research article gamma-glutamylcysteine ethyl...

Research ArticleGamma-Glutamylcysteine Ethyl Ester Protects againstCyclophosphamide-Induced Liver Injury and HematologicAlterations via Upregulation of PPAR120574 and Attenuation ofOxidative Stress Inflammation and Apoptosis

Sultan Alqahtani12 and Ayman M Mahmoud3

1College of Medicine King Saud bin Abdulaziz University for Health Science (KSAU-HS) Riyadh Saudi Arabia2King Abdullah International Medical Research Center (KAIMRC) Riyadh Saudi Arabia3Physiology Division Department of Zoology Faculty of Science Beni-Suef University Beni-Suef Egypt

Correspondence should be addressed to Ayman M Mahmoud aymanmahmoudsciencebsuedueg

Received 11 October 2016 Accepted 24 November 2016

Academic Editor Jose Luıs Garcıa-Gimenez

Copyright copy 2016 S Alqahtani and A M Mahmoud This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Gamma-glutamylcysteine ethyl ester (GCEE) is a precursor of glutathione (GSH) with promising hepatoprotective effects Thisinvestigation aimed to evaluate the hepatoprotective effects of GCEE against cyclophosphamide- (CP-) induced toxicity pointingto the possible role of peroxisome proliferator activated receptor gamma (PPAR120574) Wistar rats were given GCEE two weeks prior toCP Five days after CP administration animals were sacrificed and samples were collected Pretreatment with GCEE significantlyalleviated CP-induced liver injury by reducing serum aminotransferases increasing albumin and preventing histopathologicaland hematological alterations GCEE suppressed lipid peroxidation and nitric oxide production and restored GSH and enzymaticantioxidants in the liver which were associated with downregulation of COX-2 iNOS and NF-120581B In addition CP administrationsignificantly increased serum proinflammatory cytokines and the expression of liver caspase-3 and BAX an effect that was reversedby GCEE CP-induced rats showed significant downregulation of PPAR120574 which was markedly upregulated by GCEE treatmentThese data demonstrated that pretreatment with GCEE protected against CP-induced hepatotoxicity possibly by activating PPAR120574preventing GSH depletion and attenuating oxidative stress inflammation and apoptosis Our findings point to the role of PPAR120574and suggest that GCEE might be a promising agent for the prevention of CP-induced liver injury

1 Introduction

Drug-induced liver injury (DILI) refers to abnormalitiesin liver function tests related to the intake of medicinalcompounds [1] DILI has been the single most frequentreason for drug withdrawal from the market [2 3] Thepotential of a drug to cause hepatotoxicity is often realizedafter release onto themarket [2] and it has been estimated thatmore than a thousand drugs have been associated with liverinjury and hepatotoxicity [4 5] Cyclophosphamide (CP) isan alkylating agent commonly used in the treatment of differ-ent cancers [6] The therapeutic applications of CP havebeen associated with different side effects and organ toxicity

[7 8] CP cytotoxicity has been attributed to the toxic meta-bolites acrolein and phosphoramide produced during itsmetabolism [9] Acrolein can bind to reduced glutathione(GSH) leading to increased production of reactive oxygenspecies (ROS) and subsequently oxidative stress and lipidperoxidation [10 11] Therefore agents with free radicalscavenging and antioxidant properties can offer protectionagainst CP-induced oxidative stress and hepatotoxicity

Peroxisome proliferator activated receptor gamma(PPAR120574) is a ligand-inducible transcription factor knownto have roles in normal cell function [12] When activatedPPAR120574 heterodimerizes with retinoid X receptor (RXR)binds to specific response elements (PPREs) and promotes

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 4016209 14 pageshttpdxdoiorg10115520164016209

2 Oxidative Medicine and Cellular Longevity

the expression of target genes [13] PPAR120574 is induced duringpreadipocytes differentiation and plays a central role in lipidmetabolism glucose homeostasis inflammation and cellproliferation [14] In the liver disruption of PPARs has beenassociated with different disorders [15] On the other handactivation of PPAR120574 inhibited the fibrogenic response to liverinjury [16] and protected against drug-induced hepatotoxi-city as we recently reported [3 17 18]

Attenuation of oxidative stress through restoring GSHlevels is a well-known strategy to combat drug-inducedtoxicity For example administration of N-acetylcysteine(NAC) a precursor ofGSH protected the liver against carbontetrachloride [19] and methotrexate-induced toxicity [20]Gamma-glutamylcysteine ethyl ester (GCEE) a syntheticGSH precursor has been demonstrated to boost endogenousGSH levels and block oxidative stress in neurons [21 22]as well as cerebral endothelial cells [23] We believe thatnothing has yet been reported on the possible protectiveeffects of GCEE against CP-induced hepatotoxicity In thepresent study we asked whether GCEE can attenuate CP-induced oxidative stress apoptosis and inflammation in theliver of rats pointing to the role of PPAR120574

2 Materials and Methods

21 Chemicals Gamma-glutamyl cysteine ethyl ester(GCEE) and cyclophosphamide (CP Endoxan) were pur-chased fromBachem (Torrance CAUSA) andBaxterOncol-ogy (Dusseldorf Germany) respectively Alanine amin-otransferase (ALT) aspartate aminotransferase (AST)alkaline phosphatase (ALP) and albumin assay kits weresupplied by Spinreact (Spain) PPAR120574 nuclear factor-120581B(NF-120581B) and Bcl-2-associated X protein (BAX) antibodieswere obtained from Santa Cruz Biotechnology (USA)Cytokines assay kits were purchased from RampD Systems(USA) All other chemicals were obtained from Sigma (USA)and other standard commercial supplies

22 Experimental Animals and Treatments Male albinoWis-tar rats (10 weeks old) from the Institute of Ophthalmology(Giza Egypt) were included in the present study They weremaintained on a 12 h darklight cycle at 22 plusmn 2∘C with adlibitum access to standard laboratory diet and water Allanimal procedures related to care treatments and samplingwere in accordance with the guidelines of the InstitutionalAnimal Ethics Committee of Beni-Suef University (Egypt)

Twenty-four rats were divided randomly into threegroups of 8 rats each and allowed to adapt for 1 week priorto the experiment Group I (Control) received normal salinesolution for 16 days Group II (CP) received saline for 15 daysand 150mgkg bwt CPonday 16 [18] andGroup III (GCEE+CP) received 100mgkg bwt GCEE for 15 days and 150mgkgbwt CP on day 16

The dose route and day of CP administration wereselected based on our previous studies [18 24] Since GCEEhas been proven to be effective in vivo at doses of 10mgkg[25] and 150mgkg bwt [21] we selected a dose of 100mgkgto be tested in our study All experimental solutions wereadministered intraperitoneally

At day 21 the animals were sacrificed by cervical dis-location and various samples were collected Blood sampleswere either collected on heparinized tubes for hematologi-cal analysis or left to coagulate for serum separation Liverswere immediately excised washed in cold phosphate buf-fered saline (PBS) and weighed Samples from the liverwere fixed in 10 neutral buffered formalin for histologicaland immunohistochemical processing Other samples werehomogenized (10wv) in cold PBS for biochemical assays orkept frozen atminus80∘C for gene and protein expression analysis

23 Biochemical Assays

231 Determination of Liver FunctionMarkers Serum amin-otransferases were assayed using Spinreact (Spain) reagentkits according to the method of Schumann and Klauke[26] Serum ALP activity and albumin concentration weremeasured using Spinreact (Spain) reagent kit according to themethods of Wenger et al [27] andWebster [28] respectively

232 Determination of Oxidative Stress and AntioxidantDefenses Liver malondialdehyde (MDA) and GSH levelswere determined according to the methods of Preuss et al[29] and Beutler et al [30] respectively Liver nitric oxide(NO) was determined as nitrite using Griess reagent Super-oxide dismutase (SOD) glutathione peroxidase (GPx) andcatalase (CAT) were determined according to the methods ofS Marklund and G Marklund [31] Matkovics et al [32] andCohen et al [33] respectively

233 Determination of Proinflammatory Cytokines Tumornecrosis factor alpha (TNF-120572) and interleukin-1beta (IL-1120573)were determined in serum samples using specific rats ELISAkits (RampD Systems USA) according to the manufacturerrsquosinstructions

234 Determination of Caspase-3 Activity Liver caspase-3 activity was measured using the CaspACE assay system(Promega Madison WI USA) following the manufacturerrsquosinstructions The assay is based on the action of caspase-3 on the substrate Ac-DEVD-pNA releasing yellow chro-mophore p-nitroanilineThe activity of caspase-3 activity waspresented as percentage of corresponding control

24 Determination of Hematological Parameters Samples ofblood from all animals were collected into heparinized tubesand red blood corpuscles (RBCs) total white blood cells(WBCs) platelet count and hemoglobin (Hb) content weredetermined using an automated hematoanalyzer

25 Histopathology and Immunohistochemistry Samplesfrom the liver were immediately washed in cold PBS andfixed for histological processing and hematoxylin and eosin(HampE) staining

Liver sections were immunohistochemically stained withanti-BAX antibody Briefly the slides were deparaffinizedrehydrated and incubated in 3 hydrogen peroxide (H

2O2)

for 5minThe slides were washed in Tris-buffered saline (pH76) blocked with protein block (Novocastra) and incubated

Oxidative Medicine and Cellular Longevity 3

Table 1 Primers used for qRT-PCR

Gene GenBank accession number Sequence (51015840-31015840)

Pparg NM 001145367 F GGACGCTGAAGAAGAGACCTGR CCGGGTCCTGTCTGAGTATG

Casp3 NM 012922 F GGAGCTTGGAACGCGAAGAAR ACACAAGCCCATTTCAGGGT

BAX NM 017059 F AGGACGCATCCACCAAGAAGR CAGTTGAAGTTGCCGTCTGC

NF-120581B AF079314 F TCTCAGCTGCGACCCCGR TGGGCTGCTCAATGATCTCC

COX2 NM 017232 F TGATCTACCCTCCCCACGTCR ACACACTCTGTTGTGCTCCC

iNOS U03699 F ATTCCCAGCCCAACAACACAR GCAGCTTGTCCAGGGATTCT

120573-Actin NM 031144 F AGGAGTACGATGAGTCCGGCR CGCAGCTCAGTAACAGTCCG

with rabbit polyclonal anti-BAXThe sections were incubatedwith the secondary antibody and then horseradish peroxidaseconjugated with streptavidin Sections were then washedcounterstained with hematoxylin mounted in DPX andexamined by light microscopy

26 Gene Expression Study To study the effect of GCEE onthe mRNA expression levels of caspase-3 BAX induciblenitric oxide synthase (iNOS) cyclooxygenase-2 (COX-2)NF-120581B and PPAR120574 in the liver of CP-induced rats quan-titative RT-PCR was used as we previously reported [3]In brief total RNA was isolated from liver tissue samplesusing Invitrogen (USA) TrIzol reagent RNA was treatedwith RNase-free DNase purified using RNeasy purificationkit (Qiagen Germany) and quantified at 260 nm RNAintegrity was further confirmed using formaldehyde-agarosegel electrophoresis 2 120583g RNA was reverse transcribed intofirst strand cDNA using AMV reverse transcriptase DNAwas amplified using SYBRGreenmaster mix purchased fromFermentas The primers used to specifically amplify caspase-3 BAX COX-2 iNOS NF-120581B PPAR120574 and 120573-actin are listedin Table 1 The 2minusΔΔCt method [34] was used to analyze theobtained amplification data and the results were normalizedto 120573-actin

27 Western Blot Total liver tissue protein was extractedusing RIPA buffer supplemented with proteinase inhibitorsand Bradford reagent was used to determine protein con-centration Aliquots of the lysate containing 50 120583g proteinswere separated on SDS-PAGE electrotransferred onto PVDFmembranes followed by blocking The membranes wereprobed with PPAR120574 NF-120581B p65 and 120573-actin primary anti-bodies washed and then incubated with the proper sec-ondary antibodies The blots were developed by enhancedchemiluminescence kit (BIO-RAD USA) The intensity ofobtained bands was quantified using ImageJ normalized to120573-actin and presented as percent of control

28 Statistical Analysis Results were analyzed using Graph-Pad Prism 5 (La Jolla CA USA) and were expressed asmeans plusmn standard error of the mean (SEM) The statisticalcomparisons were made using one-way analysis of variance(ANOVA) followed by Tukeyrsquos test post hoc analysis to judgethe difference between various groups A 119875 value lt 005 wasconsidered to be statistically significant

3 Results

31 GCEE Protects against CP-Induced Liver Injury To testthe protective effect of GCEE on CP-induced hepatocellularinjury we assayed serum markers of liver function andperformed histological examination

Administration of CP induced hepatotoxicity evidencedby the significantly (119875 lt 0001) increased serum ALT (Fig-ure 1(a)) AST (Figure 1(b)) and ALP (Figure 1(c)) activitieswhen compared with the control group Pretreatment of theCP-induced rats withGCEEproduced significant (119875 lt 0001)reduction in serum aminotransferases andALP activities Onthe other hand CP-administered rats showed a significant(119875 lt 001) decline in serum albumin levels when comparedwith the corresponding control rats as depicted in Figure 1(d)Supplementation of GCEE prior to CP produced a significant(119875 lt 001) amelioration of serum albumin levels in CP-intoxicated rats

Microscopic examination of the liver sections stainedwith HampE revealed normal hepatic strands hepatocytes andsinusoids in control rats (Figure 2(a)) CP administrationto rats produced several histological alterations in the liversections such as activated Kupffer cells and hepatic vac-uolation of fat type as most of vacuoles were with clearlumen and round borders indicating hepatic steatosis (Fig-ure 2(b)) In addition CP induced periportal hepatic necrosiswith mononuclear inflammatory cells infiltration mainlymacrophages and histiocytes (Figure 2(c)) Liver sectionsfromGCEEpretreated rats showednoticeable amelioration ofthe liver histological architecture as depicted in Figure 2(d)


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Figure 1 Effect of GCEE on serum (a) ALT (b) AST (c) ALP and (d) albumin in CP-induced rats Data are expressed as mean plusmn SEM(119873 = 6) lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 CP cyclophosphamide GCEE gamma-glutamylcysteine ethyl ester ALT alanine aminotransferaseAST aspartate aminotransferase ALP alkaline phosphatase

32 GCEE Mitigates CP-Induced Hematological Alterations inRats CP-induced rats showed significant (119875 lt 001) de-crease in RBCs number when compared with the control ratsas represented in Figure 3(a)This effectwas significantly (119875 lt005) reversed in CP-induced rats pretreated with GCEE HBcontent as well was significantly (119875 lt 005) declined inthe blood of CP-induced rats (Figure 3(b)) Pretreatment ofthe rats with GCEE significantly (119875 lt 005) prevented CP-induced Hb decline

Concerning WBCs count CP-induced rats showed sig-nificant (119875 lt 001) leukopenia when compared with the con-trol rats Platelets exhibited nearly similar pattern where theirnumber was significantly (119875 lt 0001) declined in the bloodof CP-induced rats Pretreatment of the CP-induced rats withGCEE significantly prevented leukopenia (119875 lt 005) andthrombocytopenia (119875 lt 005) as depicted in Figures 3(c) and3(d) respectively

33 GCEE Attenuates CP-Induced Oxidative Stress in theLiver of Rats The protective effect of GCEE against CP-induced oxidative stress was determined through assessmentof lipid peroxidation and NO as well as antioxidant defensesIntraperitoneal administration of CP produced a significant(119875 lt 0001) increase in lipid peroxidation (Figure 4(a)) and

NO (Figure 4(b)) in the liver of rats when compared withthe control group Pretreatment of the CP-induced rats withGCEE significantly (119875 lt 0001) decreased lipid peroxidationlevels in the liver of rats Similarly GCEE pretreatmentproduced a significant (119875 lt 001) decline in liver NO levels

On the other hand CP-induced rats showed a significant(119875 lt 005) decline in liver GSH content when compared withthe corresponding control rats (Figure 4(c)) GCEE admin-istration prior to CP produced a significant (119875 lt 005) im-provement in liver GSH content The enzymatic antioxidantsexhibited a similar pattern where CP-induced rats exhibitedsignificant decrease in the activity of liver SOD (119875 lt 001Figure 4(d)) GPx (119875 lt 005 Figure 4(e)) and CAT (119875 lt001 Figure 4(f)) when compared with the control ratsGCEE administration produced significant amelioration inthe activity of SOD (119875 lt 005) GPx (119875 lt 001) and CAT(119875 lt 005) in the liver of CP-induced rats

34 GCEE Reduces CP-Induced Inflammation in the Liverof Rats Circulating levels of the proinflammatory cytokineTNF-120572 showed significant (119875 lt 0001) increase in CP-induced rats when compared with control rats (Figure 5(a))Pretreatment of the CP-induced rats with GCEE for 15days produced significant (119875 lt 0001) decrease in serum


cv

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Figure 2 Photomicrographs of HampE-stained liver sections of (a) control rats (b c) CP-induced rats revealing activated Kupffer cells (redarrow) hepatic vacuolation of fat type (black arrow) and periportal hepatic necrosis associated with mononuclear inflammatory cellsinfiltration mainly macrophages and histiocytes (blue arrow) and (d) CP-administered rats pretreated with GCEE showing noticeableamelioration of the liver histological architecture CV central vein GCEE gamma-glutamylcysteine ethyl ester

TNF-120572 levels IL-1120573 levels were significantly (119875 lt 0001)increased in serum of CP-induced rats when compared withthe control group an effect that was reversed by GCEE treat-ment (Figure 5(b))

To further confirm the anti-inflammatory effect of GCEEthe expression of COX-2 iNOS and NF-120581B was assayedin the liver of CP-induced rats COX-2 mRNA expressionshowed a significant (119875 lt 001) upregulation in the liver ofCP-induced rats when compared with the control rats (Fig-ure 5(c)) Pretreatment of the CP-induced rats with GCEEsignificantly (119875 lt 001) downregulated liver COX-2 mRNAexpression

iNOS mRNA expression revealed significant (119875 lt 001)upregulation in the liver of CP-induced rats when comparedwith the control group as represented in Figure 5(d) GCEEproduced a significant (119875 lt 001) downregulation of iNOSmRNA expression in the liver of CP-induced rats

Liver NF-120581B expression showed a significant upregula-tion in CP-induced rats at both gene (119875 lt 001 Figure 5(e))and protein levels (119875 lt 0001 Figure 5(f)) when comparedwith the control rats GCEE administered prior to CP signi-ficantly decreased NF-120581B bothmRNA (119875 lt 005) and protein(119875 lt 001) expression

35 GCEE Prevents CP-Induced Apoptosis in the Liver of RatsTo study the effect of GCEE on CP-induced apoptosis wedetermined both gene and protein expression levels of the

proapoptotic factors caspase-3 and BAX As represented inFigure 6(a) the liver of CP-induced rats showed a significant(119875 lt 001) increase in mRNA abundance of caspase-3 whencompared with the control rats Caspase-3 protein levelsshowed a similar significant (119875 lt 001) increase in liver ofCP-induced rats Pretreatment of the CP-induced rats withGCEE significantly decreased both caspase-3 mRNA expres-sion (119875 lt 001) and protein levels (119875 lt 005)

Similarly BAX mRNA expression levels showed signifi-cant (119875 lt 0001) increase in the liver of CP-induced rats whencompared with the control group (Figure 6(c)) Pretreat-ment with GCEE produced a marked (119875 lt 001) decreasein BAX mRNA expression levels in liver of the CP-inducedrats BAX protein expression levels determined by immuno-histochemistry showed a significant (119875 lt 0001) increase inthe liver of CP-induced rats when compared with the controlrats (Figure 6(d)) GCEE administered prior to CP producedmarked (119875 lt 0001) decrease in the expression of BAX pro-tein in the liver of rats

36 GCEEUpregulates PPAR120574 in the Liver of CP-Induced RatsPPAR120574mRNA abundance determined by qRT-PCR showeda significant (119875 lt 0001) decrease in the liver of CP-inducedrats as depicted in Figure 7(a) Conversely GCEE sup-plementation produced a significant (119875 lt 001) upregulationof PPAR120574 mRNA expression in the liver of CP-induced ratsPPAR120574 protein expression followed a similar pattern where


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Figure 3 Effect of GCEE on hematopoietic parameters in CP-induced rats Data are expressed as mean plusmn SEM (119873 = 6) lowast119875 lt 005 lowastlowast119875 lt001 and lowastlowastlowast119875 lt 0001 CP cyclophosphamide GCEE gamma-glutamylcysteine ethyl ester RBCs erythrocytes Hb hemoglobin WBCsleukocytes

it was significantly (119875 lt 0001) downregulated in the liverof CP-induced rats when compared with the control group(Figure 7(b)) CP-induced rats pretreated with GCEE exhib-ited marked (119875 lt 001) upregulation of liver PPAR120574 pro-tein expression

4 Discussion

Gamma-glutamylcysteine is the limiting substrate in GSHsynthesis and thus encourages product formation whenpresent In the present study we showed for the first timethat the GSHmimetic GCEE can protect against CP-inducedhepatotoxicity We assumed that this hepatoprotective activ-ity of GCEE is mediated at least in part through its ability toupregulate PPAR120574 expression

CP is an alkylating agent used for treatment of severaltypes of cancer [6 35] however its use has been limiteddue to severe toxicity [7 8] Our studies have demonstratedthat hepatotoxicity is one of the major side effects of CP[3 18 24 36] Here CP administration induced liver injuryconfirmed by increased circulating levels of liver functionmarker enzymes declined serum albumin levels andmarkedhistopathological changes of liver structures Accordingly wehave previously demonstrated increased serum ALT AST

and ALP in CP-intoxicated rats [3 18 24 36]These enzymesare used as reliable markers for the assessment of liver func-tion [37] Elevated circulating levels of these enzymes indicatehepatocellular damage induced by CP as previously reported[3 18 38] In addition CP-induced rats showed leukopeniaanemia and thrombocytopenia indicating hematopoieticdysfunction due to CP-induced bone marrow toxicity [3940] Similar findings have been reported in mice received CPat doses of 125mgkg [41]

Interestingly GCEE supplementation significantly alle-viated circulating levels of hepatic enzymes suggesting itsmembrane stabilizing potential The hepatoprotective effectof GCEE against CP was further confirmed by the improvedhistological structures of the liver and increased serum levelsof albumin Rats treated with CP developed liver dam-age characterized histologically by activated Kupffer cellshepatic vacuolation of fat type periportal hepatic necrosisand mononuclear cells infiltration mainly macrophages andhistiocytes These findings were consistent with our previousstudy [18] The decreased serum albumin in drug-inducedhepatotoxicity could be attributed to the provoked inflamma-tion and oxidative stress [42] During inflammation declinedproduction of albumin has been linked to its function as anegative acute phase protein [43] GCEEmarkedly prevented


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Figure 4 Effect of GCEE on (a) lipid peroxidation (b) nitric oxide (c) GSH (d) SOD (e) GPx and (f) CAT in liver of CP-induced rats Dataare expressed as mean plusmn SEM (119873 = 6) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 CP cyclophosphamide GCEE gamma-glutamylcysteineethyl esterMDAmalondialdehyde NO nitric oxide GSH reduced glutathione SOD superoxide dismutase CAT catalase GPx glutathioneperoxidase

histological alterations and increased serum albumin levelsconfirming its hepatoprotective activity In addition GCEEameliorates the hematopoietic parameters and hence protectsthe bone marrow against CP-induced suppression

Oxidative stress has been implicated in the hepatotoxiceffect of CP [18 24]Therefore finding a strategy to attenuateoxidative stress might grasp a key to alleviate the CP-induced hepatotoxicity The present study showed increased

levels of lipid peroxidation in the liver of CP-intoxicatedrats Excessive ROS production induced by CP can attackmembrane lipids leading to lipid peroxidation [3 7 18 24]In addition liver NOwas significantly increased as a result ofCP administration NO has been reported to be involved inCP-induced hepatotoxicity [44] It can combine with super-oxide anions producing the versatile oxidant peroxynitrite(ONOOminus) [45] ONOOminus activates NF-120581B in Kupffer cells and


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Figure 5 Effect of GCEE on serumTNF-120572 (a) and IL-1120573 (b)mRNAexpression levels of liver COX-2 (c) iNOS (d) andNF-120581B (e) and proteinexpression of liver NF-120581B-p65 (f) in CP-induced rats Data are expressed as mean plusmn SEM (119873 = 6) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001CP cyclophosphamide GCEE gamma-glutamylcysteine ethyl ester TNF120572 tumor necrosis factor alpha IL-1120573 interleukin-1beta NF-120581Bnuclear factor-kappaB iNOS inducible nitric oxide synthase COX-2 cyclooxygenase-2

subsequently increased production of the proinflammatorycytokines [46] The increased production of liver NO isa direct result of upregulated expression of iNOS as wepreviously reported in CP-induced rats [18] Moreover CP-induced rats exhibited declined liver GSH as well as activitiesof the antioxidant enzymes GSH depletion is a result of

its direct conjugation with CP metabolites [47] leading todeclined cellular defenses and necrotic cell death [48]

GCEE prevented the CP-induced lipid peroxidation NOproduction depletion of GSH and suppression of SODCAT and GPx activities in the liver of rats These findingsindicate clearly that GCEE protected against CP-induced


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Figure 6 Effect of GCEE on (a) caspase-3 mRNA expression (b) caspase-3 activity (c) BAX mRNA expression and (d) BAXimmunohistochemical staining in liver of CP-induced rats Data are expressed as mean plusmn SEM (119873 = 6) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast119875 lt 0001 CP cyclophosphamide GCEE gamma-glutamylcysteine ethyl ester BAX BCL2-associated X protein

oxidative stress through preventing GSH depletion andenhancing the enzymatic antioxidants In the same contextKobayashi et al [49] reported that GCEE protects againstischemiareperfusion-induced liver injury through prevent-ing GSH depletion More recently the study of Salama et

al [50] showed similar findings in iron-overload rat modelsupplemented with glutamyl cysteine dipeptide

In conjunction with oxidative stress increased produc-tion of inflammatory cytokines has been reported in CP-administered rats Previous studies from our laboratory


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(b)

Figure 7 Effect of GCEE on PPAR120574 (a) mRNA and (b) protein expression in liver of CP-induced rats Data are expressed as mean plusmn SEM(119873 = 6) lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 CP cyclophosphamide GCEE gamma-glutamylcysteine ethyl ester PPAR120574 peroxisome proliferatoractivated receptor gamma

showed increased production andor expression of inflam-matory cytokines following CP administration [3 18 2436] Akcay et al [51] revealed that DILI is associated withincreased production of inflammatory mediators producedby injured or immune cells-induced infiltration of leukocytesinto the site of injury In addition studies have demonstratedthat ROS augment gene expression of inflammatory media-tors and NF-120581B [52 53] and increase production of TNF-120572from Kupffer cells [54] Here CP-induced rats showed sig-nificant increase in serum TNF-120572 and IL-1120573 and liver COX-2 and iNOS This inflammatory response could be directlyconnected to the CP-induced upregulation of NF-120581B expres-sion Similar findings were showed in our previous studies[3 18 24 36] Oral administration of GCEE potentiallydecreased serum proinflammatory cytokines and COX-2 andiNOS mRNA expression in the liver of CP-induced rats Thisanti-inflammatory effect is a direct result of downregulatedNF-120581B expression and attenuated ROS production

Oxidative stress together with inflammation inducesapoptotic cell death in the liver [53] Under cell stress con-ditions hepatocytes become more susceptible to the lethaleffects of TNF120572 and Fas ligand (FasL) which bind to intra-cellular death receptors and subsequently activate caspase-8 [55] Within the mitochondria drugs or their metabolitescan cause ATP depletion excessive ROS production DNAdamage and increase permeability of the mitochondrialmembrane The resultant mitochondrial membrane perme-abilization leads to the release of cytochromeC and activationof procaspase-9 These events activate executioner caspase-3resulting in apoptotic cell death [56 57] Here CP-inducedrats showed significant increase in expression of the apoptoticmarkers caspase-3 and BAX A recent study by Germoush[58] showed significant increase in liver BAX mRNA and

protein expression in CP-induced rats These findings mightbe explained in terms of the CP-induced inflammation andoxidative stress in the liver of rats GCEE supplementationmarkedly prevented CP-induced apoptosis which is a directresult of its ability to attenuate inflammation and oxidativestress In agreement with our findings Salama et al [50]reported decreased caspase-3 activity in liver of iron-overloadrat model following treatment with glutamyl cysteine

To further explore how GCEE prevented CP-inducedoxidative stress inflammation and apoptosis expression lev-els of PPAR120574 were determined PPAR120574 is a nuclear receptorwe hypothesized to have a role in mediating the protectiveeffect of GCEE against CP-induced hepatotoxicity Previouswork from our laboratory showed declined PPAR120574 expres-sion in the liver of CP-induced rats [3 18] Interestingly wehave found a marked upregulation of liver PPAR120574 expressionin GCEE-treated rats

PPAR120574 is emerging as an important regulator of theresponse to oxidative stress and inflammation This notionhas been supported by the findings of several studies usingthe PPAR120574-specific agonists thiazolidinediones (TZDs)Together with other agonists TZDs showed beneficial ther-apeutic effects in oxidative stress-related diseases [59 60]As an example rosiglitazone induces the antioxidant enzymeheme oxygenase 1 (HO-1) in hepatocytes [61] and pioglita-zone protects against CP-induced oxidative stress in rats [60]In response to oxidative stress activation of PPAR120574 has beenreported to directly modulate the expression of several anti-oxidant genes Human mouse and rat CAT is transcrip-tionally regulated by PPAR120574 through PPREs containing thecanonical direct repeat 1 [62] located 12kb far from thetranscription initiation site [63] Furthermore PPAR120574 activa-tion promotes the expression of GPx3 [64] manganese SOD


RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus

GCEE Cyclophosphamide

PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573

Figure 8 A proposed schematic diagram for the protective mechanisms of GCEE against CP-induced hepatotoxicity PPAR120574 peroxisomeproliferator activated receptor gamma RXR retinoid X receptor PPRE PPAR response element HO-1 heme oxygenase 1 SOD superoxidedismutase GPx glutathione peroxidase CAT catalase CYP cytochrome P-450 GSH glutathione GCEE gamma-glutamylcysteine ethylester NF-120581B nuclear factor-kappaB iNOS inducible nitric oxide synthase COX-2 cyclooxygenase-2

[65] the mitochondrial uncoupling protein 2 (UCP2) [66]and HO-1 [61]

PPAR120574 has also been shown to induce anti-inflammatoryresponses through inhibiting the activation of NF-120581B result-ing in attenuation of proinflammatory cytokines production[67] PPAR120574 can transrepress NF-120581B activation via directbinding or formation of a repressor complex in the promoterof its target genes [68 69] Studies have also showed thatPPAR120574 downregulates COX-2 and iNOS [70]

Furthermore new experimental evidences suggested thepossible interaction andor coactivation of PPAR120574 andnuclear factor (erythroid-derived 2)-like 2 (Nrf2) can protectagainst CP-induced hepatotoxicity [18] Upon activationNrf2 translocates into the nucleus and promotes expressionof antioxidant and cytoprotective proteins [71] In additionNrf2 pathway has been regarded to have a central role in thecontrol of inflammation [72] and studies have shown severalanti-inflammatory agents which upregulate Nrf2 pathwayand suppress NF-120581B [18 73] Recently we have reportedthat simultaneous activation of PPAR120574 and Nrf2 in CP-induced rats significantly enhanced antioxidant defenses

downregulated NF-120581B and iNOS and prevented the produc-tion of proinflammatory cytokines [18] Through preventingoxidative stress and inflammation PPAR120574 is therefore able toprotect against apoptosis Our findings were supported by thestudies of Fuenzalida et al [74] andRen et al [75]who showedthat PPAR120574 has a prosurvival action and protects glial cellsand cardiomyocytes from oxidative stress-induced apoptosisThese antiapoptotic effects were mediated by induction ofB-cell lymphoma 2 (Bcl-2) independently of the proteinkinase B and mitogen-activated protein kinase pathways [7475]

In conclusion our study shows for the first time thatGCEE a GSH precursor confers protection against CP-induced hepatotoxicity in rats The hepatoprotective mech-anisms of GCEE are associated with activation of PPAR120574resulting in enhancement of antioxidant defenses preventionof GSH depletion and attenuation of excessive inflammatoryresponse and apoptosis (summarized mechanistic pathwaysare represented in Figure 8) Therefore GCEE has the poten-tial to provide cellular protection against CP-induced hepa-totoxicity


Abbreviations

GCEE Gamma-glutamylcysteine ethyl esterPPAR120574 Peroxisome proliferator activated receptor

gammaCP CyclophosphamideNrf2 Nuclear factor erythroid 2-related factor 2iNOS Inducible nitric oxide synthaseNF-120581B Nuclear factor-kappaBHO-1 Heme oxygenase 1ROS Reactive oxygen speciesRXR Retinoid X receptorARE Antioxidant response elementALP Alkaline phosphataseALT Alanine aminotransferaseAST Aspartate aminotransferaseMDA MalondialdehydeNO Nitric oxideGSH Reduced glutathioneSOD Superoxide dismutaseCAT CatalaseGPx Glutathione peroxidaseRBCs ErythrocytesHb HemoglobinWBCs LeukocytesBAX BCL2-associated X proteinTNF-120572 Tumor necrosis factor alphaIL-1120573 Interleukin-1betaPBS Phosphate buffered salineqRT-PCR Quantitative reverse

transcription-polymerase chain reactionANOVA One-way analysis of variance

Disclosure

Both authors participated as first author

Competing Interests

The authors have declared that no competing interests exist

References

[1] W Bleibel S KimKDrsquoSilva andE R Lemmer ldquoDrug-inducedliver injury review articlerdquo Digestive Diseases and Sciences vol52 no 10 pp 2463ndash2471 2007

[2] S RussmannG A Kullak-Ublick and I Grattagliano ldquoCurrentconcepts of mechanisms in drug-induced hepatotoxicityrdquo Cur-rent Medicinal Chemistry vol 16 no 23 pp 3041ndash3053 2009

[3] A M Mahmoud ldquoHesperidin protects against cyclophosph-amide-induced hepatotoxicity by upregulation of ppar120574 andabrogation of oxidative stress and inflammationrdquo CanadianJournal of Physiology and Pharmacology vol 92 no 9 pp 717ndash724 2014

[4] N Kaplowitz ldquoDrug-induced liver injuryrdquo Clinical InfectiousDiseases vol 38 supplement 2 pp S44ndashS48 2004

[5] W Bernal and JWendon ldquoAcute liver failurerdquoTheNew EnglandJournal of Medicine vol 369 no 26 pp 2525ndash2534 2013

[6] AMoignet ZHasanali R Zambello et al ldquoCyclophosphamideas a first-line therapy in LGL leukemiardquo Leukemia vol 28 no5 pp 1134ndash1136 2014

[7] L H Fraiser S Kanekal and J P Kehrer ldquoCyclophosphamidetoxicity characterising and avoiding the problemrdquo Drugs vol42 no 5 pp 781ndash795 1991

[8] P PapaldoM Lopez PMarolla et al ldquoImpact of five prophylac-tic filgrastim schedules on hematologic toxicity in early breastcancer patients treated with epirubicin and cyclophosphamiderdquoJournal of Clinical Oncology vol 23 no 28 pp 6908ndash6918 2005

[9] P D King and M C Perry ldquoHepatotoxicity of chemotherapyrdquoThe Oncologist vol 6 no 2 pp 162ndash176 2001

[10] M K Mohammad D Avila J Zhang et al ldquoAcrolein cytotoxi-city in hepatocytes involves endoplasmic reticulum stressmito-chondrial dysfunction and oxidative stressrdquo Toxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012

[11] S L MacAllister N Martin-Brisac V Lau K Yang and P JOrsquoBrien ldquoAcrolein and chloroacetaldehyde an examination ofthe cell and cell-free biomarkers of toxicityrdquo Chemico-BiologicalInteractions vol 202 no 1ndash3 pp 259ndash266 2013

[12] V Laudet C Hanni J Coll F Catzeflis and D StehelinldquoEvolution of the nuclear receptor gene superfamilyrdquo EMBOJournal vol 11 no 3 pp 1003ndash1013 1992

[13] G D Barish V ANarkar and RM Evans ldquoPPAR120575 a dagger inthe heart of the metabolic syndromerdquo Journal of Clinical Inves-tigation vol 116 no 3 pp 590ndash597 2006

[14] P Tontonoz and BM Spiegelman ldquoFat and beyond the diversebiology of PPAR120574rdquo Annual Review of Biochemistry vol 77 pp289ndash312 2008

[15] M Peyrou P Ramadori L Bourgoin and M Foti ldquoPPARs inliver diseases and cancer epigenetic regulation bymicroRNAsrdquoPPAR Research vol 2012 Article ID 757803 16 pages 2012

[16] L Yang S A Stimpson L Chen W W Harrington and D CRockey ldquoEffectiveness of the PPAR120574 agonist GW570 in liverfibrosisrdquo Inflammation Research vol 59 no 12 pp 1061ndash10712010

[17] AMMahmoudM O Germoush and A S Soliman ldquoBerber-ine attenuates isoniazid-induced hepatotoxicity by modulatingperoxisome proliferator-activated receptor 120574 oxidative stressand inflammationrdquo International Journal of Pharmacology vol10 no 8 pp 451ndash460 2014

[18] A M Mahmoud and H S Al Dera ldquo18120573-Glycyrrhetinic acidexerts protective effects against cyclophosphamide-inducedhepatotoxicity potential role of PPAR120574 and Nrf2 upregulationrdquoGenes amp Nutrition vol 10 no 6 article no 41 2015

[19] Y Z Maksimchik E A Lapshina E Y Sudnikovich S VZabrodskaya and I B Zavodnik ldquoProtective effects of N-acetyl-L-cysteine against acute carbon tetrachloride hepatotox-icity in ratsrdquo Cell Biochemistry and Function vol 26 no 1 pp11ndash18 2008

[20] S Akbulut H Elbe C Eris et al ldquoCytoprotective effects ofamifostine ascorbic acid and N-acetylcysteine against metho-trexate-induced hepatotoxicity in ratsrdquo World Journal of Gas-troenterology vol 20 no 29 pp 10158ndash10165 2014

[21] J Drake J Kanski S Varadarajan M Tsoras and D A But-terfield ldquoElevation of brain glutathione by 120574-glutamylcysteineethyl ester protects against peroxynitrite-induced oxidativestressrdquo Journal of Neuroscience Research vol 68 no 6 pp 776ndash784 2002

[22] A Yalcin G Armagan E Turunc S Konyalioglu and L KanitldquoPotential neuroprotective effect of 120574-glutamylcysteine ethyl


ester on rat brain against kainic acid-induced excitotoxicityrdquoFree Radical Research vol 44 no 5 pp 513ndash521 2010

[23] J Lok W Leung S Zhao et al ldquoGamma-glutamylcysteineethyl ester protects cerebral endothelial cells during injury anddecreases blood-brain barrier permeability after experimentalbrain traumardquo Journal ofNeurochemistry vol 118 no 2 pp 248ndash255 2011

[24] E M Kamel A M Mahmoud S A Ahmed and A MLamsabhi ldquoA phytochemical and computational study on flav-onoids isolated from Trifolium resupinatum L and their novelhepatoprotective activityrdquo Food amp Function vol 7 no 4 pp2094ndash2106 2016

[25] E Turunc L Kanit and A Yalcin ldquoEffect of 120574-glutamylcysteineethylester on the levels of c-fos mRNA expression glutathioneand reactive oxygen species formation in kainic acid excitotox-icityrdquo Journal of Pharmacy and Pharmacology vol 62 no 8 pp1010ndash1017 2010

[26] G Schumann and R Klauke ldquoNew IFCC reference proceduresfor the determination of catalytic activity concentrations of fiveenzymes in serum preliminary upper reference limits obtainedin hospitalized subjectsrdquo Clinica Chimica Acta vol 327 no 1-2pp 69ndash79 2003

[27] C Wenger A Kaplan F F Rubaltelli and C HammermanldquoAlkaline phosphataserdquo in Clinical Chemistry pp 1094ndash1098The C V Mosby Co St Louis Mo USA Princeton TorontoCanada 1984

[28] D Webster ldquoA study of the interaction of bromocresol greenwith isolated serum globulin fractionsrdquo Clinica Chimica Actavol 53 no 1 pp 109ndash115 1974

[29] HG Preuss S T Jarrell R Scheckenbach S Lieberman andRAAnderson ldquoComparative effects of chromium vanadiumandGymnema sylvestre on sugar-induced blood pressure elevationsin SHRrdquo Journal of the American College of Nutrition vol 17 no2 pp 116ndash123 1998

[30] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[31] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo FEBS European Journal ofBiochemistry vol 47 no 3 pp 469ndash474 1974

[32] B Matkovics L Szabo and I S Varga ldquoDetermination ofenzyme activities in lipid peroxidation and glutathione path-waysrdquo Laboratoriumi Diagnosztika vol 15 pp 248ndash249 1998(Hungarian)

[33] G CohenDDembiec and JMarcus ldquoMeasurement of catalaseactivity in tissue extractsrdquo Analytical Biochemistry vol 34 no1 pp 30ndash38 1970

[34] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCRand the 2minusΔΔ119862TmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[35] M Lawson A Vasilaras A De Vries P Mactaggart and DNicol ldquoUrological implications of cyclophosphamide and ifos-famiderdquo Scandinavian Journal of Urology and Nephrology vol42 no 4 pp 309ndash317 2008

[36] M O Germoush and A M Mahmoud ldquoBerberine mitigatescyclophosphamide-induced hepatotoxicity bymodulating anti-oxidant status and inflammatory cytokinesrdquo Journal of CancerResearch and Clinical Oncology vol 140 no 7 pp 1103ndash11092014

[37] S K Ramaiah ldquoA toxicologist guide to the diagnostic interpre-tation of hepatic biochemical parametersrdquo Food and ChemicalToxicology vol 45 no 9 pp 1551ndash1557 2007

[38] M E De Jonge A D R Huitema J H Beijnen and SRodenhuis ldquoHigh exposures to bioactivated cyclophosphamideare related to the occurrence of veno-occlusive disease of theliver following high-dose chemotherapyrdquoBritish Journal of Can-cer vol 94 no 9 pp 1226ndash1230 2006

[39] D Busse F W Busch F Bohnenstengel et al ldquoDose esca-lation of cyclophosphamide in patients with breast cancerconsequences for pharmacokinetics and metabolismrdquo Journalof Clinical Oncology vol 15 no 5 pp 1885ndash1896 1997

[40] S Vadhan-Raj ldquoManagement of chemotherapy-induced throm-bocytopenia current status of thrombopoietic agentsrdquo Seminarsin Hematology vol 46 supplement 2 pp S26ndashS32 2009

[41] J P Kap C L Byung S L Jae andH CMyung ldquoAngelica gigasNakai extract ameliorates the effects of cyclophosphamide onimmunological and hematopoietic dysfunction in micerdquo Jour-nal ofMedicinal Plants Research vol 8 no 17 pp 657ndash663 2014

[42] E M Abdella A M Mahmoud and A M El-Derby ldquoBrownseaweeds protect against azoxymethane-induced hepatic reper-cussions through up-regulation of peroxisome proliferator-activated receptor gamma and attenuation of oxidative stressrdquoPharmaceutical Biology vol 54 no 11 pp 2496ndash2504 2016

[43] J P Doweiko and D J Nompleggi ldquoThe role of albumin inhuman physiology and pathophysiology part III albumin anddisease statesrdquo Journal of Parenteral and Enteral Nutrition vol15 no 4 pp 476ndash483 1991

[44] M C Andersson G Tobin and D Giglio ldquoCholinergic nitricoxide release from the urinary bladder mucosa in cyclophos-phamide-induced cystitis of the anaesthetized ratrdquo BritishJournal of Pharmacology vol 153 no 7 pp 1438ndash1444 2008

[45] S E McKim E Gabele F Isayama et al ldquoInducible nitric oxidesynthase is required in alcohol-induced liver injury studieswith knockout micerdquoGastroenterology vol 125 no 6 pp 1834ndash1844 2003

[46] B M Matata and M Galinanes ldquoPeroxynitrite is an essentialcomponent of cytokines production mechanism in humanmonocytes through modulation of nuclear factor-120581B DNAbinding activityrdquo The Journal of Biological Chemistry vol 277no 3 pp 2330ndash2335 2002

[47] Z Yousefipour K Ranganna M A Newaz and S G MiltonldquoMechanism of acrolein-induced vascular toxicityrdquo Journal ofPhysiology and Pharmacology vol 56 no 3 pp 337ndash353 2005

[48] A Srivastava and T Shivanandappa ldquoHepatoprotective effectof the root extract of Decalepis hamiltonii against carbontetrachloride-induced oxidative stress in ratsrdquo Food Chemistryvol 118 no 2 pp 411ndash417 2010

[49] H Kobayashi T Kurokawa S Kitahara et al ldquoThe effects of120574-glutamylcysteine ethyl ester a prodrug of glutathione onischemia-reperfusion-induced liver injury in ratsrdquo Transplan-tation vol 54 no 3 pp 414ndash418 1992

[50] S A Salama M S Al-Harbi M S Abdel-Bakky and H AOmar ldquoGlutamyl cysteine dipeptide suppresses ferritin expres-sion and alleviates liver injury in iron-overload rat modelrdquo Bio-chimie vol 115 pp 203ndash211 2015

[51] A Akcay Q Nguyen and C L Edelstein ldquoMediators of inflam-mation in acute kidney injuryrdquoMediators of Inflammation vol2009 Article ID 137072 12 pages 2009

[52] R Schreck K Albermann and P A Baeuerle ldquoNuclear fac-tor kb an oxidative stress-responsive transcription factor of


eukaryotic cells (a review)rdquo Free Radical Research vol 17 no4 pp 221ndash237 1992

[53] H Jaeschke ldquoReactive oxygen and mechanisms of inflamma-tory liver injury present conceptsrdquo Journal of Gastroenterologyand Hepatology vol 26 no 1 pp 173ndash179 2011

[54] J M Bellezzo K A Leingang G A Bulla R S Britton B RBacon and E S Fox ldquoModulation of lipopolysaccharide-med-iated activation in rat Kupffer cells by antioxidantsrdquo Journal ofLaboratory and ClinicalMedicine vol 131 no 1 pp 36ndash44 1998

[55] M Li and G-T Liu ldquoInhibition of FasFasL mRNA expressionand TNF-120572 release in concanavalin A-induced liver injury inmice by bicyclolrdquoWorld Journal of Gastroenterology vol 10 no12 pp 1775ndash1779 2004

[56] J Huang W Shi J Zhang et al ldquoGenomic indicators in theblood predict drug-induced liver injuryrdquo PharmacogenomicsJournal vol 10 no 4 pp 267ndash277 2010

[57] M I Lucena E Garcıa-Martın R J Andrade et al ldquoMito-chondrial superoxide dismutase and glutathione peroxidase inidiosyncratic drug-induced liver injuryrdquoHepatology vol 52 no1 pp 303ndash312 2010

[58] M O Germoush ldquoDiosmin protects against cyclophosph-amide-induced liver injury through attenuation of oxidativestress inflammation and apoptosisrdquo International Journal ofPharmacology vol 12 no 6 pp 644ndash654 2016

[59] J M Kleinhenz D J Kleinhenz S You et al ldquoDisruptionof endothelial peroxisome proliferator-activated receptor-120574reduces vascular nitric oxide productionrdquo American Journal ofPhysiologymdashHeart andCirculatory Physiology vol 297 no 5 ppH1647ndashH1654 2009

[60] A A K El-Sheikh and R A Rifaai ldquoPeroxisome proliferatoractivator receptor (PPAR)-120574 ligand but not PPAR-120572 amelio-rates cyclophosphamide-induced oxidative stress and inflam-mation in rat liverrdquo PPAR Research vol 2014 Article ID 6263192014

[61] A Galli E Ceni T Mello et al ldquoThiazolidinediones inhibithepatocarcinogenesis in hepatitis B virus-transgenic mice byperoxisome proliferator-activated receptor 120574-independent reg-ulation of nucleophosminrdquo Hepatology vol 52 no 2 pp 493ndash505 2010

[62] G D Girnun F E Domann S AMoore andM E C RobbinsldquoIdentification of a functional peroxisome proliferator-acti-vated receptor response element in the rat catalase promoterrdquoMolecular Endocrinology vol 16 no 12 pp 2793ndash2801 2002

[63] Y OkunoMMatsuda H Kobayashi et al ldquoAdipose expressionof catalase is regulated via a novel remote PPAR120574-responsiveregionrdquo Biochemical and Biophysical Research Communicationsvol 366 no 3 pp 698ndash704 2008

[64] S S Chung M Kim B-S Youn et al ldquoGlutathione peroxidase3 mediates the antioxidant effect of peroxisome proliferator-activated receptor 120574 in human skeletal muscle cellsrdquo Molecularand Cellular Biology vol 29 no 1 pp 20ndash30 2009

[65] G Ding M Fu Q Qin et al ldquoCardiac peroxisome proliferator-activated receptor 120574 is essential in protecting cardiomyocytesfrom oxidative damagerdquo Cardiovascular Research vol 76 no2 pp 269ndash279 2007

[66] AAGupte J Z Liu Y Ren et al ldquoRosiglitazone attenuates age-and diet-associated nonalcoholic steatohepatitis in male low-density lipoprotein receptor knockout micerdquo Hepatology vol52 no 6 pp 2001ndash2011 2010

[67] Y Yu P H Correll and J P V Heuvel ldquoConjugated linoleicacid decreases production of pro-inflammatory products in

macrophages evidence for a PPAR120574-dependent mechanismrdquoBiochimica et Biophysica Acta (BBA)mdashMolecular and Cell Biol-ogy of Lipids vol 1581 no 3 pp 89ndash99 2002

[68] M Li G Pascual and C K Glass ldquoPeroxisome proliferator-activated receptor 120574-dependent repression of the induciblenitric oxide synthase generdquoMolecular and Cellular Biology vol20 no 13 pp 4699ndash4707 2000

[69] B Vandewalle E Moerman B Lefebvre et al ldquoPPAR120574-dependent and -independent effects of Rosiglitazone on lipo-toxic human pancreatic isletsrdquo Biochemical and BiophysicalResearch Communications vol 366 no 4 pp 1096ndash1101 2008

[70] M Ricote A C Li T M Willson C J Kelly and C K GlassldquoThe peroxisome proliferator-activated receptor-120574 is a negativeregulator of macrophage activationrdquo Nature vol 391 no 6662pp 79ndash82 1998

[71] A K Jaiswal ldquoNrf2 signaling in coordinated activation of anti-oxidant gene expressionrdquo Free Radical Biology and Medicinevol 36 no 10 pp 1199ndash1207 2004

[72] H K Bryan A Olayanju C E Goldring and B K Park ldquoTheNrf2 cell defence pathway keap1-dependent and -independentmechanisms of regulationrdquo Biochemical Pharmacology vol 85no 6 pp 705ndash717 2013

[73] S M Abd El-Twab W G Hozayen O E Hussein and A MMahmoud ldquo18120573-Glycyrrhetinic acid protects against metho-trexate-induced kidney injury by up-regulating the Nrf2AREHO-1 pathway and endogenous antioxidantsrdquoRenal Failure vol38 no 9 pp 1516ndash1527 2016

[74] K Fuenzalida R Quintanilla P Ramos et al ldquoPeroxisomeproliferator-activated receptor 120574 up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabi-lization and protection against oxidative stress and apoptosisrdquoJournal of Biological Chemistry vol 282 no 51 pp 37006ndash370152007

[75] Y Ren C Sun Y Sun et al ldquoPPAR gamma protects cardio-myocytes against oxidative stress and apoptosis via Bcl-2 upreg-ulationrdquo Vascular Pharmacology vol 51 no 2-3 pp 169ndash1742009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the expression of target genes [13] PPAR120574 is induced duringpreadipocytes differentiation and plays a central role in lipidmetabolism glucose homeostasis inflammation and cellproliferation [14] In the liver disruption of PPARs has beenassociated with different disorders [15] On the other handactivation of PPAR120574 inhibited the fibrogenic response to liverinjury [16] and protected against drug-induced hepatotoxi-city as we recently reported [3 17 18]

Attenuation of oxidative stress through restoring GSHlevels is a well-known strategy to combat drug-inducedtoxicity For example administration of N-acetylcysteine(NAC) a precursor ofGSH protected the liver against carbontetrachloride [19] and methotrexate-induced toxicity [20]Gamma-glutamylcysteine ethyl ester (GCEE) a syntheticGSH precursor has been demonstrated to boost endogenousGSH levels and block oxidative stress in neurons [21 22]as well as cerebral endothelial cells [23] We believe thatnothing has yet been reported on the possible protectiveeffects of GCEE against CP-induced hepatotoxicity In thepresent study we asked whether GCEE can attenuate CP-induced oxidative stress apoptosis and inflammation in theliver of rats pointing to the role of PPAR120574

2 Materials and Methods

21 Chemicals Gamma-glutamyl cysteine ethyl ester(GCEE) and cyclophosphamide (CP Endoxan) were pur-chased fromBachem (Torrance CAUSA) andBaxterOncol-ogy (Dusseldorf Germany) respectively Alanine amin-otransferase (ALT) aspartate aminotransferase (AST)alkaline phosphatase (ALP) and albumin assay kits weresupplied by Spinreact (Spain) PPAR120574 nuclear factor-120581B(NF-120581B) and Bcl-2-associated X protein (BAX) antibodieswere obtained from Santa Cruz Biotechnology (USA)Cytokines assay kits were purchased from RampD Systems(USA) All other chemicals were obtained from Sigma (USA)and other standard commercial supplies

22 Experimental Animals and Treatments Male albinoWis-tar rats (10 weeks old) from the Institute of Ophthalmology(Giza Egypt) were included in the present study They weremaintained on a 12 h darklight cycle at 22 plusmn 2∘C with adlibitum access to standard laboratory diet and water Allanimal procedures related to care treatments and samplingwere in accordance with the guidelines of the InstitutionalAnimal Ethics Committee of Beni-Suef University (Egypt)

Twenty-four rats were divided randomly into threegroups of 8 rats each and allowed to adapt for 1 week priorto the experiment Group I (Control) received normal salinesolution for 16 days Group II (CP) received saline for 15 daysand 150mgkg bwt CPonday 16 [18] andGroup III (GCEE+CP) received 100mgkg bwt GCEE for 15 days and 150mgkgbwt CP on day 16

The dose route and day of CP administration wereselected based on our previous studies [18 24] Since GCEEhas been proven to be effective in vivo at doses of 10mgkg[25] and 150mgkg bwt [21] we selected a dose of 100mgkgto be tested in our study All experimental solutions wereadministered intraperitoneally

At day 21 the animals were sacrificed by cervical dis-location and various samples were collected Blood sampleswere either collected on heparinized tubes for hematologi-cal analysis or left to coagulate for serum separation Liverswere immediately excised washed in cold phosphate buf-fered saline (PBS) and weighed Samples from the liverwere fixed in 10 neutral buffered formalin for histologicaland immunohistochemical processing Other samples werehomogenized (10wv) in cold PBS for biochemical assays orkept frozen atminus80∘C for gene and protein expression analysis

23 Biochemical Assays

231 Determination of Liver FunctionMarkers Serum amin-otransferases were assayed using Spinreact (Spain) reagentkits according to the method of Schumann and Klauke[26] Serum ALP activity and albumin concentration weremeasured using Spinreact (Spain) reagent kit according to themethods of Wenger et al [27] andWebster [28] respectively

232 Determination of Oxidative Stress and AntioxidantDefenses Liver malondialdehyde (MDA) and GSH levelswere determined according to the methods of Preuss et al[29] and Beutler et al [30] respectively Liver nitric oxide(NO) was determined as nitrite using Griess reagent Super-oxide dismutase (SOD) glutathione peroxidase (GPx) andcatalase (CAT) were determined according to the methods ofS Marklund and G Marklund [31] Matkovics et al [32] andCohen et al [33] respectively

233 Determination of Proinflammatory Cytokines Tumornecrosis factor alpha (TNF-120572) and interleukin-1beta (IL-1120573)were determined in serum samples using specific rats ELISAkits (RampD Systems USA) according to the manufacturerrsquosinstructions

234 Determination of Caspase-3 Activity Liver caspase-3 activity was measured using the CaspACE assay system(Promega Madison WI USA) following the manufacturerrsquosinstructions The assay is based on the action of caspase-3 on the substrate Ac-DEVD-pNA releasing yellow chro-mophore p-nitroanilineThe activity of caspase-3 activity waspresented as percentage of corresponding control

24 Determination of Hematological Parameters Samples ofblood from all animals were collected into heparinized tubesand red blood corpuscles (RBCs) total white blood cells(WBCs) platelet count and hemoglobin (Hb) content weredetermined using an automated hematoanalyzer

25 Histopathology and Immunohistochemistry Samplesfrom the liver were immediately washed in cold PBS andfixed for histological processing and hematoxylin and eosin(HampE) staining

Liver sections were immunohistochemically stained withanti-BAX antibody Briefly the slides were deparaffinizedrehydrated and incubated in 3 hydrogen peroxide (H

2O2)

for 5minThe slides were washed in Tris-buffered saline (pH76) blocked with protein block (Novocastra) and incubated















3 Results





lowastlowast


150

100

50

0

ALT

(UL

)

GCEECPControl(a)

250

200

100

150

50

0

AST

(UL

)


GCEECPControl(b)

GCEECPControl

250

200

100

150

50

0

ALP

(UL

)


(c)GCEECPControl

lowastlowast

lowastlowast

0

1

2

3

4

Alb

umin

(gd

L)

(d)









cv

(a) (b)

N

(c) (d)











GCEECPControl

0

2

4

6

8


RBCs

times10

6

(a)GCEECPControl

Hb

(gd

L)

lowastlowast

0

5

10

15

(b)

0

5

10

15

GCEECPControl

lowastlowast

lowast

times10

3W

BCs

(c)

800

600

400

200

0

GCEECPControl

lowastlowast

lowast

lowastlowastlowast

Plat

elet

stimes10

3

(d)



4 Discussion






GCEECPControl

lowastlowast


120

80

40

0

MD

A (n

mol

100

mg)

(a)GCEECPControl


No

(nm

ol100

mg)

100

80

60

20

40

0

(b)

GCEECPControl

lowastlowast

50

40

30

20

10

0

GSH

(nm

ol100

mg)

(c)GCEECPControl

lowastlowast

lowast

SOD

(Ug

)

30

20

10

0

(d)

GCEECPControl

lowastlowast

lowast

GPx

(Ug

)

25

20

15

10

5

0

(e)GCEECPControl

lowastlowast

lowast

40

30

20

10

0

CAT

(ktimes10

minus2)

(f)






GCEECPControl

200

150

100

50

0

TNF-

120572(p

gm

L)lowastlowast


(a)GCEECPControl

80

60

40

20

0


IL-1120573

(pg

mL)

(b)

GCEECPControl

3

2

1

0COX-

2 m

RNA

(rel

ativ

e to

cont

rol)


(c)GCEECPControl

25

20

15

10

05

0iNO

S m

RNA

(rel

ativ

e to

cont

rol) lowastlowast

lowastlowast

(d)

GCEECPControl

25

20

15

10

05

00NF-

120581B

mRN

A (r

elat

ive t

o co

ntro


(e)

GCEECPControl

GCEECPControl

NF-

120581B

p65

(rel

ativ

e to

cont

rol)

120573-Actin

NF-120581B p65

300

200

100

0


(f)






GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























3 Results





lowastlowast


150

100

50

0

ALT

(UL

)

GCEECPControl(a)

250

200

100

150

50

0

AST

(UL

)


GCEECPControl(b)

GCEECPControl

250

200

100

150

50

0

ALP

(UL

)


(c)GCEECPControl

lowastlowast

lowastlowast

0

1

2

3

4

Alb

umin

(gd

L)

(d)









cv

(a) (b)

N

(c) (d)











GCEECPControl

0

2

4

6

8


RBCs

times10

6

(a)GCEECPControl

Hb

(gd

L)

lowastlowast

0

5

10

15

(b)

0

5

10

15

GCEECPControl

lowastlowast

lowast

times10

3W

BCs

(c)

800

600

400

200

0

GCEECPControl

lowastlowast

lowast

lowastlowastlowast

Plat

elet

stimes10

3

(d)



4 Discussion






GCEECPControl

lowastlowast


120

80

40

0

MD

A (n

mol

100

mg)

(a)GCEECPControl


No

(nm

ol100

mg)

100

80

60

20

40

0

(b)

GCEECPControl

lowastlowast

50

40

30

20

10

0

GSH

(nm

ol100

mg)

(c)GCEECPControl

lowastlowast

lowast

SOD

(Ug

)

30

20

10

0

(d)

GCEECPControl

lowastlowast

lowast

GPx

(Ug

)

25

20

15

10

5

0

(e)GCEECPControl

lowastlowast

lowast

40

30

20

10

0

CAT

(ktimes10

minus2)

(f)






GCEECPControl

200

150

100

50

0

TNF-

120572(p

gm

L)lowastlowast


(a)GCEECPControl

80

60

40

20

0


IL-1120573

(pg

mL)

(b)

GCEECPControl

3

2

1

0COX-

2 m

RNA

(rel

ativ

e to

cont

rol)


(c)GCEECPControl

25

20

15

10

05

0iNO

S m

RNA

(rel

ativ

e to

cont

rol) lowastlowast

lowastlowast

(d)

GCEECPControl

25

20

15

10

05

00NF-

120581B

mRN

A (r

elat

ive t

o co

ntro


(e)

GCEECPControl

GCEECPControl

NF-

120581B

p65

(rel

ativ

e to

cont

rol)

120573-Actin

NF-120581B p65

300

200

100

0


(f)






GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast


150

100

50

0

ALT

(UL

)

GCEECPControl(a)

250

200

100

150

50

0

AST

(UL

)


GCEECPControl(b)

GCEECPControl

250

200

100

150

50

0

ALP

(UL

)


(c)GCEECPControl

lowastlowast

lowastlowast

0

1

2

3

4

Alb

umin

(gd

L)

(d)









cv

(a) (b)

N

(c) (d)











GCEECPControl

0

2

4

6

8


RBCs

times10

6

(a)GCEECPControl

Hb

(gd

L)

lowastlowast

0

5

10

15

(b)

0

5

10

15

GCEECPControl

lowastlowast

lowast

times10

3W

BCs

(c)

800

600

400

200

0

GCEECPControl

lowastlowast

lowast

lowastlowastlowast

Plat

elet

stimes10

3

(d)



4 Discussion






GCEECPControl

lowastlowast


120

80

40

0

MD

A (n

mol

100

mg)

(a)GCEECPControl


No

(nm

ol100

mg)

100

80

60

20

40

0

(b)

GCEECPControl

lowastlowast

50

40

30

20

10

0

GSH

(nm

ol100

mg)

(c)GCEECPControl

lowastlowast

lowast

SOD

(Ug

)

30

20

10

0

(d)

GCEECPControl

lowastlowast

lowast

GPx

(Ug

)

25

20

15

10

5

0

(e)GCEECPControl

lowastlowast

lowast

40

30

20

10

0

CAT

(ktimes10

minus2)

(f)






GCEECPControl

200

150

100

50

0

TNF-

120572(p

gm

L)lowastlowast


(a)GCEECPControl

80

60

40

20

0


IL-1120573

(pg

mL)

(b)

GCEECPControl

3

2

1

0COX-

2 m

RNA

(rel

ativ

e to

cont

rol)


(c)GCEECPControl

25

20

15

10

05

0iNO

S m

RNA

(rel

ativ

e to

cont

rol) lowastlowast

lowastlowast

(d)

GCEECPControl

25

20

15

10

05

00NF-

120581B

mRN

A (r

elat

ive t

o co

ntro


(e)

GCEECPControl

GCEECPControl

NF-

120581B

p65

(rel

ativ

e to

cont

rol)

120573-Actin

NF-120581B p65

300

200

100

0


(f)






GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















cv

(a) (b)

N

(c) (d)











GCEECPControl

0

2

4

6

8


RBCs

times10

6

(a)GCEECPControl

Hb

(gd

L)

lowastlowast

0

5

10

15

(b)

0

5

10

15

GCEECPControl

lowastlowast

lowast

times10

3W

BCs

(c)

800

600

400

200

0

GCEECPControl

lowastlowast

lowast

lowastlowastlowast

Plat

elet

stimes10

3

(d)



4 Discussion






GCEECPControl

lowastlowast


120

80

40

0

MD

A (n

mol

100

mg)

(a)GCEECPControl


No

(nm

ol100

mg)

100

80

60

20

40

0

(b)

GCEECPControl

lowastlowast

50

40

30

20

10

0

GSH

(nm

ol100

mg)

(c)GCEECPControl

lowastlowast

lowast

SOD

(Ug

)

30

20

10

0

(d)

GCEECPControl

lowastlowast

lowast

GPx

(Ug

)

25

20

15

10

5

0

(e)GCEECPControl

lowastlowast

lowast

40

30

20

10

0

CAT

(ktimes10

minus2)

(f)






GCEECPControl

200

150

100

50

0

TNF-

120572(p

gm

L)lowastlowast


(a)GCEECPControl

80

60

40

20

0


IL-1120573

(pg

mL)

(b)

GCEECPControl

3

2

1

0COX-

2 m

RNA

(rel

ativ

e to

cont

rol)


(c)GCEECPControl

25

20

15

10

05

0iNO

S m

RNA

(rel

ativ

e to

cont

rol) lowastlowast

lowastlowast

(d)

GCEECPControl

25

20

15

10

05

00NF-

120581B

mRN

A (r

elat

ive t

o co

ntro


(e)

GCEECPControl

GCEECPControl

NF-

120581B

p65

(rel

ativ

e to

cont

rol)

120573-Actin

NF-120581B p65

300

200

100

0


(f)






GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GCEECPControl

0

2

4

6

8


RBCs

times10

6

(a)GCEECPControl

Hb

(gd

L)

lowastlowast

0

5

10

15

(b)

0

5

10

15

GCEECPControl

lowastlowast

lowast

times10

3W

BCs

(c)

800

600

400

200

0

GCEECPControl

lowastlowast

lowast

lowastlowastlowast

Plat

elet

stimes10

3

(d)



4 Discussion






GCEECPControl

lowastlowast


120

80

40

0

MD

A (n

mol

100

mg)

(a)GCEECPControl


No

(nm

ol100

mg)

100

80

60

20

40

0

(b)

GCEECPControl

lowastlowast

50

40

30

20

10

0

GSH

(nm

ol100

mg)

(c)GCEECPControl

lowastlowast

lowast

SOD

(Ug

)

30

20

10

0

(d)

GCEECPControl

lowastlowast

lowast

GPx

(Ug

)

25

20

15

10

5

0

(e)GCEECPControl

lowastlowast

lowast

40

30

20

10

0

CAT

(ktimes10

minus2)

(f)






GCEECPControl

200

150

100

50

0

TNF-

120572(p

gm

L)lowastlowast


(a)GCEECPControl

80

60

40

20

0


IL-1120573

(pg

mL)

(b)

GCEECPControl

3

2

1

0COX-

2 m

RNA

(rel

ativ

e to

cont

rol)


(c)GCEECPControl

25

20

15

10

05

0iNO

S m

RNA

(rel

ativ

e to

cont

rol) lowastlowast

lowastlowast

(d)

GCEECPControl

25

20

15

10

05

00NF-

120581B

mRN

A (r

elat

ive t

o co

ntro


(e)

GCEECPControl

GCEECPControl

NF-

120581B

p65

(rel

ativ

e to

cont

rol)

120573-Actin

NF-120581B p65

300

200

100

0


(f)






GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GCEECPControl

lowastlowast


120

80

40

0

MD

A (n

mol

100

mg)

(a)GCEECPControl


No

(nm

ol100

mg)

100

80

60

20

40

0

(b)

GCEECPControl

lowastlowast

50

40

30

20

10

0

GSH

(nm

ol100

mg)

(c)GCEECPControl

lowastlowast

lowast

SOD

(Ug

)

30

20

10

0

(d)

GCEECPControl

lowastlowast

lowast

GPx

(Ug

)

25

20

15

10

5

0

(e)GCEECPControl

lowastlowast

lowast

40

30

20

10

0

CAT

(ktimes10

minus2)

(f)






GCEECPControl

200

150

100

50

0

TNF-

120572(p

gm

L)lowastlowast


(a)GCEECPControl

80

60

40

20

0


IL-1120573

(pg

mL)

(b)

GCEECPControl

3

2

1

0COX-

2 m

RNA

(rel

ativ

e to

cont

rol)


(c)GCEECPControl

25

20

15

10

05

0iNO

S m

RNA

(rel

ativ

e to

cont

rol) lowastlowast

lowastlowast

(d)

GCEECPControl

25

20

15

10

05

00NF-

120581B

mRN

A (r

elat

ive t

o co

ntro


(e)

GCEECPControl

GCEECPControl

NF-

120581B

p65

(rel

ativ

e to

cont

rol)

120573-Actin

NF-120581B p65

300

200

100

0


(f)






GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GCEECPControl

200

150

100

50

0

TNF-

120572(p

gm

L)lowastlowast


(a)GCEECPControl

80

60

40

20

0


IL-1120573

(pg

mL)

(b)

GCEECPControl

3

2

1

0COX-

2 m

RNA

(rel

ativ

e to

cont

rol)


(c)GCEECPControl

25

20

15

10

05

0iNO

S m

RNA

(rel

ativ

e to

cont

rol) lowastlowast

lowastlowast

(d)

GCEECPControl

25

20

15

10

05

00NF-

120581B

mRN

A (r

elat

ive t

o co

ntro


(e)

GCEECPControl

GCEECPControl

NF-

120581B

p65

(rel

ativ

e to

cont

rol)

120573-Actin

NF-120581B p65

300

200

100

0


(f)






GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GCEECPControl


25

20

15

10

05

00

Casp

ase-3

mRN

A

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

lowastlowastlowast

250

200

150

100

50

0

Casp

ase-3

(ng10

0m

g)

(b)

GCEECPControl


3

2

1

0BAX

mRN

A (r

elat

ive t

o co

ntro

l)

(c)

GCEECPControl

GCEECPControl

lowastlowastlowast

BAX

(rel

ativ

e to

cont

rol)

600

400

200

0

lowastlowastlowast

(d)






GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GCEECPControl


15

10

05

00

PPA

R120574m

RNA

(rel

ativ

e to

cont

rol)

(a)GCEECPControl

150

100

50

0

PPA

R120574(r

elat

ive t

o co

ntro


GCEECPControl

PPAR120574

120573-Actin

(b)








RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















RXR

PPRE

ROS

RXR

RXR

Toxic metabolites

HO-1

SOD

CAT

GPx

CYPs

GSH

APOPTOSISCox-2

iNOS

ROS

Cytochrome C

Caspase-9

Caspase-3

Caspase-8

Inactivemetabolites

Mitochondria

Nucleus


PPAR120574

PPAR120574

PPAR120574

NF-120581B

NF-120581B

TNF-120572

IL-1120573








Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Abbreviations




Disclosure


Competing Interests


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article gamma-glutamylcysteine ethyl...

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