl of lunois, luloicuIosis, nvocaidiaI infaiclion, and hvpoxia. NilioinidazoIo cvloloxicilv vas ioconlIv
capilaIizod in hvpoxic lunoi doloclion and lunoi kiIIing. NilioinidazoIos acl as snail onzvno inhililois on ono
hand and pIav as onzvno noduIalois. Livoi onzvno inhililion is uniquo foi dovoIoping diugs in diug discovoiv al
noIocuIai IovoI. Tho najoi chaIIongo is lo oxpIoio onzvno inhililoi lhal soIoclivoIv and spocificaIIv calaIvzos onzvno
inhililion in onIv alnoinaI oi disoasod Iivoi coIIs Ioaving safo noinaI coIIs in aculo slago of Iivoi danago such as
hopalic cona, lhioaloning hopalilis, hopalic cancoi. Tho succoss of onzvno inhililion nainIv doponds on
inliacoIIuIai phvsioIogicaI condilions such as pH, lonpoialuio, conconlialion of aclivo onzvno oi inhililoi,
coonzvno, co-facloi, onzvno sulslialo sliucluiaI-funclionaI confoinalion slalo, lvpo of liochonicaI ioaclion such as
coupIod, cascado oi individuaI onzvno ioaclion and finaIIv ioguIaloiv onzvno lohavioi lo conlioI nolaloIisn.
CuiionlIv, ail is dovoIoping in Iivoi lianspIanlalion using cuIluiod coIIs lo iopIaco disoasod oi non-funclionaI Iivoi
coIIs fion disoasod Iivoi. Tissuo onginooiing has oponod lho nov oppoilunilv of using condilionod nonoaxonic and
nonocIonaI Iivoi coIIs in lolh oxpoiinonlaI and pio-cIinicaI oi cIinicaI lhoiapoulics in lolh indusliios and acadonics.
RoconlIv, gono lhoiapv in Iivoi vas a gioal succoss and il viII lo a loon in nodicaI scionco of doloxificalion oi diug
nolaloIisn if and vhon cIinicaIIv accoplod. alloiv of onzvnos in hopalocvlos and ialo Iiniling onzvno ioguIaloiv
lohavioi in piosonco of addilivos oi inhililois viII coilainIv onhanco lolloi undoislanding of disoaso dovoIopnonl
and nov diug phainacoulicaI aclion in diug discovoiv. SliII lodav, Iivoi coIIs ionain as najoi iosoaich looI lo
discovoi diugs in lhoiapoulics and soaich of lianspIanlalIo disoaso iosislanl Iivoi coIIs locauso of Iivoi as soIo oigan
iosponsilIo of liIo foinalion, doloxificalion and diug nolaloIisn. Ciiihosis is a soiious hoaIlh hazaid lhal soIoIv
ioquiios funclionaI Iivoi. In fuluio, il ionains lo soo if noIocuIai lioIogv viII assisl in siIoncing gonos ON oi OII
iosponsilIo of onzvno slinuIalion oi inhililion in Iivoi as nolhod of disoaso lhoiapv and cuio. Tho najoi discovoiios
viII lo discovoiv and lolloi undoislanding of nov giovlh faclois, coIonv slinuIaling faclois affocling onzvno
ioaclions in hopalocvlos and Kupffoi coIIs as najoi onginooiod cancoi iosislanl oi ciiihosis iosislanl Iivoi coIIs. In
fuluio, chip lochnoIogv oi siIica oi poIvnoi coalod onzvno oslinalion lochniquos nav lo noio ioIialIo in snaII
sanpIo coIIoclions oi in Iivoi coIIs vilh high dogioo of accuialo onzvno oslinalion.

1. Cnnc!usInn
Tho 2nilioinidazoIo is lolh anliapaiasilic diug and iadiosonsilizoi hvpoxia naikoi in lunoi lhoiapv. Il shovs
hopalocoIIuIai cvloloxicilv. Tho 'hopalocoIIuIai hvpoxia ciiloiion dislinguishos lho nilioinidazoIo inducod
hopalocoIIuIai oxvgon dopIolion and associalod oiganoIIo changos lv onzvno IovoIs in soiun, liopsv sanpIos and
coII oiganoIIos lv oIoclion nicioscopv. IniliaIIv, gIucoso ioguIalion Ioads lo nolaloIic inlogiilv Ioss. Laloi, il nav lo
oxvgon insufficioncv and sIov coII doalh. Tho 2-nilioinidazoIo is diug of choico in hopalic infoclions and ils
doiivalivos aio onoiging choico of lunoi lioalnonl. Ils aclion nav lo ovaIualod iapidIv lv onzvno liochonicaI
oslinalions vilhoul lino consuning diug noniloiing and lhoiapoulic assav lochniquos. Lnlanoola hisloIvlica, an
inloslinaI piolozoan paiasilo can pioIifoialo, Ivso and dosliov hunan Iivoi coIIs. Wilhin lho Iasl docado, nov aninaI
nodoIs of L. hisloIvlica infoclion shovod anoolic liophozoilos causo inloslinaI disoaso and Iivoi alscoss. Roconl
sludios havo oxpandod oui undoislanding of a Iaigo nunloi of onzvno inhililion ioaclions in hopalic iogonoialion
afloi Iivoi alscoss is lioalod lv nilioinidazoIo as ionaikalIo diug lo kiII paiasilos. SliII inloiaclions lolvoon L.
hisloIvlica and hunan inloslino: and lolvoon L. hisloIvlica and hopalocvlos oi Kupffoi coIIs aio nol fuIIv knovn. In
piosonl chaploi, a schono of onzvno changos in Iivoi coIIs duiing dovoIopnonl of anoolic Iivoi alscoss and onzvno
inhililion is oxpIoiod in dolaiI as iohaliIilalivo aclion of nilioinidazoIo lo iogonoialo Iivoi coIIs. Chaiacloiislics of
onzvno ioguIaloiv lohavioi highIighl lho nolaloIic inlogiilv Ioss, onoigv dopIolion, oxidalivo phosphoivIalion, fallv
doposilion, phagocvlosis and sIov coII doalh as nain cuIpiils in dovoIopnonl of Iivoi alscoss and hvpoxia.
NilioinidazoIo inhilils sovoiaI ialo Iiniling onzvnos lo conlioI oxvgon (hvpoxia) IovoIs and koops laIanco of
NADIH, ATI, fallv acids, anino acids and pioloins. In hopalocvlos, onzvno inhililion and ioguIaloiv lohavioi of
apoplosis (caspasos, cvsloino pioloaso): cailohvdialo nolaloIizing (gIucokinaso, pviuvalo kinaso,
phosphofiuclokinaso): onoigv nolaloIisn (NADIH cvlochiono I45O Roduclaso, Ihosphodiosloiaso): DNA
svnlhosis (ThvnidvIalo Svnlholaso) dosciilo lho piocoss of Iivoi iogonoialion. In Kupffoi coIIs, onzvno inhililion of
diug nolaloIizing onzvnos (oxidoioduclasos, pioloasos, osloiasos, phosphalasos) piodicls lho Iivoi iogonoialion and
iocovoiv fion Iivoi alscoss. In Iasl docado, sovoiaI sludios iopoilod lho uso of onzvno inhililion nochanisns in
onhancing Iivoi iogonoialion and iocovoiv as looI of lissuo onginooiing and Iivoi lianspIanlalion. In concIusion,
onzvno inhililion nochanisns in Iivoi danago and iogonoialion oxpIain lho lolloi infoinalion of Iivoi
lianspIanlalion lo nainlain conpIox Iivoi coII onviionnonl in laIanco. Lnzvno inhililion nochanisns lhiov a Iighl
on chaiacloiizalion of onzvno ioguIalion in onginooiod Iivoi coIIs, anoolic Iivoi alscoss dovoIopnonl and dosiiod
dosign of laigolod nilioinidazoIo anaIoguos lo lioal Iivoi alscoss.

Acknnw!cdgcmcnts
Tho aulhoi acknovIodgos lho assislanco piovidod lv Iiofossoi Rakosh K Tandon lo piovido assislanl
iosoaich officoi jol duiing lhis voik vilh gianl assislanco fion ICMR, Nov DoIhi and Di V.S.Singh foi
guidanco and vaIualIo CounciI of Scionlific and IndusliiaI Rosoaich foIIovship and iosoaich gianl fion
ICMR, Nov DoIhi lo do Ih.D.


Rcfcrcnccs

Adans, C.L., Ahnod, I., CIaiko, L.D., O'NoiII, I., Iaiiick, I., Slialfoid, I.I., WaIIaco, R.C., Waidnan. I., Walls, M.L.
(198O) Sliucluio-aclivilv ioIalionships in lho dovoIopnonl of hvpoxic coII iadiosonsilizois. III. Lffocls of lasic
sulsliluonls in nilioinidazoIo sidochains. Inl I Radial ioI RoIal Slud Ihvs Chon Mod. VoI 38, No 6, pp 613-26.

Alualaia, S.I., aiioll, I.A., Hau, T., Ionasson, O.(1982) Anolic Iivoi alscoss. Aich Suig. VoI 117, 239-44.

Adans, L.., MacLood, I.N. (1977) Invasivo anoliasis: anolic Iivoi alscoss and ils conpIicalions. Modicino
(aIlinoio) VoI 56, 325-34.

Akgun, Y., TacviIdiz, I.H., CoIik, Y. (1999) Anolic Iivoi alscoss: changing lionds ovoi 2O voais. WoiId I Suig. VoI. 2,
1O2-O6.

AIIison, A.C., IoiIuga, I., Iivdz, H., SchoiIonnoi, H.U. (1978) Tho ioIo of naciophago aclivalion in chionic
infIannalion. Agonls Aclions. VoI 8, No 1-2, pp 27-35

Ankii, S., SloIaiskv, T., iacha, R., IadiIIa-Vaca, I., MiioInan, D. (1999) Anlisonso inhililion of oxpiossion of cvsloino
pioloinasos affocls Lnlanoola hisloIvlica-inducod foinalion of Iivoi alscoss in hanslois. Infocl. Innun. VoI 67, pp
421-422.

Ankii, S. SloIaiskv, T., MiioInan, D. (1998) Anlisonso inhililion of oxpiossion of cvsloino pioloinasos doos nol affocl
Lnlanoola hisloIvlica cvlopalhic oi haonoIvlic aclivilv lul inhilils phagocvlosis. MoI. MiciolioI. VoI 28, pp 777-785

Aoshila, K., Ronnaid, S. I., Spuizon, I. R. (1997) Iilionoclin suppoils lionchiaI opilhoIiaI coII adhosion and suivivaI in
lho alsonco of giovlh faclois. An I IhvsioI VoI. 273, No 3 Il 1, pp L684-93.

aIIingoi, I.R., Koo, I.W., Raulh, A.M. (1996) In vilio and in vivo ovaIualion of a lochnoliun-99n-IaloIod 2-
nilioinidazoIo (MS181321) as a naikoi of lunoi hvpoxia. I NucI Mod. VoI. 37, No 6, pp 1O23-31.

aIIingoi, I.R. (2OO1) Inaging hvpoxia in lunois. Sonin NucI Mod. VoI. 31, No 4, pp 321-9.

aplisla, I.M., Vvas, D., Sokoi, S. (2O11) Livoi iogonoialion: RoIo of ioonginooiing. Chaploi 11. In: Iiogioss in
MoIocuIai and LnviionnonlaI ioonginooiing- Iion AnaIvsis and ModoIing lo TochnoIogv AppIicalions. Ldiloi:
AngoIo Caipi. ISN 978-953-3O7-268-5 Inloch Opon Accoss IulIishois Conpanv.pp257-268.

aplisla, I.M., OiIando, C., MiinaIok-Sani, S.H., Siddiqui, M., AlaIa, A., Sokoi, S.(2OO9) WhoIo oigan docoIIuIaiizalion
- a looI foi lioscaffoId faliicalion and oigan lioonginooiing. Conf Iioc ILLL Lng Mod ioI Soc 2OO9, pp 6526-6529.


ainos, I.I., DoCock, K.M., RovnoIds, T.N., RaIIs, I.W. (1987) A conpaiison of anolic and pvogonic alscoss of lho
Iivoi. Modicino (aIlinoio) VoI. 66, 472-83.


aion, R., Noff, L., iovn, W., Louvaid, D., Couilov, I.I. (199O) SoIoclivo inloinaIizalion of lho apicaI pIasna nonliano
and iapid iodisliilulion of IvsosonaI onzvnos and nannoso 6-phosphalo iocoplois duiing osloocIasl inaclivalion lv
caIcilonin. I CoII Sci. VoI. 97, No 3, pp 439-47.

aiioso ,I.I., CaivaIho, R.N., IIalnaik, T. (2OO5) Kinolic anaIvsis and Iigand-inducod confoinalionaI changos in dinoiic
and lolianoiic foins of hunan lhvnidino kinaso 2.iochonisliv. VoI 44, No 12, pp 4886-96.

oininghauson, O. and Loippo, M. (1997) Nociosis voisus apoplosis as lho nochanisn of laigol coII doalh inducod lv
Lnlanoola hisloIvlica. Infocl.Innun. VoI 65, pp 3615-3621.

oiulo, L.R., Iaiah, S., McCIoIIand, R.A., Raulh, A.M. (1992) DopIolion of inliacoIIuIai gIulalhiono lv 1-nolhvI-2-
niliosoinidazoIo. Inl I Radial OncoI ioI Ihvs. VoI 22, No 4, pp 817-2O..

halnagai, R., Schiinoi, R., Linsl, M., Dockoi, K. (1981) Supoioxido ioIoaso lv zvnosan slinuIalod ial kupffoi coIIs in
vilio. Luiopoan I iochonisliv. VoI. 119, No 1, pp 171-175.

iIzoi, M., RoggoI, I., Coilos, A.L. (2OO6) RoIo of Kupffoi coIIs in hosl dofonso and Iivoi disoaso. Livoi Inl. VoI 26, No
1O, pp 1175-86.

os HI, Van Don Lijk AA, Slooionloiiv IA.(1976) AppIicalion of LLISA in soiodiagnosis of anooliasis. Tians Rov Soc
Tiop Mod Hvg. VoI 6, pp 44O.

iosnick, L., Mainigi, K.D., uccino, R., uiIoson, S.S. (197O) Sludios of dooxvlhunidino kinaso of iogonoialing ial Iivoi
and Lschoiicia coIi. Cancoi Ros.VoI. 3O, pp 25O2-25O6.

iozdon, C.., McCIoIIand, R.A., Raulh, A.M. (1997) Apoplosis and 1-nolhvI-2-nilioinidazoIo loxicilv in CHO coIIs.
i I Cancoi. VoI.76, No 2, pp 18O-8.

iouvoi, A., aioIds,

R.I., do Loouv,

A.M., Iauv,

L., IIa,s

A., Yap,

S.H., van don iook, A.M.W.C., Knook, D.L.

(1988)
IsoIalion and cuIluio of Kupffoi coIIs fion hunan Iivoi: UIliasliucluio, ondocvlosis and pioslagIandin svnlhosis
IouinaI of HopaloIogv. VoI 6, No 1, pp 36-49.

iuchhaus, I., Loflus, .I., HaII, N., Tannich, L. (2OO3) Tho inloslinaI piolozoan paiasilo Lnlanoola hisloIvlica
conlains 2O cvsloino pioloaso gonos of vhich onIv a snaII sulsol is oxpiossod duiing in vilio cuIlivalion. Lukaivolic
CoII, VoI 2, No 3, pp 5O1-5O9.

CaiIson, R.I., Lofoi, A.M. (1976) Hopalic coII inlogiilv in hvpodvnanic slalos. An I IhvsioI. VoI 231, No 5 Il. 1, pp
14O8-14.
Chang, L.M.S., oIIun, I.I. (1972) Vaiialion of dooxviilonucIoic acid poIvnoiaso aclivilios duiing ial Iivoi
iogonoialion. Tho IouinaI of ioIogicaI Chonisliv, VoI 247, pp 7948-795O

Chao, C.I., Suljock, I.R., Iohnson, R.I. (1982) NilioinidazoIo inhililion of Iaclalo pioduclion in CHO coIIs. Inl I Radial
OncoI ioI Ihvs. VoI. 8, No 3-4, pp 729-32.

Chu, T., Li, R., Hu, S., Liu, X., Wang, X. (2OO4) Iiopaialion and liodisliilulion of lochnoliun-99n-IaloIod 1-(2-
nilioinidazoIo-1-vI)-piopanhvdioxvininoanido (N2IIA) as a lunoi hvpoxia naikoi. NucI Mod ioI. VoI.31, No 2, pp
199-2O3.

Caiio, I.L., Oilan, I.C., Ro, L., IoIsnann, K., Iffoil, W., auoi, M., SuIinan, H.., Iianladosi, C.A., Mavhov, T.M., ioon,
I., Slolz, M., Singoi, M. (2O1O) SuivivaI in ciilicaI iIInoss is associalod vilh oaiIv aclivalion of nilochondiiaI liogonosis.
An I Rospii Ciil Caio Mod. VoI.182, No 6, pp 745-51.
Cohon-Tannoudji, I., Vival, V., HoiInann, I., Logiand, C., MaIlioi, I. I. (1991). RoguIalion lv piogosloiono of lho high-
affinilv slalo of nvonoliiaI lola-adionoigic iocoploi and of adonvIalo cvcIaso aclivilv in lho piognanl ial. I MoI
LndociinoI VoI 6, No 2, pp 137-45.

Covan, D.S., Malojovic, I.I., McCIoIIand, R.A., Raulh, A.M.(1994) DNA-laigolod 2-nilioinidazoIos: in vilio and in
vivo sludios. i I Cancoi. VoI 7O, No 6, pp 1O67-74.

D'AIossandio, A., ZoIIa, L. (2O11) Tho SOD vssov: sucrnxIdc disnulasos fion liochonisliv, lhiough pioloonics, lo
oxidalivo slioss, aging and nuliacoulicaIs. Lxpoil Rov Iioloonics. VoI. 8, No 3, pp 4O5-21.

Dan, C., Wako, K. (1985) Modos of ondocvlosis of Ialox pailicIos in sinusoidaI ondolhoIiaI and Kupffoi coIIs of noinaI
and poifusod ial Iivoi. Lxp CoII Ros. VoI 158, No 1, pp 75-85.

Das, I., Dolnalh, A., Munoz, M.L. (1999) noIocuIai nochanisns of palhogonosis in anooliasis. Ind I CaslioonloioI VoI.
18, No 4, pp 161-166.

Ding, A., Nalhan, C. (1988) AnaIvsis of lho nonfunclionaI iospiialoiv luisl in nuiino kupffoi coIIs. I Lxp Mod. VoI.167,
pp 1154-117O.

Duan, L., Aovagi, M., Tanaki, M., Yoshino, Y., Moiinolo, T., Wakinolo, H., Nagasaka, Y., Hiiakava, K., Ohno, K.,
Yananolo, K. (2OO4) Inpaiinonl of lolh apoplolic and cvlopioloclivo signaIings in gIiona coIIs iosislanl lo lho
conlinod uso of cispIalin and lunoi nociosis facloi aIpha. CIin Cancoi Ros. VoI. 1O, No 1 Il 1, pp 234-43.
Dusling, C.I., SoIonidis, S., Iiang, I. (2OO5) Mochanisns of suppiossing NADIH oxidaso in lho vascuIai vaII. Mon Insl
OsvaIdo Ciuz, Rio do Ianoiio, VoI. 1OO, No suppI. 1, pp 97-1O3

Ldvaids, D.I. (1981) Mochanisns of cvloloxicilv of nilioinidazoIo diugs. Iiog Mod Chon. VoI 18, pp 87-116.

Ldvaids, D.I., Knox, R.I., Knighl, R.C. (1982) Sliucluio-cvloloxicilv ioIalionships of nilioinidazoIos in an in vilio
svslon. Inl I Radial OncoI ioI Ihvs. VoI 8, No 3-4, pp 791-3.

Ldvaid, ., AIaliala, L.., CuilishIov, S.M., Wai, K., Lai, W.K., Wignoio, S.I., Adans, D.H., Affoid, S.C. (2OO7)A nov
appioach lo isoIalion and cuIluio of hunan Kupffoi coIIs IouinaI of InnunoIogicaI Molhods, VoI 326, No 1-2, pp 139-
144.

LnnoIol, I., os, C.I., onodolli, L.L., Rnko, I.H., Salo, O.(1964) Sludios on pIasna nonlianos I. ChonicaI
conposilion and onzvno conlonl of pIasna nonlianos isoIalod fion ial Iivoi. iochinica ol iophvsica Acla A VoI.
9O, No 1, pp 126-145

Lisoz, C., Kaiasu, Z., Akaica, U.S., Cunsai, I., Yuco, C., alui, Y.(2OO1) NilioinidazoIo-inducod chionic hopalilis.
Lui I CaslioonloioI HopaloI. VoI 13, No 8, pp 963-6.

Lschnann, S.M., IauIson, I., RoinoId, M., Dillnann, H., WoIz, S., RoischI, C., MachuIIa, H.I., aios, R.(2OO5) Iiognoslic
inpacl of hvpoxia inaging vilh 18I-nisonidazoIo ILT in non-snaII coII Iung cancoi and hoad and nock cancoi lofoio
iadiolhoiapv.

I NucI Mod. VoI. 46, No 2, pp 253-6O.

Ioinan, H.I., Toiios, M. (2OO2) Roaclivo oxvgon spocios and coII signaIing. An I Rosp CIin Caio Mod.VoI.166, pp S4-S8.

Iioh, M., Konno, A., Thuinan, R.C. (2OO3) IsoIalion of Livoi Kupffoi CoIIs. Cuiionl IiolocoIs in ToxicoIogv. VoI 14, pp
14.4.1-14.4.12.

Candhi, .M., Iishad, M., ChavIa, T.C., Tandon, .N.(1987) Lnzvno Iinkod pioloin-A: an LLISA foi doloclion of
anoolic anlilodv. Tiansaclions of lho RovaI Sociolv of TiopicaI Modicino and Hvgiono. VoI 81, No 2, pp 183-185.

CiInan, A. C. (1984). Cuanino nucIoolido-linding ioguIaloiv pioloins and duaI conlioI of adonvIalo cvcIaso. I CIin
Invosl VoI. 73, No 1, pp 1-4.

Cinioos, T., onlzon, L., Maijanki, I., Muiala, R., Hoisnan, M.R., Koiding, S., LskoIa, O., Haapaianla, M., Minn, H.,
SoIin, O. (2OO4) Conpaiison of lho liodisliilulion of lvo hvpoxia naikois |18I!ILTNIM and |18I!IMISO in an
oxpoiinonlaI nannaiv caicinona. Lui I NucI Mod MoI Inaging. VoI.31, No 4, pp 513-2O.

Cuongoiich, I.I., Mailin, M.V., SohI, C.D., Chong, O. (2OO9)Moasuiononl of cvlochiono I45O and NADIH-
cvlochiono I45O ioduclaso. Naluio IiolocoIs . VoI. 4, pp 1245 - 1251.

Cuoiianl, R.L., iush I., Ravdin I.I., SuIIivan I.A., MandoII, C.L.. (1981) Inloiaclion lolvoon Lnlanoola hisloIvlica
and hunan poIvnoiphonucIoai nouliophiIs. I. Infocl. Dis. VoI 143, pp 83-93.

Hanann, L. NickoI, R., Tannich, L. (1995) Tiansfoclion and conlinuous oxpiossion of holoioIogous gonos in lho
piolozoan paiasilo Lnlanoola hisloIvlica. Iioc. NalI. Acad. Sci. U. S. A. VoI. 92, pp 8975-8979.

Hoinliook, D.C., Shvan, K., SailoioIIi, A.C. (1988) NovoI 1-haIoaIkvI-2-nilioinidazoIo lioioduclivo aIkvIaling agonls.
Anlicancoi Diug Dos. VoI. 2, No 4, pp 339-5O.

Hondiiks, H.I., iouvoi, A., Knook, D.L. (199O) IsoIalion, puiificalion, and chaiacloiizalion of Iivoi coII lvpos. Molhods
LnzvnoI. VoI.19O, pp 49-58.

Houff, C., Movoi, S., ooIon, R.H.I.

(1994) IsoIalion of ial and hunan Kupffoi coIIs lv a nodifiod onzvnalic assav.
IouinaI of InnunoIogicaI Molhods, VoI 174, No 1-2, pp 61-65.

Hidi, R., Tinnoinans, S., Liu, L., Schudl, C., Donl, C., HoIgalo, S.T., Djukanovic, R. (2OOO) Ihosphodiosloiaso and
cvcIic adonosino nonophosphalo-dopondonl inhililion of T-Ivnphocvlo chonolaxis. Lui Rospii I. VoI.15, No 2, pp 342-
9.

Hodgkiss, R.I., Wolsloi, L., WiIson, C.D. (1997) Moasuiononl of hvpoxia in vivo using a 2-nilioinidazoIo (NITI). Adv
Lxp Mod ioI. VoI.428, pp 61-7.

HoiIon, C.K., Soifoil, C.I., MaIouf, C.S. (2OOO) Toxic nolionidazoIo-inducod MRI changos. Ann Ihainacolhoi. VoI.34,
No 11, pp 1273-5.

Iankovic, ., Aquino-Iaisons, C., RaIoigh, I.A., Slanliidgo, L.I., Duiand, R.L., analh, I.I., MacIhaiI, S.H., OIivo, I.L.
(2OO6) Conpaiison lolvoon pinonidazoIo linding, oxvgon oIocliodo noasuiononls, and oxpiossion of ondogonous
hvpoxia naikois in cancoi of lho uloiino coivix. Cvlonoliv CIin Cvlon. VoI. 7O, No 2, pp 45-55.

IaiuniIinla, R. and KiadoIfoi, I. (1964) Tho loxic offocl of Lnlanoola hisloIvlica on Ioucocvlos. Ann. Tiop. Mod.
IaiasiloI. VoI 58, pp 375-381.

Kalanuna, N. (2O11) Sliucluio-lasod dovoIopnonl of spocific inhililois foi individuaI calhopsins and lhoii nodicaI
appIicalions.Iioc Ipn Acad Soi Ihvs ioI Sci. VoI. 87, No 2, pp 29-39.

KausaIva, S., Kaui, S., MaIIa, N., CanguIv, N.K., Mahajan, R.C. (1996) MiciolicidaI nochanisns of Iivoi naciophagos in
oxpoiinonlaI viscoiaI Ioishnaniasis. AIMIS. VoI. 1O4, No 3, pp 171-5.
Kin, .S., aoz, C.L., AlaIa, A. (2OOO) ionaloiiaIs foi lissuo onginooiing. WoiId I UioI VoI 18, pp 2-9.
KooIing, I.L., Snilh, L.L., AIdiidgo, W.N. (1982) Tho foinalion of nixod disuIphidos in ial Iung foIIoving paiaqual
adninislialion. CoiioIalion vilh changos in inloinodiaiv nolaloIisn. iochon iophvs Acla. VoI.716, pp 249-257.
Koono, W.L. ol aI. (1986) Tho najoi nouliaI pioloinaso of Lnlanoola hisloIvlica. I. Lxp. Mod. VoI. 163, pp 536-549.
Kiin, A., ingon, A., Sloffan, A.M., WiId, M.T., KoIIoi, I., CinquaIlio, I. (1982) Lndocvlic Capacilios of Kupffoi CoIIs
IsoIalod fion lho Hunan AduIl Livoi. HopaloIogv. VoI 2, No 2, pp 216S-222S.

Knook, D.L., SIovsloi, L.C., Toulsch, H.I. (198O) High acliilv of gIucoso-6-phosphalo dohvdiogonaso in Kupffoi coIIs
isoIalod fion ial Iivoi. Hislochonisliv. VoI. 69, No 2, pp 211-6.
Knook, D.L., aikvav, C., SIovsloi, L.C. (1981) LvsosonaI onzvno conlonl of Kupffoi and ondolhoIiaI Iivoi coIIs isoIalod
fion goinfioo and cIoan convonlionaI ials. Infocl Innun. VoI 33, No 2, pp 62O-2.
Ko, C., In, Y. H., Iaik-Saigo. O. K. (1999) RoIo of piogosloiono iocoploi aclivalion in piluilaiv adonvIalo cvcIaso
aclivaling poIvpoplido gono oxpiossion in ial ovaiv. LndociinoIogv VoI. 14O, No 11, pp 5185-94.

Koch, C.I., Lvans, S.M. (2OO3) Non-invasivo ILT and SILCT inaging of lissuo hvpoxia using isolopicaIIv IaloIod 2-
nilioinidazoIos. Adv Lxp Mod ioI. VoI. 51O, pp 285-92.
KoppoIo, I.M., KoIIoi, .I., CaIdvoII-KonkoI, I.C., Lonaslois, I.I., Thuinan, R.C. (1991) Lffocl of hopaloloxic chonicaIs
and hvpoxia on hopalic nonpaionchvnaI coIIs: inpaiinonl of phagocvlosis lv Kupffoi coIIs and disiuplion of lho
ondolhoIiun in ial Iivois poifusod vilh coIIoidaI cailon. ToxicoI AppI IhainacoI. VoI 11O, No 1, pp 2O-3O.
Kunai, I., Iai, K., Iandov, H., Sundai, S.(2OO2) NADH oxidaso, NADIH oxidaso and nvoIopoioxidaso aclivilv of
viscoiaI Ioishnaniasis palionls. I. Mod. MiciolioI. VoI. 51, pp 832-836.
Kullnoi, R.L., Schunoi, W., Apanlaku, I.O. (1982) Lffocl of ondoloxin and gIucocoilicoid piolioalnonl on hoxoso
nonophosphalo shunl aclivilv in ial Iivoi. Ciic Shock VoI 9, No 1, pp 37-45..

Kvon S, 5harma R. (2OO9) RoIo of InnunoioguIalois in Niliic Oxido Iioduclion in Hvpoxia-Inducod Apoplosis in
AIvooIai CoIIs.|n| ] Canccr Prctcn|icn. VoI 3, No 3, pp 99-11O.

Kvon S, Shaina, R.(2OO9) RoIo of Lffoclois on Hvpoxia Duo lo Niliic Oxido Iioduclion in Hunan AIvooIai LpilhoIiaI
CoIIs and Oxvgon DopIolion in Hunan Hopalocvlos. |n|crna|icna| ]curna| cf Mcdica| and 8ic|cgica| |rcn|icrs VoI. 15, No 5,
pp 425-442.

Laiiov, D.(2OOO) Diug-inducod Iivoi disoasos. I HopaloI. VoI.32, No. 1 SuppI, pp 77-88.

Lassoiio, R., Iaioonvosana, N., Kuialhong, S., Soh, C-T. (1983) SingIo-dav diug lioalnonl of anolic Iivoi alscoss. An
I Tiop Mod Hvg VoI. 2, 723-26.

Mahv, I., Do asl, M., Lovoquo, I.H., CiIIail, I., Lalai, D., Maichand, I., Ciogoiio, V.(2OO4) IiocIinicaI vaIidalion of lho
hvpoxia liacoi 2-(2-nilioinidazoI-1-vI)- N-(3,3,3-|(18)I!liifIuoiopiopvI)acolanido, |(18)I!LI3. Lui I NucI Mod MoI
Inaging. VoI. 31, No 9, pp 1263-72.

Mohghini Cioigio(1959) Nov Soiios. VoI 4(9):682-692

Mailina, S.D., IsnaiI, M.S., Vosla, K.S. (2OO6) CiIoniIasl: oiaIIv aclivo soIoclivo hnshndIcstcrasc-4 inhililoi foi
lioalnonl of chionic olsliuclivo puInonaiv disoaso. Ann Ihainacolhoi. VoI.4O, No 1O, pp 1822-8.

Mailin, . R., IaindaIo, R. W. Wong, S. K. (1987) Tho ioIo of Cs in aclivalion of adonvIalo cvcIaso. iochon Soc Tians
VoI. 15, No 1, pp 19-21.

McCuskov, R.S., McCuskov, I.A. (199O) Iino sliucluio and funclion of Kupffoi coIIs.
I LIoclion Miciosc Toch. VoI.14, No 3, pp 237-46.


Mohghini Cioigio (1959) Nov Soiios. VoI 4, No 9, pp 682-692.

MoIo, T., aIIingoi, I.R., Raulh, A.M. (2OOO) RoIo of NADIH:cvlochiono I45O ioduclaso in lho hvpoxic accunuIalion
and nolaloIisn of RU59-21, a lochnoliun-99n-nilioinidazoIo foi inaging lunoi hvpoxia. iochon IhainacoI. VoI.
6O, No 5, pp 625-34.

MoIo, T., Duncan, I., aIIingoi, I.R., Raulh, A.M. (2OOO) RU59-21, a socond-gonoialion 99nTc-IaloIod 2-
nilioinidazoIo foi inaging hvpoxia in lunois. I NucI Mod. VoI. 41, No 1, pp 169-76.

MichaIopouIos, C.K.(199O) Livoi iogonoialion: noIocuIai nochanisns of giovlh conlioI. IASL I.VoI 4, pp 176-187.

MichaIopouIos, C.K, DoIiancos, M.C. (1997) Livoi iogonoialion. Scionco. VoI. 276, pp 6O-66.

MichaIopouIos, C.K.(2OO7) Livoi iogonoialion. ] Cc|| Pnusic|. VoI 213, No 2, pp 286-3OO.

MoIIoi, I., WaIIin, H., VogoI, U., Auliup, H., Rison, L., HaId, M.T., Danoshvai, ., Diagslod, L.O., IouIson, H.L., Lofl,
S. (2OO2) Mulagonicilv of 2-anino-3-nolhvIinidazo|4,5-f!quinoIino in coIon and Iivoi of ig Iuo ials: ioIo of DNA
adducls, sliand lioaks, DNA iopaii and oxidalivo slioss. Caicinogonosis. VoI 8, pp 1379-85.

Moonov, D., Hanson, L., Vacanli, I., Langoi, R., Iainoi, S., Ingloi, D. (1992) Svilching fion diffoionlialion lo giovlh in
hopalocvlos: conlioI lv oxliacoIIuIai naliix. I CoII IhvsioI VoI 151, pp 497-5O5.
MosoIon, I.W., Hav, M.I., Donnv, W.A., WiIson, W.R.(1995) N-|2-(2-nolhvI-5-nilioinidazoIvI)olhvI!-4-(2-
nilioinidazoIvI)lulananido (NSC 639862), a lisnilioinidazoIo vilh onhancod soIoclivilv as a lioioduclivo diug.
Cancoi Ros. VoI. 55, No 3, pp 574-8O

MuIcahv, R.T., Cipp, I.I., Caininali, A., aiascul, I.L., Inlach, I.L.(1989) Chonosonsilizalion al ioducod
nilioinidazoIo conconlialions lv nixod-funclion conpounds conlining 2-nilioinidazoIo and
chIoioolhvIniliosouioa. Lui I Cancoi CIin OncoI. VoI 25, No 7, pp 1O99-1O4.

Nagaoka, M.R., Kouvoundjian, M., oigos, D.R. (2OO3) Hopalic cIoaianco of lissuo-lvpo pIasninogon aclivaloi and
pIasna kaIIikioin in oxpoiinonlaI Iivoi filiosis. Livoi Inl. VoI. 23, No 6, pp 476-83.

Noaud, V., Duluisson, L., aIalaud, C., iouIac-Sago, I. (1995) UIliasliucluio of hunan Kupffoi coIIs nainlainod in
cuIluio. I Sulniciosc CvloI IalhoI. VoI.27, No 2, pp 161-7O.

Niu, I., Iiofiiovic, I., Ian, H., Vaiskunailo, R., Vovno-Yasonolskava, T. (2OO3) C Iioloin lolaganna sulunils slinuIalo
p114RhoCLI, a guanino nucIoolido oxchango facloi foi RhoA and Rac1: ioguIalion of coII shapo and ioaclivo oxvgon
spocios pioduclion. Ciic Ros VoI. 93, No 9, pp 848-56.

Noss, M.., Ianicucci, R., McCIoIIand, R.A., Raulh, A.M. (1988) Iiopaialion, loxicilv and nulagonicilv of 1-nolhvI-2-
niliosoinidazoIo. A loxic 2-nilioinidazoIo ioduclion pioducl. iochon IhainacoI. VoI.37, No 13, pp 2585-93.

Noss, M.., Ianicucci, R., McCIoIIand, R.A., Raulh, A.M. (1989) 1-MolhvI-2-niliosoinidazoIo: cvloloxic and
gIulalhiono dopIoling capaliIilios. Inl I Radial OncoI ioI Ihvs. VoI 16, No 4, pp 1O15-9.

Oll, H.C., Mallhioson, T.S., Coh, S.K., Iack, L.D., Kion, S.M., Noloff, T.I., TavIoi, D.A. (2OO8) Ioifusion docoIIuIaiizod
naliix: using naluio's pIalfoin lo onginooi a lioailificiaI hoail. Nal Mod VoI 14, pp 213-221.



IapadopouIou, M.V., Ii, M., Rao, M.K., Ioonoi, W.D. (2OO3) Roduclivo nolaloIisn of lho nilioinidazoIo-lasod
hvpoxia-soIoclivo cvloloxin NLCO-1 (NSC 7O9257). OncoI Ros. VoI.14, No 1, pp 21-9.

IiIkis, S., SchIunpf, I., IiIkis, I., CIaus, T.H. (1979) RoguIalion of phosphofiuclokinaso aclivilv lv gIucagon in isoIalod
ial hopalocvlos.
iochon iophvs Ros Connun. VoI.88, No 3, pp 96O-7.

IovoII, S.I., WiInol, A.I., LIsdon-Dov, R. (1969) SingIo and Iov dosago ioginons of nolionidazoIo in anoolic
dvsonloiv and anoolic Iivoi alscoss. Ann Tiop Mod IaiasiloI, VoI.6, 139-42.

RagIand, .D., AshIov L.S., Vaux, D.L., Iolii, W.A.. (1994) Lnlanoola hisloIvlica: laigol coIIs kiIIod lv liophozoilos
undoigo DNA fiagnonlalion vhich is nol lIockod lv cI-2.Lxp. Iaiasil. VoI 79, pp 46O-467.

RanaIho, I.S., RanaIho, L.N.Z., Caslio-o-Siva, O., ZucoIolo, S., Coiioa, I.M.A.(2OO1) Angiolonsin-convoiling onzvno
inhililion lv IisonopiiI onhancos Iivoi iogonoialion in ials. iaz. I. Mod. ioI Rosoaich VoI 34, pp 125-27.

Raniioz-LniIiano, I., IIoios-ViIIaviconcio, L.L., Sogovia, I., Aiias-Nogiolo, S. (2OO7) Niliic oxido pailicipalion duiing
anoolic Iivoi alscoss dovoIopnonl. Modicina (.Aiios) VoI. 67, No 2, pp 167-176.

Raulh, A.M., Mohindia, I.K. (1981) SoIoclivo loxicilv of 5-(3,3-dinolhvI-1-liiazono)inidazoIo-4-cailoxanido lovaid
hvpoxic nannaIian coIIs. Cancoi Ros. VoI.41, No 12 Il 1, pp 49OO-5.

Richo, I., d'Haidonaio, A.D., Sopo, S., Riou, L., Iagiol, D., VidaI, M. (2OO1) NilioinidazoIos and hvpoxia inaging:
svnlhosis of lhioo lochnoliun-99n conpIoxos loaiing a nilioinidazoIo gioup: lioIogicaI iosuIls. iooig Mod Chon
Loll. VoI. 11, No 1, pp 71-4.

Runsov, W.L., IaloI, ., Kuczvnski, ., Naiia, R.K., Chan, Y.W., Lindoi, K.L., Cvi, I., Raju, N., RanaIingan, K., Nunn,
A.D. (1994) IolonliaI of nilioinidazoIos as naikois of hvpoxia in hoail.Adv Lxp Mod ioI. VoI. 345, pp 263-7O.

RussoII, D., Svndoi, S.H. (1969) Anino svnlhosis in iogonoialing ial Iivoi: oxlionoIv iapid luinovoi of oinilhino
docailoxvIaso. MoI IhainacoI. VoI 5, No 3, pp 253-62

Snchoz-Ranioz, .L., Ranioz-CiI, M., Ranos-Mailnoz, L., Rohana, I.T. (2OOO) Lnlanoola hisloIvlica inducos
cvcIooxvgonaso-2 oxpiossion in naciophagos duiing anolic Iivoi alscoss foinalion. Aich Mod Ros. VoI.31, No.4 SuppI,
pp S122-3.

Sapoia, O., Iaono, A., Maggi, A., Ionnoi, T.I., O'NoiII, I. (1992) Induclion of nulalions in V79-4 nannaIian coIIs
undoi hvpoxic and aoiolic condilions lv lho cvloloxic 2-nilioinidazoIo-aziiidinos, RSU-1O69 and RSU-1131. Tho
infIuonco of coIIuIai gIulalhiono. iochon IhainacoI. VoI 44, No 7, pp 1341-7.
Sasagava, T., Shinizu, T., Sokiva, S., Haiaguchi, Y., Yanalo, M., Sava, Y., Okano, T.(2O1O) Dosign of piovascuIaiizod
lhioo-dinonsionaI coII-donso lissuos using a coII shool slacking nanipuIalion lochnoIogv. ionaloiiaIs. VoI 31, No 7, pp
1646-54.

SchoIzo, H. and SchuIlo, W. (1988) On lho spocificilv of a cvsloino pioloinaso fion Lnlanoola hisloIvlica. ionod.
iochin. Acla, VoI 47, pp 115-123.

Schnoing, T.M., Ranakiishnan, V. (2OO9) Whal ioconl iilosono sliucluios havo iovoaIod aloul lho nochanisn of
liansIalion. Naluio. VoI. 461, No 7268, pp 1234-42.

SchoiIonnoi, H.U., KuiiIo, R, SchIovoilach, R., ailIoll, R.R. (1999) Conoialion of O2- iadicaIs in naciophagos can lo
inhililod in vilio and in vivo lv doiivalivos of IofIunonido's piinaiv nolaloIilo. InfIann Ros. VoI.48, No SuppI 2, pp
S117-8

SchuIlo-Hoinann, R. uisch, W. CiasI-Kiaupp, . (1994) Aclivo coII doalh (apoplosis) in Iivoi lioIogv and disoaso.
Iiog. Livoi Dis. VoI 13, pp 1-35.

SovdoI, K.., SlanIov, S.L., Ii (1998) Lnlanoola hisloIvlica inducos hosl coII doalh in anolic Iivoi alscoss lv a non-
Ias-dopondonl, nonlunoi nociosis facloi u-dopondonl palhvav of apoplosis. Infocl. Innun. VoI 66, pp 298O-2983.

SovdoI, K.., Li, L., Svanson, I.L., SlonIov, S.L.(1997) Hunan inloslinaI opilhoIiaI coIIs pioduco pio-infIannaloiv
cvlokinos in iosponso lo infoclion in a SCID nouso-hunan inloslinaI xonogiafl nodoI of anoliasis. Infocl. Innun.
VoI 65, pp 1631-1639.

Shahhossoini, S., Cullikonda, S., Bhatnagar, I., Suiosh, M.R. (2OO6) Iioduclion and chaiacloiizalion of nonocIonaI
anlilodios againsl shopo filiona viius sucrnxIdc dIsmutasc and gIulalhiono-s-liansfoiaso.I Ihain Ihain Sci. VoI.
No 2, pp 165-8.
Shandoia, W.X., oIIan, I., Hashnov, R.H. Ii, ol aI. (1998) Hopalic anoliasis anong palionls in a pulIic loaching
hospilaI. Soulh Mod I VoI 91, 829-37.

Shaina, R. (2O11a) IalhophvsioIogv of anooliasis. TRLNDS in IaiasiloIogv, VoI 17, No 6, pp 28O-285.

Shaina, R.(2O11l) NilioinidazoIo RadiophainacoulicaIs in ioinaging: 1: Svnlhosis and Inaging AppIicalions.
Cuii Radiophaina. VoI 4, No 4, pp 361-378. .
Shaina R., Tandon, R.K. (2O1O) Tho HopalocoIIuIai Dvsfunclion Ciiloiia: Tho Hopalocvlo Cailohvdialo MolaloIizing
Lnzvnos and Kupffoi CoII LvsosonaI Lnzvnos in DovoIopnonl of Anoolic Livoi Alscoss (LIoclion Micioscopic -
Lnzvno Appioach). InloinalionaI I ioIogicaI Iionliois.VoI 16, No 7-8, pp 747-762.

Shaina, R. (2OO9) Tho HopalocoIIuIai Dvsfunclion Ciiloiia: Hopalocvlo Cailohvdialo MolaloIizing Lnzvnos and
Kupffoi CoII LvsosonaI Lnzvnos in 2NilioinidazoIo Lffocl on Anoolic Livoi Alscoss (LIoclion Micioscopic - Lnzvno
Appioach). Chaploi 7, in IaiasiloIogv Rosoaich Tionds (Nova). Ldilois: OIivioi Do iuvn,S. Ioolois. pp 143-159.

Shaina, R., Kvon, S., Chon, C.I. (2OO7) RoIo of innunoioguIalois as possiliIilv of lunoi Hvpoxia inducod apoplosis in
aIvooIai coIIs |n|crna|icna| ]curna| cf Canccr Prctcn|icn Vc| 3, No 3, pp 99-11O.

Shaina R, Singh VS (2OO9) Hopalocvlo CoIIuIai Lnzvnos in Cuinoa Iigs Inducod vilh Anoolic Iivoi Alscoss and
2nilioinidazoIo Tioalnonl. Nanc|ccn Rcs ], VoI. 3, No 2, pp 131-144.

Shaina R, Singh VS (2OO9) Kupffoi CoII Lnzvnos as ionaikois in Cuinoa Iigs Inducod vilh Anoolic Iivoi Alscoss
and 2NilioinidazoIo Tioalnonl. Nanc|ccn Rcs ], VoI 3, No 2, pp 119-13O.

Shaina, R., Singh, V.S. (2O1O) Ihagocvlic Aclivilv and Hoxoso Monophosphalo Shunl Aclivilv of CuIluiod Hunan
Kupffoi CoIIs Upon Zvnosan, Livlhiocvlos, Anoolic Tiophozoils, Lalox oads and NilioinidazoIo. |n| ]Mcd 8ic| |rcn|
VoI. 17, No 6,pp 521-532.

Singh, D. R., Naii, C. K., Iiadhan, D. S. (1991) ChonicaI iadiosonsilizalion lv nisonidazoIo: pioduclion and iopaii of
DNA singIo-sliand lioaks in Yoshida ascilos lunoi coIIs. Indian I Lxp ioI VoI. 29, No 7, pp 6O1-4.

SnaII, K. M., Ioilos, S. L., Rahnan, I. I. , Liggoll, S. . (2OOO) Iusion of lola 2-adionoigic iocoploi lo C aIpha s in
nannaIian coIIs: idonlificalion of a spocific signaI liansduclion spocios nol chaiacloiislic of conslilulivo aclivalion oi
piocoupIing. iochonisliv. VoI. 39, No 1O, pp 2815-21.
SpoIaiics, Z., Wu, I.X. (1997) Tunoi nociosis facloi aIpha augnonls lho oxpiossion of gIucoso-6-phosphalo
dohvdiogonaso in ial hopalic ondolhoIiaI and Kupffoi coIIs.
Lifo Sci. VoI. 6O, No 8, pp 565-71

SpoIaiics, Z., aulisla, A.I., Spilzoi, I.I. (1991) MolaloIic iosponso of isoIalod Iivoi coIIs lo in vivo phagocvlic
chaIIongo.HopaloIogv,VoI.13, No 2, pp 277-281.

SlaIIa, C. K., SlaIIa,I., von Woidoi, K., MuIIoi, , O. A., Coizoi, R., HoIIl, V., Iakols, K. H. (1989) NilioinidazoIo
doiivalivos inhilil anloiioi piluilaiv coII funclion appaionlIv lv a diiocl offocl on lho calaIvlic sulunil of lho adonvIalo
cvcIaso hoIoonzvno. LndociinoIogv VoI. 125, No 2, pp 699-7O6.
SlanIov S.L. (2OO3) Anooliasis. Tho Lancol, VoI 361, No 9362, pp 1O25-1O34.

SlanIov, S.L., Zhang, T., Rulin, D., Li, L. (1995) RoIo of lho Lnlanoola hisloIvlica cvsloino pioloinaso in anolic Iivoi
alscoss foinalion in sovoio conlinod innunodoficionl (SCID) nico. Infocl. Innun. VoI 63, pp 1587-159O.

SlanIov, S.L.Ii.(2OO1) IalhophvsioIogv of anooliasis.Tionds in IaiasiloIogv. VoI 17, No 6, pp 28O-285.

SlaizI, T.L., IiancaviIIa, A., HaIgiinson, C.C., IiancaviIIa, I.R., Ioiloi, K.A., iovn, T.H., Iulnan, C.W. (1973) Tho
oiigin, hoinonaI naluio, and aclion of hopaloliophic sulslancos in poilaI vonous lIood. Suig CvnocoI Olslol VoI 137,
pp 179-199.

Slialfoid, I.I., WiIIianson, C., Haidv, C. (1981) Cvloloxic piopoilios of a 4-nilioinidazoIo (NSC 38O87): a
iadiosonsilizoi of hvpoxic coIIs in vilio. i I Cancoi. VoI 44, No 1, pp 1O9-16.

Su, Z.I., Zhang, X., aIIingoi, I.R., Raulh, A.M., IoIIak, A., Thoinlack, I.R. (1999) Svnlhosis and ovaIualion of lvo
lochnoliun-99n-IaloIod poplidic 2-nilioinidazoIos foi inaging hvpoxia. ioconjug Chon. VoI 1O, No 5, pp 897-9O4.

Sugivana, M., Tsuzuki, K., Haianaki, N. (1993) DNA singIo-sliand lioaks and cvloloxicilv inducod lv sodiun
chionalo(VI) in hvdiogon poioxido-iosislanl coII Iinos. Mulal Ros. VoI 299, No 2, pp 95-1O2.

Sunahaia, R. K., Tosnoi, I.I., CiInan, A.C., Spiang, S. R. (1997). CivslaI sliucluio of lho adonvIvI cvcIaso aclivaloi
CsaIpha. Scionco VoI. 278, No 5345, pp 1943-7.

Talak, I., Ozaias, R., Lizin, Y., CoIik, A.I., Ozlav, C., Sonluik, H. (2OO3) OinidazoIo-inducod Iivoi danago: iopoil of
lhioo casos and ioviov of lho Iiloialuio. Livoi Inl. VoI. 23, No 5, pp 351-4.

Tandon N, Tandon HD, Iuii K. (1975) An oIoclion nicioscopic sludv of Iivoi in hopalonogaIv piosunalIv causod
lv anooliasis. Lxp MoI IalhoI VoI 22, pp 118-132.

Thonpson, I.L. Ii, IoiIonza, S., Voina, R.(1985) Anolic Iivoi alscoss: a lhoiapoulic appioach. Rov Infocl Dis VoI 7,
171-79.

Thuinan, R.C. (2OOO) Sox-ioIalod Iivoi injuiv duo lo aIcohoI invoIvos aclivalion of Kupffoi coIIs lv ondoloxin. Can I
CaslioonloioI. VoI 14, SuppI D, pp 129D-135D.

Uvgun, .L., Solo-Culioiioz, A., Yagi, H., Izanis, M.L., Cuzzaidi, M.A., ShuInan, C., MiIvid, I., ol aI. (2O1O) Oigan
ioonginooiing lhiough dovoIopnonl of a lianspIanlalIo iocoIIuIaiizod Iivoi giafl using docoIIuIaiizod Iivoi naliix. Nal
Mod 2O1O.

Vonluia-Iuioz, I., IaiiIIo-Luna, R.A., Iuonlos-AguiIai, L., Iinoda-Vzquoz, A., Muoz-Ioinndoz, L., Madiid-Rovos,
I.I., Canpos-Rodiguoz, R. (2OO3) Hunan anoolic hopalic alscoss: in silu inloiaclions lolvoon liophozoilos,
naciophagos, nouliophiIs and T coIIs. Iaiasilo InnunoI. VoI.25, No 1O, pp 5O3-11.

VoiaIa A.T., AnaloIa, I.R., CaiIos, I.M.(2O11) Iallv Livoi and Ischonia/Ropoifusion: Aio lhoio Diugs AlIo lo Miligalo
Injuiv`. Cuii Mod Chon. VoI. 18(32), pp 4987-5OO2.

Vician, M., OIojnik, I., MichaIka, I., Iakulovskv, I., ivchla, I., Coigo,I M. (2OO9) Wain Iivoi ischonia in oxpoiinonl and
IvsosonaI naikois. ialisI Lok Lislv. VoI 11O, No 1O, pp 587-91.
Vinos, R.R., Iuidv I.L., RagIand, .D., SanuoIson, I., Mann, .I., Iolii, W.A. (1995) SlalIo opisonaI liansfoclion of
Lnlanoola hisloIvlica. MoI. iochon. IaiasiloI. VoI 71, pp 265-267.

Viik, K.I., CanguIv, N.K., DiIavaii, I.., Mahajan, R.C. (1989) RoIo of IvsosonaI onzvnos in lissuo danago duiing
hopalic anooliasis. An oxpoiinonlaI sludv. Livoi. VoI. 9 No. 6, pp 338-45.

Viik, K.I., Mahajan, R.C., DiIavaii, I.., CanguIv, N.K. (1988) RoIo of lola-gIucuionidaso, a IvsosonaI onzvno, in lho
palhogonosis of inloslinaI anooliasis: an oxpoiinonlaI sludv. Tians R Soc Tiop Mod Hvg. VoI. 82, No 3, pp 422-5.

Vovlik-Hailin, S.L., iighlnan, A.O., Kiaino, M.R., Waisnoi, ., advIak, S.I. (1997) Idonlificalion of oxliaclalIo
giovlh faclois fion snaII inloslinaI sulnucosa. I CoII iochon VoI. 67, pp 478-491.
Whilnoio, C.I., Vaighoso, A.I., CuIvas, S. (1986) Roaclion of 2-nilioinidazoIo nolaloIilos vilh guanino and possilIo
lioIogicaI consoquoncos. IARC Sci IulI. VoI. 7O, pp 185-96.


WidoI, M., Walias, I., Suvinski, I., SaIvinska, L. (1982) Radiosonsilizing aliIilv and cvloloxicilv of sono 5(4)-
sulslilulod 2-nolhvI-4(5)-nilioinidazoIos. NoopIasna. VoI 29, No 4, pp 4O7-15.


Wisso, L., iaol, I., Luo, D., Zangoi, R.D., Ians, D., Ciallo, L., Voinooson, A.M. (1996) Sliucluio and Iunclion of
SinusoidaI Lining CoIIs in lho Livoi. ToxicoI IalhoI Ianuaiv VoI 24, No 1, pp 1OO-111.

Whilakoi, S. I., McMiIIan, T. I. (1992) Oxvgon offocl foi DNA doulIo-sliand lioak induclion doloininod lv puIsod-fioId
goI oIocliophoiosis. Inl I Radial ioI VoI. 61, No 1, pp 29-41.

Woilhinglon Lnzvno ManuaI. (1993) Woilhinglon iochonicaI Coipoialion, 73O Vassai Avonuo, Lakovood, Nov
Ioisov O87O1, Ldiloi: Woilhinglon, V. pp 1-1947.

Yun-Choung, K., In-Iai, L.(197O) iochonisliv of ial Iivoi iogonoialion. Tho Chung Chi IouinaI. Ip 96-1O9.

Zhang, Z., Yan, L., Wang, L., SovdoI, K.., Li, L., Ankii, S., MiioInan, D., SlanIov, S.L. (2OOO) Lnlanoola hisloIvlica
cvsloino pioloinasos vilh inloiIoukin-1 convoiling onzvno (ICL) aclivilv causo inloslinaI infIannalion and lissuo
danago in anooliasis. MoI. Miciol. VoI 37, pp 542-548.

Zhong, H., OIivo, I. L.(1997) InfIuonco of oxvgon on iadialion-inducod DNA danago in loslicuIai coIIs of C3H nico. Inl
I Radial ioI VoI. 71, No 3, pp 275-82.

Zhong, Z., Lonaslois, I.I. (2OO4)RoIo of fioo iadicaIs in faiIuio of fallv Iivoi giafls causod lv olhanoI.VoI 34, No 1, pp 49-
58.

Zhu, Y., Connoi, C.L.(1994) InloinoIocuIai associalion of IvsosonaI pioloin piocuisois duiing liosvnlhosis. I ioI
Chon. VoI. 269, No 5, pp 3846-51.

Zionoi, L.S., Lvans, S.M., Kachui, A.V., Shunan, A.L., Caidi, C.A., Ionkins, W.T., Kaip, I.S., AIavi, A., DoIlioi, W.R.
Ii, Koch, C.I.(2OO3) Noninvasivo inaging of lunoi hvpoxia in ials using lho 2-nilioinidazoIo 18I-LI5. Lui I NucI
Mod MoI Inaging. VoI. 3O, No 2, pp 259-66














AcndIx 1

Ialionls: In 1O conlioI suljocls, slooI, LLISA, soiun and Iivoi liopsios voio coIIoclod
1
. OnIv piovod 1O suljocls fioo
fion anv hopalic disoaso voio oxaninod foi onzvno oslinalions in isoIalod Iivoi coIIs fion liopsv spocinons. Olhoi
lon suljocls voio lioalod vilh nilioinidazoIo (Tinila and ZiI fion Hinduslan Lovoi Lld, onlav) lhoiapv 2 x 5 gn
ono-lino doso lhiico al 2 davs inloivaI. Al lho ond of doso, Iivoi liopsv and soiun coIIoclion vas dono.
O AII lho 1O conlioI suljocls had no cIinicaI findings of anv hopalic disoaso and shovod slooI -vo, LLISA -vo. AII 1O
shovod noinaI Iivoi and no alscoss confiinod lv uIliasound and Iivoi scan. Tho suljocls voio soIoclod of 35 + 15
voais (noan ago + sd), avoiago nonlhIv incono 175O + 1OO INR, no hopalonogaIv, no diaiihoa, fovoi and pain. Tho
suljocls afloi nilioinidazoIo lioalnonl shovod inducod hopalonogaIv insignificanl Ioss lhan 2 cn lv Iivoi scan. Tho
suljocls vilh anooliasis and anoolic Iivoi alscoss aio nol incIudod and nol shovn hoio.

Tho LLISA vas dono lv nolhod of Iiakash ol aI.|44!. Livoi liopsv vas lakon lv Manghini noodIo |45!.

iochonicaI assavs: In liopsv sonipiops and soiun sanpIos lho hopalocoIIuIai onzvnos voio oslinalod as dosciilod
oIsovhoio |Shaina, 2OO9!. AII sulslialos of onzvnos voio ollainod fion Signa ChonicaI Conpanv, Sl Louis, USA.
Tho oslinalions voio dono on UV spocliopholonoloi CociI Inc. LngIand |Shaina, 2OO9!.

LIoclion nicioscopv: Tho liopsv spocinons al sloiod in gIulaiaIdohvdo al -2OC. Ioi fixing sanpIos voio fixod in
donlaI vax and onoisod in pooI of O.1 M phosphalo luffoi conlaining O.1 M Ca
++
. Tho sanpIos voio cul 1 nn culo
lv CiIIollo lIado. Tho culos voio fixod foi 2 houis in O.2 M phosphalo luffoi in 4 changos al 3O ninulos inloivaI in
cappod viaIs. Tho viaIs voio vashod foi 2 houis in O.2 M phosphalo luffoi 4 linos al 3O ninulos inloivaI in cappod
viaIs. Tho viaIs voio posl fixod in luffoiod 1 osniun lolioxido foi 1-2 houis al 4C, dohvdialod in coId 1O olhanoI
foi aloul 5 ninulos foIIovod lv dohvdialions in coId 5O, 7O, 8O, 95 olhanoI foi aloul 5 ninulos oach and
lissuos voio kopl al ioon lonpoialuio. Iuilhoi lissuos voio dohvdialod al 1OO olhanoI foi 15 ninulos. LIoclion
Micioscopo IA nodoI vas usod foi sciooning hopalic coII danago lv opoxv iosin lIocks and osniun lolia oxido
slaining |46!.

Art nf !Ivcr cc!! Isn!atInn and cnzvmc charactcrIzatInn: Tho piocoduios of Iivoi liopsv oxcision and piopaialion of
Iivoi coIIs voio foIIovod as poi olhicaI connilloo of inslilulo as dosciilod in foIIoving soclion |Shaina, 2OO9!.

Rcagcnts Uscd:
Tho coIIagonaso onzvno vas puichasod fion Signa-AIdiich, Sl Louis, MO and pionaso onzvno vas puichasod fion
ooihingoi Moinhoin CMlH.
IsoIalion and piopaialion of Kupffoi coIIs: Tho Iivoi liopsv sanpIos (6-1O gians) voio digoslod vilh coIIagonaso
onzvno (Signa-AIdiich, Sl Louis, MO) 15 ng/nI foi 1O ninulos and lollon paIIol vas usod foi hopalocvlo

1
The patients were studied Ior ongoing other research study on amoebic hepatitis vs amoebic liver abscess by
Hepatocellular DysIunction Criteria at Liver Unit, AIIMS. The results oI hepatic damage are shown in Figure 3.
oxpoiinonls. Olhoi ionainod supoinalanl pail of digoslod Iivoi vas fuilhoi digoslod vilh 2 ng/nI pionaso onzvno
(ooihingoi Moinhoin CMlH) in O.2 Kiols HansIoil uffoi pH 7.4 conlaining 5 nn gIucoso and 3 nM caIciun
chIoiido afloi sopaialion of hopalocvlos. Tho said susponsion vas inculalod foi 45 ninulos al 37`C sliiiing 25O
iovoIulions poi ninulo. Afloi inculalion, lho coII susponsion vas fiIloiod lhiough nvIon siovo 79 79 nicions and
fiIlialo vas conliifugod and vashod lvico vilh KH conlaining 5 nM gIucoso and 3 nM caIciun chIoiido pH 7.4.
Afloi conliifugalion and finaI vashing lho fiIlialo vas conliifugod al 35O g in ico coId conliifugo. Tho sollIod paIIol
al lollon vas vashod vilh TC 199 nodiun. Tho nunloi of Kupffoi coIIs vas counlod on Naulouigh counling
chanloi and visiliIilv vas doloininod lv livpan lIuo.
Kupffoi coII fiaclionalion: Tho isoIalod Kupffoi coIIs voio dividod in lvo pails. Ono pail vas usod foi Kupffoi
coIIuIai onzvnos and olhoi pail vas usod foi in vilio oxpoiinonls. Ioi onzvno oslinalions Kupffoi coIIs fion fioshIv
haivoslod piopaialions voio sonicalod al zoio dogioo lonpoialuio in said KH luffoi in sonipiop. Laloi lhoso voio
fiaclionalod foi diffoionl coIIuIai fiaclions as dosciilod foIIoving lv nolhods |AIaliala ol aI.2OO7: Houff ol aI. 1994:
Kiin ol aI.1982!.
1. In iofiigoialod conliifugo alovo sonipiop piopaialions voio conliifugod al 1OOO g lo lako oul coII nucIoai fiaclion
and again conliifugod al 9OOO g lo lako oul nilochondiiaI fiaclion as paIIol al lollon.
2. Alovo supoinalanl vas conliifugod al 23OOO g lo isoIalo niciosonaI lodios fiaclion aIong vilh fiaclion iich in
poioxisonos.
3. Tho Iighl luilid supoinalanl vas conliifugod al 1OOOOO g lo isoIalo IvsosonaI fiaclion as cIoai vhilo paIIol al
lollon. Sonolinos niciosonos voio conlaninalod vilh Ivsosonos. Tho IvsosonaI puiilv ovaIualion vas usod lo
oxcIudo niciosonos fioo fion Ivsosonos |Wisso ol aI.1996!.

Kupffoi coIIs in cuIluios: Tho Kupffoi coII cuIluios voio nainlainod foi 48 houis afloi isoIalion fion pionaso
poifusod Iivoi liopsv sanpIos |iouvoi ol aI. 1988!. Tho in vilio cuIluiod coIIs voio scioonod foi livpan lIuo
oxcIusion |Iioh ol aI.2OO3!. Tho Kupffoi coIIs voio usod foi lhoii in vilio phagocvlic aclion upon foioign pailicIos
vhich voio shoop oivlhiocvlos, Ialox loads and zvnosan suspondod in 1O nM phosphalo luffoi saIino pH 7.4 al 4C.
Afloi haivosling Kupffoi coIIs voio addod in phosphalo luffoiod saIino pH 7.4 conlaining 4.6 x 1O
6
adhoionl coIIs and
lhioo nI IS-TC 199 nodiun in cuIluio fIasks conlaining Kupffoi coIIs in nodiukn vilh pailicIo coII ialio 1:5 foi
oivlhiocvlos, 1:4O foi Ialox loads and 1:4 foi liophozoils. Tho ialio foi zvnosan and coII vas 2O:1. Thov voio kopl up
lo 48 houis oi noio in cailogon alnosphoio (95 O2 and 5 CO2) in dosiccaloi vilh lvico changos of nodiun afloi
ovoiv 24 houis.