Integration of clinical and gene expression endpoints to explorefuran-mediated hepatotoxicity
Hisham K. Hamadeha,∗, Supriya Jayadevb, Elias T. Gaillardb, Qihong Huangb,Raymond Stollb, Kerry Blanchardb, Jeff Choua, Charles J. Tuckera, Jennifer Collinsa,
Robert Maronpota, Pierre Bushela, Cynthia A. Afsharia,1a National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
b Boehringer-Ingelheim Pharmaceuticals Inc., Ridgefield, CT, USA
Received 13 October 2003; received in revised form 17 December 2003; accepted 17 December 2003
Furan is an intermediate in the synthesis and pro-duction of organic-based compounds such as lacquers,resins, insecticides, stabilizers, and pharmaceuticals.The primary route of exposure for humans is via in-halation during occupational contact. Furan has beenclassified as a carcinogen[1] yielding a tumor inci-
∗ Corresponding author. Present address: Amgen Inc., One Am-gen Center Drive, Thousand Oaks, CA 91360-1799, USA.Tel.: +1-805-447-4818; fax:+1-805-499-2936.E-mail address: [email protected] (H.K. Hamadeh).
1 Present address: Amgen Inc., One Amgen Center Drive, Thou-sand Oaks, CA 91320-1799, USA.
dence where more than 90% of the tumors are adeno-carcinomas, and the remainder are squamous cell tu-mors. In rats, furan induces cholangiocarcinomas, thatare tumors that arise from the intrahepatic epithelium.Cholangiocarcinomas tend to grow slowly and to infil-trate the surrounding hepatic parenchyma. While theetiology of most bile duct cancers remains undeter-mined, long-standing inflammation, as with primarysclerosing cholangitis (PSC) or chronic parasitic in-fection, has been suggested to play a role by induc-ing hyperplasia, cellular proliferation, and, ultimately,malignant transformation.
This study highlights the differences in the effectsof furan on different lobes of the liver and defines geneexpression patterns that reflect the interlobular differ-
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ences observed at the microscopic level. Additionally,in this report we employ furan as a model toxicant todemonstrate the utility of visualization of histopathol-ogy and clinical chemistry data using tools and tech-niques usually reserved for the analysis of high-densitygene expression data. Previous studies have shown thepower of such techniques such as clustering, lineardiscriminant analysis, and other statistical approachesto classify compounds based on the gene expressionprofiles they induce[2–5]. Our previous analysis ofthe acute effects of methapyrilene, another rat hepato-carcinogen[6] showed a strong relationship betweengene expression patterns and pathology severity. How-ever, none of these studies has formally integrated clin-ical pathology data with the gene expression data uti-lizing analogous analysis methods. In this paper, wedemonstrate the value of combining gene expressiondata with traditional measures of adverse effects (e.g.microscopic observations, clinical chemistry) to con-struct models that contain both, continuous and cate-gorical types of data. This allows the inference of as-sociations between traditional markers of toxicity andgene expression in a relatively simple fashion and ul-timately sheds light on molecular pathways that maybe involved in early steps of furan-induced cholangio-carcinogenesis.
2. Materials and methods
2.1. In-life study
Male Sprague–Dawley rats (CRL:CD(SD) IGSVAF+) approximately 6–7 weeks of age, were ob-tained from Charles River Laboratories (Kingston,NY). Rats were housed individually in stainlesssteel wire bottom cages suspended on racks. Therats were maintained under controlled lighting (12 hlight–dark cycle), temperature (72±5◦ F) and humid-ity (50 ± 20% RH). Both water and food (certifiedrodent chow 5002, Purina, Brentwood, MO) wereavailable to the rats ad libitum except overnight priorto terminal necropsy. Forty-eight rats were assignedto 12 study groups (4 rats/group), and dosed by gav-age for 1, 3, 7 or 14 days with corn oil (vehicle),4 mg/kg per day furan, or 40 mg/kg per day furan. Ahigh dose of 40 mg/kg per day furan was chosen indirect correlation with a 16-day NTP study[1]. This
dose was chosen in order to elicit hepatotoxicity withminimal to no nephrotoxicity. A 10 times lower dose,4 mg/kg per day, was chosen as the low dose with theexpectation that minimal hepatotoxic effects wouldbe observed in the current study. Furan (99+%, pur-chased from Aldrich Chemical Co., Inc., Milwaukee,WI) dosing suspensions were prepared on a daily ba-sis under the fume hood. Stock solutions of 100 and10 mg/ml were prepared by mixing the appropriatevolumes of furan in 95% ethanol. For vehicle con-trol, a stock solution was prepared by mixing sterilede-ionized water (amount equivalent to the volume offuran used to prepare the 100 mg/ml stock solution)with 95% ethanol. Stock solutions were vortexedbriefly, bubbled with nitrogen and tightly capped un-til needed. Stock solutions were maintained in glassvials capped with threaded Teflon®-lined caps. Dos-ing suspensions were prepared by diluting the stocksolutions 1:25 in Mazola® corn oil. Suspensions weremixed, bubbled with nitrogen, tightly capped andmaintained on ice until the completion of dosing.Exposure of all solutions and suspensions to air wasminimized as much as possible. During the pretestweek (except weekends and holidays), all rats weredosed via oral gavage with corn oil once per day at avolume of 10 ml/kg. During the drug phase, animalswere dosed orally via gavage with test article or ve-hicle control (see above) once per day at a volumeof 10 ml/kg. Dosing was performed each day with aclean disposable plastic syringe equipped with a clean16-gauge× 3 in. curved stainless steel gavage needle.At the end of the exposure period, animals were eu-thanized by CO2 asphyxiation and necropsied. Liverswere examined macroscopically, weighed and col-lected. Cross-sections of liver from the caudate, rightposterior lobe, medial and left lobes were collected in10% neutral buffered formalin for histopathologicalevaluation. Remaining liver from the respective lobewas minced and snap frozen in liquid nitrogen withinseveral minutes after euthanasia for RNA isolation.Experiments were performed according to the guide-lines established in the NIH Guide for the Care andUse of Laboratory Animals.
2.2. Histopathology
Representative sections of liver from all animalswere processed and embedded in paraffin, sectioned
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(5�m), stained with hematoxylin and eosin and ex-amined microscopically. Histopathologic evaluationsof the tissue sections were conducted by a veterinarypathologist (E.T. Gaillard).
2.3. RNA isolation
Total RNA from all animals was isolated utilizingQIAGEN RNeasy Maxi Kits® (Valencia, CA) fol-lowing the standard Maxi Protocol for animal tissues.Briefly, liver sections of 700–800 mg each were placedin 15 ml of QIAGEN RLT® buffer and homogenizedusing a Vertis Cyclone® homogenizer fitted with arotor-stator probe. Homogenates were centrifugedto remove particulate debris and debris-free lysateswere mixed with equal volumes of 70% ethanol andloaded on RNeasy® silica gel columns. The sampleswere washed three times on the column and RNA wasextracted in 1.6 ml of RNase-free water. Spectropho-tometric determination (Beckman DU520 UV-VisSpectrophotometer) of 260 and 280 nm absorbancesof the RNA samples were made and used to determinepurity, concentration (�g/ml) and total yield (�g) ofRNA samples. In addition, the quality of all RNA sam-ples was verified using the Agilent Bioanalyzer (PaloAlto, CA) according to manufacturer’s instructions.
2.4. cDNA microarray manufacturing
Sequence verified rat clones cDNAs (Research Ge-netics, Huntsville, AL) (http://www.dir.niehs.nih.gov/microarray/chips.htm) were printed on glass slidesas described. cDNAs for printing were obtained byPCR amplification of inserts from purified plas-mid DNA. Double stranded PCR products werepurified, dried, and rehydrated in Array-It Spot-ting Solution Buffer (Telechem, Atlanta, GA) andspotted onto poly-l-lysine coated glass slides us-ing a robotic arrayer (Beecher Instruments, Sil-ver Spring, MD) with Telechem pins to producethe NIEHS rat chip containing∼7000 clones(http://www.dir.niehs.nih.gov/microarray/chips.htm).Detailed methods are available athttp://www.dir.niehs.nih.gov/microarray/methods.htm.
2.5. cDNA microarray analysis
cDNA targets were prepared from 35�g oftotal RNA by oligo dT-primed polymerization
using SuperScript II reverse transcriptase (LifeTechnologies, Gaithersburg, MD). Reverse tran-scription and labeling with the fluorescent dyes,Cy3 or Cy5 (Amersham Pharmacia) was per-formed as previously described (http://www.dir.niehs.nih.gov/microarray/methods.htm). Fluorescently la-beled targets were hybridized to the cDNA chip. Anal-ysis of each sample was performed in duplicate em-ploying a dye-reversal procedure. Fluorescent intensi-ties of the printed DNA probes were measured usingthe Agilent scanner with intensity data integrated over10�m2 pixels and recorded as a 16-bit gray scaleimage. Array Suite IP Lab’s 2.0 (Scanalytics, Fairfax,VA) software was used for data acquisition and im-age analysis. Images corresponding to Cy3 and Cy5fluorescent dyes were analyzed[7]. Intensity valuescorresponding to each gene on the cDNA microarraychips from the Cy3 and Cy5 channels were repre-sented as a ratio of furan-exposed and time-matched,vehicle-treated control tissue. Genes altered with eachhybridization in a statistically significant manner atthe 95% confidence level were considered signif-icantly changed for that hybridization[8]. Genesaltered in a statistically significant fashion acrossreplicate hybridizations were identified as those dif-ferentially expressed two times across duplicate mea-surements. The annotation of genes was confirmedby verification of identity using high throughput se-quencing of the corresponding clones “spotted” onthe chip.
2.6. Normalization and transformation
Pixel intensity values from the feature extractionsoftware were normalized to reduce systematic varia-tions existing in the data (Chou et al., submitted forpublication). Briefly, the mean pixel intensity val-ues from each scanning channel were individuallybackground subtracted and log base 2 transformed.Depending on the distribution of the log base 2 pixelintensity data from the two channels, a linear orpolynomial regression was performed. This regres-sion provided an adjudicator to rescale the data andmake the two channels comparable. Finally, a rescal-ing was performed across arrays to obtain multiar-ray normalization. The entire, normalized dataset isavailable athttp://www.dir.niehs.nih.gov/microarray/datasets.
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2.7. Computational analyses
We used Partek Pro 5.0 (Partek Inc. St Louis, MO)software to perform principal component analysis(PCA) to reduce the dimensionality of the data andprovide a summary of the data structure represen-tative of correlations, covariances or distances. Thegene expression data of the samples were then pro-jected in three-dimensional space with axes definedby the eigen values and eigen vectors of a matrixderived from the original data. These axes provideinformation regarding the dimensionality of the dataand the relationship between variables and the mainaxes. The first principal component (PC #1) containsthe greatest variability, the second principal compo-nent (PC #2) encompasses the next most variabilitywithin the data, which is uncorrelated and orthogonalwith the first, and so on. The reader is referred to[9]for an extensive validation and review of PCA.
We used Spotfire DecisionSite 7.1 (Spotfire Inc.,Somerville, MA) to conductK-means cluster analysison this dataset.
3. Results
3.1. Mortality and organ weights
In order to make the best biological interpretationof gene expression data it needs to be considered incontext with a comprehensive analysis of the biolog-ical sample from which the RNA was derived. In thecase of an in-life study, such as the one used here, fullclinical chemistry and pathology assessments shouldbe made and integrated with the genomics/proteomicsdata. The description of the treatment effects is tradi-tionally described in the following sections.
In this study, Sprague–Dawley animals were ex-posed to furan as described in the methods section.One animal (#280, 40 mg/kg per day, 7-day study)was sacrificed moribund on treatment day 7 due totreatment-related hepatotoxicity and/or nephrotoxic-ity. All of the remaining animals survived to the ter-mination of the study.
Statistically significant increases were observed inthe kidney to body weight ratio of the 40 mg/kg perday animals at the 3-, 7-, and 14-day time points. How-ever, the absolute kidney weights were not statistically
increased. The increased kidney to body weight ratioin the absence of an increase in the absolute kidneyweights may have been due to decreased body weightsin the 40 mg/kg per day groups and the 3-, 7-, and14-day time points. Animals treated with 4 mg/kg perday showed no discernable toxicological responses tofuran treatment in this study.
Compound-induced statistically significant in-creases in absolute liver weights and relative liver tobody weight ratios were observed in the 40 mg/kg perday groups at 1, 3, 7, and 14 days when compared totime-matched controls.
3.2. Microscopic observations
3.2.1. LiverMicroscopic, treatment-related, findings in the liver
could be broadly categorized as degenerative, inflam-matory, compensatory, and proliferative processes. Al-though each of the findings could be present in anyliver lobe, they were most common and prominent inthe right posterior and caudate lobes.
The degenerative processes affected the hepatocytesand consisted of hepatocellular degeneration and cys-tic degeneration (spongiosis hepatitis). Hepatocellulardegeneration was observed as early as the first day inall of the 40 mg/kg per day animals. It was also ob-served in all animals of the 40 mg/kg per day groupsat the 3- and 7-day time points but was only seen inone animal at the 14-day time point. Cystic degenera-tion was present in all animals in the 40 mg/kg per daygroup by the 14-day time point. Hepatocellular degen-eration was characterized by the presence of isolatedhepatocytes that were swollen and had relatively clearcytoplasm or were slightly eosinophilic. Single cellnecrosis was considered to be an integral feature ofthe hepatocellular degeneration and was therefore notdiagnosed separately. Cystic degeneration was char-acterized by variable sized cyst-like structures, whichwere lined by hepatocytes and contained either a fib-rillar to flocculent eosinophilic material and/or ery-throcytes. This finding may have represented a moreintense form of hepatocellular degeneration, affectingclusters of hepatocytes with eventual loss of those hep-atocytes.
The inflammatory process (hepatitis) occurred inthe 4 and 40 mg/kg per day groups at 1-, 3-, 7-, and14-day time points. The severity of this finding was
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greatest in the 40 mg/kg per day group at the 1- and3-day time points. At the 1-day time point, the lesionappeared to be more acute in nature as evidenced bythe presence of mononuclear cells and neutrophils.Histiocytic cells were the predominant inflamma-tory cell type at the 3-day time point. Similarly,inflammatory cells, including green pigment-ladenmacrophages, were present at the 7- and 14-day timepoints but were fewer in quantity than seen in theearlier half of the study. A modicum of fibrous con-nective tissue was associated with inflammatory cellsat the 14-day time point.
The compensatory process involved hepatocytesand consisted of hepatocellular cytomegaly, increasedmitosis of hepatocytes, and hepatocellular regener-ative hyperplasia. Each of these findings was con-sidered to be a response to the loss of hepatocytes.The aforementioned findings were not observed untilthe 3-day time point and were almost always limitedto the 40 mg/kg per day animals. Hepatocellular cy-tomegaly and hepatocellular regenerative hyperplasiawere most severe at the 14-day time point. Increasedmitotic figures were most common at the 3-day timepoint and it may have been an early marker of hep-atocellular regenerative hyperplasia. Hepatocellularcytomegaly was characterized by the presence ofhepatocytes that were enlarged due to increased cy-toplasmic volume and karyomegaly. The affectedhepatocytes also had prominent nucleoli. The pres-ence of clusters or subtle nodules of hepatocytes, thatwere slightly more basophilic and sometimes smallerthan adjacent hepatocytes, characterized hepatocellu-lar regenerative hyperplasia. Affected cells’ nucleoliwere more prominent. There was no associated com-pression of the adjacent hepatic parenchyma and thelobular architecture was maintained. In some areasof hepatocellular regenerative hyperplasia, increasednumbers of mitotic figures were present but were notdiagnosed separately because it was considered to bea feature of the regenerative hyperplasia.
Proliferative responses involved bile ducts andoval cells. Biliary hyperplasia and cholangiofibrosiswere the proliferative responses which involved thebile ducts. Both findings did not appear until thelatter half of the study (7- and 14-day time points)in the 40 mg/kg per day animals. Biliary hyperpla-sia and cholangiofibrosis peaked in frequency and/orseverity at the 14-day time point. Biliary hyperpla-
sia was characterized by increased numbers of rel-atively well-differentiated bile ducts in the hepaticparenchyma. The bile ducts were sometimes lined byslightly more immature appearing cuboidal epithe-lial cells which were relatively more basophilic andhad mildly enlarged vesicular nuclei. Cholangiofibro-sis was characterized by the presence of relativelyatypical bile duct-like structures that were lined byeither tall columnar epithelial cells or goblet cellsthat resembled interstitial epithelial cells. A mucoustype material, neutrophils and/or cellular debris waspresent in the lumina of some of these structures. Ascant amount of fibrosis was associated with someof these atypical bile duct-like structures. Oval cellhyperplasia also did not appear until the latter halfof the study in the 40 mg/kg per day animals, and itwas most severe at the 14-day time point. Oval cellhyperplasia was characterized by the presence of cellshaving oval, usually small, hyperchromatic nucleiand indistinct cytoplasm. The oval cells were eithersurrounding groups of hepatocytes or associated withfoci of cholangiofibrosis and biliary hyperplasia.
Hepatocellular vacuolization was slightly exacer-bated by treatment in the 40 mg/kg per day animals atthe 14-day time point. This finding was characterizedby the presence of variable sized, oftentimes smallvacuoles in the cytoplasm of hepatocytes with no par-ticular lobular distribution.
3.2.2. KidneyMicroscopic treatment-related findings in the kid-
ney consisted primarily of renal cortical tubular necro-sis and regeneration.
Renal cortical tubular necrosis was present in the40 mg/kg per day group at 3 and 7 days and was char-acterized by acute necrosis of individual or groups ofepithelial cells within a tubule and was variably ac-companied by granular casts.
Animals with renal cortical tubular necrosis almostalways had secondary regeneration of the tubular ep-ithelium. The overall incidence of this finding wassimilar at the 3- and 7-day time points. Renal corti-cal tubular regeneration was characterized by tubulesthat were lined by increased numbers of slightly en-larged, basophilic to pale eosinophilic epithelial cells.The nuclear to cytoplasmic ratio of the affected cellswas also increased, primarily due to a slight enlarge-ment of the nucleus. The nucleolus of affected cells
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was more prominent. A key feature that distinguishedrenal cortical tubular regeneration from renal corti-cal basophilia was the presence of necrotic epithelialcells either within the same tubule or within othertubules in the same kidney with regenerative change.In addition, renal cortical tubular regeneration wasmore widespread and less basophilic than renal cor-tical tubular basophilia, which was usually present inclusters or single tubules. Renal cortical tubule dilata-tion was considered a transient treatment-related find-ing in the 40 mg/kg per day animals at the 7-day timepoint.
For summarization purposes, major hepatic and re-nal microscopic lesions observed in rats exposed to40 mg/kg per day furan are illustrated inFig. 1.
3.3. Visualization of histopathological findings
Severities (0–4) of microscopic findings in individ-ual animals were tabulated. PCA was performed onthe data allowing for visualization of the status of ani-mals as a function of all possible lesions incurred.Fig.2 shows the spatial distribution of animals as a func-tion of (A) all lesions and (B) liver-specific lesions.Animals sacrificed at the same time point tended tocluster together. Component loadings in (A) revealedthat liver-related lesions contributed the most to prin-cipal component #1 while kidney lesions contributedmostly to the second principal component. This contri-bution was evident from the spatial position, along the2nd principal component on the PCA plot, of animalswhere kidney lesions were most prominent. Analysis
Fig. 1. Illustration of major microscopic alterations observed in rats treated with 40 mg/kg per day of furan. Largely similar lesions wereobserved in the four processed lobes with differing severity scores.
of liver-specific lesions only showed a progression to-wards increased toxicity with the high dose treatmentgroups as evidenced by their respective spatial locationthat defined a trajectory along the first principal com-ponent in the positive direction as a function of timeof exposure (Fig. 2B). However, no animals treatedwith the low dose were resolvable with this analysis,reflecting the lack of microscopic observations in theliver at this dose of furan.
3.4. Clinical chemistry findings
Statistically significant alterations in clinical chem-istry parameters were observed mostly with the highdose groups and most notably at the later time points(Table 1). Alterations in blood urea nitrogen (BUN)and creatinine were transient in nature, peaking at day3 or 7 and returning to baseline by day 14 of treatment.Elevations in alanine aminotransferase (ALT), aspar-tate aminotransferase (AST), sorbitol dehydrogenase(SDH), total bilirubin, and alkaline phosphatase (ALP)were indicative of pronounced treatment-related livertoxicities that were observed histopathologically.
Principal component analysis was performed onall clinical chemistry parameters measured in thisstudy (Fig. 3A) thus allowing the visualization offuran-exposed animals as a function of multiplevariables (serum markers). The level of blood ureanitrogen was a major contributor to the 1st principalcomponent, so we visually overlaid that measure onthe PCA plot in order to help visualize the levels ofindividual animals. Animals on the far right side of
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Fig. 2. PCA analysis of all histopathological lesions (A) or liver-specific lesions (B). The color of the cubes is indicative of the time ofexposure day 1 (red), day 3 (yellow), day 7 (blue), day 14 (green) and the size of the cubes denotes low, 4 mg/kg per day (small) andhigh, 40 mg/kg per day (large) dose exposure (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of this article).
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Table 1Clinical chemistry data from animals treated with furan at 4 or 40 mg/kg per day for 1, 3, 7, or 14 days
Averaged values reported in this table are fold change over average time-matched controls.∗ Statistically significant atP < 0.05.
the plot had the highest elevations in BUN levels, thusthe distribution of animals was heavily affected bythis measurement and was hindering the delineationof spatial distribution of furan-exposed animals based
Fig. 3. PCA of all clinical chemistry parameters (A) or excluding markers of kidney toxicity (B). (A) The color of the cubes representslevels of BUN measured in the serum of each animal (low to high is indicated in blue to red scale). (B) The color of the cubes is indicativeof the time of exposure (day 1 (red), day 3 (magenta), day 7 (blue), day 14 (green)) while size denotes low (small cubes) or high (largercubes) dose (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
on serum markers of adverse liver alterations. There-fore, a PCA was subsequently conducted excludingthe use of serum markers indicative of nephrotoxicity(e.g. BUN, creatinine) (Fig. 3B). A better separation
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Fig. 4. PCA of gene expression corresponding to the left lateral lobes of rats exposed to furan at different doses and time points. Thecolor of the cubes is indicative of the time of exposure (day 1 (red), day 3 (yellow), day 7 (blue), day 14 (green)) while size denotes low(small cubes) or high (larger cubes) dose (For interpretation of the references to color in this figure legend, the reader is referred to theweb version of this article).
of the low and high dose treated animals was thenobserved in this analysis and the various time groupsshowed relatively more resolution.
3.5. Gene expression analysis
High-density gene expression was generated onsamples derived from the left lobe of all the animals inthe study and from the rest of the lobes correspondingto animals treated with the high dose (40 mg/kg perday) for 7 days. PCA was applied to gene expressiondata derived from the left lobe (Fig. 4) showing adistinction between samples derived from rats treatedwith a low versus a high dose, as explained by the firstprincipal component. In addition, a significant distinc-tion was observed between the different time pointsat the low and high doses although the resolution wasmore pronounced with the high dose. The separationof samples based on duration of exposure is evidentin the projection of the second principal component.The data were suggestive of dose and time-dependentresponses in gene alterations, however the moreprominent parameter was dose as evidenced by thepercentage contributions to each principal component(PC#1: 71.2% versus PC#2: 9.4%).
Fig. 5 shows the utility of projecting ancillary pa-rameters such as bilirubin levels and cholangiofibrosisseverity onto gene expression visualization plots usinga continuous color scale for the former and varyingcube sizes for the latter. The first principal componentcan be viewed as a measure of toxicity where samplescloser to the far right correspond to animals incurringrelatively little toxicity while samples residing at theleft being the most adversely affected by the treatment.This polarity is confirmed by the bilirubin values pro-jected on each sample with those on the left having thehighest values. Combining gene expression data withtoxicity phenotypes provides the advantage of relay-ing multiple sources of information through one visu-alization output that then allows for potential visualcorrelations between gene(s) and various endpoints.
3.5.1. K-means clusteringK-means clustering was applied to the gene expres-
sion data from the left lobe of all animals to discoverpotential trends in the data set. Data were forced into20 major bins. Bins were classified as dose-dependentelevation or reduction in gene expression with re-spect to time-matched controls. Subgroups were fur-ther identified based on amplitude of alterations and
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Fig. 5. PCA analysis of gene expression from the left lateral lobe with projection of ancillary variables. The color indicates bilirubin levelsand the size of the cube indicates cholangiofibrosis severity.
whether genes were altered at an early versus late stagein the study.Fig. 6 shows the major trends observedwith this dataset.
Since our primary interest was to delineate and ex-ploit potential pathways important to cholangiocar-cinogenesis, we focused on the incidences of cholan-giofibrosis, which is viewed as a step preceding themalignant lesion. In the absence of more sophisticatedtechniques like laser capture microdissection (LCM),we expected gene expression alterations emanatingfrom bile ducts and cholangiocytes to be diluted due tothe relatively small population of these cells compared
Fig. 6. K-means analysis of gene expression data from the left lateral liver lobes corresponding to rats treated with furan: (A) the majortrends in the data such as dose- and time-dependent increase or decrease (high or low amplitude and early vs. late changes); (B) amagnification of the first of these trends.
to hepatocytes. Thus we studied mild to subtle, butstatistically significant, alterations in gene expressionsince a subset of those would most likely represent di-luted gene expression originating in minority cell typessuch as those of bile ducts. Gene expression alterationsof high amplitude were attributed mostly to hepatocel-lular alterations (Fig. 6). Some of the alterations weobserved are specifically described inSection 4.
3.5.2. Interlobular differencesGene expression data were generated from each of
the four lobes of the livers corresponding to animals
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treated with 40 mg/kg per day furan for 7 days. Amultivariate analysis of variance (ANOVA) was per-formed to find genes that discriminated between thefour lobes at the assayed time point. Genes wereranked based on their discriminative ability as judgedby the P-values that resulted from this analysis.Discriminating genes had the highest amplitudes ofchange relative to controls in the right lateral lobe ofexposed rats. Elevated genes represented proliferationand inflammatory pathways whereas lowered geneswere indicative of mitochondrial injury and cholesta-sis. The visualization inFig. 7 supports that a largermagnitude of change in the right lobe is consistentwith more severe toxicity, and most likely higher ex-posure, in this region of the liver.Table 2reveals the
Fig. 7. Genes that were the most discriminative between the four assayed liver lobes were identified based on a multivariate ANOVA(P < 0.001). Genes were differentially decreased (A) or increased (B) across lobes relative to respective lobes of time-matched controls.
identity of genes illustrated inFig. 7. The majority ofgenes were expressed sequence tags (ESTs).
4. Discussion
In this study, we investigated the integration ofgene expression, clinical chemistry and pathologyendpoints in order to determine if we could derive amore delineated classification of exposed tissue sam-ples. We used the model toxicant, furan, for this studybecause it induces a progressive pathology in the liver.
PCA of high-dimensional histopathology and clini-cal chemistry data is a powerful technique for quicklyvisualizing the status of animals considering all param-
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Table 2Discriminative genes illustrated inFig. 7
Gene ID (+) Gene description (+)a Gene ID (−) Gene description (−)b
AA964578 EST AA925992 ESTAA901383 EST AA819012 Pancreatic trypsinogen IIAA900530 EST AI137934 ESTAA900431 TGF-betaIIR alpha AA955059 ESTAA900215 EST AA926002 ESTAA900285 EST AI059504 ESTAA859861 Epithelial cell transmembrane protein
antigen precursor (RTI40)AI030286 EST
AA955283 EST AA819886 ESTAA956328 EST AA819206 ESTAA899900 EST AA866286 ESTAA924266 EST AA956202 ESTAA925838 EST AA817911 ESTAA819918 Type I phosphatidylinositol-4-phosphate
5-kinase alphaAI044326 EST
AA819346 EST AA819269 ESTAA901108 EST AA818798 CathepsinAA955482 EST AA858987 Protein-containing MBD 1AA956065 EST AA858969 ESTAA899765 B lymphocyte chemoattractant BLC AA866393 ESTAA901035 EST AA819889 ESTAA925877 EST AA901044 ESTAA923864 EST AA819868 ESTAA923988 EST AA901071 ESTAA900320 Integral membrane protein CII-3, nuclear
gene encoding mitochondrial proteinAA923987 EST
AA924079 EST AA859073 ESTAA956535 EST AA818526 ESTAA900914 EST AA818864 ESTAA900889 EST AA901327 ESTAA925625 EST AA955127 ESTAA923834 EST AA817906 ESTAA924087 EST AA819273 Connexin protein Cx26AA859460 48 kDa FKBP-associated protein FAP48 AA860001 Flavin-containing mono-oxygenase 1 (FMO-1)AA901194 EST AA925220 ESTAA900219 EST AA859066 ESTAA900873 Cyclin D3 AA859758 Beta-H1-globinAA924252 EST AA875273 Arix1 homeodomain proteinAA924263 EST AA900218 Metallothionein-1 (mt-1)AA900034 EST AA819806 Androgen-dependent expressed proteinAA819549 EST AA819329 ESTAA955893 EST AA900433 Long-chain acyl-CoA synthetaseAA957164 EST AA818575 ESTAI113101 EST AA998830 ESTAA900313 EST AA819237 ESTAA900902 EST AA818577 ESTAA819030 EST AA900057 ESTAA955702 EST AA818077 Placental lactogen-1AA900864 EST AA818680 Ornithine aminotransferaseAA925992 EST AA955337 EST
AA859112 ESTAA965125 ESTAA926193 ESTAA858732 Lysozyme geneAA955115 ESTAA819164 Mitochondrial IF1 protein
a Genes increased compared to lobe-matched control samples sorted in descending order of increase in the right lobe depicted inFig. 7B.b Genes decreased compared to lobe-matched control samples sorted in increasing order of decrease in the right lobe depicted inFig. 7A.
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eters measured simultaneously within an assay. One ofthe goals of this experiment was to determine whethergene expression measurements would yield increasedsensitivity in discriminating between different severi-ties of liver lesions beyond the indication of traditionalclinical chemistry parameters. Clinical chemistry wasnot as discriminative of pathology groups as gene ex-pression in the unsupervised analysis of data, mainlyby PCA.
The interpretation of gene expression changes ismostly assumed to be indicative of transcriptional me-diated affects, however, fluctuations in relative levelscould be due to loss of cell types (e.g. centrilobularnecrosis relationship to flavin mono-oxygenase, or-nithine aminotransferase changes) from the toxicantexposed tissue sample compared to the control.
However, in spite of this, insight may be gainedregarding the mechanism of organ toxicity followingcompound exposure. Construction of mathematicalmodels with different types of datasets allows visu-alization of multiple parameters from different assaysperformed on each animal in the study and enablesmore focused and specific questions to be answeredby correlating these independent data sets. For exam-ple, K-means analysis revealed groups of genes withdifferent trends of expression across dose-time space.Of special interest were genes that were elevated ordecreased relative to controls at later time points whenthe cholangiofibrosis was diagnosed microscopicallyin livers of treated rats. For example, increased levelsof O-acetyl disialoganglioside synthase (OacGD3S)and stellate cell activation-associated protein were ex-amples of genes that were slightly elevated comparedto time-matched controls at days 7 and 14 in livers ofrats treated with 40 mg/kg per day furan.
Increased expression ofO-acetyl disialogangliosidesynthase during rat liver fibrogenesis relates to stellatecell activation[10], a key step in liver fibrogenesis.O-acetyl disialoganglioside synthase (OAcGD3S)was identified as one of the significantly elevated fac-tors in cultured activated hepatic stellate cells by bothNorthern and Western blot analyses. In addition, insitu hybridization revealed OAcGD3S mRNA expres-sion in areas of ductular proliferation in experimentalrat models of fibrosis and in human cirrhotic livers.Stellate cell activation-associated protein (STAP) is anovel cytoplasmic protein with molecular weight of21 kDa. Biochemical characterization of recombinant
rat STAP revealed that STAP is a heme protein exhibit-ing peroxidase activity toward hydrogen peroxide andlinoleic acid hydroperoxide, both of which have beenreported to trigger stellate cell activation and con-sequently promote progression of liver fibrosis[11].STAP was dramatically induced, in vivo, in activatedstellate cells isolated from fibrotic liver and in stellatecells undergoing in vitro activation during primaryculture [11]. The expression of STAP protein andmRNA was augmented in a time-dependent manner inthioacetamide-induced fibrotic liver. Immunoelectronmicroscopy and proteome analysis detected STAP instellate cells but not in other hepatic constituent cells.The relatively low degree of elevation of these genesmight be explained in this case by the localized sitesof expression of STAP and OAcGD3S, namely, thestellate cells, which are a much smaller populationrelative to hepatocytes. Thus those genes should notbe dismissed due to their relatively low fold of change.
Considering genes that were elevated at later timepoints with high amplitude, we observed alterationsin numerous genes indicative of fibrosis and tissue re-modeling including procollagen types I and III. Thesegenes are precursors to collagen type I and III pro-teins, which have been suggested as serum markers offibrogenesis in patients with chronic liver disease andanimal models of liver fibrosis[12,13]. In addition,we observed a time- and dose-dependent increase inmRNA levels of tissue inhibitor of metalloproteinase-1(TIMP-1) relative to controls. TIMP-1 has tightcontrol over matrix metalloproteinase-1 (MMP-1),which belongs to a family of secreted zinc proteasescapable of degrading collagen and other extracellu-lar matrix components. Recent studies suggest thatMMP-1 may participate in pathological responsesinvolved in liver fibrosis[14]. Hepatic stellate cells(HSC) can express TIMP-1, leading to the hypothesisthat matrix degradation is inhibited during progres-sive fibrosis [15]. This hypothesis is supported byfindings that overexpression of TIMP-1 enhancesexperimental fibrosis and that spontaneous recoveryfrom liver fibrosis is associated with a diminution ofTIMP-1 expression and an increase in collagenaseactivity with consequent matrix degradation[16].Liu and coworkers employed an antisense strategyagainst TIMP-1 as the target gene and found an in-crease in the enzymatic activity of MMP-1 relativeto controls and a decrease in the amounts of collagen
182 H.K. Hamadeh et al. / Mutation Research 549 (2004) 169–183
I, III relative to controls as determined by Westernblotting [17].
Other markers of fibrosis observed at later timepoints included retinol-binding protein, whose levelswere decreased relative to control. This is consistentwith the etiology of fibrogenesis and activation ofstellate cells. Quiescent stellate cells from normalliver are considered to be the storage and metabolismsite of Vitamin A. Following liver injury of any eti-ology, stellate cells undergo a process termed activa-tion, which represents a transition into proliferative,fibrogenic, proinflammatory and contractile myofi-broblasts, thereby losing their Vitamin A homeostaticproperties leading to a decline in the expression ofgenes associated with that pathway[18].
The incidence of cholangiofibrosis in furan treatedanimals was of interest to us because it is consideredto be a precursor to cholangiocarcinomas observed inlonger-term studies with furan[19]. The lobe inci-dence of cholangiofibrosis was in agreement with anearlier report that found preferential induction of smallintestinal metaplasia and subsequent cholangiofibrosisin the caudate and right liver lobes[19].
Finally, this is one of the first studies to comparegene expression changes in different lobes of the liverfollowing hepatotoxicant exposure. Our data indicate(Fig. 7) that while we saw similar gene expressionchanges in different lobes of the liver, the magnitudeof the induction/repression was higher in the right lobe(followed by caudate). This is consistent with the inter-lobar differences observed via histopathological anal-ysis, where increased severity of lesions was observedin the right lobe in our study and previous studies[19–21]. One of the potential explanations for the in-creased right and caudate lobe severity would be due toincreased blood flow to these lobes[22]. Another pos-sible explanation could be due to the physical proxim-ity of these lobes to the stomach-gastrointestinal tract,associated with leaking of furan into the intraperi-toneal space could lead to higher exposure in theselobes. Further analysis of the genes found to discrim-inate between the four assayed liver lobes might aidin revealing molecular mechanism(s) underlying theobserved differential severity in lesion developmentacross different lobes of rat liver. Since the majorityof the discriminative genes were found to be ESTs,advancements in gene ontology will allow the betterinterpretation of such data. We would like to high-
light the importance of performing histopathology andgene expression on the same lobe of the liver. Investi-gators who do not conduct concurrent histopathologyand gene expression on the same lobes could be ledastray in their interpretation of the results, and over-or under-estimation of toxicity.
In conclusion, we have found that furan induces aprogression of pathological changes in the rat liverthat may be monitored by both traditional and ge-nomic endpoints. We have shown that incorporationof genomic endpoints into the interpretation of clini-cal findings can lead to better separation of individualsamples. Further studies of other compounds in thismanner will most likely lead to the elucidation of setsof genes that can serve as biomarkers of disease eti-ology and provide enhanced classification above tra-ditional clinical markers.
Acknowledgements
We would like to thank Dr. Ray Tennant and RickPaules for their support of this project. We would alsolike to thank Dr. Tom Downey for modifications andsupport provided via the Partek Pro package. Finally,we thank Julie Foley of NIEHS for her advice on theconduct on various aspects of this study.
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