5-Lipoxygenase Deficiency Reduces Acetaminophen-Induced ... · 2 BioMedResearchInternational withthedevelopmentofhepaticedemaanddysfunction[8]. Furthermore,LTB4 andthe5-LOpathwaywerereportedto

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 627046 13 pageshttpdxdoiorg1011552013627046

Research Article5-Lipoxygenase Deficiency Reduces Acetaminophen-InducedHepatotoxicity and Lethality

Miriam S N Hohmann1 Renato D R Cardoso1 Felipe A Pinho-Ribeiro1

Jefferson Crespigio1 Thiago M Cunha2 Joseacute C Alves-Filho2 Rosiane V da Silva1

Phileno Pinge-Filho1 Sergio H Ferreira2 Fernando Q Cunha2

Rubia Casagrande3 and Waldiceu A Verri Jr1

1 Department of Pathology Biological Science Centre State University of Londrina Rodovia Celso Garcia Cid Pr 445Km 380 Cx Postal 6001 86051-990 Londrina PR Brazil

2 Department of Pharmacology Ribeirao Preto Medical School University of Sao Paulo Avenida Bandeirantes 390014049-900 Ribeirao Preto SP Brazil

3 Department of Pharmaceutical Sciences Health Sciences Centre State University of Londrina Rodovia Celso Garcia Cid Pr 445Km 380 Cx Postal 10011 86051-990 Londrina PR Brazil

Correspondence should be addressed to Waldiceu A Verri Jr waldiceujryahoocombr

Received 30 April 2013 Revised 4 September 2013 Accepted 6 September 2013

Academic Editor Chun-Ming Wong

Copyright copy 2013 Miriam S N Hohmann et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation At present theparticipation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed 5-LO deficient(5-LOminusminus) mice and background wild type mice were challenged with APAP (03ndash6 gkg) or saline The lethality liver damageneutrophil and macrophage recruitment LTB

4

cytokine production and oxidative stress were assessed APAP induced adose-dependent mortality and the dose of 3 gkg was selected for next experiments APAP induced LTB

4

production in theliver the primary target organ in APAP toxicity Histopathological analysis revealed that 5-LOminusminus mice presented reducedAPAP-induced liver necrosis and inflammation compared with WT mice APAP-induced lethality increase of plasma levels ofaspartate aminotransferase and alanine aminotransferase liver cytokine (IL-1120573 TNF-120572 IFN-120574 and IL-10) superoxide anion andthiobarbituric acid reactive substances productionmyeloperoxidase andN-acetyl-120573-D-glucosaminidase activity Nrf2 and gp91phoxmRNA expression and decrease of reduced glutathione and antioxidant capacitymeasured by 221015840-azinobis(3-ethylbenzothiazoline6-sulfonate) assay were prevented in 5-LOminusminusmice compared toWTmiceTherefore 5-LO deficiency resulted in reducedmortalitydue to reduced liver inflammatory and oxidative damage suggesting 5-LO is a promising target to reduce APAP-induced lethalityand liver inflammatoryoxidative damage

1 Introduction

The 5-lipoxygenase (5-LO) pathway converts arachidonicacid into leukotrienes (LTs) specifically LTB

4

and cysteinyl-LTs (LTC

4

LTD4

and LTE4

) [1] and studies indicate thatthis pathway is responsible for developing and sustaininginflammation [2] All LTs display a variety of proinflamma-tory actions but LTB

4

in particular is one of the most potentchemotactic agents and activating factors for leukocytes [3]

In fact LTB4

has been implicated in the pathophysiology ofacute and chronic inflammatory diseases [4 5]

In this context many studies have investigated the par-ticipation of 5-LO and its products in different experimentalmodels of liver injury 5-LOpathway andLTs have been impli-cated in hepatic inflammation and liver damage Elevatedproduction of LTs in the liver was detected in rats with CCl

4

-induced liver injury [6 7] In hepatic ischemia and reper-fusion injury LT production was enhanced and associated

2 BioMed Research International

with the development of hepatic edema and dysfunction [8]Furthermore LTB

4

and the 5-LO pathway were reported tobe involved in the pathogenesis of experimental liver fibrosisand inflammatory necrosis [7 9 10]Thus 5-LO products areimportant mediators of hepatic inflammation and cell injury

Considering that LTs are important mediators of liverinflammation and damage 5-LO pathway and its productsmight also be involved in acetaminophen (APAP)-inducedliver injury APAP is a widely used over-the-counter analgesicand antipyreticwith few side effectswhen taken at therapeuticdoses However APAP intoxication can result in severe liverdamage characterized by centrilobular liver necrosis and inmore severe cases acute liver failure and eventually death[11] Studies have shown that APAP overdose has become themost common cause of acute liver failure in many Westerncountries [12 13]

It is well established that APAP-induced liver injurydepends on the metabolic conversion of APAP to thehighly reactive metabolite N-acetyl-p-benzoquinone imine(NAPQI) by cytochrome P-450 (CYP) enzymes primarilyCYP2E1 and to a lesser extent CYP1A2 CYP2A6 andCYP3A4 [14 15] In normal conditions this metabolite isreadily detoxified by hepatic reduced glutathione (GSH)However after toxic dose of APAP GSH is depleted and asa result NAPQI accumulates and covalently binds to hepa-tocellular proteins initiating liver injury [16 17] Althoughprotein binding is the initiator of toxicity and cellular injurysecondary processes amplify and propagate it Initial hepa-tocellular damage caused by NAPQI can lead to the releaseof damage-associated molecular patterns (DAMPS) such ashigh-mobility group box 1 protein (HMGB1) heat shockproteins DNA fragments and others [18] which can directlyactivate nonparenchymal hepatic cells Kupffer cells (KC)and other innate immune cells [19 20] In addition thesecells can also be activated by potent chemotactic factors(ie LTB

4

) released by injured hepatocytes [21] Activatedcells can release a wide range of inflammatory mediatorssuch as reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) [22] proinflammatory cytokines [18] andLTs [6] amplifying the oxidative stress and inflammatoryresponses consequently promoting further hepatic injury[23] Moreover the excessive ROS and RNS productionduring inflammation contributes to the depletion of GSH[24] which could result in further NAPQI accumulation [17]

Neutrophil and macrophage accumulation in the liverhas also been associated with APAP-induced liver injury[25 26] and several other experimental animal models ofliver injury such as ischemia-reperfusion injury [27 28]and endotoxemia [29] Neutrophils and macrophages undercertain circumstances can severely aggravate tissue damageIn APAP-induced hepatotoxicity excessive neutrophil andmacrophage activity can contribute to liver inflammation andinjury [25 26]

Although many studies have demonstrated that 5-LOpathway is involved in the pathogenesis of different modelsof liver inflammation at present the participation of 5-LOin APAP-induced hepatotoxicity and liver damage has notbeen addressed In this context the aim of the currentstudy was to use 5-LO deficient mice to investigate the role

of 5-LO on APAP-induced lethality and liver damage Inaddition it aimed to demonstrate the mechanisms by which5-LO deficiency ameliorates the events mentioned previ-ously focusing on neutrophil and macrophage recruitmentcytokine production and oxidative stress in the liver

2 Methods

21 Animals Male 5-lipoxygenase deficient (5-LOminusminus) andwild type (WT SV129) mice weighing 20ndash25 g were used 5-LOminusminus andWTmice originally fromThe Jackson Laboratory(Bar Harbor ME USA) were gifted from Dr Marc LeePeters-Golden (University of Michigan) weighing 20ndash25 gMice were housed at 21∘C on a 12 h lightdark cycle instandard clear plastic cages with food and water ad libitumAnimals were handled humanely and all procedures con-cerning animal care and use were approved by the Researchand Ethics Committee of State University of Londrina (pro-cess 29612010-10) and carried out in accordance with itsguidelines

22 Experimental Design In dose-response experimentsWT mice (119899 = 10 per group) were treated orally with 03 12 3 and 6 gkg of APAP suspended in saline (200mgmL)In APAP-induced lethality experiments 5-LOminusminus and WTmice (119899 = 10 per group) were treated orally with 3 gkgof APAP suspended in saline (200mgmL of saline) orequal volume of saline (control group) In these survivalexperiments mice were observed every 6 h during 72 hand any that showed extreme distress or became moribundwere sacrificed For subsequent experiments animals wereanesthetized and sacrificed 12 h after APAP treatment Bloodsamples were collected by cardiac puncture to determinethe enzymatic activities of aspartate aminotransferase (AST)and alanine aminotransferase (ALT) For liver analysis theabdomen of the mice was opened and the left lobe of theliver was quickly excised A portion of the tissue was storedat minus20∘C in 50mM K

2

HPO4

buffer (pH 60) containing05 hexadecyl trimethylammonium bromide (HTAB) formyeloperoxidase (MPO) andN-acetyl-120573-D-glucosaminidase(NAG) activity determination and the remaining tissue wasstored in saline at minus80∘C for LTB

4

and cytokine (TNF-120572IL-1120573 IFN-120574 and IL-10) quantification The midsections ofthe left lobe of the liver were collected and processed forhistopathology analysis In order to assess hepatic oxidativestress the liver was first perfused with 10mL of ice-cold 115KCl solution through the portal vein For quantitative PCR(qPCR) analysis the 115 KCl solution used for liver perfu-sion was treated with 01 vv diethylpyrocarbonate (DEPC)for 24 hours at 37∘C and then autoclaved to inactivate DEPCThe left lobe was excised in the same manner and a portionof the perfused tissue was separated for 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonate ABTS) assay whereas theremaining tissue was immediately stored at minus80∘C for laterassessment of reduced glutathione (GSH) thiobarbituric acidreactive substances (TBARS) levels nitroblue tetrazolium(NBT) reduction and mRNA expression for gp91phox andtranscription factor Nrf2

BioMed Research International 3

23 Enzymatic Markers of Liver Injury Blood was col-lected into microtubes containing 50 120583L of the anticoagu-lant ethylenediamine tetraacetic acid (EDTA) (5000 IUmL)and centrifuged (200timesg 10min 4∘C) and the plasma wasseparated In order to determine enzymatic activities ofAST and ALT plasma samples were processed according tothe manufacturerrsquos instructions (Labtest Diagnostico SABrazil) Results were presented as UL

24 Histopathology For liver histopathology analysis mid-sections of the left lobes of the liver were fixed in 10 bufferedneutral formalin solution for 24 h and embedded in paraffinwax and 5 120583m sections were prepared and stained withhematoxylinmdasheosin (HampE) HampE stained liver sections wereexamined and scored by a pathologist using lightmicroscopyand the degree of necrosis and inflammation was determinedas previously described by Yaman et al [30] The degree ofnecrosis was classified on a scale of 0ndash3 [normal 0 (0)mild 1 (1ndash25) moderate 2 (26ndash49) severe 3 (50ndash100)]and expressed as the mean of 10 high power fields (HPFs)chosen at randomThe degree of inflammation was evaluatedin the same 10 HPFs and classified on a scale of 0ndash3 [noinflammation 0 (mean of inflammatory cells in 10HPFs = 0)weak inflammation 1 (mean of inflammatory cells in 10HPFs= 1ndash10) moderate inflammation 2 (mean of inflammatorycells in 10 HPFs = 11ndash49) and severe inflammation 3 (meanof inflammatory cells in 10 HPFs = 50 and over)]

25 Cytokines and LTB4

Levels Frozen liver samples werehomogenized in 500 120583L of saline using a turrax T10 basic(IKA Staufen Germany) in an ice bath The samples werecentrifuged (800timesg 10min 4∘C) and with the resultingsupernatant IL-1120573 TNF-120572 IFN-120574 IL-10 (eBioscience) andLTB4

(Cayman Chemical Ann Arbor MI USA) levelswere determined by ELISA according to the manufacturerrsquosinstructions Results were presented as pgmg of liver

26 MPO Activity MPO is an enzyme abundantly presentin the azurophilic granules of neutrophils that has beenused as a biochemical marker of neutrophil infiltration intovarious tissues [24 31] thus in the present study MPOcolorimetric assay was used to assess neutrophil migrationto the liver Frozen samples were homogenized as describedabove and centrifuged (16100timesg 2min 4∘C) The resultingsupernatant was assayed spectrophotometrically for MPOactivity determination at 450 nm Briefly 5120583L of the super-natant was mixed with 200 120583L 50mM phosphate buffer (pH60) containing 0167mgmL o-dianisidine dihydrochlorideand 0015 hydrogen peroxideThe results were presented asthe MPO activity (Umg of liver) [24 31]

27 NAG Activity NAG activity was determined by anadapted colorimetric method previously described by Hori-nouchi et al [32] Briefly 20 120583L of supernatant previouslydescribed in MPO activity was placed in a 96-well platefollowed by the addition of 80 120583L of 50mM phosphate bufferpH 60The reaction was initiated by the addition of 224mM4-nitrophenyl N-acetyl-120573-D-glucosaminide The plate was

incubated at 37∘C for 10min and the reaction was stoppedby the addition of 100 120583L of 02M glycine buffer pH 106Theenzymatic activity was determined spectrophotometricallyat 400 nm (Multiskan GO Microplate SpectrophotometerThermoScientific Vantaa Finland) NAG activity of sampleswas presented as NAG activity (ODmg of liver)

28 GSH GSH levels were determined spectrophotometri-cally by an adaptedmethod described by Sedlack and Lindsay[33] The frozen perfused liver samples were homogenizedin cold 002M EDTA The homogenate was treated with50 trichloroacetic acid and centrifuged (1500timesg 15min)and to the supernatant 04M Tris-HCl pH 89 was addedNext samples were vortex-mixed and 10mM dithiobis-nitrobenzoic acid was added followed by vortex-mixingSamples were allowed to stand for 5 minutes before beingread at 412 nm Standard curve was prepared using differentconcentrations of GSH in addition to the other reagentsmentioned before Results were presented as nmol GSHmgof liver

29 ABTS Assay The perfused liver samples collected wereimmediately processed and homogenates (20 wv) wereprepared with ice-cold 115 KCl Samples were centrifuged(200timesg 10min 4∘C) andwith the resulting supernatant totalantioxidant capacity of liver was assessed by ABTS assay[34] This assay is based on the ability of the antioxidantsmolecules to quench ABTS radical cation (ABTSsdot+) a blue-green chromophore with characteristic absorption at 734 nmcompared with that of TroloxThe antioxidants present in theliver samples when added to ABTSsdot+ reduce it into ABTSwhich results in decolorization ABTSsdot+ was produced byreacting ABTS stock solution (ABTS dissolved in water toa 7mM concentration) with 245mM potassium persulfate(final concentration) and allowing the mixture to stand inthe dark at room temperature for 12ndash16 h before use For thestudy ABTSsdot+ solutionwas diluted in phosphate buffer pH 74to reach an absorbance of 08 (plusmn002) at 734 nm 10 120583L of thesamples was added to 1mL of the diluted ABTSsdot+ solutionsamples were vortex-mixed and allowed to stand for 6minThe samples were read in spectrophotometer at 734 nm Astandard curve was prepared using different concentrationsof Trolox Because this is a Trolox equivalent antioxidantcapacity (TEAC) assay results were presented as 120583mol Troloxequivalentmg of liver

210 NBT Reduction The superoxide anion production wasdetermined by the reduction of the redox dye NBT [35]Frozen liver sampleswere homogenizedwith 500120583L of salineand 50 120583L of the homogenate was placed in a 96-well platefollowed by the addition of 100 120583l of NBT solution (1mgmL)and incubation for 1 h at 37∘C The supernatant was carefullyremoved and the formazan precipitated was then solubilizedby adding 120120583L of 2M KOH and 140 120583L of DMSO Theoptical density wasmeasured bymicroplate spectrophotome-ter reader (Multiskan GO Microplate SpectrophotometerThermoScientific Vantaa Finland) at 600 nm The weight of


samples was used for data normalization and results werepresented as NBT reduction (ODmg of liver)

211 Lipid Peroxidation Lipid peroxidation in the liver wasassessed by determining TBARS levels using an adaptedmethod previously described by Guedes et al [36] Forthis assay trichloroacetic acid (10) was added to thehomogenate to precipitate proteins followed by centrifuga-tion (1000timesg 3min 4∘C) The protein-free supernatant wasseparated and thiobarbituric acid (067) was added Themixture was kept in water bath (15min 100∘C)Malondialde-hyde (MDA) an intermediate product of lipid peroxidationwas determined by difference between absorbances at 535 and572 nm using a microplate spectrophotometer reader Theresults were presented as TBARS (nmol MDAmg of liver)

212 Quantitative Polymerase Chain Reaction (qPCR) qPCRwas performed as previously described [37] Samples werehomogenized in TRIzol reagent and total RNAwas extractedby using the SV Total RNA Isolation System (Promega)All reactions were performed in triplicate using the fol-lowing cycling conditions 50∘C for 2min 95∘C for 2minfollowed by 40 cycles of 95∘C for 15 s and 60∘C for 30 sqPCR was performed in a LightCycler Nano Instrument(Roche Mississauga ON USA) sequence detection systemby using the Platinum SYBR Green qPCR SuperMix UDG(Invitrogen USA) The primers used were gp91phox sense51015840-AGCTATGAGGTGGTGATGTTAGTGG-31015840 antisense51015840-CACAATATTTGTACCAGACAGACTTGAG-31015840 Nrf2sense 51015840-TCACACGAGATGAGCTTAGGGCAA anti-sense 51015840-TACAGTTCTGGGCGGCGACTTTAT 120573-actinsense 51015840-AGCTGCGTTTTACACCCTTT-31015840 antisense 51015840-AAGCCATGCCAATGTTGTCT-31015840 The expression of 120573-actin mRNA was used as a control for tissue integrity in allsamples

213 Sleeping Time Induced by Pentobarbital APAP-induced-toxicity is highly dependent on metabolic conversion ofAPAP to NAPQI by CYP enzymes therefore the effect of5-LO deficiency on hepatic microsomal cytochrome P450activity was investigated by assessing pentobarbital-inducedsleeping time in 5-LOminusminus and WT mice 5-LOminusminus andWT mice were treated with pentobarbital diluted in saline(50mgkg ip) and the duration of sleep (min) of eachanimal was analyzed Loss of righting reflex to recovery wasrecorded as the sleeping time [38]

214 Statistical Analysis The results are expressed as meanplusmn SEM Survival rates were estimated by the Kaplan-Meiermethod and statistical analysis was carried out by the log-rank test to test for equality of the survival curves Statisticaldifferences were compared by Studentrsquos t-test or by one-wayANOVA followed by Bonferronirsquos multiple comparison testFor categorical variables the Kruskal-Wallis test followedby Dunnrsquos test was performed All statistical analyses wereperformed using Graph Pad Prism 5 (La Jolla CA) The levelof significance was set at 119875 lt 005 Studies were conductedtwo to three times and mean data are shown

3 Results

31 APAP Induces Dose-Dependent Lethality and LTB4

Pro-duction in the Liver To determine the dose of APAP nec-essary to induce significant lethality in this strain a dose-response study was performed WT mice were treated orallywith APAP (03 1 2 3 and 6 gkg) or equal volume of salineand lethality was assessed Saline and 03 gkg of APAP didnot induce death in any of the animals (Figure 1(a)) Theadministration of 10 gkg of APAP induced 15 lethality in6 h 25 in 18 h 30 in 24 h and 35 in 48 h which wasmaintained until the end of the experiment The adminis-tration of 2 gkg induced similar lethality 20 in 12 h and35 in 66 h which was also maintained Mice treated with3 gkg of APAP presented 45 and 70 mortality within 6 hand 12 h respectively and a little over 95 in 24 h which wasmaintained Finally mice were treated with 6 gkg of APAPto assure that 3 gkg of APAP was the submaximal lethaldose in this experimentalmodelThe administration of 6 gkgof APAP induced 100 mortality in 6 h thus consideredinadequate (Figure 1(a)) Therefore 3 gkg of APAP wasselected for the following experiments addressing the hepaticmechanisms triggered by a lethal dose of APAP

In order to determine 5-LO participation in APAP hep-atotoxicity the effect of the toxic dose of APAP on hepaticlevels of LTB

4

was assessed In this contextWTmice received3 gkg of APAP or equal volume of saline per oral andafter 12 h animals were sacrificed and liver samples werecollected for assessment of LTB

4

levels It was observed thatAPAP induced a sim10-fold increase of LTB

4

levels in the livercompared to saline (Figure 1(b)) Twelve h was selected sinceit is an intermediary time point between intoxication anddeath (Figure 1(b))

32 5-LO Participates in APAP-Induced Lethality 5-LOminusminusand WT mice were treated with APAP (3 gkg) or equalvolume of saline per oral and survival rates during APAPintoxication were determined (Figure 2) APAP adminis-tration induced significant mortality in WT mice withapproximately 45 lethality in 6 h 75 in 12 h and 100 in24 h However APAP induced significantly lower mortalityin 5-LOminusminus mice compared to WT mice 5 lethality in 6 h15 in 12 h 60 in 24 h and 90 in 54 h in 5-LOminusminus miceSaline did not induce death in any of the animals Twelve hwas selected for the next experiments investigating themech-anisms involved in APAP-induced intoxication because thegreatest difference betweenWT and 5-LOminusminus was observed atthis time point

33 APAP-Induced Histopathological Changes in the LiverWere Reduced in 5-LOminusminusMice APAP (3 gkg) or equal vol-ume of saline per oral was administrated to 5-LOminusminus andWT mice and after 12 h liver histopathological analysis wasperformed and representative images of liver histology wereobtained Histopathology analysis of the liver demonstratedthat APAP induced significantly higher degree of liver necro-sis (Figure 3(a)) and inflammation (Figure 3(b)) in WT micewhen compared to 5-LOminusminus mice Saline did not induce


0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100

Saline

Time (h)

Sur

viva

l (

)

03 gkg APAP10 gkg APAP


(a)

Saline APAP0

50

100

150

200

250

300

350

400

450

500

LTB 4

(pg

mg

liver

)

lowast

(b)

Figure 1 Acetaminophen (APAP) induces dose-dependent lethality and LTB4

production in the liver (a) WTmice were treated with APAP(03 1 3 and 6 gkg) or saline per oral and lethality was assessed The lethality induced by APAP was monitored at 6 h intervals during72 h 119899 = 10 representative of three separate experiments (b) WT mice were treated with APAP (3 gkg per oral) or saline and after 12 hliver samples were collected for the determination of LTB

4

levels by ELISA Values are mean plusmn SEM 119899 = 5 representative of two separateexperiments lowast119875 lt 005 compared to saline group Studentrsquos t-test

necrosis (Figure 3(a)) or inflammation (Figure 3(b)) in theliver of WT and 5-LOminusminus mice In representative imagesof liver histology it can be observed that APAP inducedsignificantly larger area of necrosis in the liver of 5-LOminusminusmice when compared to WT mice (Figures 3(e) and 3(f)resp) No apparent difference was observed in the liversamples of WT and 5-LOminusminus mice receiving only saline(Figures 3(c) and 3(d))

34 APAP-Induced Increase in Plasmatic AST and ALT LevelsWas Reduced in 5-LOminusminus Mice APAP (3 gkg) or equalvolume of saline per oral was administrated in 5-LOminusminus andWT mice and after 12 h APAP-induced liver damage wasestimated by plasmatic AST and ALT level determinationAPAP significantly increased plasma levels of both enzymesinWTmice when compared to control group receiving salinebut not in 5-LOminusminusmice (Figures 4(a) and 4(b))There was nosignificant difference inAST andALT levels betweenWT and5-LOminusminus mice receiving saline

35 APAP-Induced Increase in MPO and NAG Activity WasReduced in 5-LOminusminus Mice The MPO and NAG activity wereused as an indirect marker of neutrophilmacrophage andmacrophage presence respectively in hepatic tissue 12 h afteroral administration of APAP (3 gkg) or equal volume ofsaline APAP induced a significant increase of MPO andNAG activity in WT mice compared to saline (Figure 5) Onthe other hand MPO and NAG activity were reduced in 5-LOminusminus mice compared to those in WT receiving APAP No

significant difference was found between MPO and NAGactivity of WT and 5-LOminusminus mice that received saline

36 APAP-Induced Cytokine Production in the Liver WasReduced in 5-LOminusminus Mice Mice were treated with APAP(3 gkg) or equal volume of saline per oral and after 12 h liversamples were collected and cytokine levels were determinedInWTmice APAP induced significant increase of hepatic IL-1120573 TNF-120572 IFN-120574 and IL-10 production compared to saline(Figures 6(a) 6(b) 6(c) and 6(d) resp) In 5-LOminusminus micehowever APAP did not increase cytokine production Therewas no significant difference in cytokine levels between WTand 5-LOminusminus that received saline

37 APAP-Induced Oxidative Stress in the Liver Was Reducedin 5-LOminusminus Mice Mice were treated with APAP (3 gkg) orequal volume of saline per oral and after 12 h liver sampleswere collected to determine superoxide anion production(NBT reduction) lipid peroxidation (TBARS levels) GSHlevels and antioxidant capacity by ABTS assay WT micetreated with APAP presented significant increase of super-oxide anion production (Figure 7(a)) and lipid peroxidation(Figure 7(b)) and decrease of GSH levels (Figure 7(c)) andantioxidant capacity (Figure 7(d)) compared to saline WTmice which was not observed in 5-LOminusminus mice treated withAPAP There was no significant difference between WT and5-LOminusminus that received saline

38 APAP-Induced 11989211990191119901ℎ119900119909 mRNA Expression Was Reducedand Transcription Factor Nrf2 mRNA Expression Was


lowast

0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100

WT + APAPWT + saline

Time (h)

Surv

ival

()

5-LOminusminus + APAP5-LOminusminus + saline

Figure 2 5-LO participates in acetaminophen (APAP)-inducedlethality WT mice and 5-LOminusminus mice were treated with APAP(3 gkg) or saline per oral The lethality induced by APAP wasmonitored at 6 h intervals during 72 h 119899 = 10 representative of threeseparate experiments lowast119875 lt 0001 compared to WT mice treatedwith APAP Kaplan-Meier method followed by the log-rank test

Enhanced in 5-LOminusminus Mice Mice were treated with APAP(3 gkg) or equal volume of saline per oral and after 12 hliver samples were collected to determine gp91phox and Nrf2mRNA expression by qPCR WT mice treated with APAPpresented significant increase of gp91phox mRNA expression(Figure 8(a)) compared to saline which was not observedin 5-LOminusminus mice treated with APAP On the other handAPAP-induced Nrf2 mRNA expression was enhanced in5-LOminusminus mice compared to WT mice treated with APAP(Figure 8(b)) There was no significant difference betweenWT and 5-LOminusminus that received saline

39 Pentobarbital-Induced Sleeping Time Was Similar in WTand 5-LOminusminus Mice Mice were treated with sodium pento-barbital (50mgkg intraperitoneal route) and sleeping timewas assessed WT (1428 plusmn 1010min) and 5-LOminusminus (12825plusmn 725min) mice did not present significant difference inpentobarbital-induced sleeping time (119875 = 0363) (Table 1)Therefore 5-LOminusminusmice did not present significant alterationin drug metabolism by CYP enzymes

4 Discussion

In most studies hepatotoxicity is induced in mice by admin-istrating 300ndash750mgkg of APAP [39 40] however in thisstudy a higher dose was used Dose-response studies carriedout in wild type (WT Sv129) mice demonstrated that 3 gkg

Table 1 Effect of 5-LO deficiency on pentobarbital (50mgkg ip)-induced sleeping time in mice

Groups Sleeping time (min)WT 1428 plusmn 10105-LOminusminus 12825 plusmn 725Data as mean plusmn SEM 119899 = 10 per group 119875 = 0363 versus WT (Studentrsquos119905-test)

of APAP is the submaximal lethal dose in this experimentalmodelThe route of administration is certainly a contributingfactor for this difference since in the present study APAP wasadministered per oral and not by intraperitoneal route [3940] Another factor is that food restriction or fasting enhancessusceptibility toAPAP toxicity byCYP2E1 induction enhanc-ingATP andGSHdepletion [41 42] whichwas not the case ofthe present study Furthermore the dose of APAP necessaryto induce hepatotoxicity may also vary depending on micestrains In Swiss mice for example 15 gkg of APAP per oralinduced a similar profile as 3 gkg of APAP in Sv129 mice(data not shown)

APAP induced sim10-fold increase of LTB4

productionin the liver In agreement 5-LO deficient (5-LOminusminus) micepresented lower lethality rates compared to WT mice Themarkedly higher lethality in WT mice lined up well with thehigher degree of necrosis and liver damage in these mice asassessed by liver histopathology analysis and plasma levels ofAST and ALT Furthermore APAP-induced increase ofMPOand NAG activity and cytokine production was reduced in 5-LOminusminus mice APAP-induced oxidative stress was also reducedin 5-LOminusminus mice compared to WT mice as observed byreduction of GSH depletion lipid peroxidation superoxideproduction and increased total antioxidant capacity Further-more therewere reduced gp91phox and increasedNrf2mRNAexpression in 5-LOminusminus mice compared to those in WT mice

APAP induced 5-LO-dependent increase of the biochem-ical markers of neutrophils and macrophages MPO andNAG activity Excessive neutrophil and macrophage activitycan contribute to perpetuation of inflammatory responsesadditional liver damage and even liver failure [25 26] byreleasing a series of proinflammatory molecules such ascytokines [43] reactive oxygen species (ROS) [25] andproteases [44] that are responsible for further tissue damageand inflammation Previous evidence [25 26] together withthe present results suggest that increased 5-LO-dependentneutrophil and macrophage recruitmentactivity may con-tribute to liver damage induced by APAP

In the present study APAP-induced IL-1120573 TNF-120572 IFN-120574 and IL-10 production in WT mice was reduced in 5-LOminusminus mice suggesting that 5-LO products are involved inthe production of cytokines induced by APAP Cytokines arecritical mediators of APAP hepatotoxicity Previous studiesreport that the enhanced release of TNF-120572 and IL-1120573 maybe responsible for further hepatic damage caused by NAPQI[45] Interestingly TNF120572 and IL-1120573 induce hepatic neutrophiland macrophage recruitment and activation [46 47] IFN-120574 participates in APAP-induced liver injury by mediating


5-LOminusminusWT

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f nec

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(a)

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Saline

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(b)

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WT mice

(c)

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Saline

(d)

APAP

(e)

APAP

(f)

Figure 3 Acetaminophen (APAP) induces 5-LO-dependent histopathological changes in the liver WT and 5-LOminusminus mice were treated withAPAP (3 gkg) or saline per oral and after 12 h liver samples were collected and processed for histopathology analysis The degree of livernecrosis (a) (119899 = 5 for saline groups 119899 = 11 for WT APAP group and 119899 = 14 for 5-LOminusminus APAP group) and inflammation (b) (119899 = 4-5 forsaline groups 119899 = 10 for WT APAP group and 119899 = 2 for 5-LOminusminus APAP group) were assessed lowastP lt 005 compared to saline-treated WTand 5-LOminusminus mice and 119875 lt 005 compared to APAP-treated WTmice Kruskal-Wallis test was followed by Dunnrsquos multiple comparison test(cndashf) Representative images of histopathological changes in the liver (HampE 40times) (c) WT mice treated with saline (d) 5-LOminusminus mice treatedwith saline (e) WT mice treated with APAP and (f) 5-LOminusminus mice treated with APAP


0

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APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)

Figure 4 Acetaminophen (APAP) induces 5-LO-dependent liver damage WT and 5-LOminusminus mice were treated with APAP (3 gkg) or salineper oral and after 12 h blood samples were collected to assess liver damage bymeasuring plasma levels of (a) aspartate aminotransferase (AST)and (b) alanine aminotransferase (ALT) Values are mean plusmn SEM 119899 = 5 representative of two separate experiments lowast119875 lt 005 comparedto saline-treatedWT and 5-LOminusminus mice and 119875 lt 005 compared to APAP-treatedWTmice One-way ANOVA was followed by Bonferronirsquosmultiple comparison test

0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)

Figure 5 Acetaminophen (APAP) induces 5-LO-dependent neutrophil and macrophage recruitment Neutrophil and macrophagerecruitment to the liver was assessed by myeloperoxidase (MPO) and N-acetyl-120573-D-glucosaminidase (NAG) activity determination in theliver 12 h after APAP (3 gkg) or saline per oral treatment of WT and 5-LOminusminus mice Values are mean plusmn SEM 119899 = 5 representative of twoseparate experiments lowast119875 lt 005 compared to saline-treated WT and 5-LOminusminus mice and 119875 lt 005 compared to APAP-treated WT miceOne-way ANOVA was followed by Bonferronirsquos multiple comparison test

leukocyte infiltration hepatocyte apoptosis and nitric oxideand cytokine (IL-1120572 IL-1120573 IL-6 and TNF-120572) production[39]Therefore it is conceivable that these cytokinesmay con-tribute to the increase of neutrophil andmacrophagemarkersin the liver liver damage and lethality in APAP intoxication

On the other hand IL-10 is a potent anti-inflammatorycytokine capable of downregulating inflammation and isupregulated during severe liver damage as a protectivemechanism against exacerbated tissue injury [48] This is apossible explanation as to why increased IL-10 production


0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)

Figure 6 Acetaminophen (APAP) induces 5-LO-dependent induction of cytokine production in the liverWT and 5-LOminusminusmicewere treatedwith APAP (3 gkg) or saline per oral and after 12 h liver samples were collected to determine (a) IL-1120573 (b) TNF-120572 (c) IFN-120574 and (d) IL-10levels by ELISA Values are mean plusmn SEM 119899 = 5 representative of two separate experiments lowast119875 lt 005 compared to saline-treated WT and5-LOminusminus mice and 119875 lt 005 compared to APAP-treatedWTmice One-way ANOVAwas followed by Bonferronirsquos multiple comparison test

was not observed in 5-LOminusminus mice after administration ofAPAP 5-LOminusminus mice presented significantly reduced liverdamage and inflammation when compared to WT micethus the endogenous upregulation of IL-10 was not observedFurthermore although it has been suggested that IL-10 maysuppress proinflammatory cytokine production in the liver[49] in our study IL-10 levels were not increased in 5-LOminusminusmice suggesting that the reduction of IL-1120573 TNF-120572 and IFN-120574 production observed in 5-LOminusminus mice was not dependenton the increased IL-10 production

Another important finding of our study was that 5-LO deficiency improves antioxidant status in the liver ofmice treated with APAP APAP-induced increase of super-oxide anion production (NBT assay) and lipid peroxidation

(TBARS assay) and depletion of reduced glutathione (GSH)levels and overall oxidative buffering capacity of the liver(ABTS assay) of WT mice were prevented in 5-LOminusminus miceFurthermore a previous study reported that GSH levelscorrelate with ABTS profile as observed in the present study[50] The production of superoxide anion by phagocytessuch as macrophages and neutrophils is a crucial step inoxidative stress leading to lipid peroxidation and depletionof GSH and the overall endogenous antioxidant systems Infact APAP-induced increase of NADPH oxidase subunitgp91phox mRNA expression in the liver of WT mice wasnot observed in 5-LOminusminus mice Furthermore inflammationinduces the expression of the transcription factorNrf2 whichis responsible for inducing the expression of antioxidant


0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)

Figure 7 Acetaminophen (APAP) induces hepatic oxidative stress in a 5-LO-dependent manner WT and 5-LOminusminus mice were treated withAPAP (3 gkg) or saline per oral and after 12 h liver samples were collected to determine superoxide anion production (nitroblue tetrazolium(NBT) reduction) (a) lipid peroxidation (thiobarbituric acid reactive substances (TBARS)) levels (b) (c) reduced glutathione (GSH) levelsand (d) antioxidant capacity by 221015840-azinobis(3-ethylbenzothiazoline 6-sulfonate ABTS) assay Values aremeanplusmn SEM 119899 = 5 representativeof two separate experiments lowast119875 lt 005 compared to saline-treated WT and 5-LOminusminus mice and 119875 lt 005 compared to APAP-treated WTmice One-way ANOVA was followed by Bonferronirsquos multiple comparison test

molecules including GSH [51] In the present study 5-LOdeficiency resulted in an even greater expression of Nrf2mRNAcompared to that inWTmice which further indicatesan active role of 5-LO products during APAP intoxicationto consume and limit antioxidant systems In agreementin acute lung injury mediated by oxidative stress andinflammation inhibition of 5-LO by MK-886 significantlyattenuated GSH depletion and lipid peroxidation in tissues[52] Moreover 5-LO deficiency inhibited leukocyte-derivedROSproduction andprotected against degeneration of retinalcapillaries in amousemodel of diabetic retinopathy [53]Thisis consistent with the role that 5-LO plays in ROS generation

by for instance activating NADPH oxidase resulting insuperoxide anion production [54] It is also important toconsider the interactive system in which cytokines induceoxidative stress by stimulation of NADPH oxidase and ROSinduce the activation of Nuclear Factor kappa B (NF120581B) andconsequently cytokine production [24]Therefore it is possi-ble that there is also an association between the inhibition ofcytokine production and preservation of antioxidant systemsobserved in 5-LOminusminus mice

The protection conferred by 5-LO deficiency in APAP-induced lethality was more evident in the first 12 h followingAPAP administration Afterwards although 5-LOminusminus mice


00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)

Figure 8 5-LO deficiency reduces acetaminophen (APAP)-induced increase of gp91phox mRNA expression and increases transcription factorNrf2 mRNA expression The mRNA expression for gp91phox (a) and Nrf2 (b) in the liver was assessed 12 h after APAP (3 gkg) or saline peroral treatment of WT and 5-LOminusminus mice Values are mean plusmn SEM 119899 = 4 representative of two separate experiments lowast119875 lt 005 comparedto saline-treatedWT and 5-LOminusminus mice and 119875 lt 005 compared to APAP-treatedWTmice One-way ANOVA was followed by Bonferronirsquosmultiple comparison test

presented less severe lethality when compared to WT miceprogressive lethality did occur This might be related to thelack of lipoxin (LX) production in 5-LOminusminus mice since thesynthesis of these important lipid mediators is dependent on5-LO [55] LXs present dual role in inhibiting inflammationand promoting resolution of the inflammation which isessential for resolution of acute inflammatory processes andreturn to homeostasis [56]Therefore 5-LO inhibition seemsto be more beneficial in the early stages of APAP intoxicationwhen LT contribution to liver damage is critical Moreoverthe indirect inhibition of the 5-LO pathway may eventuallybe more beneficial in APAP intoxication since the inhibitorof 5-LO activating protein (FLAP) Bay-X-1005 significantlyreduces LT biosynthesis and stimulated LX formation result-ing in further protection against CCl

4

-induced liver injury[9]

It is noteworthy that the sleeping time induced by pen-tobarbital was similar comparing WT and 5-LOminusminus mice Inagreement 5-LOminusminus andWTmice do not present differencesin liverCYP content and cytochrome c reductase activity [57]Thus the reduction of APAP-induced lethality and hepato-toxicity was not related to reduction of NAPQI formation byimpaired activity of CYP

In conclusion the current study demonstrates that 5-LOparticipates in APAP-induced liver damage and lethality byenhancing LTB

4

production in the liver A lethal dose ofAPAP induced liver necrosis and inflammation macrophageand neutrophil recruitment cytokine production and oxida-tive stress in the liver all of which are reduced or abolished in5-LOminusminusmice therefore elucidating the participation of 5-LOin these mechanisms of APAP hepatotoxicity Furthermore

our findings suggest that inhibition of 5-LO may be apotential strategy to reduce the lethality and liver damageproduced by APAP intoxication and possibly other typesof liver damage that are mediated by similar mechanismsFinally although 5-LO deficiency did not abolish the lethalityofAPAP it increased the survival rates following the ingestionof a lethal dose of APAP and prevented liver damage whichmight add to the current therapeutic approaches to reduceAPAP intoxication-induced death

Acknowledgments

The authors appreciated the technical support of GiulianaB Francisco and Pedro S R Dionısio Filho This work wassupported by grants from SETIFundacao Araucaria ParanaState Government Fundacao de Amparo a Pesquisa doEstado de Sao Paulo (FAPESP) ConselhoNacional deDesen-volvimento Cientıfico e Tecnologico (CNPq) and Coorde-nadoria de Aperfeicoamento de Pessoal de Nıvel Superior(CAPES) Brazil Miriam S N Hohmann received a Brazilianfellowship from Departamento de Ciencia e Tecnologia daSecretaria de Ciencia Tecnologia e Insumos Estrategicos(DecitSCTIE)Ministerio da Saude (MS) (DecitSCTIEMS)by means of CNPq and Fundacao Araucaria

References

[1] B Samuelsson S-E Dahlen and J A Lindgren ldquoLeukotrienesand lipoxins structures biosynthesis and biological effectsrdquoScience vol 237 no 4819 pp 1171ndash1176 1987


[2] C D Funk ldquoProstaglandins and leukotrienes advances ineicosanoid biologyrdquo Science vol 294 no 5548 pp 1871ndash18752001

[3] A W Ford-Hutchinson M A Bray and M V DoigldquoLeukotriene B a potent chemokinetic and aggregating sub-stance released from polymorphonuclear leukocytesrdquo Naturevol 286 no 5770 pp 264ndash265 1980

[4] M Chen B K Lam A D Luster et al ldquoJoint tissuesamplify inflammation and alter their invasive behavior vialeukotriene B4 in experimental inflammatory arthritisrdquo Journalof Immunology vol 185 no 9 pp 5503ndash5511 2010

[5] F G Al-Amran N R Hadi and A M Hashim ldquoLeukotrienebiosynthesis inhibition ameliorates acute lung injury followinghemorrhagic shock in ratsrdquo Journal of Cardiothoracic Surgeryvol 6 no 1 article no 81 2011

[6] L Alric C Orfila N Carrere et al ldquoReactive oxygen intermedi-ates and eicosanoid production by Kupffer cells and infiltratedmacrophages in acute and chronic liver injury induced in ratsby CCl4rdquo Inflammation Research vol 49 no 12 pp 700ndash7072000

[7] E Titos J Claria A Planaguma et al ldquoInhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in ratKupffer cells implications for liver fibrosisrdquoThe FASEB Journalvol 17 no 12 pp 1745ndash1747 2003

[8] Y Takamatsu K Shimada K Chijiiwa S Kuroki K Yam-aguchi and M Tanaka ldquoRole of leukotrienes on hep-atic ischemiareperfusion injury in ratsrdquo Journal of SurgicalResearch vol 119 no 1 pp 14ndash20 2004

[9] E Titos J Claria A Planaguma et al ldquoInhibition of 5-lipoxygenase-activating protein abrogates experimental liverinjury role of Kupffer cellsrdquo Journal of Leukocyte Biology vol78 no 4 pp 871ndash878 2005

[10] R Horrillo A Planaguma A Gonzalez-Periz et al ldquoCom-parative protection against liver inflammation and fibrosisby a selective cyclooxygenase-2 inhibitor and a nonredox-type 5-lipoxygenase inhibitorrdquo Journal of Pharmacology andExperimental Therapeutics vol 323 no 3 pp 778ndash786 2007

[11] L F Prescott ldquoHepatotoxicity of mild analgesicsrdquo British Jour-nal of Clinical Pharmacology vol 10 supplement 2 pp 375Sndash377S 1980

[12] A M Larson J Polson R J Fontana et al ldquoAcetaminophen-induced acute liver failure results of aUnited Statesmulticenterprospective studyrdquo Hepatology vol 42 no 6 pp 1364ndash13722005

[13] D G N Craig C M Bates J S Davidson K G Martin PC Hayes and K J Simpson ldquoOverdose pattern and outcomein paracetamol-induced acute severe hepatotoxicityrdquo BritishJournal of Clinical Pharmacology vol 71 no 2 pp 273ndash2822011

[14] J R Mitchell D J Jollow and W Z Potter ldquoAcetaminopheninduced hepatic necrosis I Role of drug metabolismrdquo Journalof Pharmacology and Experimental Therapeutics vol 187 no 1pp 185ndash194 1973

[15] C J Patten P E Thomas R L Guy et al ldquoCytochromeP450 enzymes involved in acetaminophen activation by rat andhuman liver microsomes and their kineticsrdquo Chemical Researchin Toxicology vol 6 no 4 pp 511ndash518 1993

[16] D J Jollow J R Mitchell and W Z Potter ldquoAcetaminopheninduced hepatic necrosis II Role of covalent binding in vivordquoJournal of Pharmacology and Experimental Therapeutics vol187 no 1 pp 195ndash202 1973

[17] J R Mitchell D J Jollow and W Z Potter ldquoAcetaminopheninduced hepatic necrosis IV Protective role of glutathionerdquoJournal of Pharmacology and Experimental Therapeutics vol187 no 1 pp 211ndash217 1973

[18] B VMartin-Murphy M P Holt and C Ju ldquoThe role of damageassociated molecular pattern molecules in acetaminophen-induced liver injury in micerdquo Toxicology Letters vol 192 no 3pp 387ndash394 2010

[19] R F Schwabe E Seki and D A Brenner ldquoToll-Like ReceptorSignaling in the Liverrdquo Gastroenterology vol 130 no 6 pp1886ndash1900 2006

[20] P Jeannin S Jaillon and Y Delneste ldquoPattern recognitionreceptors in the immune response against dying cellsrdquo CurrentOpinion in Immunology vol 20 no 5 pp 530ndash537 2008

[21] D L Laskin andK J Pendino ldquoMacrophages and inflammatorymediators in tissue injuryrdquoAnnual Review of Pharmacology andToxicology vol 35 pp 655ndash677 1995

[22] S L Michael N R Pumford P R Mayeux M R Niesmanand J A Hinson ldquoPretreatment of mice with macrophageinactivators decreases acetaminophen hepatotoxicity and theformation of reactive oxygen and nitrogen speciesrdquoHepatologyvol 30 no 1 pp 186ndash195 1999

[23] R A Roberts P E Ganey C Ju L M Kamendulis I Rusynand J E Klaunig ldquoRole of the Kupffer cell in mediating hepatictoxicity and carcinogenesisrdquo Toxicological Sciences vol 96 no1 pp 2ndash15 2007

[24] D A Valerio S R Georgetti D A Magro et al ldquoQuercetinreduces inflammatory pain inhibition of oxidative stress andcytokine productionrdquo Journal of Natural Products vol 72 no11 pp 1975ndash1979 2009

[25] Z-X Liu D Han B Gunawan and N Kaplowitz ldquoNeutrophildepletion protects against murine acetaminophen hepatotoxic-ityrdquo Hepatology vol 43 no 6 pp 1220ndash1230 2006

[26] A-C Dragomir J D Laskin and D L Laskin ldquoMacrophageactivation by factors released from acetaminophen-injuredhepatocytes potential role of HMGB1rdquo Toxicology and AppliedPharmacology vol 253 no 3 pp 170ndash177 2011

[27] Y Zhai R W Busuttil and J W Kupiec-Weglinski ldquoLiverischemia and reperfusion injury new insights intomechanismsof innate-adaptive immune-mediated tissue inflammationrdquoAmerican Journal of Transplantation vol 11 no 8 pp 1563ndash1569 2011

[28] H Jaeschke A Farhood and C W Smith ldquoNeutrophilscontribute to ischemiareperfusion injury in rat liver in vivordquoFASEB Journal vol 4 no 15 pp 3355ndash3359 1990

[29] H Jaeschke A Farhood andCW Smith ldquoNeutrophil-inducedliver cell injury in endotoxin shock is a CD11bCD18-dependentmechanismrdquo American Journal of Physiology vol 261 no 6 ppG1051ndashG1056 1991

[30] H Yaman E Cakir E O Akgul et al ldquoPentraxin 3 as apotential biomarker of acetaminophen-induced liver injuryrdquoExperimental and Toxicologic Pathology vol 65 no 1-2 pp 147ndash151 2013

[31] P P Bradley D A Priebat R D Christensen and G RothsteinldquoMeasurement of cutaneous inflammation estimation of neu-trophil content with an enzyme markerrdquo Journal of InvestigativeDermatology vol 78 no 3 pp 206ndash209 1982

[32] C D Horinouchi D A Mendes S Soley Bda et al ldquoCombre-tum leprosumMart (Combretaceae) potential as an antiprolif-erative and anti-inflammatory agentrdquo Journal of EthNopharma-cology vol 145 no 1 pp 311ndash319 2013


[33] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 pp 192ndash205 1968

[34] V Katalinic D Modun I Music and M Boban ldquoGenderdifferences in antioxidant capacity of rat tissues determined by221015840-azinobis (3-ethylbenzothiazoline 6-sulfonate ABTS) andferric reducing antioxidant power (FRAP) assaysrdquo ComparativeBiochemistry and Physiology Part C vol 140 no 1 pp 47ndash522005

[35] H Watanuki K Ota A C M A R Tassakka T Kato and MSakai ldquoImmunostimulant effects of dietary Spirulina platensison carp Cyprinus carpiordquo Aquaculture vol 258 no 1ndash4 pp157ndash163 2006

[36] R P Guedes L Dal Bosco C M Teixeira et al ldquoNeuropathicpain modifies antioxidant activity in rat spinal cordrdquo Neuro-chemical Research vol 31 no 5 pp 603ndash609 2006

[37] WAVerri Jr A TGGuerrero S Y Fukada et al ldquoIL-33medi-ates antigen-induced cutaneous and articular hypernociceptionin micerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 7 pp 2723ndash2728 2008

[38] V Darias S Abdala D Martin-Herrera M Luisa Tello and SVega ldquoCNS effects of a series of 124-triazolyl heterocarboxylicderivativesrdquo Pharmazie vol 53 no 7 pp 477ndash481 1998

[39] Y Ishida T Kondo T OhshimaH Fujiwara Y Iwakura andNMukaida ldquoA pivotal involvement of IFN-120574 in the pathogenesisof acetaminophen-induced acute liver injuryrdquo FASEB Journalvol 16 no 10 pp 1227ndash1236 2002

[40] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[41] L-Q Qin YWang J-Y Xu T Kaneko A Sato and P-YWangldquoOne-day dietary restriction changes hepatic metabolism andpotentiates the hepatotoxicity of carbon tetrachloride andchloroform in ratsrdquo Tohoku Journal of Experimental Medicinevol 212 no 4 pp 379ndash387 2007

[42] D J Antoine D P Williams A Kipar H Laverty and BKevin Park ldquoDiet restriction inhibits apoptosis and HMGB1oxidation and promotes inflammatory cell recruitment duringacetaminophen hepatotoxicityrdquoMolecular Medicine vol 16 no11-12 pp 479ndash490 2010

[43] J Scholmerich ldquoInterleukin in acute pancreatitisrdquo ScandinavianJournal of Gastroenterology Supplement vol 31 no 219 pp 37ndash42 1996

[44] M Faurschou and N Borregaard ldquoNeutrophil granules andsecretory vesicles in inflammationrdquoMicrobes and Infection vol5 no 14 pp 1317ndash1327 2003

[45] M E Blazka J LWilmer S DHolladay R EWilson andM ILuster ldquoRole of proinflammatory cytokines in acetaminophenhepatotoxicityrdquo Toxicology and Applied Pharmacology vol 133no 1 pp 43ndash52 1995

[46] C Cover J Liu A Farhood et al ldquoPathophysiological roleof the acute inflammatory response during acetaminophenhepatotoxicityrdquo Toxicology and Applied Pharmacology vol 216no 1 pp 98ndash107 2006

[47] W A Verri Jr T M Cunha S H Ferreira et al ldquoIL-15mediates antigen-induced neutrophil migration by triggeringIL-18 productionrdquo European Journal of Immunology vol 37 no12 pp 3373ndash3380 2007

[48] C Ju T P ReillyM Bourdi et al ldquoProtective role of kupffer cellsin acetaminophen-induced hepatic injury in micerdquo ChemicalResearch in Toxicology vol 15 no 12 pp 1504ndash1513 2002

[49] C R Gardner J D Laskin D M Dambach et al ldquoReducedhepatotoxicity of acetaminophen in mice lacking induciblenitric oxide synthase potential role of tumor necrosis factor-120572and interleukin-10rdquo Toxicology and Applied Pharmacology vol184 no 1 pp 27ndash36 2002

[50] H-M Kang and M E Saltveit ldquoAntioxidant capacity of lettuceleaf tissue increases after woundingrdquo Journal of Agricultural andFood Chemistry vol 50 no 26 pp 7536ndash7541 2002

[51] K Chan X-D Han and Y W Kan ldquoAn important func-tion of Nrf2 in combating oxidative stress detoxification ofacetaminophenrdquo Proceedings of the National Academy of Sci-ences of theUnited States of America vol 98 no 8 pp 4611ndash46162001

[52] M Takahashi ldquoOxidative stress and redox regulation on in vitrodevelopment of mammalian embryosrdquo Journal of Reproductionand Development vol 58 no 1 pp 1ndash9 2012

[53] R A Gubitosi-Klug R Talahalli Y Du J L Nadler and T SKern ldquo5-Lipoxygenase but not 1215-lipoxygenase contributesto degeneration of retinal capillaries in a mouse model ofdiabetic retinopathyrdquo Diabetes vol 57 no 5 pp 1387ndash13932008

[54] C H C Serezani D M Aronoff S Jancar and M Peters-Golden ldquoLeukotriene B4 mediates p47phox phosphorylationand membrane translocation in polyunsaturated fatty acid-stimulated neutrophilsrdquo Journal of Leukocyte Biology vol 78no 4 pp 976ndash984 2005

[55] N Chiang C N Serhan S-E Dahlen et al ldquoThe lipoxinreceptor ALX potent ligand-specific and stereoselective actionsin vivordquo Pharmacological Reviews vol 58 no 3 pp 463ndash4872006

[56] C N Serhan ldquoControlling the resolution of acute inflamma-tion a new genus of dual anti-inflammatory and proresolvingmediatorsrdquo Journal of Periodontology vol 79 no 8 pp 1520ndash1526 2008

[57] W P Beierschmitt J DMcNeish R J Griffiths ANagahisaMNakane and D E Amacher ldquoInduction of hepatic microsomaldrug-metabolizing enzymes by inhibitors of 5-lipoxygenase (5-LO) studies in rats and 5-LO knockout micerdquo ToxicologicalSciences vol 63 no 1 pp 15ndash21 2001


with the development of hepatic edema and dysfunction [8]Furthermore LTB

4

and the 5-LO pathway were reported tobe involved in the pathogenesis of experimental liver fibrosisand inflammatory necrosis [7 9 10]Thus 5-LO products areimportant mediators of hepatic inflammation and cell injury

Considering that LTs are important mediators of liverinflammation and damage 5-LO pathway and its productsmight also be involved in acetaminophen (APAP)-inducedliver injury APAP is a widely used over-the-counter analgesicand antipyreticwith few side effectswhen taken at therapeuticdoses However APAP intoxication can result in severe liverdamage characterized by centrilobular liver necrosis and inmore severe cases acute liver failure and eventually death[11] Studies have shown that APAP overdose has become themost common cause of acute liver failure in many Westerncountries [12 13]

It is well established that APAP-induced liver injurydepends on the metabolic conversion of APAP to thehighly reactive metabolite N-acetyl-p-benzoquinone imine(NAPQI) by cytochrome P-450 (CYP) enzymes primarilyCYP2E1 and to a lesser extent CYP1A2 CYP2A6 andCYP3A4 [14 15] In normal conditions this metabolite isreadily detoxified by hepatic reduced glutathione (GSH)However after toxic dose of APAP GSH is depleted and asa result NAPQI accumulates and covalently binds to hepa-tocellular proteins initiating liver injury [16 17] Althoughprotein binding is the initiator of toxicity and cellular injurysecondary processes amplify and propagate it Initial hepa-tocellular damage caused by NAPQI can lead to the releaseof damage-associated molecular patterns (DAMPS) such ashigh-mobility group box 1 protein (HMGB1) heat shockproteins DNA fragments and others [18] which can directlyactivate nonparenchymal hepatic cells Kupffer cells (KC)and other innate immune cells [19 20] In addition thesecells can also be activated by potent chemotactic factors(ie LTB

4

) released by injured hepatocytes [21] Activatedcells can release a wide range of inflammatory mediatorssuch as reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) [22] proinflammatory cytokines [18] andLTs [6] amplifying the oxidative stress and inflammatoryresponses consequently promoting further hepatic injury[23] Moreover the excessive ROS and RNS productionduring inflammation contributes to the depletion of GSH[24] which could result in further NAPQI accumulation [17]

Neutrophil and macrophage accumulation in the liverhas also been associated with APAP-induced liver injury[25 26] and several other experimental animal models ofliver injury such as ischemia-reperfusion injury [27 28]and endotoxemia [29] Neutrophils and macrophages undercertain circumstances can severely aggravate tissue damageIn APAP-induced hepatotoxicity excessive neutrophil andmacrophage activity can contribute to liver inflammation andinjury [25 26]

Although many studies have demonstrated that 5-LOpathway is involved in the pathogenesis of different modelsof liver inflammation at present the participation of 5-LOin APAP-induced hepatotoxicity and liver damage has notbeen addressed In this context the aim of the currentstudy was to use 5-LO deficient mice to investigate the role

of 5-LO on APAP-induced lethality and liver damage Inaddition it aimed to demonstrate the mechanisms by which5-LO deficiency ameliorates the events mentioned previ-ously focusing on neutrophil and macrophage recruitmentcytokine production and oxidative stress in the liver

2 Methods

21 Animals Male 5-lipoxygenase deficient (5-LOminusminus) andwild type (WT SV129) mice weighing 20ndash25 g were used 5-LOminusminus andWTmice originally fromThe Jackson Laboratory(Bar Harbor ME USA) were gifted from Dr Marc LeePeters-Golden (University of Michigan) weighing 20ndash25 gMice were housed at 21∘C on a 12 h lightdark cycle instandard clear plastic cages with food and water ad libitumAnimals were handled humanely and all procedures con-cerning animal care and use were approved by the Researchand Ethics Committee of State University of Londrina (pro-cess 29612010-10) and carried out in accordance with itsguidelines

22 Experimental Design In dose-response experimentsWT mice (119899 = 10 per group) were treated orally with 03 12 3 and 6 gkg of APAP suspended in saline (200mgmL)In APAP-induced lethality experiments 5-LOminusminus and WTmice (119899 = 10 per group) were treated orally with 3 gkgof APAP suspended in saline (200mgmL of saline) orequal volume of saline (control group) In these survivalexperiments mice were observed every 6 h during 72 hand any that showed extreme distress or became moribundwere sacrificed For subsequent experiments animals wereanesthetized and sacrificed 12 h after APAP treatment Bloodsamples were collected by cardiac puncture to determinethe enzymatic activities of aspartate aminotransferase (AST)and alanine aminotransferase (ALT) For liver analysis theabdomen of the mice was opened and the left lobe of theliver was quickly excised A portion of the tissue was storedat minus20∘C in 50mM K

2

HPO4

buffer (pH 60) containing05 hexadecyl trimethylammonium bromide (HTAB) formyeloperoxidase (MPO) andN-acetyl-120573-D-glucosaminidase(NAG) activity determination and the remaining tissue wasstored in saline at minus80∘C for LTB

4

and cytokine (TNF-120572IL-1120573 IFN-120574 and IL-10) quantification The midsections ofthe left lobe of the liver were collected and processed forhistopathology analysis In order to assess hepatic oxidativestress the liver was first perfused with 10mL of ice-cold 115KCl solution through the portal vein For quantitative PCR(qPCR) analysis the 115 KCl solution used for liver perfu-sion was treated with 01 vv diethylpyrocarbonate (DEPC)for 24 hours at 37∘C and then autoclaved to inactivate DEPCThe left lobe was excised in the same manner and a portionof the perfused tissue was separated for 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonate ABTS) assay whereas theremaining tissue was immediately stored at minus80∘C for laterassessment of reduced glutathione (GSH) thiobarbituric acidreactive substances (TBARS) levels nitroblue tetrazolium(NBT) reduction and mRNA expression for gp91phox andtranscription factor Nrf2



















3 Results




4


4


4





0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100

Saline

Time (h)

Sur

viva

l (

)



(a)

Saline APAP0

50

100

150

200

250

300

350

400

450

500

LTB 4

(pg

mg

liver

)

lowast

(b)



4










lowast

0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100


Time (h)

Surv

ival

()





4 Discussion










5-LOminusminusWT

00

05

10

15

20

25

30APAP

Saline

lowast

Deg

ree o

f nec

rosis

(a)

5-LOminusminusWT

00

05

10

15

20

25

30

APAP

Saline

lowast

Deg

ree o

f infl

amm

atio

n

(b)

Saline

WT mice

(c)

5-LOminusminus mice

Saline

(d)

APAP

(e)

APAP

(f)



0

10

20

30

40

Saline

APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)


0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)





0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References














































































3 Results




4


4


4





0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100

Saline

Time (h)

Sur

viva

l (

)



(a)

Saline APAP0

50

100

150

200

250

300

350

400

450

500

LTB 4

(pg

mg

liver

)

lowast

(b)



4










lowast

0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100


Time (h)

Surv

ival

()





4 Discussion










5-LOminusminusWT

00

05

10

15

20

25

30APAP

Saline

lowast

Deg

ree o

f nec

rosis

(a)

5-LOminusminusWT

00

05

10

15

20

25

30

APAP

Saline

lowast

Deg

ree o

f infl

amm

atio

n

(b)

Saline

WT mice

(c)

5-LOminusminus mice

Saline

(d)

APAP

(e)

APAP

(f)



0

10

20

30

40

Saline

APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)


0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)





0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References


































































3 Results




4


4


4





0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100

Saline

Time (h)

Sur

viva

l (

)



(a)

Saline APAP0

50

100

150

200

250

300

350

400

450

500

LTB 4

(pg

mg

liver

)

lowast

(b)



4










lowast

0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100


Time (h)

Surv

ival

()





4 Discussion










5-LOminusminusWT

00

05

10

15

20

25

30APAP

Saline

lowast

Deg

ree o

f nec

rosis

(a)

5-LOminusminusWT

00

05

10

15

20

25

30

APAP

Saline

lowast

Deg

ree o

f infl

amm

atio

n

(b)

Saline

WT mice

(c)

5-LOminusminus mice

Saline

(d)

APAP

(e)

APAP

(f)



0

10

20

30

40

Saline

APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)


0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)





0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References





























































0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100

Saline

Time (h)

Sur

viva

l (

)



(a)

Saline APAP0

50

100

150

200

250

300

350

400

450

500

LTB 4

(pg

mg

liver

)

lowast

(b)



4










lowast

0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100


Time (h)

Surv

ival

()





4 Discussion










5-LOminusminusWT

00

05

10

15

20

25

30APAP

Saline

lowast

Deg

ree o

f nec

rosis

(a)

5-LOminusminusWT

00

05

10

15

20

25

30

APAP

Saline

lowast

Deg

ree o

f infl

amm

atio

n

(b)

Saline

WT mice

(c)

5-LOminusminus mice

Saline

(d)

APAP

(e)

APAP

(f)



0

10

20

30

40

Saline

APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)


0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)





0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References





























































lowast

0 6 12 18 24 30 36 42 48 54 60 66 720

10

20

30

40

50

60

70

80

90

100


Time (h)

Surv

ival

()





4 Discussion










5-LOminusminusWT

00

05

10

15

20

25

30APAP

Saline

lowast

Deg

ree o

f nec

rosis

(a)

5-LOminusminusWT

00

05

10

15

20

25

30

APAP

Saline

lowast

Deg

ree o

f infl

amm

atio

n

(b)

Saline

WT mice

(c)

5-LOminusminus mice

Saline

(d)

APAP

(e)

APAP

(f)



0

10

20

30

40

Saline

APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)


0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)





0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References





























































5-LOminusminusWT

00

05

10

15

20

25

30APAP

Saline

lowast

Deg

ree o

f nec

rosis

(a)

5-LOminusminusWT

00

05

10

15

20

25

30

APAP

Saline

lowast

Deg

ree o

f infl

amm

atio

n

(b)

Saline

WT mice

(c)

5-LOminusminus mice

Saline

(d)

APAP

(e)

APAP

(f)



0

10

20

30

40

Saline

APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)


0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)





0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References





























































0

10

20

30

40

Saline

APAP

AST

(UL

)

lowast

5-LOminusminusWT

(a)

Saline

APAP

0

10

20

30

40

50

60

ALT

(UL

)

lowast

5-LOminusminusWT

(b)


0

5

10

15

20

25

30

Saline

APAP

MPO

activ

ity (U

times10

minus3m

g of

live

r)

lowast

5-LOminusminusWT

(a)

000

002

004

006

008

Saline

APAP

NAG

activ

ity (O

Dm

g of

live

r)

lowast

5-LOminusminusWT

(b)





0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References





























































0

50

100

150

200

250

300

Saline

APAP

lowast

IL-1120573

(pg

mg

of li

ver)

(a)

0

50

100

150

200

250

300

Saline

APAP

lowast

TNF-120572

(pg

mg

of li

ver)

(b)

0

1

2

3

4

5

6

7

8

9

10

Saline

APAP

lowast

IFN

-120574(p

gm

g of

live

r)

5-LOminusminusWT

(c)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Saline

APAP

lowast

IL-10

(pg

mg

of li

ver)

5-LOminusminusWT

(d)






0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References





























































0

2

4

6

8

10

12

Saline

APAP

NBT

redu

ctio

n (O

Dm

g of

live

r)

lowast

(a)

00

05

10

15

20

25

30

Saline

APAP

TBA

RS (n

mol

MD

Am

g of

live

r)

lowast

(b)

0

5

10

15

20

Saline APAP

lowast

5-LOminusminusWT

(120583m

ol T

rolo

x Eq

mg

of li

ver)

(c)

00

02

04

06

08

10

12

Saline

APAP

(nm

ol G

SHm

g of

live

r)

lowast

5-LOminusminusWT

(d)






00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References





























































00

05

10

15

20

Saline

APAP

lowast

5-LOminusminusWT

gp91

phox

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(a)

0

1

2

3

4

5

6

7

Saline

APAP

lowast

5-LOminusminusWT

Nrf2

mRN

A ex

pres

sion

(nor

mal

ized

to120573

-act

in)

(b)



4




4



Acknowledgments


References
















































































































































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