drug - induced liver 
 injury ( dili ) is a leading cause of acute liver 
 failure , thus constituting a major reason 
 for drug candidates failing during development or withdrawal from 
 the market . because of drug - related toxicity ,
many drug candidates 
 that may otherwise be potentially efficacious in the treatment of 
 disorders or diseases have been discontinued ; this precipitous discontinuation 
 represents a major setback to a larger population which may benefit 
 from further development of these drug candidates .
in addition , from 
 the pharmaceutical industry s perspective , the resultant regulatory 
 actions have driven up the development cost to meet acceptable safety 
 requirements .
drugs that cause dili rarely do so at doses tolerated 
 well by most patients at frequencies of less than 1 in 10,000 patients , 
 and generally do not exhibit dose - dependency .
consequently , they often 
 escape detection preclinically and even in phase ii / iii trials due 
 to its limited patient pool versus the much larger population exposed 
 to the drug . establishing definitive prediction 
 of drugs that exhibit dili can be difficult and signals an urgent 
 need for innovative approaches that can rigorously detect early signs 
 of potential hepatotoxicity in drug candidates .
current toxicological studies using normally healthy animals 
 are often insufficient in addressing the mechanisms behind human diseases 
 and are relatively insensitive in detecting drug - induced toxicity 
 effects .
therefore , considerations of 
 animal models with underlying genetic abnormalities relevant to human 
 diseases may be a useful strategy in revealing and evaluating potential 
 drug - induced adverse effects .
recently , transgenic mice of heterozygous 
 genetic knockouts are proposed as alternative animal models in a diverse 
 range of research fields including toxicity assessment due to their 
 intermediate phenotypes .
troglitazone , 
 a once - marketed first - generation thiazolidinedione 
 used in the treatment of type - ii diabetes mellitus was withdrawn from 
 the market due to unacceptable idiosyncratic hepatotoxicity risks . in early drug safety assessments and even in 
 long - term studies , troglitazone did not cause hepatotoxicity in normal 
 healthy rodents and monkeys .
this highlights the shortcomings 
 and remarkable difficulty in predicting idiosyncratic dili at the 
 standard preclinical animal testing phase .
numerous 
 attempts to explain the mechanisms underlying troglitazone - induced 
 liver toxicity ( tili ) or to recapitulate the human scenario have been 
 unsuccessful , and in vitro results and ensuing hypotheses 
 provided little mechanistic relevance to address clinical tili .
we and others have demonstrated that the sod2 mouse exhibits higher sensitivity 
 toward the mitochondrial damaging effects of drugs , including troglitazone .
the sod2 mouse 
 model presents an interesting phenotype that is clinically silent 
 yet amenable to unmasking potential drug - induced adverse reactions 
 of normally mild drugs , thereby representing a useful model in drawing 
 correlations between increased mitochondrial oxidative stress and 
 drug - induced adverse effects .
two - dimensional liquid chromatography  difference 
 gel electrophoresis on the sod2 hepatic mitochondrial proteome ( henceforth referred to as 
 mitoproteome ) revealed molecular changes that recapitulate the clinical 
 features of tili in a time - dependent fashion .
encouraged by the involvement of specific mitochondrial proteins 
 in troglitazone - induced hepatotoxicity and the superior comprehensiveness 
 of mass spectrometry - based proteomics , we sought to deepen the coverage of mitochondrial protein changes 
 between sod2 and sod2 mice , as well 
 as to track sod2 mitoproteome changes with troglitazone administration ( figure 1 ) . integrating quantitative proteomics , toxicological 
 end points , and topological changes , we found fragilities in mitochondrial 
 glutathione ( mgsh ) transport and oxidative - stress - induced dysregulation 
 of lipid - associated proteins as crucial nodes that underlie the transition 
 from early compensatory responses to late hepatic injury in the sod2 mouse .
furthermore , 
 we show that deficiency in mgsh transport by dicarboxylate ion carrier 
 ( dic ) accelerates troglitazone - induced cytotoxicity .
this quantitative 
 systems approach represents a new and powerful way toward understanding 
 dili with major implications for its early prediction .
flow - chart summary of 
 the itraq experimental designs of 4-plex 
 and 8-plex systems .
( a ) quantification of proteins differentially 
 expressed in the sod2 and sod2 hepatic 
 mitochondria using the 4-plex itraq channels .
( b ) quantitative shotgun proteomics 
 using the 8-plex itraq labels to elucidate and identify mitoproteome 
 dynamics over two periods of daily vehicle ( veh ) or troglitazone ( trg ) 
 dosing . the experiment was performed in technical replicates .
nc , no change ; ns , 
 not significant ; s , significant ; scx , strong cation exchange ; rp , 
 reverse phase .
all protocols involving 
 animals were in compliance with the institutional animal care and 
 use committee and in accordance with the guidelines of the national 
 advisory committee for laboratory animal care and research .
heterozygous sod2/j mice , congenic in the c57bl/6 background , were obtained from jackson 
 laboratory ( bar harbor , me ) .
a breeding colony was established by 
 crossing male sod2 with female wild type sod2 mice .
female sod2 mice were randomly divided into four groups ( n = 36 ) and injected daily intraperitoneally with 9% solutol 
 hs-15 ( 10 l / g body weight ) or troglitazone ( 30 mg / kg body weight ; 
 cayman chemical , ann arbor , mi ) for 14 or 28 days . after 14 or 28 
 days of treatment , the mice were anesthetized with pentobarbital ( 60 
 mg / kg , intraperitoneally ) , and immediately after necropsy , livers 
 were excised ; one portion of liver sample for use in histopathological 
 evaluation was fixed in 4% neutral buffered formalin while the remaining 
 portion was used to prepare mitochondrial fractions .
blood was drawn 
 via cardiac puncture ; serum was prepared by allowing blood to clot 
 for 30 min and centrifuging at 2000 g at 4 c 
 for 10 min ; and the supernatant was decanted for analysis .
the mitochondrial fraction was obtained 
 by centrifugation of liver homogenates first at 800 g and then twice at 8000 g .
the enriched mitochondrial 
 pellet was resuspended in wash buffer ( 210 mm mannitol , 70 mm sucrose , 
 5 mm hepes , 1 mm egta , ph 7.4 ) and gently layered onto a modified 
 discontinuous percoll gradient .
a gradient was generated by layering 
 1.5 ml of 60% , 4 ml of 32% , 1.5 ml of 23% , and 1.5 ml of 15% ( v / v ) 
 percoll in wash buffer .
after centrifugation at 134000 g at 4 c for 1 h , purified mitochondria were recovered from 
 the 60/32% interface , which was then diluted with 4 volumes of wash 
 buffer before being centrifuged at 12000 g at 4 c . 

the purified mitochondrial proteins were resuspended in lysis buffer 
 ( 7 m urea , 2 m thiourea , 4% ( w / v ) chaps ) with halt protease inhibitor 
 cocktail , phosstop phosphatase inhibitor ( pierce , rockford , il ) , dnase 
 1 , and rnase a ( roche applied science , mannheim , germany ) , with protein 
 content being determined and then with storage as described previously .
two itraq experiments 
 were performed : ( i ) 
 4-plex system for the sod2 versus sod2 comparative study and ( ii ) 8-plex system for the troglitazone study . 

4-plex and 8-plex itraq labeling was performed 
 according to manufacturers protocols ( ab sciex , ca ) .
the labeled 
 samples were combined and passed through a strong cation exchange 
 cartridge ( ab sciex ) and seppak ( millipore ) , vacuum - dried , and stored 
 at 80 c until further use .
each of the itraq - labeled 
 peptide mixtures was separated by 2d - lc using an ultimate dual gradient 
 lc system ( dionex - lc packings ) equipped with a probot maldi spotting 
 device .
the lc fractions were mixed directly with maldi matrix solution 
 ( 7 mg / ml -cyano-4-hydroxycinnamic acid and 130 g / ml 
 ammonium citrate in 75% acn ) at a flow rate of 5.4 l / min via 
 a 25 nl mixing tee ( upchurch scientific ) before they were spotted 
 onto a 192-well stainless steel maldi target plate ( ab sciex ) using 
 a probot micro precision fraction collector ( dionex - lc packings ) at 
 a speed of 5 s / well .
the samples on the maldi target plates 
 were analyzed using a 4700 proteomics analyzer mass spectrometer ( ab 
 sciex ) .
ms / ms analyses were performed using nitrogen at a collision 
 energy of 1 kv and a collision gas pressure of 1  10 torr .
one thousand shots were accumulated for each ms spectrum . 
 for ms / ms ,
6000 shots were combined for each precursor ion with a 
 signal - to - noise ( s / n ) ratio greater than or equal to 100 . for precursors 
 with a s / n ratio between 50 and 100 ,
no smoothing was 
 applied before peak detection for both ms and ms / ms , and the peaks 
 were deisotoped . for ms / ms , only the peaks from 60 to 20 da below 
 each precursor mass and with s /
peak density was limited to 30 peaks per 200 da , and the 
 maximum number of peaks was set to 125 .
cysteine methanethiolation , 
 n - terminal itraq labeling , and itraq - labeled lysine were selected 
 as fixed modifications ; methionine oxidation was considered as a variable 
 modification .
precursor error tolerance 
 was set to 100 ppm ; ms / ms fragment error tolerance was set to 0.4 
 da .
itraq data analyses were performed 
 using proteinpilot ( ab sciex ) by searching the spectra against the 
 international protein index ( ipi ) mouse database ( version 3.48 ) concatenated 
 with a randomized  decoy  version of itself , restricted 
 to tryptic peptides .  instantaneous 
fdr estimation 
 was set at 5% ( supporting information figure s1a ) . as a further refinement
, proteins have to meet the criteria of 
 ( i ) proteinpilot unused protscore of 2.0 ( 99% c.i . ) and ( ii ) 
 two or more tryptic peptides with at least one unique peptide of high 
 confidence ( > 99% ) . the itraq fold change ( r )
is 
 a 
 relative ratio of either ( i ) sod2 over sod2 or 
 ( ii ) trg over veh . for quantification purposes , ms and ms / ms spectra 

were manually examined and detected sequences rejected on the basis 
 of one or more of the following criteria : ( i ) c.i .
< 99% , ( ii ) 
 partially cleaved tryptic sequences or internal miscleavages , except 
 when proline is the c - terminus of lysine or arginine , ( iii )  crowding  

of one or more adjacent precursor peaks whose intensity is half or 
 more than the matched monoisotopic precursor peak , within a precursor 
 ion tolerance window of 5 da , ( iv ) unlabeled itraq , ( v ) nonspecific 
 itraq labeling , ( vi ) plausible ptms , and ( vii ) weak itraq reporter 
 ion intensity ( threshold at fifth percentile , < 3500 relative intensity 
 for 4-plex and < 1500 for 8-plex ; supporting 
 information figure s1b ) . to achieve accurate and reliable quantification
, 
 cutoff was tailed at the fifth percentile to restrict peptides of 
 low reporter ion signals because they tend to result in less robust 
 quantification .
proteins that have p - values 0.05 
 in at least one data set and showed consistent changes in the replicates 
 were considered as significantly altered in the expression level . 

antibodies against ndufs3 
 ( mitosciences , invitrogen , carlsbad , ca ) , sdhb , cox iv ( molecular 
 probes , invitrogen ) , acadm , dic , msra , sod2 , mt - cox1 , tomm20 , uqcrfs1 
 ( abcam ) , and cytochrome c ( bd pharmingen , san jose , 
 ca ) were used .
bands were visualized by utilizing peroxidase - conjugated 
 secondary antibodies and advanced chemiluminescence ( ge healthcare , 
 uppsala , sweden ) .
total cytochrome c levels were 
 determined from liver extracts . to detect activation of the mitogen - activated 
 protein kinase ( mapk ) signaling cascade ,
equal amounts of liver lysates 
 were separated and subjected to immunblotting with antibodies specific 
 for ask1 , phospho - ask1 , phospho - ask1 , jnk , phospho - jnk , p38 mapk , 
 and phospho - p38 mapk ( cell signaling 
 technology , beverly , ma ) . to assess the transcriptional regulatory 
 circuits leading to expression of mitochondria gene products , liver 
 lysates were immunoblotted using antibodies against nrf-1 , err 
 ( abcam ) , pgc-1 ( santa cruz biotechnology ) , pgc-1 ( aviva 
 systems biology , san diego , ca ) , proliferator - activated receptor gamma 
 ( ppar ) , pfoxo3a , and foxo3a ( cell signaling 
 technology ) .
quantification of band densities was obtained by scanning 
 the blots with a gs-800 densitometer ( bio - rad ) .
mrm was employed 
 as an alternative validation platform where commercial antibodies 
 were not available .
in addition , mrm provided the opportunity to detect 
 selected proteins missed in the itraq assay , especially lower abundant 
 proteins .
individually , 80 g of peptides from sod2 and sod2 mice liver were labeled with light and 
 heavy mtraq channels ( ab sciex ) and subsequently mixed in a 1:1 ratio . 

the peptides were analyzed using the mrm technique on a tempo nano - lc 
 ( ab sciex ) coupled to a q trap 4000 configuration ( ab sciex ) .
five 
 microliters of sample was injected and desalted online using a reprosil 
 c18-aq trap column ( 5 m  0.3 mm i.d . ; sge analytical 
 science , victoria , australia ) , running at 0.1% formic acid in water 
 for 5 min at 20 l / min .
the peptides were then eluted from a 
 chromolith caprod c18 column ( 0.1 mm i.d .  150 mm ; emd darmstadt , 
 germany ) with a linear gradient starting from 5% b to 30% b ( a , 0.1% 
 formic acid in water ;
b , 0.1% formic acid in acn ) for 30 min at 0.3 
 l / min .
the ion source parameters were optimized at an ion spray 
 voltage of 2800 v , with ion source gas 1 at 40 v , and with the interface 
 heater temperature at 150 c .
the mrm transitions of the peptides 
 were generated from tiqam software ( www.proteomecenter.org ) .
peptides were excluded where there were cysteines and methionines 
 and peptides with two and more consecutive basic amino acids on either 
 the c- or n - terminus .
in addition , the molecular masses of the peptides 
 selected were between 400 and 1200 da .
the masses of the precursor 
 and product ions were then modified on the basis of the mtraq labeling 
 technique .
a total of 430 mrm transitions were used to detect and 
 quantify 25 proteins .
the mrm transitions , the dwell time and collision 
 energy of the mtraq - labeled peptides are illustrated in supporting information table s1 . in order to 
 confirm the presence of the protein , at least 2 mrm transitions from 
 each of its
serum alt activity was measured 
 using a cobas c111 analyzer ( roche ) . 
 for histopathological analysis ,
livers from the same set of mice used 
 in the itraq experiments were formalin - fixed and paraffin - embedded . 

the fixed tissues were subsequently processed with an automatic tissue 
 processor ( leica tp 1020 , germany ) and embedded in paraffin blocks . 

two micrometer tissue sections were stained with hematoxylin and eosin 
 and analyzed by light microscopy .
for ihc , the formalin - fixed , 
 paraffin - embedded sections were dewaxed and rehydrated , and antigen 
 retrieval was performed by heating in sodium citrate buffer ( 10 mm 
 sodium citrate , 0.05% tween 20 , ph 6.0 ) for 20 min .
sections 
 were then blocked and immunostained using an envision + system - hrp 
 labeled polymer kit ( dako , denmark ) .
anti - phospho - foxo3a antibodies ( cell signaling technology ) were incubated overnight 
 at 4 c , and detection of antigens was performed using 3,3-diaminobenzidine . 

the sections were counterstained using lillie s modified mayers 
 hematoxylin , dehydrated , mounted , and viewed under a light microscope 
 ( carl zeiss , oberkochen , germany ) .
monochlorobimane features 
 a high selectivity for gsh and is conjugated to glutathione ( gsh ) 
 by a glutathione - s - transferase - catalyzed reaction .
the fluorescence 
 was determined at 380/460 nm , and gsh levels were calculated from 
 a standard curve formed from glutathione positive controls .
cytochrome 
 c ( r & d systems , minneapolis , mn ) and nitrotyrosine ( kamiya biomedical , 
 seattle , wa ) elisa was performed according to the manufacturer s 
 instructions .
mitochondrial 
 aco2 activity was determined from isolated mitochondria using an aconitase 
 activity assay ( cayman chemicals ) , which measures the formation of 
 nadph from nadp+ concomitant with the conversion of citrate to isocitrate 
 and of isocitrate to -ketoglutarate .
8% nondenaturing gels were used to monitor aco2 higher 
 molecular weight aggregates ( > 250 kda ) from mitochondrial extracts . 

12.5% denaturing gels were used to monitor intact ( 83 kda ) and cleaved 
 aco2 ( 40 kda ) .
antibodies specifically targeting residues 
 767780 of the carboxy terminus of aco2 ( a generous gift from 
 dr . bi xuezi ) were used for immunoblotting 
 analysis .
bands were visualized by utilizing peroxidase - conjugated 
 secondary antibodies and advanced chemiluminescence ( ge healthcare ) .
hepg2 cells were cultured in the presence of dulbecco s 
 modified eagle s medium ( dmem ; sigma ) , 100 iu penicillin/g 
 streptomycin , and 10% fetal bovine serum at 37 c in a humidified 
 incubator with 5% co2 .
the cells were washed with pbs and 
 treated with various concentrations of troglitazone , dissolved in 
 0.1% dmso , at different time - points in serum - free dmem ( 1 , 2 , and 
 5 h ) . at 5 h ,
100 m troglitazone was determined as the cytotoxic 
 dose , which is consistent with previous studies .
hepg2 cells were seeded ( 0.25  10/well ; 
 24-wells ) , and at 7080% confluency , they were transfected 
 with a pool of four predesigned sirna targeting slc25a10 ( 50 nm , on - targetplus smartpool sirna , dharmacon , thermo scientific , 
 waltham , ma ) for 18 h using 2.5 l of transit - tko as the transfection 
 reagent ( mirus bio , madison , wi ) in the absence of streptomycin .
d-001830 - 20 , thermo scientific ) , 
 scrambled nontargeting sirna pool ( catalog no .
d-001810 - 10 , thermo 
 scientific ) , dmem + 0.1% dmso , and dmem + transit - tko ( mock ) served 
 as controls .
small molecule inhibition via 20 mm butylmalonate ( sigma ) 
 or antioxidant gsh - ethyl ester ( sigma ) was added to hepg2 cells for 
 1 h prior to the addition of troglitazone .
cell viability was measured 
 by the formation of formazan using the mts ( promega , madison , wi ) 
 assay at 490 nm .
we performed our network - based 
 profiling utilizing the proteomics expansion pipeline or pep as described 
 previously .
briefly , the mitoproteome 
 data set identified formed the basis of our seed selection .
a seed 
 here is a high - confidence identified protein defined by the criteria 
 as stated above .
this selection process reduced the original 277 identified 
 proteins to a set of 113 seeds .
we built an expanded mouse ppin by 
 merging data from two data sources : mppdb and intnetdb .
protein 
 interaction ( ppi ) database , and we used the reference set of mouse 
 ppi data collected over five ppi databases : dip , bind , mips , mint , 
 and intact .
hence , we used intnetdb to obtain interologs from human ppin to mouse , 
 and we merged this with the mppdb data set .
the resultant network 
 of 10307 nodes and 124866 edges was then subfiltered for edges between 
 mitochondrial and its associated proteins from mitocarta ( http://www.broadinstitute.org/pubs/mitocarta/ ) .
this subfiltered network , which we henceforth refer to as the 
 mitonetwork , was utilized for functional analysis of our proteomics 
 data .
ingenuity pathway 
 analysis ( ipa ; version 7.1 ) was used for the analysis of over - represented 
 biological pathways .
right - tailed fisher s exact test ( p < 0.05 ) is used for multiple testing of over - represented 
 pathways . to detect differences in distribution of fold changes , we 
 performed student s t - test on the wild type 
 versus sod2 mouse ( p < 0.05 ) and 1-way anova with bonferroni 
 posthoc test for the toxicological study ( 14 and 28 days ; p < 0.05 ) .
distances between samples 
 were determined using euclidean distance , and cluster distances were 
 determined using ward s method .
gene ontology ( go ) enrichment 
 analysis was performed using go - term finder .
terms 
 were identified using the standard hypergeometric test ; significance 
 was defined at padjusted < 0.01 using 
 the bonferroni multiple testing correction .
metabolic 
 process , cellular metabolic process , and primary metabolic process  
 were considered broad terms and were not included in the analysis . 

unique annotation terms were extracted by pairwise comparisons between 
 all identified expression clusters . for the prediction of troglitazone 
 induction on ppar target genes ,
corresponding matched dna genomic 
 sequences were obtained and screened using matinspector ( version 8.0 ) 
 for ppar response elements ( ppres ) .
genomic sequences and transcription 
 start sites were obtained from the ensembl database ( release 49 ) , 
 and regions spanning 5000 bp upstream and downstream of the transcriptional 
 starting sites were extracted to accommodate for genes with unusual 
 ppre locations . only the v$pero matrix 
 family was included in the search . to increase the stringency , core 
 similarity and matrix similarity scores for each prediction
this gave rise to 325 potential ppre sites in 
 76 genes / proteins ( 14 days , 136 genes with 507 predicted sites ; 28 
 days , 148 genes with 584 sites ; noting several common genes between 
 the two time courses ) . to test
if the presence of a predicted ppre 
 site gives rise to larger fold changes , we split the expression data 
 into two categories on the basis of bespoke ppre analyses , and we 
 compared their fold distributions . as the fold distributions are non - normal , 
 significant differences were determined using the nonparametric mann 
the monoallelic loss of sod2 provided the opportunity 
 to examine how the mitoproteome copes with the damaging effects of 
 free radicals under diminished protection .
liver mitochondria from sod2 and sod2 mice 
 ( n = 4 per group ) were compared , and 321 mitochondrial 
 proteins were identified with high confidence ( figure 2a ) . we confirmed the quantitative specificity of ms to detect sod2 haplodeficiency ( r+/:+/+  0.53 ; figure 2b ) .
itraq has been 
 reported to suppress quantification , in particular with the larger 
 fold changes . therefore , while the observed 
 itraq ratio of sod2 follows that of the expected ratio , we can not 
 rule out potential underestimation of the larger fold changes .
the 
 expression of several selected proteins was confirmed by immunoblotting 
 and mrm , demonstrating the accuracy of our ms quantification ( supporting information figure 2a and b ) .
silver 
 staining of whole proteomes run on 1d sds - page gel acted as loading 
 controls ( supporting information figure 2c ) .
vertical dotted lines denote 
 significant fold change cut - offs , defined as r+/:+/+  1.2 for up - regulation and 0.833 
 for down - regulation , and horizontal dotted lines denoted p  0.05 .
the inset shows the proportion of the sod2 mitoproteome that was unchanged , 
 downregulated , or upregulated relative to sod2 .
( b ) ms / ms spectrum of the m / z region of reporter ions generated from itraq 
 labeled sod2 tryptic peptide .
compensatory responses to maintain mitochondrial functionality were evidenced by the up - regulation of a larger 
 proportion of the mitochondrial proteins ( 25% ) compared to down - regulated 
 proteins ( 13% ; supporting information table 
 s2 ) .
ros levels are modulated in young sod2 mice , and 
 we asked if this ros homeostatic maintenance is maintained in part 
 by the antioxidant enzymes housed within the mitochondria .
gsh , a 
 major low - molecular - weight antioxidant is regulated by a network of 
 enzymes and oxidoreductases to maintain its homeostasis .
depletion 
 of mgsh is a hallmark of endogenous and chemically induced oxidant 
 stress , and in the absence of de novo gsh synthesis 
 in the mitochondria , maintenance of the mgsh pool is crucial to ensure 
 mitochondrial redox balance . the up - regulation 
 of gstk1 , mgst1 ( supporting information table s2 ) , and gpx1 suggests increased 
 recycling of mgsh to restore redox equilibrium with the partial ablation 
 of sod2 . proteins with iron
 sulfur ( [ fe  s ] ) 
 clusters are known for their exquisite sensitivity to oxidative stress 
 and used if these mitochondrial proteins are affected .
because of 
 their low abundance , we measured fxn and fdx1 levels by mrm and found 
 lower levels in sod2 liver ( supporting information figure 
 2b ) . under oxidative stress , [ fe  s ] proteins may be damaged 
 and degraded , or their biogenesis interrupted . given the many roles of [ fe  s ] proteins , which include 
 stabilizing structures and acting as electron carriers and regulatory 
 sensors , it is crucial to maintain a 
 steady pool of [ fe  s ] proteins with nondisrupted [ fe  s ] 
 clusters .
concordantly , we found enrichment of tst ( r+/:+/+  
 1.46 ) , whose role includes the formation of [ fe  s ] clusters . 

similar levels of endogenous mgsh , mitochondrial protein carbonyl , 
 and nitrotyrosyl adducts in sod2 and sod2 mice were observed ( student s t - test , p = 0.19 , p = 0.11 , and p = 0.92 respectively ; supporting information figure 3a ) , reflecting gsh - induced protection from oxidative stress .
go enrichment analysis of the up- and down - regulated proteins revealed 
 statistically significant alteration of several  biological 
 processes  associated with the loss of a single sod2 allele ( supporting information figure 3b ) .
expectedly , electron transport chain and redox homeostasis were 
 implicated ( padj = 5.24 
  10 and 3.61  10 , respectively ) , consistent with increased mitochondrial oxidative 
 stress and respiration .
many biochemical functions 
 were also perturbed , including fatty acid metabolism ( padj = 1.95  10 ) .
glutamine is a precursor of gsh , and 
 the overrepresentation of glutamine metabolism ( padj =  10 ) 
 and gsh metabolism ( padj = 8.66  10 ) provided further evidence 
 of gsh perturbation .
taken together , our data suggest that there is 
 attenuation of ros due to an upregulated antioxidant defense , and 
 also discrete molecular aberrations that are present that may sensitize 
 the sod2 mouse 
 to clinically silent drug toxicities .
a hallmark of tili 
 is the delayed onset of liver injury , which could abruptly progress 
 to life - threatening irreversible liver failure .
troglitazone administration in sod2 ( wild type ) mice did not result in hepatic 
 injuries , and because sod2 mice below the age of 20 weeks 
 accumulate clinically silent oxidative stress , we focused on studying troglitazone effects on the sod2 hepatic mitochondria . 

histological examination of sod2 liver displayed a two - phase response that mirrors the delayed 
 onset of liver injury characteristic of idiosyncratic tili in humans  an 
 early nontoxic phase and a later sustained phase of liver injury , 
 exemplified by confluent areas of cytoplasmic vacuolation and hepatocytic 
 death ( figure 3a ) .
serum alanine aminotransferase 
 levels significantly increased ( 187% ) in sod2 treated with 28 days troglitazone 
 relative to solutol administered sod2 mice ( figure 3b ) .
( a ) representative hematoxylin 
 and eosin liver sections prepared from solutol , and troglitazone - treated sod2 mice dosed over 
 either 14 days or 28 days at 100 magnification .
insets at the 
 top left are observations made at 400. black arrows indicate 
 vacuolation of hepatocytes .
( b ) serum alanine aminotransferase levels 
 of sod2 mice 
 administered with 14 days or 28 days troglitazone .
the total 
 number of proteins that make up a pathway is displayed on the top 
 of columns .
the ratio is defined as the number of proteins analyzed 
 against the total number of proteins in a pathway .
veh , vehicle - administered ; trg , troglitazone - administered . to understand the associated mitoproteome 
 changes
, we performed 
 8-plex itraq to interrogate the mitochondrial profiles across 14 and 
 28 days drug administration ( 30 mg / kg daily , i.p . ) .
a total of 314 
 proteins were identified ( supporting information table s3 ) , and hierarchical clustering ( hcl ) showed that temporal - defined 
 proteins were classified into three major clusters ( figure 3c ) . by immunoblotting
, we validated the differential 
 expression of selected mitochondrial proteins and found good correspondence 
 with ms - quantification ( supporting information 
 figure 4a ) .
we evaluated the attributes of detected proteins 
 against mitocarta , and they followed 
 the distribution of annotated mitochondrial proteins ( supporting information figure 4b ) .
the detected 
 proteins have the tendency to describe their functional clusters substantially , 
 covering 44 out of 48 go slim terms annotated to the mitochondrial 
  biological process  ( supporting 
 information figure 4c ) .
pathway analysis revealed that these 
 perturbed clusters include mitochondrial dysfunction , fatty acid metabolism , 
 and oxidative stress ( figure 3c , supporting information figure 5 and table s4a ) , concordant with biochemical end points supporting the association 
 of mitochondrial oxidative stress in troglitazone hepatotoxicity .
protein networks can reflect interdependencies 
 between proteins 
 and potentially identify functionally important groups of proteins 
 and/or important proteins ( hubs ) .
to 
 better understand the emergent modular properties of the mitoproteome 
 with troglitazone exposure , we applied a network - cleaned clique - enrichment 
 approach on our proteomics data set and derived an 
 extended mouse specific mitochondrial network of 798 nodes , and 1145 
 edges from the single largest connected component and 1206 nodes , 
 2103 edges ( supporting information figure 6a ) .
the well - defined topological interconnectivity of the 14 and 28 
 days ppins suggests a transitory response from an early adaptor phase 
 to a stressed phase with prolonged troglitazone administration . at 
 14 days
, there were few major changes in the mitoproteome , of which 
 we observed slight perturbation in lipid metabolism , specific to the 
 troglitazone s intended pharmacological action to normalize 
 glucose and lipid levels .
in contrast , at 28 days , as a consequence 
 of prolonged troglitazone treatment , there were widespread mitoproteome 
 changes , including cell death , fatty acid metabolism perturbation , 
 and responses to oxidative - stress clusters ( supporting 
 information figure 6b and table s4b and c ) .
troglitazone 
 induces the expression of ppar - responsive lipid metabolism 
 genes , and this motivated us to investigate 
 any inadvertent  off - target  troglitazone effects on 
 fatty acid metabolism . to test this ,
a position - weight matrix was 
 applied to predict ppre sequences among the differentially regulated 
 proteins . within expectations , at 14 days , proteins with ppre motifs 
 ( supporting information table 5 )
were up - regulated 
 relative to ppre - absent proteins ( wilcoxon p < 
 0.01 ) .
in contrast , at 28 days , the protein expressions of both groups 
 were similar ( wilcoxon p = 0.196 ; supporting information figure 6c ) , suggesting a lesser degree 
 of troglitazone - induced regulation on ppre protein expressions and 
 also off - target ppar - dependent toxicity .
these data suggest that perturbations 
 in lipid metabolism partially contribute to the tili , together with 
 other perturbed clusters .
however , we do not preclude the possibility 
 that certain determinants of toxicity may interact and overlap with 
 pharmacologically mediated changes .
we hypothesized that 
 prolonged troglitazone exposure leads to accumulated 
 oxidative stress that results in a transition from redox equilibrium 
 to disequilibrium breaches .
therefore , we assessed the ramifications 
 of troglitazone administration on the mitochondrial redox proteins . 

we found sod2 up - regulation ( r28days:14days  1.29 ) despite intrinsic heterozygosity of sod2 ( figure 4a ) , concomitant with 
 the up - regulation of prdx3 , glrx5 , and gpx1 , proteins that were involved 
 in maintaining redox homeostasis ( r28days:14days  1.44 , 1.21 , and 1.23 , respectively ) . to further investigate 
 the impact of ros - induced mitochondrial stress caused by troglitazone , 
 we examined aco2 , a mitochondrial matrix [ fe  s ] tricarboxylic 
 acid cycle enzyme that displays exquisite sensitivity to ros .
aco2 
 activity decreased by 30% in 14 days and > 55% with 28 days of troglitazone 
 treatment , while aco2 levels remain basal ( r14days  0.98 and r28days  1.08 ; supporting information figure 7a ) . by immunoblotting ,
40 
 kda aco2 fragments suggest ros - induced cleavage of aco2 with 14 days 
 of troglitazone administration , consistent with proteolytic degradation 
 by lonp1 under moderate stress . continued 
 troglitazone administration caused the aggregation of aco2 ( supporting information figure 7b ) , which is reflective 
 of increased , chronic o2 in 
 the sod2 mouse 
 liver mitochondria .
other indices of 
 oxidative damage , such as mitochondrial nitrotyrosine adducts , no 
 levels ( 1-way anova , p < 0.05 , p < 0.001 , respectively ; supporting information 
 figure 7c and d ) , and protein carbonyls , increased significantly after 28 days of troglitazone administration , 
 with a concomitant mgsh depletion ( 1-way anova , p < 0.05 ; figure 4b ) and increase of cytochrome c release into the cytosol ( 1-way anova , p < 0.01 ; supporting information figure 7f ) , a key initial step in the apoptosis process .
itraq 
 channels 116 and 114 constitute 14 days of troglitazone administration , 
 channels 115 and 113 constitute 14 days of control treatment , while 
 channels 121 and 118 constitute 28 days of troglitazone administration , 
 and channels 119 and 117 constitute 28 days of control treatment . 

( b ) 
 measurements of mitochondrial gsh levels of sod2 mice after 14 and 28 days troglitazone 
 administration .
n = 36 per group ; 1-way anova with bonferroni post - test ; veh , 
 vehicle - administered ; trg , troglitazone - administered .
< 0.05 for 28 days 
 treated compared with 14 days treated ; * p < 0.05 
 and * * * p < 0.001 for 28 days treated compared 
 with 28 days vehicle .
( d ) 
 hepg2 cells were transfected with a pool of sirnas , and cell viability 
 was measured by mtt assay .
nontargeting sirna acted as negative control , 
 and gapd sirna acted as positive control .
dose response assays were 
 normalized to 0.1% dmso , and troglitazone - treated sirna assays were 
 normalized to mock .
p < 0.001 relative to dmem + 0.1% dmso ; * * * p < 0.0001 relative to 100 m troglitazone ( 2 h ) after normalizing 
 to 0.1% dmso ; p < 0.0001 relative 
 to dic sirna .
bm , butylmalonate ; ctrl , nontargeting sirna ; mock , transfection 
 reagent ; gsh - ee , gsh - ethyl ester ; trg , troglitazone .
depletion of mgsh may be due to the down - regulation 
 of dicarboxylate 
 carrier ( dic ; slc25a10 ; r28days:14days  0.71 ) , one of the two mgsh import proteins . because of the importance in maintaining mgsh homeostatic levels 
 through active transport , we investigated the functional relevance 
 of troglitazone - induced dic down - regulation via genetic and pharmacological 
 assays .
attenuation of slc25a10 ( figure 4b ) by rnai in hepg2 cells treated with 100 m 
 troglitazone accelerated troglitazone - induced cytotoxicity from 5 
 to 2 h ( figure 4c ; 1-way anova , p < 0.0001 relative to 2 h troglitazone treatment ) .
treatment with 
 gapd - sirna or nontargeting sirna in the presence of troglitazone confirmed 
 the  toxic - enhancement  effect of dic knockdown .
similarly , 
 incubation with butylmalonate , a dic inhibitor , in the presence of 
 troglitazone resulted in increased cytotoxicity ( 1-way anova , p < 0.0001 relative to 2 h troglitazone incubation ) . 

when cells were pretreated with gsh - ethyl ester , troglitazone - induced 
 cytotoxicity was blunted ( 1-way anova , p < 0.001 ) . 
 therefore , dic is necessary for the maintenance of mgsh transport 
 and attenuates the cytotoxic effects of troglitazone . taken together , 
 these data suggest that chronic troglitazone exposure in sod2 mouse liver leads to decreased 
 mgsh import and a buildup of ros , potentially leading to intolerable 
 mitochondrial damage and triggering the abrupt progression of hepatocytic 
 death .
ros are 
 potent stressors to jnk and p38 activation , and their involvement 
 in troglitazone - induced cytotoxicity has been noted previously .
we observed ask1 activation , the upstream activator of jnk ( phosphorylation 
 of thr845 and dephosphoryation of inhibitory residue ser83 ) and jnk1/2 
 activation but not p38 after 28 days of troglitazone administration 
 ( figure 5a ) .
by contrast , jnk1/2 were minimally 
 phosphorylated in control and 14 days treated groups ( figure 5a ) .
therefore , accumulated mitochondrial oxidative stress 
 enhanced with troglitazone led to ask1-jnk activation in vivo but not p38 in the sod2 mice .
( a ) liver homogenates were subjected to immunoblotting 
 with pask1 , pask1 , ask1 , pjnk , jnk1/2 , 
 pp38 , and p38 antibodies . elevated oxidative stress from 4 weeks of 
 troglitazone administration induced ask1 and jnk1/2 activation .
the 
 right panel shows mean normalized density values relative to the corresponding 
 nonphosphorylated form .
( b ) immunoblot analysis of transcriptional 
 regulators and coactivators involved in regulating nuclear - encoded 
 mitochondrial proteins in sod2 and sod2 mice .
( c ) liver homogenates were subjected to immunoblotting with 
 foxo3a , pfoxo3a , pgc-1 , pgc-1 , err , 
 nrf-1 , and ppar antibodies .
( d ) liver sections were subjected 
 to immunohistochemistry by incubating with pfoxo3a antibody and counterstained .
extended administration of sod2 mice with troglitazone 
 induced the nuclear translocation of foxo3a in areas of hepatocytic 
 degeneration but not the vehicle ( solutol ) .
( e ) proposed schematic diagram 
 showing that extended troglitazone administration coupled to increased 
 mitochondrial oxidative stress in sod2 mice primed ask1-jnk - foxo3a activation , 
 which in turn resulted in mitoproteome reconfiguration and dysregulated 
 lipid metabolism , enhancing mitochondrial ros regulation in a feedback 
 loop .
jnk has been implicated in the activation of foxo3a under 
 sustained 
 oxidative stress in antagonism to akt .
notably , in vertebrates and c. elegans , foxo3a 
 acts as a transcriptional factor in the up - regulation of sod2 as a 
 form of oxidative - stress defense . as shown in 
 figure 5b ,
endogenous foxo3a was inherently 
 activated in the sod2 mouse liver ( indicated by moderate dephosphorylation at ser253 ) , 
 possibly in response to elevated ros . in sod2 mouse , however , foxo3a remained inactivated 
 ( phosphorylated ) . administering sod2 mice with troglitazone for 28 days resulted 
 in markedly reduced p - foxo3a and its retention in 
 the cytosol ( figure 5c and d ) . using immunohistochemistry
, 
 we showed that , in areas of confluent hepatocytic degeneration , activation 
 of foxo3a occurred in a dephosphorylation - dependent manner .
in contrast , 
 in the neighboring areas denoted by surviving hepatocytes , foxo3a 
 remained phosphorylated at ser253 and was retained in the cytoplasm 
 ( figure 5d ) .
besides foxo3a , several 
 crucial transcriptional factors or activators 
 of nuclear - encoded gene regulatory programs were evaluated in their contributions in governing the observed 
 differential expression of the sod2 mitochondrial proteins in the absence and presence of troglitazone . 

given that in certain cell types pgc-1 drives the gene expression 
 of sod2 under pro - oxidant status and that troglitazone restores pgc-1 levels , we investigated if troglitazone - treated sod2 mitoproteome changes were 
 pgc-1 and pgc-1-dependent .
consistent with the lack 
 of increase of lrpprc levels ( r28days:14days  0.96 ) , which up - regulates pgc-1 and pgc-1 , pgc-1 and pgc-1 remained at similar 
 levels throughout the study and were independent of the form of treatment 
 ( figure 5c ) .
hepatocyte ppar expression 
 has been reported to increase with troglitazone , but our immunoblot analysis revealed no significant differences , 
 consistent with another pharmacoproteomic study .
in addition , no significant difference was observed with 
 duration or drug - treatment for two other well - established transcriptional 
 regulators of nuclear - encoded mitochondrial proteins , nrf-1 and err- 
 ( figure 5c ) . taken together ,
our results demonstrate 
 that a compromised intrinsic mitochondrial antioxidant defense combined 
 with prolonged troglitazone exposure leads to the specific activation 
 of ask1-jnk and foxo3a , driving a reconfiguration of the liver mitoproteome . 
 despite compensatory measures such as the upregulation of important 
 antioxidant mitochondrial proteins , oxidative damage due to the lack 
 of sustained mgsh import
in 
 this study we described an integrative toxicoproteomics approach 
 on a mouse model of compromised mitochondrial antioxidant defense 
 for the study of the idiosyncratic hepatotoxicant , troglitazone . by 
 combining high throughput ms - based mitoproteome - wide profiling , biochemical 
 end points , and network biology
, we demonstrated that the sod2 hepatic mitoproteome 
 followed a two - phase response to repeated troglitazone administration 
 that cumulated in liver injuries by the fourth week .
this integrative 
 approach identified the combined deterioration of key fragile nodes 
 including attenuated dic levels and a dysfunctional mgsh transport 
 system that lead to the eventual toxicity of troglitazone .
inhibition 
 of dic through pharmacological or genetic means sensitized cells to 
 troglitazone - induced cytotoxicity and was reversed when pretreated 
 with a gsh donor .
the mgsh node is intrinsically perturbed by 
 a compromised mitochondrial 
 ros defense in the sod2 mouse , and this fragile node conferred a point of sensitization 
 for troglitazone to expose its toxic effect .
data documented by us 
 and others suggest that dic may be a key protein in exemplifying tili : 
 ( i ) its temporal inverse differential expression parallels the two - phase 
 change observed at tissue level ; ( ii ) dic knockdown in c. 
 elegans increases ros production , which could result in further 
 oxidative damage ; and ( iii ) the unaffected 
 levels of ogc , together with dic , account for gsh transport into liver 
 mitochondria .
association studies on 
 troglitazone - prescribed type ii diabetes mellitus in japanese patients 
 with elevated serum liver transaminases revealed gene - polymorphisms 
 with combined gstt1 and gstm1 genotypes , and such a correlation 
 suggested that these acquired genetic factors at least partially predisposed 
 certain groups of patients to tili .
importantly , this is consistent 
 with our results and implicated clinical associations of silent genetic 
 abnormalities in ros detoxification and mgsh transport with tili . 

it would be interesting to test for the potential convergence in mgsh 
 cluster perturbation of other dili toxicants implicated with mitochondrial 
 dysfunction .
however , to more accurately reflect mgsh status , determination 
 of coenzyme a and coenzyme a  glutathione mixed disulfide may 
 be employed because measurements of mgsh may not entirely account 
 for intramitochondrial thiol / disulfide exchanges with mgsh .
activation of p38 and jnk has been previously 
 implicated in troglitazone - induced 
 cytotoxicity .
here we demonstrated in vivo jnk activation in mediating ros - induced cell death 
 and that mgsh depletion could trigger a signaling crosstalk between 
 ros - mediated ask1-jnk and foxo3a , plausibly through jnk - dependent 
 phosphorylation of 14 - 3 - 3 .
this led to the activation 
 of transcriptional programs to reconfigure the mitoproteome , including 
 increased sod2 expression to attenuate troglitazone - induced oxidative 
 stress .
in addition , the recurring themes of lipid metabolism suggest 
 that continual troglitazone exposure might perturb lipid metabolism 
 as collateral damage . this is crucial because free fatty acids can 
 enhance mitochondrial - generated ros by inhibiting oxidative phosphorylation 
 and by interfering with electron flow , further exacerbating oxidative damage .
application of proteomic 
 studies will help to elucidate the complexity 
 behind idiosyncratic - dili as a probable multifactorial disease and 
 perhaps offer better therapeutic design .
deeper coverage by ms - based 
 proteomics strengthens such efforts by unbiasedly capturing the complexities 
 and dynamics of proteome changes at a global scale .
it has been proposed 
 that differences in the proportion of abnormal mitochondria and sod2 mitochondria in individual 
 cells ( heteroplasmy ) have different consequences on the viability 
 of a cell or the ability to function optimally , also known as the 
 threshold effect .
undoubtedly , single 
 cell proteomics can be applied to assess if heteroplasmic cells harboring 
 more mitochondrial mutations have a higher propensity to initiate 
 drug - induced toxic cascades . at a time 
 when drug - induced toxicities are a growing concern , platforms have 
 been initiated to modernize the drug 
 development process and drug safety evaluation , including to improve 
 our understanding of the mechanisms underlying dili and for its better 
 prediction .
broadly , this present study may represent a powerful step 
 forward in using a systems approach in a mouse model of underlying 
 genetic abnormality to advance our understanding of the risk factors 
 in other idiosyncratic toxic drugs and tests of hepatotoxic signals .
 troglitazone , 
 a first - generation thiazolidinedione of antihyperglycaemic 
 properties , was withdrawn from the market due to unacceptable idiosyncratic 
 hepatotoxicity . despite intensive research , 
 the underlying mechanism 
 of troglitazone - induced liver toxicity remains unknown . 
 here we report 
 the use of the sod2+/ mouse model of silent mitochondrial oxidative - stress - based 
 and quantitative mass spectrometry - based proteomics to track the mitochondrial 
 proteome changes induced by physiologically relevant troglitazone 
 doses . by quantitative untargeted proteomics , 
 we first globally profiled 
 the sod2+/ hepatic 
 mitochondria proteome and found perturbations including gsh metabolism 
 that enhanced the toxicity of the normally nontoxic troglitazone . 
 
 short- and long - term troglitazone administration in sod2+/ mouse led to a mitochondrial 
 proteome shift from an early compensatory response to an eventual 
 phase of intolerable oxidative stress , due to decreased mitochondrial 
 glutathione ( mgsh ) import protein , decreased dicarboxylate ion carrier 
 ( dic ) , and the specific activation of ask1-jnk and foxo3a with prolonged 
 troglitazone exposure . 
 furthermore , mapping of the detected proteins 
 onto mouse specific protein - centered networks revealed lipid - associated 
 proteins as contributors to overt mitochondrial and liver injury when 
 under prolonged exposure to the lipid - normalizing troglitazone . by 
 integrative toxicoproteomics 
 , we demonstrated a powerful systems approach 
 in identifying the collapse of specific fragile nodes and activation 
 of crucial proteome reconfiguration regulators when targeted by an 
 exogenous toxicant . 
Introduction
Experimental Procedures
Results
Discussion