CONCLUSIONS TCDD responsiveness of HL1-1 cell was confirmed with CYP1A1 mRNA expression induction by QRT-PCR

TCDD-elicited temporal and dose response microarray analysis has identified the conserved classical responses in accordance with literature data

Gene expression changes verified by QRT-PCR correlate well with the microarray data

Comparative studies across models and species confirmed that some toxicity-related, conserved and model- specific responses were observed in HL1-1 cells

The limited differential gene expression conservation between human and mouse model systems provides further evidence for species-specific AhR regulons.

INTRODUCTION

TCDD is the prototypical ligand of the aryl hydrocarbon receptor (AhR) and an ubiquitous and bioaccumulative environmental contaminant that causes various adverse and biological effects in animals and humans including endocrine, immuno- and hepato- toxicity, teratogenesis, and multi-site tumor promotion. Various in vivo and in vitro model systems have been utilized to investigate the molecular networks and mechanisms responsible for the diverse toxic responses. An in vitro model system derived from human target tissue is generally expected to be more predictive of human toxicity. Human stem cells provide an attractive in vitro alternative, which may more closely mimic human responses, and be a potentially unlimited source of human cells. Toxicogenomic analysis provides extensive global differential gene expression profile information, that can be used to more accurately predict a compound's toxicity and support quantitative risk assessments. In addition, 'omics' technologies may facilitate the development of predictive toxicity screening assays as well as more comprehensive computational modeling. Application of toxicogenomics to human liver stem cells will provide expression profiles that can be used to investigate species-specific responses and the identification putative biomarkers.

ABSTRACT

Time course and dose response studies with the human liver cell line, HL1-1, which possesses stem cell characteristics, were conducted to assess gene expression responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For the dose response study, cells were treated for with 0.001, 0.01, 0.1, 1, 10 or 100 nM TCDD or DMSO vehicle control for 12 hrs. The time course study involved treating cells with 10 nM TCDD or vehicle for 1, 2, 4, 8, 12, 24 or 48 hrs. TCDD-elicited changes in gene expression were monitored using human cDNA microarrays consisting of 7,844 genes. Empirical Bayes analysis identified 251 genes that were differentially expressed at one or more time points. Most of these genes also exhibited dose-dependent responses. Dose- and time- dependent induction of CYP1B1, ALDH1A3 and SLC7A5 gene expressions were confirmed by QRT-PCR. Comparisons of the HL1-1 temporal responses with human HepG2 and mouse Hepa1c1c7 hepatoma cell lines identified 75 genes and 18 orthologs common to HL1-1 cells, respectively. Further comparison of temporal gene expression in HL1-1 with hepatic tissue from immature ovariectomized C57BL/6 mice treated with 30 mg/kg TCDD identified 32 commonly regulated orthologous genes, primarily associated with signal transduction and transcriptional regulation. Metabolism and transport related genes were also commonly regulated between all comparisons. This comparative analysis further demonstrates the species- and model-specific AhR-mediated gene expression elicited by TCDD.

Email: kimsun5@msu.edu Supported by NIGMS R21 GM075838 Web: www.bch.msu.edu/~zacharet/

Assess TCDD responsiveness of human adult liver stem cells with CYP1A1 quantitative real-time PCR (QRT-PCR)

Comprehensively assess TCDD induced temporal and dose dependent changes in gene expression

Determine and verify primary response genes via cycloheximide (CHX) co-treatment studies

Comparatively evaluate the model-specific and model-conserved gene expression responses elicited by TCDD

OBJECTIVES

QRT-PCR CONFIRMATION OF MICROARRAY DATA

Figure 6 Quantitative real-time PCR (QRT-PCR) verification of microarray results in time course and dose response studies. Three putative primary TCDD-responsive genes (CYP1B1, ALDH1A3 and SLC7A5) were examined for microarray result verification. Fold changes were calculated relative to time-matched vehicle controls. Bar (left axis) and lines (right axis) represent QRT-PCR and cDNA microarray data, respectively. Results are represented as the average of three biological replicates. Error bars for QRT-PCR data represent the SEM for the average fold change. *p < 0.05 for QRT-PCR.

FUNCTIONAL CATEGORIZATION OF PUTATIVE PRIMARY RESPONSE GENES

559553112048

Unclassifiable

Secondary

response

Primary

responseUnclassifi

ableSecondary

response

Primary

response

12 hr CHX Study (203 active genes)4 hr CHX Study (79 active genes)

559553112048

Unclassifiable

Secondary

response

Primary

responseUnclassifi

ableSecondary

response

Primary

response

12 hr CHX Study (203 active genes)4 hr CHX Study (79 active genes)

Table 1: Functional categorization of putative primary response genes elicited by TCDD (Part of whole table)

T/V T+C/V C/V T/V T+C/V C/V

Metabolizing 1543 CYP1A1d BC023019 cytochrome P450, family 1, subfamily A, polypeptide 1 13 506.7 4135.9 28.0 222.2 26794.4 512.9Enzymes 1545 CYP1B1 AA448157 cytochrome P450, family 1, subfamily B, polypeptide 1 7 9.0 9.0 1.3 11.1 14.9 2.9

224 ALDH3A2 AA633569 aldehyde dehydrogenase 3 family, member A2 6 10.0 8.3 -1.2 5.5 15.1 -1.4220 ALDH1A3 AA465614 aldehyde dehydrogenase 1 family, member A3 3 8.6 7.5 -1.1 5.3 12.0 -1.3

Cell growth/ 23189 KANK1 AA464605 KN motif and ankyrin repeat domains 1 6 2.2 2.1 1.0 1.6 1.7 -1.3Differentiation 5069 PAPPA R02529 pregnancy-associated plasma protein A, pappalysin 1 3 2.5 2.4 1.6 1.7 5.8 1.7

113130 CDCA5 W00895 cell division cycle associated 5 2 8.6 14.8 1.4 2.8 33.3 2.15270 SERPINE2 N57754 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 0 2.9 2.6 1.6 2.0 7.1 1.91490 CTGF AA598794 connective tissue growth factor 2 -2.3 -2.2 1.0 -1.7 -1.9 1.7

Lipid metabolism 51084 CRYL1 H99932 crystallin, lambda 1 7 2.4 2.7 1.0 2.2 10.1 1.023646 PLD3 H15746 phospholipase D family, member 3 6 1.6 2.9 1.6 1.1 2.7 -1.1

Apoptosis 79370 BCL2L14 AA620708 BCL2-like 14 (apoptosis facilitator) 9 2.1 2.5 1.2 1.8 5.0 1.3317 APAF1 N51014 apoptotic peptidase activating factor 1 -1.2 -1.2 -1.1 -1.7 -2.2 -1.7

Regulation of 83463 MXD3 H95253 MAX dimerization protein 3 13 2.4 2.8 -1.1 2.1 9.2 1.0transcription 10522 DEAF1 AA425806 deformed epidermal autoregulatory factor 1 (Drosophila) 13 1.2 1.1 1.0 1.7 1.9 1.3

22938 SNW1 H17512 SNW domain containing 1 8 2.0 1.6 1.1 2.6 2.8 1.410062 NR1H3 H61726 nuclear receptor subfamily 1, group H, member 3 8 2.4 1.8 1.2 3.3 3.7 1.8

2002 ELK1 AA844141 ELK1, member of ETS oncogene family 7 2.5 4.9 2.0 2.4 9.5 2.822936 ELL2 AA284232 elongation factor, RNA polymerase II, 2 4 1.4 -1.1 -1.4 2.1 1.5 -1.3

9792 SERTAD2 AA489839 SERTA domain containing 2 1 1.6 6.7 2.8 2.0 8.7 4.410370 CITED2 AA115076 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 3 -1.9 -1.7 -1.1 1.4 2.3 1.0

Transport 29066 CLCN3 AA885491 zinc finger CCCH-type containing 7A 13 1.6 1.6 1.0 1.3 1.7 -1.16583 SLC22A4 N39791 solute carrier family 22 (organic cation transporter), member 4 13 2.2 4.1 1.9 2.2 7.5 3.26566 SLC16A1 AA043133 solute carrier family 16 (monocarboxylic acid transporters), member 1 11 2.4 2.2 1.1 2.0 4.5 1.56513 SLC2A1 H58872 solute carrier family 2 (facilitated glucose transporter), member 1 9 3.3 6.5 1.4 1.7 8.2 1.0

55937 APOM N74679 apolipoprotein M 8 1.2 1.2 1.0 2.0 1.1 -1.223788 MTCH2 AA463946 mitochondrial carrier homolog 2 (C. elegans) 4 2.9 5.0 1.0 1.6 5.8 -1.123788 MTCH2 AA463946 mitochondrial carrier homolog 2 (C. elegans) 4 -1.6 -1.6 1.1 -1.4 -1.7 1.2

Regulation of 1965 EIF2S1 W60015 eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa 5 1.6 3.3 2.6 1.8 9.4 5.3translation 1983 EIF5 AA669443 eukaryotic translation initiation factor 5 9 -1.6 -1.3 1.0 -1.8 -2.0 -1.4

• • • • • • • • • • • •• • • • • • • • • • • •

a. Funtional categories was performed using an in-house Gene Ontology toolb. DRE identified in -10kb to transcriptional start site (TSS) and 5' UTRc. Expression fold changes determined by microarray analysis and numbers in colored font indicate |Fold change| > 1.5 d. Gene expressiion data was measured by QRT-PCR

Functional

Category aEntrezGeneId

GeneSymbol

12 hr Fold

ChangecGenBankAccession

Gene NameDRE

Count b

4 hr Fold

Changec

4848 5353

2222

4hr4hr 12hr12hr

2626 3131

Primary responsePrimary response

4848 5353

2222

4hr4hr 12hr12hr

2626 3131

Primary responsePrimary response

Figure 7: Identification of putative primary TCDD responsive genes from CHX co-treatment study. Microarray analysis identified 79 and 203 TCDD-responsive genes at 4 and 12 hr, respectively. CHX co-treatment analysis resulted 48 and 53 genes classified as putative primary responsive genes, 20 and 95 genes were classified to putative secondary responsive genes, and 11 and 55 genes were unclassifiable at 4 and 12 hr, respectively. The Venn diagram illustrates that 79 of the putative primary responsive genes were identified in 4 and 12 hour time points CHX co-treatment studies.

79 putative primary response genes

EXPERIMENTAL DESIGN

Figure 1 HL1-1 TCDD time course, dose response and cycloheximide (CHX) co-treatment study designs. Time course study: HL1-1 cells were treated with either 10 nM TCDD or 0.1% DMSO and harvested at 1, 2, 4, 8, 12, 24, or 48 hrs post-treatment. Dose response study: HL1-1 cells were treated with 0.001, 0.01, 0.1, 1, 10, 100 nM TCDD or 0.1 % DMSO vehicle and harvested 12 hrs post-treatment (as indicated †). CHX co-treatment study: 10 mg/mL CHX was treated 1 hr precedent to 10 nM TCDD or 0.1% DMSO treatment. Each combination of treatment group was harvested at 4 and 12 hrs post-treatment (as indicated *). N=3 for each study.

A. TIME COURSE DESIGN B. DOSE RESPONSE DESIGN Figure 2 Microarray experimental designs for (A) time course, (B) dose response and (C) cycloheximide co-treatment study. (A) Temporal gene expression changes were analyzed using an independent reference design that results in two independent labeling of each sample. Numbers indicate time of cell harvesting (hours), T indicates TCDD treatment and V indicates DMSO vehicle treatment. (B) Dose response gene expression changes were analyzed using a spoke design. Each dose treatment sample was compared with independent vehicle control. (C) Cycloheximide co-treatment gene expression changes were analyzed using a 2x2 factorial design. This array design allows for multiple comparisons to identify significant changes in gene expression between treatments. Each arrow represents a single microarray where arrow heads represent Cy5-labeled samples and tails represent Cy3-labeled samples and double headed arrows indicate dye swap labeled on different arrays.

C. CHX STUDY DESIGN

00 4848

DDosed with TCDD orosed with TCDD orDMSO vehicleDMSO vehicle

Cell HarvestCell Harvest

11 22 4*4* 88 1212†** 2424 (hr)(hr)--11

CHX*CHX*

† : Dose response study* : CHX co-treatment study

00 4848

DDosed with TCDD orosed with TCDD orDMSO vehicleDMSO vehicle

Cell HarvestCell Harvest

11 22 4*4* 88 1212†** 2424 (hr)(hr)--11

CHX*CHX*

† : Dose response study* : CHX co-treatment study

4T4T 24T24T12T12T8T8T1T1T 2T2T 48T48T

1V1V 2V2V 4V4V 8V8V 12V12V 24V24V 48V48V

4T4T4T4T 24T24T24T24T12T12T12T12T8T8T8T8T1T1T1T1T 2T2T2T2T 48T48T48T48T

1V1V1V1V 2V2V2V2V 4V4V4V4V 8V8V8V8V 12V12V12V12V 24V24V24V24V 48V48V48V48V V1V1

10nM10nM

1nM1nM

0.1nM0.1nM

0.01nM0.01nM

0.001nM0.001nM

100nM100nM

V6V6V5V5

V2V2V3V3

V4V4

V1V1

10nM10nM

1nM1nM

0.1nM0.1nM

0.01nM0.01nM

0.001nM0.001nM

100nM100nM

V6V6V5V5

V2V2V3V3

V4V4

VV TT

CHXCHXCHXCHX+T+T

VV TT

CHXCHXCHXCHX+T+T

T :TCDDT :TCDD

V :DMSO vehicleV :DMSO vehicle

CHX :cycloheximideCHX :cycloheximide

Cy3 Cy5Cy3 Cy5

T :TCDDT :TCDD

V :DMSO vehicleV :DMSO vehicle

CHX :cycloheximideCHX :cycloheximide

Cy3 Cy5Cy3 Cy5

Figure 5 Hierarchical clustering of 273 active features expression profiles by gene and time in the TCDD time course study. Three time-dependent clusters were formed; early (2 and 4hrs), middle (8 and 12hrs), and late (24 and 48hrs). The early and middle time points show significant differences in their expression pattern, suggesting the characteristic differences in expression profiles between primary and secondary response genes.

Figure 4 Number of genes exhibiting significant expression changes in the TCDD time course study. The number of active genes showed steady increase between 1 and 8 hr, followed by a decrease at 12 hr but further increases at 24 and 48 hr.

1hr 2hr 4hr 8hr 12hr 24hr 48hr1hr 2hr 4hr 8hr 12hr 24hr 48hr

MICROARRAY DATA: TIME COURSE

TEMPORAL AND DOSE-DEPENDENT AHR-MEDIATED GENE EXPRESSION ANALYSIS IN HUMAN LIVER ADULT STEM CELLS

S Kim1,2,3, E Dere1,2, LD Burgoon1,2,3, CC Chang2,4 & TR Zacharewski1,2,3.1Department of Biochemistry & Molecular Biology, 2National Food Safety & Toxicology Center,

3Center for Integrative Toxicology, 4Department of Pediatrics & Human Development, Michigan State University, East Lansing, MI, 48824

TCDD INDUCES CYP1A1 mRNA IN HL1-1 CELLS

Figure 3 QRT-PCR verification of CYP1A1 gene expression levels from the dose response (A) and time course (B) studies in HL1-1 cells treated with TCDD. Based on CYP1A1 induction levels, the EC50 for CYP1A1 expression was 8.30 nM. Error bars represent the SEM for the average fold change. * p < 0.05.

MODEL COMPARISON OF CONSERVED EXPRESSION RESPONSES

HL1-1251

HepG21057

74177 983

• Cell cycle progression and arrest- CDCA5↑, CDKN1C↓, MCM3AP↓

• Matrix metalloproteinase- MMP13↑

HL1-1 vs. Hepa1c1c7 HL1-1 vs. Mm liver

• Transcription factors• Signal transductions• Lipid metabolism

• ID3 : Mm liver↑ / HL1-1 & Hepa1c1c7↓• SLC12A7 : HL1-1↑ / Hepa1c1c7 & Mm liver↓

Divergent responses

HL1-1251

Hepa1c1c7770

18233 752

HL1-1251

Mm Liver1465

32219 1433

• 55 genes were regulated in the same direction

- 13 putative primary responsive genes

were included

• 19 genes were divergently regulated

- No putative primary responsive gene

- Signal transduction, development related genes

HL1-1 vs. HepG2

A B

Figure 8: Comparative analysis of temporal gene expression profiles among human liver stem cell (HL1-1), human hepatoma cell line (HepG2), mouse hepatoma cell line (Hepa1c1c7) and hepatic tissue of C57BL/6 mice (Mm liver) studies. 251 active genes were identified from HL1-1 time course study, and 1,057, 770 and 1,465 active genes were identified from HepG2, Hepa1c1c7 and mouse liver, respectively. 75, 18 and 32 genes were identified as conserved between model systems compared with HL1-1 cell study.