Perturbation of bile acid homeostasis is an early pathogenesis event of drug induced liver injury in rats
Makoto Yamazaki a ,Manami Miyake a ,Hiroko Sato a ,Naoya Masutomi a ,Naohisa Tsutsui a ,
Klaus-Peter Adam b ,Danny C.Alexander b ,Kay A.Lawton b ,Michael V.Milburn b ,John A.Ryals b ,Jacob E.Wulff b ,Lining Guo b ,⁎
a Mitsubishi Tanabe Pharma Corporation,Kisarazu,Chiba 292-0818,Japan b
Metabolon Inc.,617Davis Drive,Suite 400,Durham,NC 27713,USA
a b s t r a c t
a r t i c l e i n f o Article history:
Received 4September 2012Revid 3January 2013Accepted 18January 2013
Available online 27January 2013Keywords:Metabolomics Biomarker Hepatotoxicity Oxidative stress
Mechanism of action
Drug-induced liver injury (DILI)is a signi ficant consideration for drug development.Current preclinical DILI asssment relying on histopathology and clinical chemistry has limitations in nsitivity and discordance with human.To gain insights on DILI pathogenesis and identify potential biomarkers for improved DILI detec-tion,we performed untargeted metabolomic analys on rats treated with thirteen known hepatotoxins causing various types of DILI:necrosis (acetaminophen,bendazac,cyclosporine A,carbon tetrachloride,ethi-onine),cholestasis (methapyrilene and naphthylisothiocyanate),steatosis (tetracycline and ticlopidine),and idiosyncratic (carbamazepine,chlorzoxasone,flutamide,and nimesulide)at two dos and two time points.Statistical analysis and pathway mapping of the nearly 1900metabolites pro filed in the plasma,urine,and liver revealed diver time and do dependent metabolic cascades leading to DILI by the hepatotoxins.The most consistent change induced by the hepatotoxins,detectable even at the early time point/low do,was the signi ficant elevations of a panel of bile acids in the plasma and urine,suggesting that DILI impaired hepatic bile acid uptake from the circulation.Furthermore,bile acid amidation in the hepatocytes was altered depending on the verity of the hepatotoxin-induced oxidative stress.The alteration of the bile acids was most evident by the necrosis and cholestasis hepatotoxins,with more subtle effects by the steatosis and idiosyncratic hepatotoxins.Taking together,our data suggest that the perturbation of bile acid homeostasis is an early event of DILI.Upo
n further validation,lected bile acids in the circulation could be potentially ud as nsitive and early DILI preclinical biomarkers.
©2013Elvier Inc.All rights rerved.
Introduction
Drug induced liver injury (DILI)is a major challenge for both clini-cians and pharmaceutical rearchers.DILI has been associated with a wide variety of drugs and accounts for a major portion of acute liver fail-ure (Ramachandran and Kakar,2009).It is the leading cau of drug withdraws from the market (Temple and Himmel,2002)and is also among the main reasons for drug candidate attrition (Schuster et al.,2005).Becau of the signi ficant impact of DILI,there is clearly a need to improve DILI detection during the drug developmental process.
DILI can be generally classi fied as hepatocellular,cholestatic,or mixed.Its histological outcomes consist of a wide spectrum of conditions,ranging from steatosis,hepatitis,to acute liver failure (Ramachandran and Kakar,2009).The current pre-clinical asssment for DILI relies pri-marily on histopathology and clinical chemistry bad on a panel of rum biomarkers,including aspartate aminotransfera (AST),alanine aminotransfera (ALT),sorbitol dehydrogena (SDH),glutamate d
ehy-drogena (GLDH)(for hepatocellular damage),alkaline phosphata (ALP),γ-glutamyltransfera (GGT),and total bilirubin (TBil)(for hepa-tobiliary dysfunction)(Boone et al.,2005).However,there are consider-able limitations associated with the standard tests.Histopathology is typically examined at a single end point and may lack suf ficient nsi-tivity.Changes with the liver clinical markers may be contributed by factors other than DILI (Ennulat et al.,2010).Additionally,the current preclinical DILI asssment only correlates with approximately 50%of the cas of human liver toxicity;thus leading to fal positives or fal negatives (Olson et al.,2000).The limitations in nsitivity and speci ficity have prompted the discovery of new DILI biomarkers that can complement the current methodology.
Toxicology and Applied Pharmacology 268(2013)79–89
Abbreviations:ALP,alkaline phosphata;ALT,alanine aminotransfera;ANIT,1-naphthylisothiocyanate;APAP,acetaminophen;AST,aspartate aminotransfera;BDZ,bendazac;CBZ,carbamazepine;CCL4,carbon tetrachloride;CSA,cyclosporine A;CZS,chlorzoxasone;DILI,drug-induced liver injury;ETN,DL-ethionine;FTM,flutamide;GGT,gamma-glutamyl transfera;GLDH,glutamate dehydrogena;MTP,methapyrilene;NMS,nimesulide;SDH,sorbitol dehydrogena;TBil,total bilirubin;
TCP,ticlopidine;TTC,tetracycline.
⁎Corresponding author.Fax:+19195721721.E-mail address: (L.
Guo).0041-008X/$–e front matter ©2013Elvier Inc.All rights rerved.
dx.doi/10.1016/j.taap.2013.01.018
Contents lists available at SciVer ScienceDirect
Toxicology and Applied Pharmacology
j o u rn a l ho m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /y t a a p
The key to predicting the progression and outcome of hepatic injury is understanding the underlying mechanisms.Various studies have been conducted to discover biomarkers for improved DILI detection using technologies such as genomics,proteomics,and metabolomics (Andrade et al.,2009;Beger et al.,2010;Collins et al.,2010).However, many of the studies analyzed only a single or a very limited number of hepatotoxins at a single do/time point,thereby restricting the scope of thefindings.More extensive efforts toward developing DILI bio-markers carried out by various consortia,such as InnoMed PredTox, Critical Path Liver Toxicity Biomarker Study(LTBS),and the Consortium for Metabonomic Toxicology(COMET),have yet to come to full fruition.
Here we report the u of untargeted metabolomics to gain insights into DILI induced pathogenesis and explore the feasibility of identifying early and nsitive DILI biomarkers.Rats were dod with div
er types of hepatotoxins that are known to cau hepatic necrosis,cholestasis,or steatosis in rats,as well as with hepatotoxins that do not cau obrv-able injury in rats but are known to be associated with liver injury in humans(idiosyncratic hepatotoxins).We were able to identify pertur-bation of bile acid homeostasis as a common and early event of DILI by the diver hepatotoxins.Negative controls such as testicular and muscular toxins did not significantly alter the levels of major plasma bile acids.This understanding led to the identification of specific bile acid species as potential early and more nsitive DILI biomarkers in rats.
Materials and methods
蓝天沙发Drugs,dosing regimen,and animal studies.Thirteen compounds that are known to cau liver toxicity were lected for this study.For each compound,a low do and a high do were administrated.The list of the hepatotoxins,dos,administration method,and vehicles were summarized in Table1.Crl:CD(SD)male rats of six weeks of age ud in this study were from Charles River Japan,Inc.(Atsugi Breeding Center,Tokyo,Japan).All the experimental animals were houd in stainless-steel cages in a room that was lit for12h(7:00–19:00)daily, ventilated with an air-exchange rate of10–20times per hour,and maintained at20–26°C with a relative humidity of30–70%.All animals were allowed free access to water and food(CRF-1,sterilized by radia-tion,Oriental Yeast Co.,Tokyo,J
apan)except for a4-hour fasting period prior to plasma collection.After a5-day acclimatization period,the rats were randomly assigned to experimental groups(n=5for each group). For each compound,two groups of animals received the low do, two groups of animals received the high do,and the other two groups of animals received a corresponding vehicle control.The plasma (anti-coagulated with sodium EDTA),urine,and liver samples were collected on day2and day5.Urine was collected over24h with the collection vesls surrounded by dry ice.After urine collection,rats were maintained under fasting conditions for4h after which plasma samples were collected.The animals were then euthanized to collect liver for pathological examination.All procedures of animal studies were performed in accordance with the rule of the Institutional Animal Care and U Committee at the study facility.
Metabolomic profiling.The metabolomic platform consisted of three independent methods:ultrahigh performance liquid chromatography/ tandem mass spectrometry(UHLC/MS/MS2)optimized for basic species, UHLC/MS/MS2optimized for acidic species,and gas chromatography/ mass spectrometry(GC/MS).The detailed descriptions of the platform, including sample processing,instrument configuration,data acquisition, and metabolite identification and quantitation,were published previ-ously(Evans et al.,2009;Ohta et al.,2009).Esntially,the samples
were extracted and split into three equal aliquots for analysis by the three methods.For the two LC methods,chromatographic paration followed by full scan mass spectra was carried out to record reten-tion time,molecular weight(m/z)and MS/MS2of all detectable ions prented in the samples.For GC,the samples were derivatized using bistrimethyl-silyl-triflouroacetamide.The retention time and molecular weight(m/z)for all detectable ions were measured.The metabolites were identified by comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time,molecular weight(m/z),pre-ferred adducts,and in-source fragments as well as their associated MS/MS2spectra.
Data imputation and statistical analysis.For statistical analysis,the missing values for a given metabolite were imputed with the ob-rved minimum detection value bad on the assumption that they were below the limits of instrument detection nsitivity.Statistical anal-ysis of the data was performed using JMP(SAS,) and“R”(cran.r-project/).Welch's two-sample t-tests were performed on the log-transformed data to compare the treatment and control groups for each drug do at each time point.Multiple com-parisons were accounted for with the fal discovery(FDR)rate method, and each FDR was estimated using q-values.For the convenience of data visualization,the raw area counts for each biochemical were re-scaled by dividing the value f
or a specific biochemical in each sample by the medi-an value obrved for that specific biochemical.
Quantitative determination of glycocholate and taurocholate in the plasma and cholate in the urine.For absolute quantitation,the metabolites were analyzed by isotope dilution UHPLC-MS/MS.The range of quanti-tation was0.0250to5.00μg/mL for cholate,0.0400to16.0μg/mL for glycocholate,and0.0500to20.0μg/mL for taurocholate.Samples with higher levels were appropriately diluted with PBS to be covered by the calibration range.The calibration standard and the internal stan-dard solutions were prepared in methanol/water(4:1).For the plasma sample preparation,50.0μL/mL of rat plasma was spiked with20.0μL internal standard solution(4.00μg/mL glycocholate-D4, 5.00μg/mL taurocholate-D5,)and subjected to protein precipitation by vigorously mixing with200μL methanol.Following centrifugation, 1.0μL of the supernatant was injected onto a Agilent1290/Sciex5500QTrap LC-MS/MS system.For the urine sample preparation,50.0μL/mL of rat urine was spiked with20.0μL internal standard solution(1.00μg/mL cholate-D4)and vigorously mixed with50.0μL methanol.Following centrifugation,1.0μL of the supernatant was injected onto a Agilent 1290/Sciex5500QTrap LC-MS/MS system.
The chromatographic conditions were identical for plasma and urine analys and included the follo
wing:Acquity C18BEH column,1.7μm, 2.1×50mm(Waters);mobile pha A:6.5mM ammonium bicarbonate in water;mobile pha B:6.5mM ammonium bicarbonate in methanol/ water(95:5);flow rate:0.550mL/min;gradient:initial,30%mobile pha A;0.7min,10%pha mobile A,linear;1.00min,10%pha mo-bile A;linear;1.05min30%pha mobile A,linear;retention times
Table1
Hepatotoxins and their dos ud in this study.All hepatotoxins were administrated
with oral gavage.CMC-Na:sodium carboxymethyl cellulo.
Hepatotoxin Abbreviation Dos
(mg/kg/day)
Vehicle
1-naphthyl isothiocyanate ANIT1550Corn oil
Acetaminophen APAP50010000.5%CMC-Na
Bendazac BDZ3001000Corn oil
Carbamazepine CBZ1503000.5%CMC-Na
Carbon tetrachloride CCL4100300Corn oil
Chlorzoxasone CZS50010000.5%CMC-Na
Cyclosporine A CSA50100Corn oil
DL-ethionine ETN1252500.5%CMC-Na
Flutamide FTM501500.5%CMC-Na
Methapyrilene MTP501500.5%CMC-Na
Nimesulide NMS1002000.5%CMC-Na
Tetracycline TTC100020000.5%CMC-Na
Ticlopidine TCP1503000.5%CMC-Na
80M.Yamazaki et al./Toxicology and Applied Pharmacology268(2013)79–89
were 0.95min for cholate,0.82min for glycocholate,and 0.80min for taurocholate.The mass spectrometer was t as follows:multiple re-action monitoring,negative ionization mode;turbo spray ESI source;spray voltage:−4500V;source temperature:550°C,vaporizing/curtain gas:N 2;collision gas:N 2;monitored transitions:cholate:m/z 407.3–>343.3,cholate-D 4:m/z 411.3–>347.3,collision energy:−48V,each;glycocholate:m/z 464.3–>74.0;glycocholate-D 4:m/z 468.3–>74.0,collision energy:−90V,each;taurocholate:m/z 514.3–>124.0,taurocholate-D 5:m/z 519–>124.0,collision energy:−66V,each.The peak areas of the respective product ions were measured against the peak area of the respective internal standard product ions.Quantitation was performed using a weighted linear least squares regression analysis generated from forti fied calibration stan-dards prepared immediately prior to each run.
Results
Experimental design,histology,and clinical chemistry
Among the 13hepatotoxins ud in this study (Table 1),1-naphthylisothiocyanate (ANIT)(Kossor et al.,1993),acetamino-phen (APAP)(Hinson et al.,2010),bendazac (BDZ)(Alcalde et al.,1996),carbon te
胡萝卜炒玉米trachloride (CCL4)(Weber et al.,2003),cyclosporine A (CSA)(Ande et al.,2010),DL-ethionine (ETN)(Skordi et al.,2007),methapyrilene (MTP)(Mercer et al.,2009),tetracycline (TTC)(Breen et al.,1975),and ticlopidine (TCP)(Sossai et al.,1998)were lected becau of their known abilities to induce various types of liver injury in rats.In addition,four compounds,carbamazepine (CBZ),flutamide (FTM),chlorzoxasone (CZS),and nimesulide (NMS),do not cau
Table 2
Summary of histopathology,clinical chemistry,and the hepatotoxin classi fications.Toxicant a
Do
Histopathology b Clinical chemistry Toxicant
classi fication c
Day 2
Day 5
Day 2Day 5AST ALT ALP γ-GT TBil AST ALT ALP γ-GT TBil U/L
U/L U/L U/L mg/dL U/L U/L U/L U/L mg/dL APAP
V –
–134.632.21193.00.4380.078135.832.81198.8 1.3180.066Necrosis d L +(1/5)–135.441.21262.00.9120.092124.835.81373.2 1.1460.070H +(4/5)–210.2e 51.0e 1586.60.9060.122111.035.81160.8 1.3700.102BDZ
V ––
130.534.81381.3 1.010.07514533.81194.80.5020.072L –+(1/5)12635.42728.6e 0.8320.08151.249.6e 1801.40.970.056H –+(3/5)118.6387404.2e 1.2320.054138.6109.2e 4326.4e 1.0360.036ETN
V –
–137361204.2 1.3340.078134.632.41154.4 1.3620.068L +(2/5)–
141.836.81302.60.7820.104e 151.642.612780.9980.204e H +(3/5)+(3/5)24.2 4.51330.4650.01727.9 4.3185.70.5430.024MTP
V ––
14835.61276.40.9580.06812932.41148.80.860.06L –
+(5/5)139.237.21150.2 1.080.072240.6e 63.6e 1119.2 1.8080.150e H +(3/5)+(5/5)335e 153.2e 1486 1.0840.280e 471.6e 196.6e 1921.0e 8.456e 0.578e TCP
V ––134.632.211930.4380.078135.832.81198.8 1.3180.066L ––
14638.21056.40.7260.082137.240.41233.8 1.380.086H –+(5/5)118.228.81058.4 1.0660.100e 111.647.4e 1456.8 2.546e 0.116e ANIT
V ––
130.534.81381.3 1.010.07514533.81194.80.5020.072Cholestasis f
L –
+(5/5)136.433.41351.20.7960.076163.463.2e 1453.8 1.420e 0.216e H +(5/5)+(5/5)549.4e 197.8e 1765.47.344e 1.742e 431.6e 120.2e 1439.4 5.760e 4.884e CSA
V ––130.534.81381.3 1.010.07514533.81194.80.5020.072L ––102.628.61218.80.9960.154e 104.829.81399.40.8040.248e H ––79.4e 251244.40.9060.206e 58.6e 23.21341.80.6060.31e CCL4
过生日专用配图
V –
–
13736.61241.8 1.2240.07812034.81124 1.1020.07Steatosis g
L +,++(5/5)+++(5/5)127.836.21496.60.9260.084147.641.21187.80.9880.078H ++(5/5)+++(5/5)140.6391539.6 1.1240.092170.644.81193.2 1.0220.088TTC
V –
–
梦见自己洗衣服128.632.81184.2 1.0060.076123.830.61214.40.8140.066L +(1/5)+(2/5)156.831.81057.2 1.1580.08141.635.21114.80.7360.056H –+(4/5)121.833.81218.80.8720.066119.233.81087.60.690.06CBZ V ––134.632.211930.4380.078135.832.81198.8 1.3180.066Idiosyncratic
L ––13233.21213.6 1.0160.086134.433.81062 1.0960.044H ––127.6351235.60.6440.08613839.41130.2 1.2860.06CZS
V ––14835.61276.40.9580.06812932.41148.80.860.06L ––152.6371230.20.9180.072120.633.61114.80.6380.084H ––126.836.41283.80.660.068118.634.81149.60.7160.088FTM
V ––134.632.21193.00.4380.078135.832.81198.8 1.3180.066L ––119.234.21209.80.6100.078126.433.41121.0 1.0440.070H ––138.830.01063.00.9000.092137.037.61030.6 1.5760.074NMS
V ––128.632.81184.2 1.0060.076123.830.61214.40.8140.066L ––136.635.411810.9180.1121.840.810020.9220.054H
朵字怎么写
–
–
141.2
36.2
1495.6
1.16
0.138e
126.8
37.5
889.3
1.583e
0.053
V:vehicle;L:low do;H:high do.AST:aspartate aminotransfera;ALT:alanine aminotransfera;ALP:alkaline phosphata;γ-GT:gamma-glutamyl transfera;Tbil:total bilirubin.a
Hepatotoxin abbreviations:APAP,acetaminophen;BDZ,bendazac;MTP,methapyrilene;TCP,ticlopidine;ETN:DL-ethi
onine;ANIT,1-naphthylisothiocyanate;CSA,cyclosporine A;CCL4,carbon tetrachloride;TTC,tetracycline;CBZ,carbamazepine;CZS,chlorzoxasone;FTM,flutamide;NMS,nimesulide.b
–:no abnormal histopathology;+:slight;++:moderate;+++:rver.The number of animals with the main abnormal findings per experimental group (n=5)examined is shown in the parenthesis.The detailed histopathology summary for each toxicant can be found in the Supplemental data.c
The classi fication of the toxicants was bad on both histopathological obrvations and clinical chemistry data.d
Compounds in this class generally caud hepatocellular degeneration,single cell death,and/or necrosis.The histopathology generally accompanied by the dominant increa in ALT and moderate increa in total bilirubin.e
Statistically signi ficant compared to the vehicle control (p b 0.05).f
Compounds in this class caud marked increa in total bilirubin compared with ALT.Hepatocellular damage and bile duck injury were often obrved.g张怎么组词
Compounds that primarily caud vacuolation/fatty degeneration of hepatocytes in the liver.
81
M.Yamazaki et al./Toxicology and Applied Pharmacology 268(2013)79–89
apparent toxicity to rats but are known to cau idiosyncratic liver injury and dysfunction in humans.Carbamazepine is an antiepileptic drug; various degrees of hepatotoxicity,from hepatitis to liver failure,have been reported to be associated with carbamazepine treatment (Kalapos,2002).Flutamide,an anti-androgen drug often ud for pros-tate cancer treatment,is known to cau rare but vere liver dysfunc-tion(Wysowski and Fourcroy,1996).Chlorzoxasone is a skeletal muscle relaxant that bears rious warnings becau of liver toxicity (Temple,2006).Nimesulide is a non-steroidal anti-inflammatory drug (NSAID)that has been reported to cau higher incidences of liver toxicity than other NSAIDs(Aithal and Day,2007).
For each hepatotoxin,male rats in groups of5were administrated with a low do and a high do daily,and plasma,urine,and liver tissue were analyzed at days2and5.We lected the dos so the histopathology results were abnt/subtle at the early time point/ low do and more apparent at the later point/high do.This allowed us to capture biochemical alterations at dos and/or times below the level necessary to cau histologic lesions and gain insights on the cas-cade of metabolic pertur
bations leading to DILI pathogenesis.Provided in Table2is a summary of the histopathology and clinical chemistry data.The detailed results are prented in the Supplemental material (“Supplemental data histopathology”and Supplemental Table A). Bad on the indications,the13compounds were classified into four groups consisting of necrosis,cholestasis,steatosis,and idiosyncratic.In general,the classifications were consistent with the established lit-erature(Alcalde et al.,1996;Ande et al.,2010;Breen et al.,1975; Hinson et al.,2010;Kossor et al.,1993;Mercer et al.,2009;Skordi et al.,2007;Sossai et al.,1998;Weber et al.,2003).
Necrosis
APAP,BDZ,ETN,MTP,and TCP caud hepatocellular damage such as hepatocellular degeneration,single cell death and/or necrosis.In-terestingly,APAP-induced necrosis was obrved for both dos on day2but was abnt on day5.Similar results in adaptation to APAP treatment have been reported previously and were attributed to the up regulation of cellular antioxidant production for detoxification (O'Brien et al.,2000).With the exception of ETN,which incread bil-irubin but had no effect on the liver enzymes,the hepatocellular dam-age caud by this group of compounds was generally accompanied by significant increas of ALT.
Cholestasis
Both ANIT and CSA caud marked elevations in total bilirubin. Degeneration of biliary epithelium and elevations of ALT,AST and GGT were obrved in ANIT-treated rats.However,no abnormal his-topathology or alterations in the liver enzymes were obrved for CSA
treatments.
0.2 1.0 5.0
Median = 1.0
微信海Fig.1.Fold-change heat map showing metabolomic effects in plasma,urine,and liver of rats following
administration of various hepatotoxins.Each hepatotoxin was given in low and high dos,or as a vehicle control,and samples were collected after two days andfive days.Metabolomic data for each compound was scaled to the median detected value(defined as1.0)among all samples for that hepatotoxin.Heat map cells reprent the fold change,defined as the ratio of the group means(n=5),for a test group relative to its corresponding time vehicle control.Saturated red color indicates at least afive-fold increa in mean levels of a compound relative its vehicle control for the same day.Saturated green color indicates a decrea to20%or less of the vehicle control level.White cells indicate compounds which were not detected in any of the samples for a particular hepatotoxin.The four columns within each hepatotoxin group are the in order,low do/vehicle,day2;low do/vehicle,day5;high do/vehicle,day2;high do/vehicle,day5.Hepatotoxin ab-breviations:ANIT,1-naphthylisothiocyanate;APAP,acetaminophen;BDZ,bendazac;CBZ,carbamazepine;CCL4,carbon tetrachloride;CSA,cyclosporine A;CZS,chlorzoxasone;ETN: DL-ethionine;FTM,flutamide;MTP,methapyrilene;NMS,nimesulide;TCP,ticlopidine;TTC,tetracycline.
82M.Yamazaki et al./Toxicology and Applied Pharmacology268(2013)79–89
Steatosis
Both CCL4and TTC primarily caud vacuolation and fatty degenera-tion of hepatocytes in the liver.However,no signi ficant changes in the liver enzymes or bilirubin were detected with either agent.
Idiosyncratic
This group consisted of CBZ,CZS,FTM,and NMS.Except for slight hypertrophy of hepatocytes by CBZ and NMS and transient increas of total bilirubin on day 2and elevated γ-GT on day 5by NMS,the 4compounds did not cau abnormal histopathology or clinical chemistry.
As shown in Table 2,DILI induced by the hepatotoxins were generally more evident at the high do/later time point,and either abnt or subtle at the low do/early time point.Metabolomic pro files and statistical analysis
Nearly 1900metabolites from plasma,urine,and liver tissues were pro filed (494,684,and 681metabolites from the plasma,urine,and liver,respectively)using the untargeted metabolomic platform.The metabolites were mapped into general biochemical pathways as illus-trated in the Kyoto Encyclopedia of Genes and Genomes (KEGG)(jp/kegg/).The visualization of the metabolomic data by heat map is displayed in Fig.1.A full annotated version is provided in Sup
plemental Table B.The fold-changes of the metabolites between the treated groups and their controls at each do and each time point were calculated as the ratio of their group means.The statis-tical signi ficance of the changes was analyzed by Welch's t test,with p ≤0.05deemed to be signi ficant.The full statistical table is included in the supplemental results (Supplemental Table C).
As shown in Fig.1and Supplemental Table S3,each hepatotoxin induced its unique metabolomic changes in the liver,plasma,and urine across many biochemical pathways,likely due to the combina-tion of the pharmacological actions and toxicological effects.Becau the main objective was to identify biomarkers associated with broad class of DILI,we focud our analysis on the biochemical pathways commonly altered by all or most of the hepatotoxins.The changes of bile acids in the plasma,liver,and urine,and the biosynthesis of glutathione and taurine in the liver (Supplementary Table C)were found to meet the
criteria.
glycochenodeoxycholate
glycocholate
taurochenodeoxycholate
taurocholate
cholate
12-dehydrocholate hyodeoxycholate 3-dehydrocholate taurodeoxycholate glycodeoxycholate
taurolithocholate 3-sulfate 6-beta-hydroxylithocholate
lithocholate beta-muricholate
hyocholate
粽子的图片tauro-beta-muricholate
alpha-muricholate taurocholenate sulfate
deoxycholate chenodeoxycholate /
glycochenodeoxycholate
glycocholate
taurochenodeoxycholate
taurocholate
cholate
12-dehydrocholate hyodeoxycholate taurodeoxycholate glycodeoxycholate taurolithocholate 3-sulfate 6-beta-hydroxylithocholate
beta-muricholate tauro-beta-muricholate
alpha-muricholate taurocholenate sulfate chenodeoxycholate taurolithocholate
glycocholate taurocholate
cholate 3-dehydrocholate glycochenodeoxycholate
alpha-muricholate
taurochenodeoxycholate
7-ketodeoxycholate systemic circulation
A
B
Fig.2.Bile acid biosynthesis and circulation (A)and Fold-change heat map showing the perturbations of bile acid pro files in the plasma,urine,and liver by the hepatotoxins (B).The explanation of the heat map can be found in Fig.1legend.
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84M.Yamazaki et al./Toxicology and Applied Pharmacology268(2013)79–89
