review article oxidative stress in the healthy and wounded ...review article oxidative stress in the...

Review ArticleOxidative Stress in the Healthy and Wounded HepatocyteA Cellular Organelles Perspective

Tommaso Mello12 Francesca Zanieri1 Elisabetta Ceni1 and Andrea Galli12

1Department of Biomedical Clinical and Experimental Sciences ldquoMario Seriordquo University of Florence 50134 Florence Italy2Careggi University Hospital 50134 Florence Italy

Correspondence should be addressed to Tommaso Mello tommasomellounifiit

Received 25 July 2015 Accepted 10 September 2015

Academic Editor Pablo Muriel

Copyright copy 2016 Tommaso Mello et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions Thiscontrol is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporalconfinement of the oxidative species The diverse cellular compartments each although structurally and functionally relatedactively maintain their own redox balance which is necessary to fulfill specialized tasks Many fundamental cellular processes suchas insulin signaling cell proliferation and differentiation and cell migration and adhesion rely on localized changes in the redoxstate of signal transducers which is mainly mediated by hydrogen peroxide (H

2O2) Therefore oxidative stress can also occur long

before direct structural damage to cellular components by disruption of the redox circuits that regulate the cellular organelleshomeostasis The hepatocyte is a systemic hub integrating the whole body metabolic demand iron homeostasis and detoxificationprocesses all of which are redox-regulated processes Imbalance of the hepatocytersquos organelles redox homeostasis underlies virtuallyany liver disease and is a field of intense research activity This review recapitulates the evolving concept of oxidative stress in thediverse cellular compartments highlighting the principle mechanisms of oxidative stress occurring in the healthy and woundedhepatocyte

1 Introduction

11 RedoxHomeostasis andOxidative Stress Accurate controlof the cell redox state which is mandatory for maintain-ing the structural integrity and physiological functions isachieved both by a fine-tuned balance between prooxidantand anti-oxidant molecules and by spatial and temporalconfinement of the oxidative species This tight regulation ismainly achieved by controlling the steady-state productionand the subcellular compartmentalization of reactive oxy-gen (ROS) and reactive nitrogen species (RNS) prooxidantenzymes such as NADHNAPDH oxidases (NOX) and glu-tathione peroxidases (Gpx) and that of several antioxidantsystems such as reducedoxidized glutathione (GSHGSSG)reducedoxidized cysteine (CysCySS) thioredoxin (Trx)peroxiredoxin (Prx) superoxide dismutase (SOD) and cata-lase

While it has long been recognized that an imbalancebetween pro- and anti-oxidants is harmful to cells and is

a central mechanism in the development of several patholo-gies including neurodegeneration atherosclerosis diabetescancer and aging the importance of ROS as second mes-sengers in the cell physiology is a relatively recent acquisi-tion Indeed many fundamental cellular processes such asinsulin signaling cell proliferation and differentiation andcell migration and adhesion just to name a few rely onlocalized changes in the redox state of signal transducersmainly mediated by hydrogen peroxide (H

2O2) [1]

The widespread notion of oxidative stress is that anexcessive production of prooxidants or exhaustion of thecellular anti-oxidant defenses can lead to oxidative damageto proteins nucleic acids carbohydrates and lipids in whichradical ROS or RNS are generally thought to play a majorrole However since the activities of many proteins involvedin the cellular signaling are regulated by the redox state oftheir oxidizable thiol residues which act as redox-sensitivemolecular-switches [2] oxidative stress can also occur inthe absence of direct structural damage by disruption of

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 8327410 15 pageshttpdxdoiorg10115520168327410

2 Oxidative Medicine and Cellular Longevity

Protein folding

Endoplasmic reticulumMitochondria

Insulin

IRSLysosomes

Plasma membraneNOX

ROS

PTPs

Signaling

ROS

Intracellular Signaling

Peroxisomes

ROS

ROS

ETC

ROS

ROS

ROS

TCA

Autophagy

CYP

Xenobiotics

ROS

Lipids rafts

120573-oxidation

120573-oxidation

Fe2+

Ca2+

Figure 1 Sites of physiologically produced ROS Plasma membrane localized ROS bursts deactivate PTPs and allow signal transduction(ie by insulin or IGF-1) after tyrosine kinase receptor activation Mitochondria produce ROS during cellular respiration and metabolicactivity ROS are produced in the ER during protein folding and detoxification by the cytochrome P450 systems Lysosomes are requiredfor iron metabolism and the removal of damaged cellular components through autophagy Peroxisomes produce ROS during metabolic ordetoxification activities

the redox circuits that regulate many signaling pathways[3] Among ROS hydrogen peroxide is supposed to playa major role either directly or indirectly in the regulationof the thioldisulphide redox switches [4] because (i) thesereactions typically require a two-electron transfer (ii) H

2O2

is kinetically restricted and thus can be highly selective in sub-strate oxidation and (iii) H

2O2is generated following growth

factor cytokine or hormone signaling However the detailedmolecular mechanisms leading to selective thiol oxidation inredox-sensitive proteins by H

2O2are still mostly obscure and

are the focus of intense research activity A growing bodyof data suggests that altered redox signaling precedes andcontributes substantially more than direct radical damage tothe development of several human pathologies

The concept of ldquooxidative stressrdquo introduced 30 years ago[5] evolved over time from the original oxidative damage tothe cell structure and subsequent stress response to includethat of alteration of signaling pathways redox homeostasisand redox adaptation to stress [6 7]

Consequently oxidative stress is not necessarily harmfuland antioxidants are not utterly beneficial In fact manyclinical trials failed to prove the efficacy of low-molecularweight antioxidants in the treatment of several pathologiesand the use of the antioxidants selenium beta-carotene and

vitamin E was even found to increase overall mortality in alarge meta-analysis [8]

Our understanding of the redox landscape of the cell israpidly evolving and thanks to the recent development ofspecific redox probes [9ndash12] we are beginning to unravela complex spatial and temporal organization of the redoxfluxes in the living cells Compartmentalization of the redoxcircuitry is crucial to maintain physiology and is a key tounderstand the alterations of the redox homeostasis occur-ring in disease

The liver is the main metabolic organ and plays a funda-mental role in whole body detoxification and blood streamfiltering Most detoxification processes (drugs alcohol andendo- and xenobiotics) are carried out through oxidativereactions by the cytochrome P450 (CYP) isoenzymes whichgenerate superoxide anion (O

2

∙minus) (Figure 1) Derangementof the liver metabolic processes such as those occurringby fatty acids overload in NAFLD results in increasedROS production by increased electron transfer during mito-chondrial 120573-oxidation as well as increased CYP2E1 expres-sion and activity [13] Strong induction of CYP2E1 alsooccurs due to excessive consumption of ethanol whose toxicmetabolite acetaldehyde (AcCHO) generates oxidative stressthrough a number of direct and indirect mechanisms [6 14

Oxidative Medicine and Cellular Longevity 3

ETC Signaling

ROS

ROS

ROS

ROS

TCA

AcCHO

FFA

GSH

AcCHO

Endoplasmic reticulum

Mitochondria

Insulin

IRSLysosomes

Plasma membraneNOX

ROS

PTPs

ROS

ROS

MAMs

Virus

CYP

ETOH

ETOH

ROS

Peroxisomes

ROS

FFA

ETOHIron

Lipids rafts

clustering

Autophagy

UPR

ROS

ROS

ROSAltered

intracellular signaling

Lipids rafts clusters

120573-oxidation

120573-oxidation

Fe2+

Fe2+

Fe2+

Ca2+

Ca2+

Ca2+

Figure 2Mechanisms of enhanced ROS production during hepatocyte damage Ethanolmetabolism promotes strong ROS production in theER by the inducible CYP It impairs GSH import in the mitochondria preventing ROS removal It also impairs 120573-oxidation promoting lipidaccumulation ETOH induces lipid-raft clustering and increases iron uptake promoting Fe2+ leakage from lysosomes and increased Fe2+ loadsin mitochondria and ER resulting in ROS production Ethanol also reduced the autophagic removal of damaged cellular components Viralinfection challenges the ER protein folding process leading to ROS production and Ca2+ leakage in the cytosol and mitochondria IncreasedMAMs formation promotes Ca2+ efflux from ER into mitochondria increasing mitochondrial ROS production

15] (Figure 2) Hepatotropic viruses cause direct oxidativedamage to the endoplasmic reticulum (ER) of hepatocytestriggering an inflammatory response which propagates theoxidative stress-induced damage to neighboring cells Acti-vation of the inflammatory signaling pathways in the hepa-tocyte (ie due to translocation of bacterial toxins from thegut or inflammatory cytokines release from visceral fat inobese subjects) is a trait drsquounion molecular mechanism thatsynergistically propagates the oxidative stress within differentcellular compartments regardless of the initiating agent

The liver also plays a central role in heme catabolismand iron recycling which poses an additional threat sinceiron catalyzes the formation of ROS through the Fenton andHaber-Weiss reactions (see Section 12) Iron accumulationin the liver is associated with increased oxidative stressand can occur as a consequence of genetic disorders (asin hemochromatosis) but also secondarily to other liverdiseases such as NAFLD [16ndash18] or HCV infection [19 20]significantly contributing to the progression of disease

Therefore the liver has an enormous potential for thegeneration of oxidative species and virtually any noxiatargeting the liver results in elevated oxidative stress thatwhen chronic promotes the fibroproliferative response and

progression through fibrosis cirrhosis and eventually hepa-tocellular carcinoma The onset and progression of chronicliver disease require a complex interplay among differentcellular components of the liver hepatocytes cholangiocytesKupffer cells sinusoidal endothelial cells and hepatic stellatecells mostly orchestrated through a proinflammatory andprofibrogenic crosstalk in which oxidative stress mediatorssuch as H

2O2or nitric oxide (∙NO) are active players

As we become more and more aware of the complexity ofthe redox signaling underlying crucial metabolic regulationscell fate decision mechanisms and intercellular communica-tion it is easy to foresee that the ldquoredox hepatologyrdquo field willshape the liver biology research in the next future

This review recapitulates the evolving concept of oxida-tive stress in diverse cellular compartments highlighting theprinciple mechanisms of oxidative stress occurring in thehealthy and wounded hepatocyte

12 Mechanism of Reactive Oxygen and Nitrogen Species(RONS) Mediated Toxicity Several ROS (O

2

∙minus ∙OH andH2O2) and RNS (∙NO ONOOminus) are generated inside the

cells under physiological and pathological conditions Thebiological activity of RONS toward cellular substrate is not


equivalent the hydroxyl radical (∙OH) has an indiscriminatereactivity toward most biological substrates and is the mostrelevant ROS involved in oxidative DNA damage while O

2

∙minusthe most abundant mitochondrial ROS preferentially reactswith iron-sulfur clusters in target proteins and is effectivelyconverted to H

2O2 which is the principal oxidant of low pKa

cysteine residues (Cys) acting as sulfur switches in redox-sensitive proteins [21]

Nitric oxide is a highly diffusible signaling molecule gen-erated by Nitric Oxide Synthases (eNOS iNOS and mNOS)in the cytoplasm extracellular space and possibly mitochon-dria [22] and can react with redox-sensitive cysteine residuesin proteins forming nitrosothiols a mechanism of redoxsensing analog to hydrogen peroxide (H

2O2) [23] ∙NO does

not appear to be toxic at physiological concentrations [24]but can readily react with superoxide anion and generateperoxynitrite (ONOOminus) a spontaneous reaction occurring atsuch a fast rate that outperforms SOD capability of removingO2

∙minus Peroxynitrite is therefore formed whenever ∙NO andO2

∙minus are produced simultaneously [25] it is highly reactivetoward iron-sulfur clusters (present in several metabolicenzymes like mitochondrial aconitase and alcohol dehydro-genase) can oxidize protein thiols and promote tyrosinenitration in target proteins (ie Complexes I II III andV of the mitochondrial Electron Transport Chain (ETC))thereby impairing both the redox- and phosphorylation-dependent cellular signaling A major mechanism of perox-ynitrite toxicity is mediated by lipid peroxidation that causesthe degradation of membranes through radical reactionsleading to changes in membrane permeability and fluidityFinally oxidative damage to DNA is a hallmark of high-level oxidative stress not only in the nuclei but also inmitochondria [25]

Moreover while highly reactive radical ROS and RNSare generally diffusion-limited some ROS and RNS caneasily diffuse through biological membranes thanks to theirnon-low-polar nature (H

2O2 ∙NO and peroxynitrous acid

ONOOH) and to dedicated transporter such as aquapor-ins (for H

2O2) or the HCO

3

minusClminus anion exchanger (forONOOminus) The pKa for the couple ONOOONOOH is 68very close to physiological pH thus implying that bothmechanisms of peroxynitrite diffusion are relevant in vivo[26] Therefore depending on the given RONS involvedoxidative stress can elicit localized alteration of the redoxstate localized structural damage or spread among differentcellular compartments and neighboring cells An elucidatingmechanism of such event is the ROS-Induced ROS Release(RIRR) [27] that is the temporarily opening ofmitochondriapermeability transition pore (mPTP) that elicit an amplifiedROS production after an oxidative challenge ROS releasedduring RIRR may spread to neighboring mitochondria anddepending on the level of ROS release either promotemitophagy and removal of nonfunctional mitochondria ortrigger a ROS avalanche that can lead to cell death Ofimportance RIRR is considered to be themainmechanism ofhepatocyte damage during ischemiareperfusion injury thatoccurs following hepatic surgery or transplantation

Iron overload constitutes a source of oxidative stressof particular relevance in the liver since hepatocytes and

Kupffer cells are the main cell type devoted to iron storagein the body Iron is an essential component of oxygen sensingproteins oxygen transport systems and iron-sulfur contain-ing enzymes [28] it is a transition metal readily convertedbetween the reduced ferrous (Fe2+) and the oxidized ferric(Fe3+) forms The majority of iron in biological complexesis kept as Fe3+ while iron reduction to Fe2+ is crucial for itsmobilization and transport through membranes loading onferritin and heme synthesis [28]

In the hepatocyte iron is stored in the cytoplasm ERmitochondria and lysosomes largely as ferritin-bound Fe3+About 02ndash5 of the total cellular iron is considered asintracellular transiently mobile ldquolabile poolrdquo either ldquofreerdquoiron or loosely bound ldquochelatablerdquo iron both mainly inthe form of redox-active Fe2+ [29] The ldquolabile poolrdquo ironis potentially toxic since it can catalyze the formation ofdangerous ∙OH radical through the Fenton (1) and Haber-Weiss (3) reactions

Fe2+ +H2O2997888rarr Fe3+ +OHminus + ∙OH (1)

Fe3+ +O2

∙minus 997888rarr Fe2+ +O2

(2)

The net reaction O2

∙minus +H2O2

997888rarr OHminus +O2+ ∙OH

(3)

Therefore leakage of Fe2+ from the lysosome due toaltered membrane permeability as well as reduction of Fe3+by superoxide (2) can catalyze the production of ROSand promote lipid peroxidation and severe cellular damage(Figure 2) Mitochondria are particularly susceptible to iron-mediated oxidative stress due to the high production rateof O2

∙minus and its dismutation product H2O2during cellular

respiration in close proximity to several Fe-S containingenzymes [30]

2 Mitochondria

Among cellular organelles mitochondria account for thelargest amount of electron transfer to oxygen thanks to theelectron transfer chain (ETC) complexes IndashV ETC com-plex I (NADH ubiquinone oxidoreductase) and complexII (ubiquinone cytochrome c oxidoreductase) as well asother mitochondrial enzymes such as 120572-ketoglutarate dehy-drogenase pyruvate phosphate dehydrogenase fatty acylCoA dehydrogenase and glycerol 3-phosphate dehydroge-nase [31] can produce O

2

∙minus as byproduct [32] releasingit within the mitochondrial matrix Moreover H

2O2is

produced by the monoamine oxidases (MAOs) located inthe outermitochondrialmembrane [33] (Figure 1)Thereforemitochondria are often referred to as amajor ROSproductionsite although in fact whether high ROS leakage occursin the mitochondria at least in a physiological setting isstill highly debated [34 35] Estimates of H

2O2produc-

tion as a measure of O2

∙minus leakage vary from 2 [36]to 01-02 [31 32 34] of total O

2consumption and vary

largely depending on tissue origin experimental settings andthe specific substrate fed to the mitochondria For liver


mitochondria the rate of ROS leakage could be even lowerthan 01 [32] In fact sincemitochondria are physiologicallyprone to produce high ROS levels due to the oxidativephosphorylation process they are also well equipped witha large array of antioxidant systems and radical scavengerssuch as Mn-Superoxide Dismutase (Mn-SOD) CuZn-SODGSH glutathione peroxidase tioredoxin-2 peroxiredoxinsglutaredoxins and also catalase [37] Mn-SOD (SOD2) inthemitochondrial matrix readily catalyzes the dismutation ofO2

∙minus to H2O2 which in turn is eliminated by glutathione per-

oxidase using reduced glutathione (GSH) as hydrogen donorOxidized glutathione (GSSG) is then reduced by NADPH-dependent glutathione reductase Superoxide released in theintermembrane space by the ETC complex III is scavengedby CuZn-SOD (SOD1) followed again by GPx and GSHto eliminate H

2O2 Since GSH is only synthetized in the

cytosol [38] and the mitochondrial pool of GSH (mGSH) isreplenished by importing GSH produced in the cytoplasm[39] the GSHGSSG redox state inside the mitochondria isheavily controlled byGSH import through the 2-oxoglutaratecarrier and the dicarboxylate carrier [40ndash43]

Two major enzymatic antioxidant systems collaborate inthe mitochondrial matrix the GSH-dependent glutathioneperoxidase and the NADPH-dependent thioredoxin-2 sys-tems each with specific cofactors

It must be noted that although abundant GSH hasvery limited spontaneous antioxidant activity but very highaffinity for GPx Within the mitochondria GPx1 [44 45]and Gpx4 are the most abundant with GPx1 representingover one-third of total GPx activity in the liver [46] Gpx1is the major isoform localized both in the mitochondrialmatrix and in the intermembrane space and ismainly devotedto H2O2detoxification while GPx4 preferentially reduces

lipid peroxides thereby preventing membrane damage tomitochondria [47]

Nevertheless a number of molecular mechanisms pro-mote mitochondrial ROS overproduction or decreasedantioxidant defense under nonphysiological condition [48]The alteration of the redox homeostasis of mitochondriais well documented in several human pathologies such asNAFLD viral infection and toxic events (Figure 2)

Chronic alcohol feeding depletes the mGSH in severalanimal models [49ndash51] leading to enhanced ROS produc-tion and mitochondrial damage The mechanism underlyingmGSH depletion involves cholesterol accumulation in theinner mitochondrial membrane that results in excess mem-brane rigidity and impaired GSH carriers functionality thusdisrupting GSH import from the cytosol (Figure 2) In factrestoring the membrane fluidity but not increasing cyto-plasmic levels of GSH by N-acetylcysteine administration(NAC) recovers mGSH pool and ameliorates liver damagein alcohol-fed rats [45 50] The importance of GSH importin the mitochondria can be appreciated considering thatseveral antioxidant systems depend upon mGSH and thatthemitochondrial GSHGSSG redox state is evenmaintainedin a more reduced steady-state redox potential than in thecytoplasm [52] thus requiring energy expenditure for GSHimport

The absence of protective histones incomplete DNArepair mechanisms and the close proximity to ROS produc-tion site renders mitochondrial DNA (mDNA) sensitive tooxidative damage increasing the risk of double-strand breaksand somatic mutations with increased ROS production [53]Indeed a single dose of alcohol proved effective in inducingmassive mitochondrial DNA degradation through a ROS-dependent pathway [54] The acute degradation of mDNAis then followed by an overshoot of mDNA synthesis as acompensatory mechanism However repeated administra-tion of alcohol (binge drinking) accumulated DNA damageand blocked the adaptive response of mDNA resynthesisresulting in prolonged hepatic mDNA depletion [55] mDNAencodes 13 proteins involved in the ETC two rRNA and allthe tRNA necessary for translating the 13 encoded proteinsMutations in the mDNA therefore may produce dysfunc-tional ETC complexes increase ROS production and exposethe mitochondria to new damage in a vicious circle [53]Indeed mitochondrial DNA depletion and mutation havebeen described in patients with alcoholic and nonalcoholicsteatohepatitis [56 57]

Acetaldehyde (AcCHO) produced by ethanolmetabolismis readily detoxified by aldehyde dehydrogenase 1 (ALDH1)in the cytosol and by ALDH2 in the mitochondria Acetalde-hyde oxidation to acetate generates NADH and reduces theNAD+NADH ratio possibly impairing mitochondrial 120573-oxidation which requires NAD+ (Figure 2)

Chronic alcohol administration reduces ALDH activitytherefore promoting AcCHO accumulation and inducingadduct formation with lipids proteins and mDNA [58 59]

Failure to efficiently remove AcCHO exposes mitochon-dria to protein lipid and DNA adduct formation such asMDA 4-NHE and mixed MAA adducts [15] Moreover 4-HNE a lipid peroxidation derivative can directly inhibitALDH2 thus promoting AcCHO accumulation in the mito-chondrion in a endangering loop [60]

Consistently the inactivating polymorphism ALDH2lowast2common in East Asia confers reduced alcohol tolerance andis associated with increased risk of gastrointestinal cancerVery recently by the use of a knock-in mice harboring theALDH2 (E487K) mutation Jin and colleagues recapitulatedthe ALDH2lowast2 human phenotype including intolerance toacute or chronic alcohol administration impaired clearanceof AcCHO increased DNA damage and susceptibility tocancer development [61]

Many of the abovementioned findings also apply tothe mitochondria of NASH patients which have alteredmorphology [62 63] reduced or mutated mDNA content[57] reduced oxidative phosphorylation [64] and increasedROS production

However the molecular mechanisms initiating the mito-chondrial dysfunction in NASH are different and originateby an overwhelming induction of mitochondrial 120573-oxidationrather than its inhibition as inASHThis is consistent with theincreased expression of UCP-2 observed in the mitochondriaof several obesity and NASH animal models and in theexpansion of peroxisomal 120573-oxidation found in humansTheincreased electron flux through the ETC produces oxidative


stress which is strongly associated with the severity of NASH(Figure 2)

Depletion of mGSH occurs in NASH animal modelssimilar to ASH [65] and in NASH patients that have reducedlevels of GSH SOD and catalase and increased proteinoxidation a hallmark of increased oxidative stress [66] Inprinciple targeting oxidative stress is potential therapeuticoption for oxidative liver diseases [67] Of notice mGSHdepletion can affect also the outcome of potential therapeuticantioxidant treatments such as the use of SOD mimeticsin steatohepatitis Indeed the use of SOD2 mimetics in acontext of mGSH depletion results in increased H

2O2levels

and increases liver injury in animal models of steatohepatitishighlighting the importance of a combinatory strategy in thetargeting of oxidative stress mechanisms [68]

3 Endoplasmic Reticulum

Endoplasmic reticulum (ER) is a master intracellular organ-elle responsible for protein synthesis folding modificationand trafficking In addition the ER plays a crucial role incalcium homeostasis and in regulating the biosynthesis ofsteroids lipids and carbohydrates [69]

During the folding process a protein may be oxidizedto form disulfide bonds isomerized to allow polypeptiderearrangement or reduced to allow unfolding and subsequentdegradation [70]

The ER lumen has a high ratio of oxidized to reducedglutathione (GSSGGSH) (145) which creates an oxidizingenvironment that promotes disulfide bond formation Theelectron transport required for this process is driven bya protein pathway that involves two ER-located enzymesprotein disulphide isomerase (PDI) and ER oxidoreductin 1(ERO1) [71] PDI directly accepts electrons leading to theoxidation of cysteine residues and the formation of disul-phide bonds In turn ERO1 oxidizes PDI through a flavin-dependent reaction and transfers electrons to molecularoxygen as final acceptor The use of molecular oxygen as theterminal electron recipient leads to the production of ROSmainly hydrogen peroxide contributing to cellular oxidativestress [72]

It has been estimated that about 25of theROS generatedin a cell derive from ER disulfide bond formation duringoxidative protein folding thus making ER the major site ofROS production [73] (Figure 1)

Furthermore additional oxidative stress can result fromthe depletion of reduced glutathione that is consumed duringthe reduction of unstable and improperly formed disulphidebonds [74]Therefore an increase in the protein-folding loadin the ER can lead to the accumulation of ROS [75] Cells haveevolved several strategies to oppose the ER accumulationof unfolded and misfolded proteins which are collectivelyreferred to as the UPR (unfolded protein response)

Under normal physiological conditions the unfolded ormisfolded proteins are directed to degradative pathways torestore the ER homeostasis however if the unfolded proteinproduction overwhelm the ER buffering capacity the UPRcan activate a cascade of intracellular events resulting in

cell death [76 77] The UPR is of major importance inhepatocytes which are rich in ER content and responsible forthe synthesis of proteins cholesterol bile acids and phospho-lipids [78] And it is characterized by the activation of threedistinct signal transduction pathways the inositol requiring1 (IRE1) pathway the protein kinase RNA-like ER kinase(PERK) pathway and the activating transcription factor 6(ATF6) pathway Under nonstressed condition these threeproteins are kept inactive by binding to a chaperone proteinBiPGRP78 which is the master regulator of the UPR Understressed condition (due to for example accumulation ofmisfolded or unfolded proteins depletion of ER calciumcontent or increase of free cholesterol in the ER lumen)BiPGRP78 dissociates from the UPR transducers resultingin activation of their respective signaling pathways

Briefly the activated IRE1120572 removes a 26-bp intron fromthe XBP1 mRNA resulting in the production of a splicedXBP1 protein (XBP1s) XBP1s is a transcription factor thatregulates the expression of several genes involved in UPRand ER-assisted degradation (ERAD) to help restore ERhomeostasis [79]The IRE1120572Xbp1 pathway is also critical forhepatic lipid homeostasis since it activates the transcriptionof master adipogenic regulators such as PPAR120574 and CEBPs[80] In addition IRE1120572 induces the activation of stresskinases JNK and p38 MAPK that promote apoptosis [81]

The PERK pathway activates an antioxidant programfocused on ATF4 and nuclear factor-erythroid-derived 2-(NF-E2-) related factor 2 (NRF2) [82 83] NRF2 is a keyplayer in antioxidant response After PERK-mediated phos-phorylation NRF2 translocates to the nucleus and acti-vates the transcription of a set of antioxidant and oxidant-detoxifying enzymes including NAD(P)H-quinone oxidore-ductase (NQ01) heme oxygenase 1 (HO1) and glutathioneS-transferase (GST) [84 85] In addition NRF2 and ATF4induce the transcription of genes whose products areinvolved in the maintenance of glutathione cellular levelthe main redox buffer in the cell [82 83 86 87] Theoverall antioxidant effect of the PERK pathway is supportedby the finding that a potent ER-stress-inducing chemicaltunicamycin induces only weak accumulation of ROS inwild-type cells whereas this treatment induces a toxic accu-mulation of ROS in cells that lack PERK [75]

Dissociation of BiPGRP78 from ATF6120572 leads to itstranslocation to the Golgi where this protein is processedinto its active form [88] The activated ATF6 translocates tothe nucleus and functions as a transcription factor promotingthe expression of downstream target genes involved in ERstress including XBP1 GADD153 (also known as CHOP aproapoptotic transcription factor that plays a critical role inER stress-mediated apoptosis) and ER chaperones [89 90]ATF6120572 is also a regulator of gluconeogenesis [91]

All together these three pathways mitigate the ER stressby reducing global protein synthesis increasing misfolded orunfolded protein degradation and simultaneously increas-ing the specific expression of proteins that help maintainingthe protein folding process in the ER lumen as well as ERintegrity [92ndash94]

Under pathological andor stressful conditions in whichthe demand of protein synthesis folding andor repair is


increased the UPR efficiency decreases resulting in theaccumulation of unfolded protein andmisfolded proteins andER damage [93 95ndash98]

Moreover an overactivation of the UPR leads to asustained activity of ERO1 as well as induction of ERO1120573expression [99] resulting in an increased H

2O2production

[100] that is found in several liver diseases such as NASHASH and viral infection (Figure 2)

As a first-line response duringUPR activation ER-relatedPERK pathway attenuates general mRNA translation andactivates the Nrf2 transcription factor [71 83 92 101 102]that translocates to the nucleus and activates antioxidantresponsive element-dependent gene expression [71 75 92 93101] However in NASH the UPR-induced Nrf2-mediatedresponse is downregulated [75] Impaired Nrf2 activity isassociatedwithmitochondrial depolarizationdysfunction aswell as increased hepatic free fatty acid levels fatty liverand NASH development [102ndash104] Moreover NASH-relatedaccumulation of misfolded proteins and related unmiti-gated ER stress also induces increased ROS productionand macromolecules oxidation in the ER lumen throughPDI leading to intracellular depletion of reduced glutathione[71 72 105] Indeed when oxidized PDI with ERO1 actsin the oxidative folding of proteins by allowing properdisulfide bond formation When reduced PDI breaks andrearranges disulfides in the nascent proteins until the reducedglutathione pool is depleted [71 73] Furthermore both ERstress and oxidative damage prompt calcium leak from theER leading tomitochondrial calciumaccumulationwhich inturn promotes exacerbated mitochondrial ROS productionfurther amplifying ER stress [72 104 106] (Figure 2) Ithas been recently suggested that elevated levels of palmiticacid would compromise the ER ability to maintain calciumstores resulting in the stimulation ofmitochondrial oxidativemetabolism ROS production and ultimately cellular dys-function [75] Therefore it appears that ER stress may occurearlier than the onset of mitochondrial dysfunction ROSaccumulation and apoptosis [107 108] Moreover SREBP-1themaster regulator of triglycerides and cholesterol synthesisis kept inactive at the ER by interaction with insulin inducedgene proteins (INSIGs) During ER stress proteolytic degra-dation of Insig-1 releases SREBP [96] which is subsequentlyprocessed in theGolgi and finally directed to the nuclei whereit activates the transcription of the lipogenic program Inturn excess fatty acids and cholesterol promote ER stressthus the reinforced cycle of ER stress oxidative stress andlipogenesis-induced lipotoxicity fuels the pathogenesis ofNASH [78]

Alcoholic liver disease (ALD) is certainly related to anexcessive production of ROS from ethanol metabolism andthe consequent oxidative stress within the hepatocytes [109110] Twometabolic pathways are involved in the degradationof ethanol First ethanol is oxidized into acetaldehyde byalcohol dehydrogenase (ADH) followed by production ofacetate by means of acetaldehyde dehydrogenase (ALDH)Acetaldehyde mediates most of the toxic effect of alcohol[15 111 112] The second pathway of ethanol degradationwhich is largely inducible operates through the microso-mal ethanol-oxidizing system (MEOS) cytochrome P450

CYP2E1 the main cytochrome P450 isoform induced byethanol consumption is located at the membrane of ER[113ndash116] making it the master mechanism of ER ethanol-induced ROS production Ethanol oxidation by CYP2E1generates O

2

∙minus and H2O2promoting membrane lipoper-

oxidation Moreover ethanol administration and ROS pro-duction increase free iron which catalyzes the productionof strong oxidants such as hydroxyl radical (OH∙) ferrousoxide (FeO) and hydroxyethyl radical (CH

3CHOH) This

damaging mechanism is also common to lysosomes andmitochondria (Figure 2)

The UPR overactivation and ROS production occur alsoin Hepatitis C and B but the process that induces theseresponses is different from other liver diseases

Hepatitis C virus (HCV) replication in infected hostcells is dependent on several viral proteins that are foldedin the ER and synthesized in ribonucleoprotein complexesin association with the ER [117] HCV replication has beenshown to cause ER stress and its gene products such as CoreE2 NS5A and NS4B have also been demonstrated to induceUPR and ROS production [118 119]

The protein aggregates that are formed in the viralreplication induce all three different pathways of UPR whichsustain viral replication alleviating ER stress [118 119] ROSproduction during HCV infection also occurs due to alteredintracellular Ca+2 homeostasis For example Core viralprotein perturbs the intracellular calcium both by inducingER Ca2+ release and by stimulating the Ca2+ uniporter inmitochondria increasing ROS production in mitochondriaand opening of the mPTP [120] Similarly NS5A perturbsCa2+ signaling and elevates ROS production inmitochondrialeading to activation of transcription factors such as NF-120581Band STAT that are involved inHCV-mediated hepatocarcino-genesis [121] NF-120581B activation is also stimulated by NS4B amechanism requiring Ca2+-induced ROS production [122]

Similar mechanisms of UPR activation and ROS produc-tion also occur in HBV infection [123] (Figure 2)

4 Lysosomes

Autophagy is a fundamental mechanism of cell adaptationto stress allowing removal of damaged molecules and cel-lular components by degradation in the lysosomal compart-ment which is of particular importance for the removal ofnonfunctional mitochondria (mitophagy) (Figure 1) Alter-ations of the autophagic pathway play a major role in theonset and perpetuation of several chronic diseases includ-ing neurodegenerative and metabolic disorders as well ascancer chemoresistance Most of the processes associatedwith RONS production also stimulate autophagy [124] Forinstance starvation through inhibition of mTOR pathwaystimulates autophagy and increases mitochondrial ROS pro-duction H

2O2oxidizes a redox-sensitive thiol of Atg4 which

then promotes LC3-I conversion to LC3-II and maturationof the autophagosome [125] Consistently autophagy is stim-ulated in vivo by mitochondrial superoxide production asseen by experimental downregulation of MnSOD whichincreases O

2

∙minus and reduces H2O2[126] However it is not


clear whether O2

∙minus directly stimulates autophagy or morelikely induces lipid peroxidation and mitochondrial dam-age which in turn activate autophagy This second line ofthought is supported by the observation that in nutrient-deprived hepatocytes mitochondrial membrane depolariza-tion precedes the formation of the autophagosome [127] anddefective mitophagy results in accumulation of dysfunctionalmitochondria increases oxidative stress and promotes tissueliver damage and cancer [128] Chronic ethanol feeding isassociated with decreased intralysosomal hydrolases contentand reduced proteasomal activity due to impaired cathepsin Ltrafficking in the lysosome [129 130] Oxidative stress can alsoharm lysosomal membranes resulting in elevated cytosoliclevels of cathepsin B due to lysosomal leakage [131]

The effect of ethanol metabolism on autophagy is quitedebated and controversial results have been reported [132]Using LC3-GFP transgenic mice two groups described thatbinge- [133] acute- or chronic-ethanol administration [134]promoted autophagosome formation in vivo The increasein autophagosome formation was paralleled by inhibition ofmTORC1 signaling pathway [133]

In contrast using a CYP2E1 knock-out or knock-in miceand parallel in vitro model Wu and colleagues showed thatbinge ethanol administration reduced macroautophagy inCYP2E1 KI mice and cells but not in KO mice [135] Despitethe different autophagy status which could be due to the useof different transgenic models and different binge drinkingprotocols inhibition of autophagy enhanced ethanol toxicitywhile pharmacological promotion of macroautophagy bycarbamazepine as well as rapamycin was shown to havea therapeutic potential in animal models of acute ethanol-induced toxicity [133] as well as ASH [135] and NASH [136]

Since ethanol is known to reduce the proteolytic activityof lysosomes it is possible that the degradative part ofautophagy that is the autophagolysosome formation maybe impaired in chronic ethanol feeding despite increasedautophagosome formation leading to defective removal ofdamaged cellular components [137] (Figure 2)

This mechanism could be relevant also for the pathogen-esis of NAFLD In vitro experiments showed that autophagywas increased in hepatoma cells exposed to FFA mimickingthe ldquofirst hitrdquo ofNASHpathogenesis However after a ldquosecondhitrdquo of H

2O2or TNF120572 (oxidative damage and inflammation)

the autophagic flux diminished despite enhanced autophago-some formation [138]

An important mechanism of ROS induced liver injury ismediated by the release of ldquolabilerdquo iron (Fe2+) that occurs indamaged lysosomes during ischemiareperfusion [139]

Lysosomes are a major storage site of iron which entersthe hepatocyte through transferrin mediated endocytosissubsequent endosome acidification iron reduction andrelease from transferrin Fe2+ is then released in the cytosolor delivered tomitochondria in controlled amount (Figure 1)Moreover lysosomes can significantly increase their ironcontent by reparative autophagic uptake of damaged mito-chondria peroxisomes or cytosolic iron-loaded ferritin [29]Labile iron is transported to mitochondria where it catalyzesthe Fenton reaction with H

2O2(see Section 12) producing

the deleterious hydroxyl (OH∙) and hydroxyperoxyl (OOH∙)radicals which damagemitochondriamembranes and inducethe opening of the permeability transition pore triggeringthe RIRR response and eventually cell death (Figure 2) Fe2+induced ROS formation can represent a potent mechanismof damage amplification not only within the cellular com-partments but also at the cell and organ level In fact manyhuman diseases especially liver diseases are associated withincreased serum ferritin levels that arise due to hepatocytedeath Increased ferritin uptake in hepatocytes is associatedwith abnormal endosome clustering and induces lysosomalmembrane permeability and promotes lipid peroxidationdepletion of GSH and reduction of GSHGSSG ratio Ferritinaccumulation triggersmacroautophagywhich is abolished byFe chelation confirming the mechanistic role of Fe-inducedROS formation in the onset of ferritin toxicity Also inthis model pharmacological inhibition of macroautophagystrongly enhanced ferritin toxicity further substantiating theconcept that induction of autophagy is a generalized defensemechanism against ROS-mediated cellular damage [140]

5 Peroxisomes

Peroxisomes are ubiquitous organelles involved in catabolicoxidative reactions xenobiotic detoxification and bile saltsynthesis In mammals peroxisomes carry on the 120572-oxida-tion of very-long chain fatty acids that cannot directly enterthe mitochondria for 120573-oxidation[141]

Several peroxisomal oxidases produce ROS primarilyH2O2 but also ∙NO since iNOS was detected in peroxisomes

of hepatocytes [142] Peroxisomes are not only a source ofROS but also a powerful ROS disposal compartment thanksto a large array of antioxidant enzymes mainly catalase butalso GPx Mn-SOD and CuZn-SOD and peroxyredoxin-1and peroxyredoxin-5 [143] (Figure 1)

Peroxisomes are highly dynamic structures that undergoenlargement elongation and increase in number in responseto several xenobiotic or physiologic stimuli

Peroxisomes can support mitochondria in the 120573-oxida-tion of fatty acids [144] a process mediated by the activationof the nuclear receptor PPAR120572 [145] Accordingly peroxi-somes are elevated in several mice models of NAFLD [146]and proliferation and enlargement of peroxisomes are alsodescribed in patients with fatty liver [147] (Figure 2)

Activation of PPAR120572 and subsequent induction of perox-isomal 120573-oxidation is a potent inducer of H

2O2production

which is not paralleled by an equal increase in antioxidantenzymes mainly catalase leading to oxidative stress andsubstantially contributing to the hepatocarcinogenic effectsof PPAR120572 activators observed in mice but not in humans[148]

Disruption of the peroxisomal structure and function inthe hepatocyte as in the PDX5 knock-out mice resultedin structural alteration and reduced functionality of mito-chondria increased proliferation of ER and lysosomes andaccumulation of lipid droplets Surprisingly no sign of signif-icant oxidative stress was found although metabolic rear-rangement toward a more glycolytic setting was observed[149]


6 Plasma Membrane

At the plasma membrane interface key signal transductionevents originate following the ligand-receptor interactionSignal transduction is biologically encoded by phosphory-lation of key Tyr or SerThr residues that induce confor-mational and functional changes in transducing proteinsThe coordinated activities of protein kinases and proteinphosphatases ensure the substrate temporal and spatial con-finement required to obtain specificity for signal transductionevents Signal transduction occurs not only by means of thewell-known ldquophosphorylation coderdquo but also by regulationof redox-sensitive Cys residues in signaling proteins Suchldquoredox-coderdquo [21] adds a level of complexity and flexibility tosignaling circuitry

The best characterized redox regulation of kinase sig-naling and likely the most relevant to the hepatocyte isthe insulin pathway Insulin binding to its receptor acti-vates intrinsic kinase activity and initiates a phosphorylationcascade through the PI3KAKTmTOR pathway In parallelinsulin promotes the deactivation of protein phosphatases(PTEN PP2A and SH2P) a mechanism requiring localizedH2O2burst at the plasmamembrane generated by extracellu-

lar NOX [150ndash153] (Figure 1)Adiponectin which potentiates insulin action activates

5-LOX H2O2bursts and deactivates PTP1B [154]

Exogenous H2O2administration can therefore modulate

insulin signaling through inhibition of Protein TyrosinePhosphatases (PTP) as recently demonstrated by Iwakamiet al [155]

The ROS-mediated inactivation of PTP is a generalphysiological mechanism necessary for efficient signal trans-duction of several if not all MAPK family members [2]including p38 [9 156] JNK [157] and ERK12 [158] (Figure 1)

Removal of signaling H2O2and reduction of oxidized

Cys in PTP are ensured by cytoplasmic antioxidants suchas GSH thioredoxin 1 [159] and peroxiredoxin-2 [152] thusterminating the signaling activity

Although H2O2is required for normal signal trans-

duction excess oxidative stress can activate stress-sensitiveSerThr kinase such as JNK ERK1 and IKKb (inhibitor of NF-120581B) and p70S6k all of which are client of the redox-regulatedprotein phosphatase PP2A Stress sensitive kinases take partin insulin resistance by inhibitory phosphorylation of IRS-1[160] (Figure 2)

Clearly redox signaling is highly intertwined with phos-phorylation signaling and a high level of spatial and temporalconfinement must be fulfilled to ensure the proper biologicalresponse

Very recently Hua and colleagues uncovered the coor-dinated redox mechanisms initiating and terminating hepa-tocyte proliferation during liver ontogenesis or regenerationafter partial hepatectomy [161] They found that sustainedelevated H

2O2levels are required for the activation of ERK

signaling and trigger a shift from quiescence to prolifer-ation while sustained decreased H

2O2levels activate p38

signaling and trigger a shift from proliferation to quiescencePharmacological lowering of H

2O2levels reduces the extent

of fetal hepatocyte proliferation and delays the onset ofliver regeneration Chemical augmentation of H

2O2levels

in adult hepatocytes triggers proliferation and delays thetermination of liver regeneration Although the authors didnot map the precise cellular location of H

2O2production

the mechanism triggering the H2O2-induced hepatocyte

proliferation during embryogenesis and liver regenerationinvolves early induction of NADPH Oxidase 4 (NOX4) andthe adaptor protein p66shc [161] which is known to benecessary for NOX assembly at the plasma membrane [162]Interestingly NOX4 recruitment at the plasma membraneand localized H

2O2production are also required for IGF-

I signal transduction to Src a mechanism mediated bythe scaffold protein SHPS-1 [163] suggesting that NOX4assembly at the plasma membrane could promote specificsignal transduction by redox regulation of different clienteffectors

In hepatocytes membrane fluidity and lipid-raft cluster-ing have been described to mediate ethanol-induced oxida-tive stress ROS produced by ethanol metabolism increaseplasma membrane fluidity and determine an increase in low-molecular weight iron triggering massive ROS productionand membrane peroxidation [164] These event are associ-ated with lipid-raft clustering intraraft disulfide-bond andperoxidation phospholipase C recruitment inside the lipid-raft and activation of a signaling pathway that modulateiron content in the lysosomal compartment [165] (Figure 2)Consistently reducing lipid-raft clustering and oxidation byDHA administration [166] or increasing plasma membranefluidity by benzo(a)pyrene administration [167] respectivelyalleviated or worsened the ethanol toxicity mediated byincreased lysosomal iron leakage Since membrane lipid raftsare required for peroxisome biogenesis [168] it would beinteresting to assess the effect of ethanol-induced lipid-raftclustering on peroxisome structure and function

7 Concluding Remarks

Redox homeostasis in the cell is achieved by accurate com-partmentalization of prooxidants and RONS buffering sys-tems Each cellular compartment differs in the overall redoxstate as well as in the concentration and oxidative steady-stateof its antioxidant pairs Lysosomes and ER are relatively moreoxidizing than cytoplasm while more reducing conditionsare found in the nuclei and mitochondria Each of thesecompartments is actively maintained in a nonequilibriumredox state to fulfill specific tasks At the same time cellularorganelles are highly interconnected by a complex networkof physical connections signaling pathways intracellulartrafficking routes and metabolic activities

Oxidative stress can arise by different mechanism in anycellular compartment and spread to other cellular organellesthrough any of the physical or functional connections

For instance mild alterations of plasma membrane per-meability induced by ethanol metabolism promote clus-tering of lipid raft and increase ferritin uptake that pro-mote membrane lipid peroxidation and free iron leakagefrom the lysosome Free iron is eventually transported to


the mitochondria where it triggers a burst of oxidative stressand opening of the mPTP propagating the oxidative damagethrough the RIRR mechanism (Figure 2)

Perturbation of the network of physical and functionalconnections between cellular organelles is not only a conse-quence of pathological oxidative stress but can be indeed theprimary causal mechanism of oxidative stress and morbidityIn a recent paper Arruda and colleagues by using an elegantin vivo strategy demonstrated that artificially increasing thephysical interaction between mitochondria and ER (MAMsmitochondrial-associated ER membranes) in the liver led toaltered calcium load in themitochondria increased oxidativestress and metabolic alterations promoting obesity andmetabolic dysfunction [169] Rearrangement of ER aroundmitochondria seems to be an early event after HFD treatmentand likely reflects a transient adaptation of this physiologi-cally highly dynamic process (Figure 2)

The recent development of genetically encoded ROSsensor paved the way for a new era of exploration in the redoxcell biology allowing real-time detection and measure of theredox dynamics in living cells These technological advancesare contributing to unravel the complex spatial and temporaldynamics of H

2O2bursts that acting as switches can activate

or deactivate signaling components thus interconnecting theredox-based and kinase-based signaling pathways

Aswe deepen the understanding of the redox homeostasisin the different cellular compartment the concept of ldquooxida-tive stressrdquo evolves and alterations of redox mechanisms areincreasingly being recognized in human diseases revealingthe potential for new therapeutic opportunities

Abbreviations

IRS Insulin receptor substrateNOX NADPH OxidasePTPs Protein Tyrosine PhosphatasesETC Electron Transport ChainTCA Tricarboxylic acid cycleFFA Free fatty acidsGSH Reduced GlutathioneCYP Cytochrome P450 systemUPR Unfolded protein responseMAMs Mitochondria associated membranesETOH EthanolAcCHO Acetaldehyde

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work is supported by Italian Ministry of Health (GrantGR-2009-1600315) and Ente Cassa di Risparmio di Firenze(Grant 20130673)

References

[1] D P Jones ldquoRadical-free biology of oxidative stressrdquo TheAmerican Journal of PhysiologmdashCell Physiology vol 295 no 4pp C849ndashC868 2008

[2] P D Ray B-W Huang and Y Tsuji ldquoReactive oxygen species(ROS) homeostasis and redox regulation in cellular signalingrdquoCellular Signalling vol 24 no 5 pp 981ndash990 2012

[3] D P Jones and Y-M Go ldquoRedox compartmentalization andcellular stressrdquo Diabetes Obesity and Metabolism vol 12 sup-plement 2 pp 116ndash125 2010

[4] C C Winterbourn ldquoAre free radicals involved in thiol-basedredox signalingrdquo Free Radical Biology andMedicine vol 80 pp164ndash170 2015

[5] H Sies ldquoOxidative stress a concept in redox biology andmedicinerdquo Redox Biology vol 4 pp 180ndash183 2015

[6] L L Ji M-C Gomez-Cabrera and J Vina ldquoExercise andhormesis activation of cellular antioxidant signaling pathwayrdquoAnnals of the New York Academy of Sciences vol 1067 no 1 pp425ndash435 2006

[7] M Schieber and N S Chandel ldquoROS function in redoxsignaling and oxidative stressrdquo Current Biology vol 24 no 10pp R453ndashR462 2014

[8] G Bjelakovic D Nikolova L L Gluud R G Simonettiand C Gluud ldquoMortality in randomized trials of antioxidantsupplements for primary and secondary prevention systematicreview and meta-analysisrdquo Journal of the American MedicalAssociation vol 297 no 8 pp 842ndash857 2007

[9] R-M Liu J Choi J-H Wu et al ldquoOxidative modificationof nuclear mitogen-activated protein kinase phosphatase 1 isinvolved in transforming growth factor 1205731-induced expressionof plasminogen activator inhibitor 1 in fibroblastsrdquoThe Journalof Biological Chemistry vol 285 no 21 pp 16239ndash16247 2010

[10] M Bencina ldquoIllumination of the spatial order of intracellularpH by genetically encoded pH-sensitive sensorsrdquo Sensors vol13 no 12 pp 16736ndash16758 2013

[11] K A Lukyanov and V V Belousov ldquoGenetically encodedfluorescent redox sensorsrdquo Biochimica et Biophysica Acta vol1840 no 2 pp 745ndash756 2014

[12] S G Rhee T-S Chang W Jeong and D Kang ldquoMethods fordetection and measurement of hydrogen peroxide inside andoutside of cellsrdquoMolecules and Cells vol 29 no 6 pp 539ndash5492010

[13] V Zambo L Simon-Szabo P Szelenyi E Kereszturi GBanhegyi and M Csala ldquoLipotoxicity in the liverrdquo WorldJournal of Hepatology vol 5 no 10 pp 550ndash557 2013

[14] E Ceni T Mello and A Galli ldquoPathogenesis of alcoholicliver disease role of oxidative metabolismrdquo World Journal ofGastroenterology vol 20 no 47 pp 17756ndash17772 2014

[15] T Mello E Ceni C Surrenti and A Galli ldquoAlcohol inducedhepatic fibrosis role of acetaldehyderdquo Molecular Aspects ofMedicine vol 29 no 1-2 pp 17ndash21 2008

[16] P Dongiovanni A L Fracanzani S Fargion and L ValentildquoIron in fatty liver and in the metabolic syndrome a promisingtherapeutic targetrdquo Journal of Hepatology vol 55 no 4 pp 920ndash932 2011

[17] N Fujita and Y Takei ldquoIron overload in nonalcoholic steato-hepatitisrdquo Advances in Clinical Chemistry vol 55 pp 105ndash1322011

[18] J E Nelson H Klintworth and K V Kowdley ldquoIronmetabolism in Nonalcoholic Fatty Liver Diseaserdquo CurrentGastroenterology Reports vol 14 no 1 pp 8ndash16 2012


[19] M Arciello M Gori and C Balsano ldquoMitochondrial dys-functions and altered metals homeostasis new weapons tocounteract HCV-related oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 971024 10 pages2013

[20] K Hino S Nishina and Y Hara ldquoIron metabolic disorder inchronic hepatitis C mechanisms and relevance to hepatocar-cinogenesisrdquo Journal of Gastroenterology and Hepatology vol28 no 4 pp 93ndash98 2013

[21] D P Jones and H Sies ldquoThe redox coderdquo Antioxidants amp RedoxSignaling 2015

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[24] A-L Levonen R P Patel P Brookes et al ldquoMechanisms of cellsignaling by nitric oxide and peroxynitrite from mitochondriato MAP kinasesrdquo Antioxidants and Redox Signaling vol 3 no2 pp 215ndash229 2001

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[28] L Tandara and I Salamunic ldquoIron metabolism current factsand future directionsrdquo Biochemia Medica vol 22 no 3 pp 311ndash328 2012

[29] N Bresgen and P M Eckl ldquoOxidative stress and the homeo-dynamics of iron metabolismrdquo Biomolecules vol 5 no 2 pp808ndash847 2015

[30] R Lill B Hoffmann S Molik et al ldquoThe role of mitochondriain cellular iron-sulfur protein biogenesis and iron metabolismrdquoBiochimica et Biophysica ActamdashMolecular Cell Research vol1823 no 9 pp 1491ndash1508 2012

[31] E B Tahara F D T Navarete and A J Kowaltowski ldquoTissue- substrate- and site-specific characteristics of mitochondrialreactive oxygen species generationrdquo Free Radical Biology andMedicine vol 46 no 9 pp 1283ndash1297 2009

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[33] N Kaludercic A Carpi T Nagayama et al ldquoMonoamineoxidase B prompts mitochondrial and cardiac dysfunction inpressure overloaded heartsrdquo Antioxidants and Redox Signalingvol 20 no 2 pp 267ndash280 2014

[34] K Staniek and H Nohl ldquoAre mitochondria a permanent sourceof reactive oxygen speciesrdquo Biochimica et Biophysica ActamdashBioenergetics vol 1460 no 2-3 pp 268ndash275 2000

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[38] O W Griffith and A Meister ldquoOrigin and turnover of mito-chondrial glutathionerdquo Proceedings of the National Academy ofSciences of the United States of America vol 82 no 14 pp 4668ndash4672 1985

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[40] Z Chen and L H Lash ldquoEvidence for mitochondrial uptake ofglutathione by dicarboxylate and 2-oxoglutarate carriersrdquo TheJournal of Pharmacology and Experimental Therapeutics vol285 no 2 pp 608ndash618 1998

[41] Z Chen D A Putt and LH Lash ldquoEnrichment and functionalreconstitution of glutathione transport activity from rabbitkidney mitochondria Further evidence for the role of thedicarboxylate and 2-oxoglutarate carriers in mitochondrialglutathione transportrdquo Archives of Biochemistry and Biophysicsvol 373 no 1 pp 193ndash202 2000

[42] O Coll A Colell C Garcıa-Ruiz N Kaplowitz and J CFernandez-Checa ldquoSensitivity of the 2-oxoglutarate carrierto alcohol intake contributes to mitochondrial glutathionedepletionrdquo Hepatology vol 38 no 3 pp 692ndash702 2003

[43] H M Wilkins K Marquardt L H Lash and D A LinsemanldquoBcl-2 is a novel interacting partner for the 2-oxoglutaratecarrier and a key regulator of mitochondrial glutathionerdquo FreeRadical Biology and Medicine vol 52 no 2 pp 410ndash419 2012

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[49] J C Fernandez-Checa C Garcıa-Ruiz M Ookhtens and NKaplowitz ldquoImpaired uptake of glutathione by hepatic mito-chondria from chronic ethanol-fed rats tracer kinetic studies invitro and in vivo and susceptibility to oxidant stressrdquoThe Journalof Clinical Investigation vol 87 no 2 pp 397ndash405 1991


[50] J C Fernandez-ChecaMOokhtens andNKaplowitz ldquoEffectsof chronic ethanol feeding on rat hepatocytic glutathione Rela-tionship of cytosolic glutathione to efflux and mitochondrialsequestrationrdquo The Journal of Clinical Investigation vol 83 no4 pp 1247ndash1252 1989

[51] T Hirano N Kaplowitz H Tsukamoto S Kamimura and J CFernandez-Checa ldquoHepatic mitochondrial glutathione deple-tion and progression of experimental alcoholic liver disease inratsrdquo Hepatology vol 16 no 6 pp 1423ndash1427 1992

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disease patientsrdquo Clinical Science vol 106 no 3 pp 261ndash2682004

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[76] X Shen K Zhang and R J Kaufman ldquoThe unfolded proteinresponsemdasha stress signaling pathway of the endoplasmic retic-ulumrdquo Journal of Chemical Neuroanatomy vol 28 no 1-2 pp79ndash92 2004

[77] P I Merksamer and F R Papa ldquoThe UPR and cell fate at aglancerdquo Journal of Cell Science vol 123 no 7 pp 1003ndash10062010

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[79] D Acosta-Alvear Y Zhou A Blais et al ldquoXBP1 controls diversecell type- and condition-specific transcriptional regulatorynetworksrdquoMolecular Cell vol 27 no 1 pp 53ndash66 2007

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[81] D Ron and S R Hubbard ldquoHow IRE1 reacts to ER stressrdquo Cellvol 132 no 1 pp 24ndash26 2008

[82] H P Harding Y Zhang H Zeng et al ldquoAn integrated stressresponse regulates amino acid metabolism and resistance tooxidative stressrdquoMolecular Cell vol 11 no 3 pp 619ndash633 2003

[83] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[84] J Mathers J A Fraser M McMahon R D C Saunders JD Hayes and L I McLellan ldquoAntioxidant and cytoprotective


responses to redox stressrdquo Biochemical Society Symposium vol71 pp 157ndash176 2004

[85] D D Zhang ldquoMechanistic studies of the Nrf2-Keap1 signalingpathwayrdquo Drug Metabolism Reviews vol 38 no 4 pp 769ndash7892006

[86] S B Cullinan and J A Diehl ldquoPERK-dependent activation ofNrf2 contributes to redox homeostasis and cell survival fol-lowing endoplasmic reticulum stressrdquoThe Journal of BiologicalChemistry vol 279 no 19 pp 20108ndash20117 2004

[87] J B DuRose D Scheuner R J Kaufman L I Rothblum andM Niwa ldquoPhosphorylation of eukaryotic translation initia-tion factor 2120572 coordinates rRNA transcription and translationinhibition during endoplasmic reticulum stressrdquoMolecular andCellular Biology vol 29 no 15 pp 4295ndash4307 2009

[88] X Chen J Shen and R Prywes ldquoThe luminal domain ofATF6 senses endoplasmic reticulum (ER) stress and causestranslocation of ATF6 from the er to the Golgirdquo Journal ofBiological Chemistry vol 277 no 15 pp 13045ndash13052 2002

[89] S Oyadomari and M Mori ldquoRoles of CHOPGADD153 inendoplasmic reticulum stressrdquoCell DeathampDifferentiation vol11 no 4 pp 381ndash389 2004

[90] S J Marciniak C Y Yun S Oyadomari et al ldquoCHOP inducesdeath by promoting protein synthesis and oxidation in thestressed endoplasmic reticulumrdquo Genes and Development vol18 no 24 pp 3066ndash3077 2004

[91] Y Wang L Vera W H Fischer and M Montminy ldquoTheCREB coactivator CRTC2 links hepatic ER stress and fastinggluconeogenesisrdquo Nature vol 460 no 7254 pp 534ndash537 2009

[92] G S Hotamisligil ldquoEndoplasmic reticulum stress and theinflammatory basis of metabolic diseaserdquo Cell vol 140 no 6pp 900ndash917 2010

[93] D Ron and P Walter ldquoSignal integration in the endoplasmicreticulum unfolded protein responserdquo Nature Reviews Molecu-lar Cell Biology vol 8 no 7 pp 519ndash529 2007

[94] S Fu S M Watkins and G S Hotamisligil ldquoThe role ofendoplasmic reticulum in hepatic lipid homeostasis and stresssignalingrdquo Cell Metabolism vol 15 no 5 pp 623ndash634 2012

[95] U Ozcan Q Cao E Yilmaz et al ldquoEndoplasmic reticulumstress links obesity insulin action and type 2 diabetesrdquo Sciencevol 306 no 5695 pp 457ndash461 2004

[96] J Lee and U Ozcan ldquoUnfolded protein response signaling andmetabolic diseasesrdquo The Journal of Biological Chemistry vol289 no 3 pp 1203ndash1211 2014

[97] X-Q Zhang C-F Xu C-H Yu W-X Chen and Y-M LildquoRole of endoplasmic reticulum stress in the pathogenesis ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 20 no 7 pp 1768ndash1776 2014

[98] C Hetz ldquoThe unfolded protein response controlling cell fatedecisions under ER stress and beyondrdquo Nature Reviews Molec-ular Cell Biology vol 13 no 2 pp 89ndash102 2012

[99] C X C Santos L Y Tanaka J Wosniak and F R MLaurindo ldquoMechanisms and implications of reactive oxygenspecies generation during the unfolded protein response rolesof endoplasmic reticulum oxidoreductases mitochondrial elec-tron transport and NADPH oxidaserdquo Antioxidants amp RedoxSignaling vol 11 no 10 pp 2409ndash2427 2009

[100] C M Haynes E A Titus and A A Cooper ldquoDegradation ofmisfolded proteins prevents ER-derived oxidative stress and celldeathrdquoMolecular Cell vol 15 no 5 pp 767ndash776 2004

[101] S B Cullinan and J A Diehl ldquoCoordination of ER andoxidative stress signaling the PERKNrf2 signaling pathwayrdquo

International Journal of Biochemistry and Cell Biology vol 38no 3 pp 317ndash332 2006

[102] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[103] S Chowdhry M H Nazmy P J Meakin et al ldquoLoss ofNrf2 markedly exacerbates nonalcoholic steatohepatitisrdquo FreeRadical Biology and Medicine vol 48 no 2 pp 357ndash371 2010

[104] H Sugimoto K Okada J Shoda et al ldquoDeletion of nuclearfactor-E

2-related factor-2 leads to rapid onset and progression

of nutritional steatohepatitis in micerdquo The American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 298 no2 pp G283ndashG294 2010

[105] R J Kaufman S H Back B Song J Han and J HasslerldquoThe unfolded protein response is required to maintain theintegrity of the endoplasmic reticulum prevent oxidative stressand preserve differentiation in beta-cellsrdquoDiabetes Obesity andMetabolism vol 12 no 2 pp 99ndash107 2010

[106] K Zhang ldquoIntegration of ER stress oxidative stress and theinflammatory response in health and diseaserdquo InternationalJournal of Clinical and Experimental Medicine vol 3 no 1 pp33ndash40 2010

[107] R A Egnatchik A K Leamy D A Jacobson M Shiota andJ D Young ldquoER calcium release promotes mitochondrial dys-function and hepatic cell lipotoxicity in response to palmitateoverloadrdquoMolecularMetabolism vol 3 no 5 pp 544ndash553 2014

[108] A K Leamy R A Egnatchik M Shiota et al ldquoEnhancedsynthesis of saturated phospholipids is associated with ER stressand lipotoxicity in palmitate treated hepatic cellsrdquo Journal ofLipid Research vol 55 no 7 pp 1478ndash1488 2014

[109] A I Cederbaum Y Lu and D Wu ldquoRole of oxidative stress inalcohol-induced liver injuryrdquo Archives of Toxicology vol 83 no6 pp 519ndash548 2009

[110] H Cichoz-Lach and A Michalak ldquoOxidative stress as a crucialfactor in liver diseasesrdquo World Journal of Gastroenterology vol20 no 25 pp 8082ndash8091 2014

[111] A I Cederbaum ldquoAlcoholmetabolismrdquoClinics in Liver Diseasevol 16 no 4 pp 667ndash685 2012

[112] A Ambade and P Mandrekar ldquoOxidative stress and inflamma-tion essential partners in alcoholic liver diseaserdquo InternationalJournal of Hepatology vol 2012 Article ID 853175 9 pages 2012

[113] A Louvet and P Mathurin ldquoAlcoholic liver disease mech-anisms of injury and targeted treatmentrdquo Nature ReviewsGastroenterology amp Hepatology vol 12 no 4 pp 231ndash242 2015

[114] C S Lieber ldquoAlcohol its metabolism and interaction withnutrientsrdquo Annual Review of Nutrition vol 20 pp 395ndash4302000

[115] C S Lieber ldquoAlcoholic fatty liver its pathogenesis and mecha-nism of progression to inflammation and fibrosisrdquo Alcohol vol34 no 1 pp 9ndash19 2004

[116] Y Lu and A I Cederbaum ldquoCYP2E1 and oxidative liver injuryby alcoholrdquo Free Radical Biology andMedicine vol 44 no 5 pp723ndash738 2008

[117] T Hugle F Fehrmann E Bieck et al ldquoThe hepatitis C virusnonstructural protein 4B is an integral endoplasmic reticulummembrane proteinrdquo Virology vol 284 no 1 pp 70ndash81 2001

[118] L Kong S Li M Huang et al ldquoThe roles of endoplasmicreticulum overload response induced by HCV and NS4Bprotein in human hepatocyte viability and virus replicationrdquoPLOS ONE vol 10 no 4 Article ID e0123190 2015


[119] C Vasallo and P Gastaminza ldquoCellular stress responses inhepatitis C virus infection mastering a two-edged swordrdquoVirusResearch 2015

[120] Y Li D F Boehning T Qian V L Popov and S A WeinmanldquoHepatitis C virus core protein increases mitochondrial ROSproduction by stimulation of Ca2+ uniporter activityrdquo TheFASEB Journal vol 21 no 10 pp 2474ndash2485 2007

[121] G Gong G Waris R Tanveer and A Siddiqui ldquoHumanhepatitis C virus NS5A protein alters intracellular calciumlevels induces oxidative stress and activates STAT-3 and NF-120581Brdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 17 pp 9599ndash9604 2001

[122] S Li L Ye X Yu et al ldquoHepatitis C virus NS4B inducesunfolded protein response and endoplasmic reticulumoverloadresponse-dependent NF-kappaB activationrdquo Virology vol 391no 2 pp 257ndash264 2009

[123] K Awe C Lambert and R Prange ldquoMammalian BiP controlsposttranslational ER translocation of the hepatitis B virus largeenvelope proteinrdquo FEBS Letters vol 582 no 21-22 pp 3179ndash3184 2008

[124] M Dodson V Darley-Usmar and J Zhang ldquoCellular metabolicand autophagic pathways traffic control by redox signalingrdquoFree Radical Biology and Medicine vol 63 pp 207ndash221 2013

[125] R Scherz-Shouval E Shvets E Fass H Shorer L Gil and ZElazar ldquoReactive oxygen species are essential for autophagy andspecifically regulate the activity of Atg4rdquo The EMBO Journalvol 26 no 7 pp 1749ndash1760 2007

[126] Y Chen M B Azad and S B Gibson ldquoSuperoxide is the majorreactive oxygen species regulating autophagyrdquo Cell Death andDifferentiation vol 16 no 7 pp 1040ndash1052 2009

[127] S P Elmore T Qian S F Grissom and J J Lemasters ldquoThemitochondrial permeability transition initiates autophagy in rathepatocytesrdquoThe FASEB Journal vol 15 no 12 pp 2286ndash22872001

[128] A Takamura M Komatsu T Hara et al ldquoAutophagy-deficientmice develop multiple liver tumorsrdquo Genes and Developmentvol 25 no 8 pp 795ndash800 2011

[129] K K Kharbanda D L McVicker R K Zetterman and TM Donohue Jr ldquoEthanol consumption reduces the proteolyticcapacity and protease activities of hepatic lysosomesrdquo Biochim-ica et Biophysica Acta vol 1245 no 3 pp 421ndash429 1995

[130] K K Kharbanda D L McVicker R K Zetterman and TM Donohue Jr ldquoEthanol consumption alters trafficking oflysosomal enzymes and affects the processing of procathepsinL in rat liverrdquo Biochimica et Biophysica Acta General Subjectsvol 1291 no 1 pp 45ndash52 1996

[131] T M Donohue T V Curry-McCoy A A Nanji et al ldquoLysoso-mal leakage and lack of adaptation of hepatoprotective enzymecontribute to enhanced susceptibility to ethanol-induced liverinjury in female ratsrdquo Alcoholism Clinical and ExperimentalResearch vol 31 no 11 pp 1944ndash1952 2007

[132] Y Li S Wang H Ni H Huang and W Ding ldquoAutophagy inalcohol-induced multiorgan injury mechanisms and potentialtherapeutic targetsrdquo BioMed Research International vol 2014Article ID 498491 20 pages 2014

[133] W X Ding M Li X Chen et al ldquoAutophagy reduces acuteethanol-induced hepatotoxicity and steatosis in micerdquo Gas-troenterology vol 139 no 5 pp 1740ndash1752 2010

[134] P G Thomes C S Trambly G M Thiele et al ldquoProteasomeactivity and autophagosome content in liver are reciprocallyregulated by ethanol treatmentrdquo Biochemical and BiophysicalResearch Communications vol 417 no 1 pp 262ndash267 2012

[135] D Wu X Wang R Zhou L Yang and A I CederbaumldquoAlcohol steatosis and cytotoxicity the role of cytochromeP4502E1 and autophagyrdquo Free Radical Biology andMedicine vol53 no 6 pp 1346ndash1357 2012

[136] C-W Lin H Zhang M Li et al ldquoPharmacological promotionof autophagy alleviates steatosis and injury in alcoholic andnon-alcoholic fatty liver conditions in micerdquo Journal of Hepa-tology vol 58 no 5 pp 993ndash999 2013

[137] A Dolganiuc P G Thomes W-X Ding J J Lemasters and TM Donohue Jr ldquoAutophagy in alcohol-induced liver diseasesrdquoAlcoholism Clinical and Experimental Research vol 36 no 8pp 1301ndash1308 2012

[138] P Jiang Z Huang H Zhao and T Wei ldquoHydrogen peroxideimpairs autophagic flux in a cell model of nonalcoholic fattyliver diseaserdquo Biochemical and Biophysical Research Communi-cations vol 433 no 4 pp 408ndash414 2013

[139] X Zhang and J J Lemastersn ldquoTranslocation of iron fromlysosomes to mitochondria during ischemia predisposes toinjury after reperfusion in rat hepatocytesrdquo Free Radical Biologyand Medicine vol 63 pp 243ndash253 2013

[140] M A Krenn M Schurz B Teufl K Uchida P M Eckl andN Bresgen ldquoFerritin-stimulated lipid peroxidation lysosomalleak andmacroautophagy promote lysosomal lsquometastabilityrsquo inprimary hepatocytes determining in vitro cell survivalrdquo FreeRadical Biology and Medicine vol 80 pp 48ndash58 2015

[141] J K Ready and G P Mannaerts ldquoPeroxisomal lipidmetabolismrdquo Annual Review of Nutrition vol 14 pp 343ndash370 1994

[142] P A Loughran D B Stolz Y Vodovotz S C Watkins R LSimmons and T R Billiar ldquoMonomeric inducible nitric oxidesynthase localizes to peroxisomes in hepatocytesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 102 no 39 pp 13837ndash13842 2005

[143] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp 1755ndash1766 2006

[144] R J A Wanders P Vreken S Ferdinandusse et al ldquoPeroxi-somal fatty acid 120572- and 120573-oxidation in humans enzymologyperoxisomalmetabolite transporters and peroxisomal diseasesrdquoBiochemical Society Transactions vol 29 no 2 pp 250ndash2672001

[145] J K Reddy ldquoPeroxisome proliferators and peroxisomeproliferator-activated receptor 120572 biotic and xenobioticsensingrdquo American Journal of Pathology vol 164 no 6 pp2305ndash2321 2004

[146] B Knebel S Hartwig J Haas et al ldquoPeroxisomes compensatehepatic lipid overflow in mice with fatty liverrdquo Biochimica etBiophysica Acta vol 1851 no 7 pp 965ndash976 2015

[147] J C Collins I H Scheinberg D R Giblin and I SternliebldquoHepatic peroxisomal abnormalities in abetalipoproteinemiardquoGastroenterology vol 97 no 3 pp 766ndash770 1989

[148] S Yu S Rao and J K Reddy ldquoPeroxisome proliferator-activated receptors fatty acid oxidation steatohepatitis andhepatocarcinogenesisrdquo Current Molecular Medicine vol 3 no6 pp 561ndash572 2003

[149] R Dirkx I Vanhorebeek K Martens et al ldquoAbsence ofperoxisomes in mouse hepatocytes causes mitochondrial andER abnormalitiesrdquoHepatology vol 41 no 4 pp 868ndash878 2005

[150] N Bashan J Kovsan I Kachko H Ovadia and A RudichldquoPositive and negative regulation of insulin signaling by reactiveoxygen and nitrogen speciesrdquo Physiological Reviews vol 89 no1 pp 27ndash71 2009


[151] B J Goldstein K Mahadev X Wu L Zhu and H MotoshimaldquoRole of insulin-induced reactive oxygen species in the insulinsignaling pathwayrdquo Antioxidants and Redox Signaling vol 7 no7-8 pp 1021ndash1031 2005

[152] J Kwon S-R Lee K-S Yang et al ldquoReversible oxidation andinactivation of the tumor suppressor PTEN in cells stimu-lated with peptide growth factorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no47 pp 16419ndash16424 2004

[153] S-R Lee K-S Yang J Kwon C Lee W Jeong and S GRhee ldquoReversible inactivation of the tumor suppressor PTENby H2O2rdquo The Journal of Biological Chemistry vol 277 no 23

pp 20336ndash20342 2002[154] T Fiaschi F Buricchi G Cozzi et al ldquoRedox-dependent

and ligand-independent trans-activation of insulin receptor byglobular adiponectinrdquo Hepatology vol 46 no 1 pp 130ndash1392007

[155] S Iwakami H Misu T Takeda et al ldquoConcentration-dependent dual effects of hydrogen peroxide on insulin signaltransduction in H4IIEC hepatocytesrdquo PLoS ONE vol 6 no 11Article ID e27401 2011

[156] K A Robinson C A Stewart Q N Pye et al ldquoRedox-sensitiveprotein phosphatase activity regulates the phosphorylation stateof p38 protein kinase in primary astrocyte culturerdquo Journal ofNeuroscience Research vol 55 no 6 pp 724ndash732 1999

[157] H Kamata S-I Honda S Maeda L Chang H Hirata andM Karin ldquoReactive oxygen species promote TNF120572-induceddeath and sustained JNK activation by inhibiting MAP kinasephosphatasesrdquo Cell vol 120 no 5 pp 649ndash661 2005

[158] C C Wentworth A Alam R M Jones A Nusrat and A SNeish ldquoEnteric commensal bacteria induce extracellular signal-regulated kinase pathway signaling via formyl peptide receptor-dependent redox modulation of dual specific phosphataserdquoTheJournal of Biological Chemistry vol 286 no 44 pp 38448ndash38455 2011

[159] S R Lee K S Kwon S R Kim and S G Rhee ldquoReversibleinactivation of protein-tyrosine phosphatase 1B in A431 cellsstimulated with epidermal growth factorrdquo The Journal of Bio-logical Chemistry vol 273 pp 15366ndash15372 1998

[160] S Boura-Halfon and Y Zick ldquoPhosphorylation of IRS proteinsinsulin action and insulin resistancerdquo American Journal ofPhysiology Endocrinology and Metabolism vol 296 no 4 ppE581ndashE591 2009

[161] H Bai W Zhang X Qin et al ldquoHydrogen peroxide mod-ulates the proliferationquiescence switch in the liver duringembryonic development and posthepatectomy regenerationrdquoAntioxidants amp Redox Signaling vol 22 no 11 pp 921ndash937 2015

[162] E R Galimov ldquoThe role of p66shc in oxidative stress andapoptosisrdquo Acta Naturae vol 2 no 4 pp 44ndash51 2010

[163] G Xi X-C Shen C Wai and D R Clemmons ldquoRecruitmentof Nox4 to a plasmamembrane scaffold is required for localizedreactive oxygen species generation and sustained Src activationin response to insulin-like growth factor-Irdquo Journal of BiologicalChemistry vol 288 no 22 pp 15641ndash15653 2013

[164] O SergentM Pereira C BelhommeMChevanne LHuc andD Lagadic-Gossmann ldquoRole for membrane fluidity in ethanol-induced oxidative stress of primary rat hepatocytesrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 313 no 1 pp104ndash111 2005

[165] P Nourissat M Travert M Chevanne et al ldquoEthanol inducesoxidative stress in primary rat hepatocytes through the early

involvement of lipid raft clusteringrdquo Hepatology vol 47 no 1pp 59ndash70 2008

[166] F Aliche-Djoudi N Podechard A Collin et al ldquoA role for lipidrafts in the protection afforded by docosahexaenoic acid againstethanol toxicity in primary rat hepatocytesrdquo Food and ChemicalToxicology vol 60 pp 286ndash296 2013

[167] A Collin K Hardonniere M Chevanne et al ldquoCooperativeinteraction of benzo[a]pyrene and ethanol on plasma mem-brane remodeling is responsible for enhanced oxidative stressand cell death in primary rat hepatocytesrdquo Free Radical Biologyand Medicine vol 72 pp 11ndash22 2014

[168] J Woudenberg K P Rembacz M Hoekstra et al ldquoLipidrafts are essential for peroxisome biogenesis in HepG2 cellsrdquoHepatology vol 52 no 2 pp 623ndash633 2010

[169] A P Arruda B M Pers G Parlakgul E Guney K Inouye andG SHotamisligil ldquoChronic enrichment of hepatic endoplasmicreticulum-mitochondria contact leads to mitochondrial dys-function in obesityrdquo Nature Medicine vol 20 pp 1427ndash14352014

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


Protein folding

Endoplasmic reticulumMitochondria

Insulin

IRSLysosomes

Plasma membraneNOX

ROS

PTPs

Signaling

ROS

Intracellular Signaling

Peroxisomes

ROS

ROS

ETC

ROS

ROS

ROS

TCA

Autophagy

CYP

Xenobiotics

ROS

Lipids rafts

120573-oxidation

120573-oxidation

Fe2+

Ca2+

Figure 1 Sites of physiologically produced ROS Plasma membrane localized ROS bursts deactivate PTPs and allow signal transduction(ie by insulin or IGF-1) after tyrosine kinase receptor activation Mitochondria produce ROS during cellular respiration and metabolicactivity ROS are produced in the ER during protein folding and detoxification by the cytochrome P450 systems Lysosomes are requiredfor iron metabolism and the removal of damaged cellular components through autophagy Peroxisomes produce ROS during metabolic ordetoxification activities

the redox circuits that regulate many signaling pathways[3] Among ROS hydrogen peroxide is supposed to playa major role either directly or indirectly in the regulationof the thioldisulphide redox switches [4] because (i) thesereactions typically require a two-electron transfer (ii) H

2O2

is kinetically restricted and thus can be highly selective in sub-strate oxidation and (iii) H

2O2is generated following growth

factor cytokine or hormone signaling However the detailedmolecular mechanisms leading to selective thiol oxidation inredox-sensitive proteins by H

2O2are still mostly obscure and

are the focus of intense research activity A growing bodyof data suggests that altered redox signaling precedes andcontributes substantially more than direct radical damage tothe development of several human pathologies

The concept of ldquooxidative stressrdquo introduced 30 years ago[5] evolved over time from the original oxidative damage tothe cell structure and subsequent stress response to includethat of alteration of signaling pathways redox homeostasisand redox adaptation to stress [6 7]

Consequently oxidative stress is not necessarily harmfuland antioxidants are not utterly beneficial In fact manyclinical trials failed to prove the efficacy of low-molecularweight antioxidants in the treatment of several pathologiesand the use of the antioxidants selenium beta-carotene and

vitamin E was even found to increase overall mortality in alarge meta-analysis [8]

Our understanding of the redox landscape of the cell israpidly evolving and thanks to the recent development ofspecific redox probes [9ndash12] we are beginning to unravela complex spatial and temporal organization of the redoxfluxes in the living cells Compartmentalization of the redoxcircuitry is crucial to maintain physiology and is a key tounderstand the alterations of the redox homeostasis occur-ring in disease

The liver is the main metabolic organ and plays a funda-mental role in whole body detoxification and blood streamfiltering Most detoxification processes (drugs alcohol andendo- and xenobiotics) are carried out through oxidativereactions by the cytochrome P450 (CYP) isoenzymes whichgenerate superoxide anion (O

2

∙minus) (Figure 1) Derangementof the liver metabolic processes such as those occurringby fatty acids overload in NAFLD results in increasedROS production by increased electron transfer during mito-chondrial 120573-oxidation as well as increased CYP2E1 expres-sion and activity [13] Strong induction of CYP2E1 alsooccurs due to excessive consumption of ethanol whose toxicmetabolite acetaldehyde (AcCHO) generates oxidative stressthrough a number of direct and indirect mechanisms [6 14


ETC Signaling

ROS

ROS

ROS

ROS

TCA

AcCHO

FFA

GSH

AcCHO


Mitochondria

Insulin

IRSLysosomes

Plasma membraneNOX

ROS

PTPs

ROS

ROS

MAMs

Virus

CYP

ETOH

ETOH

ROS

Peroxisomes

ROS

FFA

ETOHIron

Lipids rafts

clustering

Autophagy

UPR

ROS

ROS

ROSAltered



120573-oxidation

120573-oxidation

Fe2+

Fe2+

Fe2+

Ca2+

Ca2+

Ca2+










2





2





2O2) [23] ∙NO does







2O2) or the HCO

3






Fe3+ +O2


(2)

The net reaction O2

∙minus +H2O2


(3)




2 Mitochondria


2


2O2is


2O2produc-


























2O2levels


















2O2production





2



3CHOH) This






4 Lysosomes




2



clear whether O2












5 Peroxisomes







2O2production




6 Plasma Membrane




















2O2levels


2O2production

















Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ETC Signaling

ROS

ROS

ROS

ROS

TCA

AcCHO

FFA

GSH

AcCHO


Mitochondria

Insulin

IRSLysosomes

Plasma membraneNOX

ROS

PTPs

ROS

ROS

MAMs

Virus

CYP

ETOH

ETOH

ROS

Peroxisomes

ROS

FFA

ETOHIron

Lipids rafts

clustering

Autophagy

UPR

ROS

ROS

ROSAltered



120573-oxidation

120573-oxidation

Fe2+

Fe2+

Fe2+

Ca2+

Ca2+

Ca2+










2





2





2O2) [23] ∙NO does







2O2) or the HCO

3






Fe3+ +O2


(2)

The net reaction O2

∙minus +H2O2


(3)




2 Mitochondria


2


2O2is


2O2produc-


























2O2levels


















2O2production





2



3CHOH) This






4 Lysosomes




2



clear whether O2












5 Peroxisomes







2O2production




6 Plasma Membrane




















2O2levels


2O2production

















Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2





2O2) [23] ∙NO does







2O2) or the HCO

3






Fe3+ +O2


(2)

The net reaction O2

∙minus +H2O2


(3)




2 Mitochondria


2


2O2is


2O2produc-


























2O2levels


















2O2production





2



3CHOH) This






4 Lysosomes




2



clear whether O2












5 Peroxisomes







2O2production




6 Plasma Membrane




















2O2levels


2O2production

















Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































2O2levels


















2O2production





2



3CHOH) This






4 Lysosomes




2



clear whether O2












5 Peroxisomes







2O2production




6 Plasma Membrane




















2O2levels


2O2production

















Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2O2production





2



3CHOH) This






4 Lysosomes




2



clear whether O2












5 Peroxisomes







2O2production




6 Plasma Membrane




















2O2levels


2O2production

















Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















clear whether O2












5 Peroxisomes







2O2production




6 Plasma Membrane




















2O2levels


2O2production

















Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















6 Plasma Membrane




















2O2levels


2O2production

















Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Abbreviations




Acknowledgments


References



























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article oxidative stress in the healthy and wounded ...review article oxidative stress in the...

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