JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 JPET FastThis Forward. article has not Published been copyedited on andDecember formatted. The 23, final 2005 version as mayDOI:10.1124/jpet.105.097014 differ from this version.

JPET #97014

MICROARRAY ANALYSIS OF MOUSE EAR TISSUE EXPOSED TO BIS-(2-

CHLOROETHYL) SULFIDE: EXPRESSION PROFILES CORRELATE WITH

TREATMENT EFFICACY AND AN ESTABLISHED CLINICAL ENDPOINT

James F. Dillman III, Alison I. Hege, Christopher S. Phillips, Linda D. Orzolek, Albert J.

Sylvester, Carol Bossone, Claudia Henemyre-Harris, Robyn C. Kiser, Young W. Choi,

John J. Schlager and Carol L. Sabourin Downloaded from

Cell and Molecular Biology Branch, U.S. Army Medical Research Institute of Chemical jpet.aspetjournals.org Defense, Aberdeen Proving Ground, MD, 21010, USA (J.F.D., A.I.H., C.S.P., L.D.O.,

A.J.S., J.J.S.).

at ASPET Journals on September 24, 2021

Analytical Toxicology Division, U.S. Army Medical Research Institute of Chemical

Defense, Aberdeen Proving Ground, MD 21010, USA (C.B.).

Physiology and Immunology Branch, U.S. Army Medical Research Institute of Chemical

Defense, Aberdeen Proving Ground, MD, 21010, USA (C.H-H.).

Medical Research and Evaluation Facility, Battelle Memorial Institute, Columbus, OH,

USA (R.C.K., Y.W.C., C.L.S.).

1

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014

Running Title: Correlating drug efficacy and gene expression

Corresponding Author:

James F. Dillman III, Ph.D.

Cell and Molecular Biology Branch

U.S. Army Medical Research Institute of Chemical Defense

3100 Ricketts Point Rd

Aberdeen Proving Ground, MD 21010-5400 Downloaded from

Phone: 410.436.1723

Fax: 410.436.1960 jpet.aspetjournals.org Email: [email protected]

Text page count: 27 at ASPET Journals on September 24, 2021

Table count: 4

Figure count: 6

Reference count: 38

Abstract word count: 249

Introduction word count: 347

Discussion word count: 1180

Recommended section assignment: Toxicology

Non-standard abbreviations: SM, sulfur mustard; PCA, principal component analysis;

OHV, octyl homovanillamide; RMA, robust multiarray averaging; GO, ;

CEES, chloroethyl ethyl sulfide

2 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014

ABSTRACT

Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent alkylating agent.

Three treatment compounds have been shown to limit SM damage in the mouse ear vesicant model: dimercaprol, octyl homovanillamide and indomethacin. Microarrays were used to determine gene expression profiles of biopsies taken from mouse ears following exposure to SM in the presence or absence of treatment compounds. Mouse

ears were topically exposed to SM alone or were pretreated for 15 min with a treatment Downloaded from compound and then exposed to SM. Ear tissue was harvested 24 hr after exposure for ear weight determination, the endpoint used to evaluate treatment compound efficacy. jpet.aspetjournals.org RNA extracted from the tissues was used to generate microarray probes for gene expression profiling of therapeutic responses. Principal component analysis of the gene expression data revealed partitioning of the samples based on treatment compound and at ASPET Journals on September 24, 2021

SM exposure. Patterns of gene responses to the treatment compounds were indicative of exposure condition and were phenotypically anchored to ear weight. Pretreatment with indomethacin, the least effective treatment compound, produced ear weights close to those treated with SM alone. Ear weights from animals pretreated with dimercaprol or octyl homovanillamide were more closely associated with exposure to vehicle alone.

Correlation coefficients between gene expression level and ear weight revealed involved in mediating responses to both SM exposure and treatment compounds.

These data provide a basis for elucidating the mechanisms of response to SM and drug treatment, and also provide a basis for developing strategies to accelerate development of effective SM medical countermeasures.

3 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

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INTRODUCTION

Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent bifunctional alkylating agent capable of modifying and crosslinking cellular macromolecules such as DNA and by nucleophilic attack (Papirmeister et al., 1991). SM exposure can produce debilitating pulmonary, ocular, and cutaneous injuries. Following cutaneous exposure to SM, there is a dose-dependent latent phase of 8-24 h that precedes clinical

expression of tissue damage. Erythema occurs initially and is followed by vesication Downloaded from due to separation at the epidermal-dermal junction. This results in large fluid-filled lesions that are long lasting and slow to heal (Papirmeister et al., 1991; Petrali and jpet.aspetjournals.org Oglesby-Megee, 1997). The formation of blisters is accompanied by a potent inflammatory response, observed as increased production of inflammatory mediators and infiltration of the exposure area by activated immune cells (Sabourin et al., 2002; at ASPET Journals on September 24, 2021

Ricketts et al., 2000; Sabourin et al., 2000; Tsuruta et al., 1996; Rikimaru et al., 1991).

The mouse ear vesicant model (MEVM) was developed to rapidly screen for potential SM treatment compounds (Casillas et al., 1997). In this animal model, ear weight was determined as a measure of edema and inflammation and was utilized as a rapid, cost-effective endpoint to determine compound efficacy. Three potential antivesicant treatment compounds were identified using this screening paradigm: 2,3- dimercapto-1-propanol (dimercaprol, British anti-Lewisite), 2-(4-hydroxy-3-methoxy- phenyl)-N-octyl-ethanamide (octyl homovanillamide, OHV) and 1-(4-chlorobenzoyl)-5- methoxy-2-methyl-1H-indole-3-acetic acid (indomethacin) (Casillas et al., 2000).

Dimercaprol has been employed as a treatment for exposure to the chemical warfare agent Lewisite (another vesicant) and also for heavy metal poisoning. Octyl

4 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 homovanillamide is a vanilloid receptor antagonist, and indomethacin is a classic non- steroidal anti-inflammatory compound.

The mechanism of toxicity of SM is not well characterized, and previous studies have used DNA microarray technology to gain greater insight into the molecular pathways perturbed by SM exposure (Rogers et al., 2004; Sabourin et al., 2004).

Based on these studies, we hypothesized that gene expression profiling of mouse ear

skin exposed to SM alone or pretreated with one of these treatment compounds would Downloaded from provide important insight into the mechanism of cutaneous toxicity of SM and might identify genes and biological pathways involved in the response to SM exposure. jpet.aspetjournals.org at ASPET Journals on September 24, 2021

5 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

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METHODS

SM exposure and drug treatments

Outbred male CD-1 mice (28 to 33 g from Charles River Laboratories, Portage, MI) were anesthetized with a combination of ketamine (60 mg/kg) and xylazine (12 mg/kg) administered i.p. A single 5 µL application of SM (0.08 mg, 0.5 µmoles) in methylene chloride was applied topically to the inner surface of the right ear. The left ear (vehicle control) was exposed to 5 µL of methylene chloride only. For drug treatment, animals Downloaded from were administered 10 µl of dimercaprol (6.25 dose, 50.3 µmoles), 30 µl of OHV (0.585 mg, 1.995 µmoles), or 20 µl of indomethacin (1.34 mg, 3.74 µmoles) in ethanol 15 jpet.aspetjournals.org minutes prior to SM challenge. Each drug compound was tested at the maximum tolerated dose. A group of animals received drug in ethanol on the right ear and an

equal volume of ethanol (drug vehicle) on the left ear. A group of untreated, unexposed at ASPET Journals on September 24, 2021 animals served as naïve controls. At 24 hr post-exposure, animals were euthanized and an 8 mm diameter ear punch biopsy was obtained. The ear punch biopsy was weighed and immediately frozen in liquid nitrogen. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of

Laboratory Animals by the Institute of Laboratory Animal Resources, National Research

Council, in accordance with the stipulations mandated for an AAALAC International accredited facility.

Microarray procedures

All microarray experiments were performed using Affymetrix Mouse 430A oligonucleotide arrays, as described at

6 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 http://www.affymetrix.com/support/technical/datasheets/ mouse430_datasheet.pdf

(Affymetrix, Santa Clara, CA). RNA was isolated from frozen mouse ear biopsies using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The quality and amount of RNA was monitored throughout processing with an Agilent Bioanalyzer (Agilent, Palo Alto, CA) and a NanoDrop® ND-1000 UV-Vis

Spectrophotometer (Nanodrop Technologies, Rockland, DE). Purified RNA was used to

prepare biotinylated target RNA, with minor modifications from the manufacturer’s Downloaded from recommendations (http://www.affymetrix.com/support/technical/manual/ expression

_manual .affx). Briefly, 10 µg of total RNA was used to generate first-strand cDNA by jpet.aspetjournals.org using a T7-linked oligo(dT) primer. After second-strand synthesis, in vitro transcription was performed with biotinylated nucleotides (Enzo Kits from Affymetrix), resulting in approximately 100-fold amplification of cRNA. The target cRNA generated from each at ASPET Journals on September 24, 2021 sample was processed as per manufacturer's recommendation using an Affymetrix

GeneChip Instrument System (http://www.affymetrix.com/support/technical/ manual/expression_manual.affx). Briefly, spiked controls were added to 15 µg of fragmented cRNA before overnight hybridization using 10 µg of cRNA. Arrays were then washed and stained with streptavidin-phycoerythrin before being scanned on an Agilent

GeneArray Scanner. After scanning, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface. The 3'/5' ratios for GAPDH were between 0.8-1.72 and the ratios for β- were between 1.07-2.71. BioB spike controls were found to be present on 44 out of 64 chips that were scanned (75.0%, four were called marginal and not included in the calculation), with BioC, BioD and CreX also present in increasing intensity. When scaled

7 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 to a target intensity of 150 (using Affymetrix Microarray Suite 5.0 array analysis software), scaling factors for all arrays were between 0.559-2.243.

Microarray data analysis

Sample sizes for each treatment group are indicated in the figure legends.

Scanned output files from each array were inspected for quality control as described

above. Raw signal intensities were normalized using the robust multi-array averaging Downloaded from

(RMA) algorithm (Irizarry et al., 2003). The RMA normalized data were imported into

Partek Pro 6.0 (Partek, St. Charles, MO) and analyzed by principal component analysis jpet.aspetjournals.org (PCA) to determine the significant sources of variability in the data. A correlation coefficient (r) between signal intensities and ear weight was calculated for each gene and a p-value determined. A set of genes with r ≥ 0.90 was used to determine gene at ASPET Journals on September 24, 2021 pathways and molecular networks highly correlated with ear weight (a measure of drug efficacy). Onto-Express was used to screen for significant pathways modulated by SM exposure (Khatri et al., 2002).

Quantitative real-time PCR (Q-PCR)

All quantitative real-time PCR (Q-PCR) was performed with Taq-Man PCR reagents and analyzed using the ABI 7500 Sequence Detection System (Applied

Biosystems, Foster City, CA). All primers and probes used for Q-PCR analysis were designed using ABI Prism Primer Express V2.0 (Applied Biosystems) and are listed in

Supplementary Table 1. Primers and probes for each gene were optimized individually

8 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 for maximum amplification efficiency. A validation experiment was performed to demonstrate that each target gene and endogenous control in a multiplex reaction maintained equal efficiencies (data not shown). Total RNA was purified as described above and DNAse-I-treated on a purification column according to the manufacturer’s protocol (Qiagen, Valencia, CA). The reverse transcription reaction was carried out using 1 µg of total RNA (final concentration 50 ng/µl) using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). After completion of cDNA synthesis, all Downloaded from reactions were diluted to a final RNA input concentration of 5 ng/µl. For each gene analyzed, the experimental samples being tested (three biological replicates for each jpet.aspetjournals.org treatment group) were run in triplicate (three technical replicates) along with the corresponding no-template control and no-amplification control. The primer and probe

pair concentrations used for each gene are as follows: GAPDH (endogenous control), at ASPET Journals on September 24, 2021

50 nM forward primer, 50 nM reverse primer, 200 nM VIC probe; Fgfr-3, 800 nM forward primer, 800 nM reverse primer, 100 nM Fam probe; Krt1-17, 300 nM forward primer, 50 nM reverse primer, 100 nM Fam probe; L-myc, 300 nM forward primer, 300 nM reverse primer, 100 nM Fam probe. Amplification reactions were carried out using the instrument default cycle conditions. GAPDH was used as our internal reference gene to calculate the ∆Ct for each sample assayed. The ∆∆Ct was then calculated based on the average ∆Ct of the naïve control samples. The fold change in gene expression was

∆∆ determined as 2- Ct (Applied Biosystems, 2001). Dixon’s outlier test (extreme value test) was applied to all fold change values for each gene investigated.

9 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014

RESULTS

Evaluation of treatment compounds in the mouse ear vesicant model

Mouse ears were exposed to SM or pretreated with test compounds prior to SM exposure as described. Ear weights measured from 8 mm punch biopsies revealed that

SM exposure significantly increases mouse ear weight after 24 hrs compared with

control ear weights (Fig 1). Ear weights following pretreatment with indomethacin were Downloaded from decreased compared with SM alone, but were still significantly greater than the unexposed controls. In contrast, the weights of ears pretreated with OHV or jpet.aspetjournals.org dimercaprol prior to SM exposure were not significantly different from the control ear weights. Treatment compounds and vehicles alone did not significantly affect ear weight 24 hrs after exposure. at ASPET Journals on September 24, 2021

Gene expression profiling of mouse ears pretreated with candidate compounds and exposed to sulfur mustard

Treated and exposed mouse ear biopsies were processed, after obtaining ear weights, for analysis using oligonucleotide microarrays. Data from probed and scanned arrays were normalized using the RMA algorithm and then analyzed by PCA (Fig 2A,

B). PCA reduces the complexity of high-dimensional data and simplifies the task of identifying patterns and sources of variability in a large data set. The samples are represented by the points in the three-dimensional plot. The distance between any pair of points is related to the similarity between the two observations in high-dimensional space. Samples that are near each other in the plot are similar in a large number of

10 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 variables (i.e. expression level of individual genes). Conversely, samples that are far apart in the plot are different in a large number of variables. The PCA revealed that the drug-only controls, naïve controls, and ethanol vehicle controls partitioned away from the SM-exposed samples and the methylene chloride controls. In addition, SM-exposed samples partitioned away from all the controls. Of the three treatment compounds evaluated, the mouse ears pretreated with indomethacin prior to SM exposure

partitioned closest to the SM-exposed samples. In contrast, the mouse ears pretreated Downloaded from with OHV or dimercaprol prior to SM exposure partitioned closer to the methylene chloride control samples. This partitioning of the pretreated samples was also observed jpet.aspetjournals.org when looking at ear weights. The SM-exposed ears weighed more 24 hrs after exposure when compared with controls (Fig 1). Ears pretreated with indomethacin also showed a statistically significant increase in ear weight compared with control ear at ASPET Journals on September 24, 2021 weights (Fig 1) and partitioned closer to the SM-exposed ears. In contrast, OHV and dimercaprol pretreated ear weights are not significantly different 24 hr after SM exposure compared to control ear weights (Fig 1) and partition closer to methylene chloride controls. When visualized using ellipsoids representing a Euclidean space two standard deviations from the mean of each group, considerable overlap between the

SM-exposed samples and the indomethacin pretreated samples was observed (Fig 2B).

The OHV and dimercaprol pretreated samples do not overlap with the SM-exposed samples to the same extent, but do overlap with the methylene chloride-exposed control group (Fig 2B).

Identification of genes whose expression profile correlates with ear weight

11 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

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The gene expression profiles were analyzed and a correlation coefficient between signal intensity (representing gene expression level) and ear weight was determined for each gene. Genes showing a positive correlation or a negative correlation with ear weight to an r ≥ 0.90 are reported in Table 1 and Table 2 respectively. To determine the molecular functions and biological processes that are highly correlated with ear weight, the genes with r ≥ 0.90 were mapped to the Gene

Ontology™ (GO, The Gene Ontology Consortium, 2000) using the web-based search Downloaded from engine Onto-Express (Khatri et al., 2002). Onto-Express translates lists of differentially regulated genes identified in high throughput gene expression experiments into jpet.aspetjournals.org functional profiles based on the GO, and a statistical significance value is calculated.

Table 3 summarizes genes that represent the molecular functions most highly correlated with ear weight, and Table 4 summarizes the genes that represent the at ASPET Journals on September 24, 2021 biological processes most highly correlated with ear weight. An examination of the biological processes most highly correlated with ear weight (based on genes with a r ≥

0.90) reveals several major categories of biological processes: cell cycle regulation, inflammation, signal transduction, and cytoskeletal and cell adhesion processes. The gene expression profiles of genes that are classified in each of these biological processes are shown in Figure 3 (cell cycle regulation), Figure 4 (inflammation), Figure

5 (signal transduction), and Figure 6 (cytoskeletal and cell adhesion processes).

Validation of selected microarray results by Q-PCR

The reliability of our microarray data was confirmed using Q-PCR analysis of several genes whose expression levels were highly correlated with ear weight. As

12 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 shown in Supplementary Figure 1, the relative expression levels of Krt-17, Fgfr3, and L- myc based on Q-PCR analysis were consistent with the expression profiles determined by microarray analysis.

Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

13 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014

DISCUSSION

To gain insight into the molecular mechanism of SM toxicity, we utilized oligonucleotide microarrays to determine the gene expression profile of mouse ear skin exposed to SM. These results were compared with gene expression profiles determined from ears that were pretreated with one of three treatment compounds

(dimercaprol, OHV, or indomethacin). Principal component analysis revealed that the

gene expression profiles of dimercaprol- and OHV-pretreated tissues were similar to Downloaded from profiles from tissues exposed to vehicle alone (methylene chloride). In contrast, the gene expression profile of indomethacin-pretreated tissues was similar to that of tissues jpet.aspetjournals.org exposed only to SM. These profiles complement the ear weight data, which show that indomethacin is the least effective treatment compound for attenuating post-exposure increases in mouse ear weight. Statistical analysis of the gene expression profiles at ASPET Journals on September 24, 2021 revealed specific sets of genes that were highly correlated with SM-induced changes in ear weight (r ≥ 0.90). Gene ontology mapping of these gene sets revealed several major biological processes that were affected concomitantly with increased ear weight.

These were cell cycle regulation, inflammation, signal transduction, and cytoskeletal and cell adhesion processes.

Previous work has examined changes in gene expression following exposure to

SM. The systems studied include mouse ear (Sabourin et al., 2004; Rogers et al.,

2004; Sabourin et al., 2000), pig skin (Sabourin et al., 2002), cultured human keratinocytes (Platteborze, 2003; Schlager et al., 2002), HepG2 cells transfected with various stress gene reporter constructs (Schlager and Hart, 2000), and Jurkat cells exposed to the mustard-related compound chloroethyl ethyl sulfide (CEES) (Zhang et

14 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 al., 2002). Our previous studies show that vanilloids reduce skin edema and cytokine mRNA expression following SM exposure in a mouse model (Sabourin et al., 2003;

Sabourin et al., 2004). This is the first study to characterize SM-induced changes in gene expression in relation to a characterized endpoint of SM exposure (ear weight) and also to examine the effect of treatment compounds on SM-induced gene expression using a global gene expression approach. These results provide important

details that were previously lacking, and indicate that gene expression profiles may help Downloaded from to uncover the mechanisms of an observed phenotypic response (e.g., increased ear weight). Interestingly, few of the genes found to be highly correlated with increased ear jpet.aspetjournals.org weight showed what would normally be considered significant changes in gene expression (>2-fold change). Our results suggest that many of the genes highly correlated with a clinical endpoint may not show large fold-changes in gene expression. at ASPET Journals on September 24, 2021

Further investigation with a broader time course and dose response may reveal more dynamic changes in the expression levels of these genes.

Cell cycle regulation has been previously implicated as a biological process affected by SM exposure. This has been attributed to the ability of SM to crosslink DNA by alkylation at the N-7 of guanine (Papirmeister et al., 1991). The cell cycle has been shown to stop at the G1/S transition after SM exposure (Smith et al., 1993). p53 is an important component of the G1/S cell cycle checkpoint. Rosenthal et al. (1998) showed that p53 accumulates after SM exposure in cultured human keratinocytes. Schlager and Hart (2000) showed increases in activity of the p53 promoter response element in

HepG2 cells transfected with reporter constructs. We recently showed that rat pulmonary tissue exposed to SM via an intravenous route elicits a robust p53 response

15 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

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(Dillman et al., 2005). We also reported that p53 is phosphorylated on serine 15 within

15 minutes of SM exposure in cultured human keratinocytes (Minsavage and Dillman,

2005). Interestingly, it has been shown that there is a high incidence of lung cancer in former mustard gas workers, and there are p53 mutations in many of these cancers

(Easton et al., 1988; Manning et al., 1981; Nishimoto et al., 1998; Takeshima et al.,

1994; Tokuoka et al., 1986). The relationship between p53 activation, the cell cycle,

and the genes identified in SM-exposed mouse skin and rat lung remains to be Downloaded from determined.

Upregulation of cytokines and chemokines following SM exposure has been well jpet.aspetjournals.org characterized in a number of recent studies (Arroyo et al., 1999; Lardot et al., 1999;

Sabourin et al., 2000; Sabourin et al., 2002; Sabourin et al., 2003; Sabourin et al.,

2004). Related to this, a number of cell signaling pathways have been identified that at ASPET Journals on September 24, 2021 are activated or altered following SM exposure. Pharmacological inhibition of the p38

MAP kinase (MAPK14) has been reported to block upregulation of cytokine production in response to SM exposure in cultured keratinocytes (Dillman et al., 2004). In addition the transcription factor NF-κB, which is involved in both inflammation and apoptosis, is activated following SM exposure in cultured cells (Atkins et al., 2000; Schlager and Hart,

2000). In other systems, NF-κB has been shown to be activated by CEES in guinea pig lung (Chatterjee et al., 2003), and the Akt pathway is perturbed by CEES exposure in

Jurkat cells (Zhang et al., 2002). It is not clear how CEES exposure relates to SM exposure, but it is clear that signaling pathways associated with inflammatory response are activated by SM exposure. These signaling pathways may be potential therapeutic

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JPET #97014 targets for attenuating cytokine production and the subsequent recruitment of activated immune cells to the site of injury.

The is an important target of SM exposure. Several studies have identified as direct targets of SM alkylation (Dillman et al., 2003; van der

Schans et al., 2002). In addition, cross-linking of keratin filaments by SM, but not by other alkylating agents (e.g., CEES), has been observed (Dillman et al., 2003). This

alkylation results in collapse of the keratin network and Downloaded from subsequent adverse changes in cellular morphology (Werrlein and Madren-Whalley,

2000, 2003). Furthermore, exposure to SM results in changes in the keratin proteins jpet.aspetjournals.org that are expressed in cultured human keratinocytes (Rosenthal et al., 1998). Western blotting of keratinocytes exposed to SM revealed that keratin-5 (K5) and keratin-14

(K14) protein levels decrease, and keratin-1 (K1) and keratin-10 (K10) protein levels at ASPET Journals on September 24, 2021 increase. K5/K14 are associated with proliferating cells in the basal layer of the epidermis, while K1/K10 are associated with terminally differentiating cells in the suprabasal layers of the epidermis. SM exposure also results in perturbation of the actin cytoskeleton (Werrlein et al., 2005). Our data regarding altered gene expression are in agreement with these observations that cytoskeletal proteins are adversely affected by SM exposure. Although direct effects of SM on the network have not been reported to date, we observe changes in the mRNA levels of microtubule-based motor proteins from the family (Figure 6), suggesting that changes in microtubule-based motility may result from SM exposure.

In conclusion, our results demonstrate that gene expression profiling can provide additional insight into the pathways important in SM-induced injury and which pathways

17 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 may be potential targets for future development of antivesicant therapeutics. Our results suggest that gene expression profiling can be predictive of drug efficacy and imply that this could be an important tool in predictive toxicology and drug development.

These approaches are of particular importance in the discovery and testing of pharmacological treatments for toxicant exposure, since in these cases traditional clinical trials for regulatory approval are not an option. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

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ACKNOWLEDGEMENTS

We thank Robyn Lee for statistical consultation; Rich Sweeney for help with data management; and Gary Minsavage and Robert Werrlein for critical reading of the manuscript. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

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FOOTNOTES

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals of the Institute of

Laboratory Animal Resources, National Research Council.

Downloaded from

The data discussed in this publication have been deposited in NCBI’s Gene Expression

Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO jpet.aspetjournals.org Series accession number GSE2950.

Preprint requests: at ASPET Journals on September 24, 2021

James F. Dillman III, Ph.D.

Cell and Molecular Biology Branch

U.S. Army Medical Research Institute of Chemical Defense

3100 Ricketts Point Rd

Aberdeen Proving Ground, MD 21010-5400

1Current address for J.J.S.:

Biosciences and Protection Division

Human Effectiveness Directorate

2729 R St., Area B, Bldg 837

Wright-Patterson AFB, OH 45434

27 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

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LEGENDS FOR FIGURES

Figure 1. Pretreatment with compounds attenuated the increase in mouse ear weight induced by SM exposure. Mouse ears were pretreated with 6.25 mg dimercaprol, 1.34 mg indomethacin, or 0.6 mg OHV in ethanol and exposed 15 min later to 0.08 mg of SM in methylene chloride. After 24 hr, the weight of an 8-mm ear biopsy

was determined for each sample. The SM-exposed group and the SM + indomethacin Downloaded from groups had significantly greater ear weights than all other groups (p<0.05, based on a general linear model analysis). Sample sizes were: Unexposed (naïve), n=5; SM jpet.aspetjournals.org exposed, n=5; SM+indomethacin, n=6; SM+OHV, n=5; SM+ dimercaprol, n=6; indomethacin alone, n=4; OHV alone, n=5; dimercaprol alone, n=4; ethanol alone, n=3; methylene chloride alone, n=21. The error bars represent standard deviation. *p<0.05. at ASPET Journals on September 24, 2021

Figure 2. Principal component analysis (PCA) of gene expression profiles reveals partitioning of samples based on treatment group. Mouse ears were treated as described, ear weights were determined for an 8-mm biopsy of each sample, and the biopsy tissue was processed for analysis with oligonucleotide microarrays. The gene expression values were normalized using the robust multi-array averaging algorithm and then visualized using a principal component analysis (PCA) (A). Each sphere represents the gene expression profile of an individual sample. Point color corresponds to treatment group and point size indicates ear weight (three groups with weight cutoffs indicated in the legend). (B) PCA showing ellipsoids encompassing an area around each treatment group corresponding to two standard deviations from the mean of the

28 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #97014 group. The axes correspond to principal component 1 (PC1, x-axis, 41.3% of information content), PC 2 (y-axis, 12.8% information content), and PC3 (z-axis, 5.5% information content).

Figure 3. Gene expression profiles of individual cell cycle control and cellular growth genes that are highly correlated with SM-induced ear weight gain. Genes

with expression patterns that were highly correlated (r ≥ 0.9) with ear weight were Downloaded from identified by gene expression profiling. Gene ontology-based analysis was used to determine significantly altered biological processes. Individual genes were graphed jpet.aspetjournals.org according to treatment group and gene expression signal intensity. Error bars represent standard deviation. The data were analyzed for statistical significance using a 1-way

ANOVA with a Dunnet’s post test to look for treatment groups significantly different from at ASPET Journals on September 24, 2021 the unexposed control group. *p<0.05, ***p<0.001.

Figure 4. Gene expression profiles of individual inflammation and immune response genes that are highly correlated with SM-induced ear weight gain.

Genes with expression patterns that were highly correlated (r ≥ 0.9) with ear weight were identified by gene expression profiling. Gene ontology-based analysis was used to determine significantly altered biological processes. Individual genes were graphed according to treatment group and gene expression signal intensity. Error bars represent standard deviation. The data were analyzed for statistical significance using a 1-way

ANOVA with a Dunnet’s post test to look for treatment groups significantly different from the unexposed control group. *p<0.05, ***p<0.001.

29 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

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Figure 5. Gene expression profiles of individual signal transduction genes that are highly correlated with SM-induced ear weight gain.

Genes with expression patterns that were highly correlated (r ≥ 0.9) with ear weight were identified by gene expression profiling. Gene ontology-based analysis was used to determine significantly altered biological processes. Individual genes were graphed according to treatment group and gene expression signal intensity. Error bars represent

standard deviation. The data were analyzed for statistical significance using a 1-way Downloaded from

ANOVA with a Dunnet’s post test to look for treatment groups significantly different from the unexposed control group. *p<0.05, ***p<0.001. jpet.aspetjournals.org

Figure 6. Gene expression profiles of individual cytoskeletal and cell adhesion genes that are highly correlated with SM-induced ear weight gain. at ASPET Journals on September 24, 2021

Genes with expression patterns that were highly correlated (r ≥ 0.9) with ear weight were identified by gene expression profiling. Gene ontology-based analysis was used to determine significantly altered biological processes. Individual genes were graphed according to treatment group and gene expression signal intensity. Error bars represent standard deviation. The data was analyzed for statistical significance using a 1-way

ANOVA with a Dunnet’s post test to look for treatment groups significantly different from the unexposed control group. *p<0.05, ***p<0.001.

30 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version.

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Table 1. Genes showing a positive correlation between increased ear weight and expression level (r≥0.90).

Probe ID Accession Gene Name r p-value (r) Number 1448169_at NM_010664 keratin complex 1, acidic, gene 18 0.96 2.59E-24 1420450_at NM_019471 matrix metalloproteinase 10 0.95 4.16E-22 1448562_at NM_009477 uridine phosphorylase 1 0.92 1.29E-18 1418349_at L07264 diphtheria toxin receptor 0.92 1.91E-18 1427364_a_at S64539 ornithine decarboxylase, structural 0.92 1.92E-18 1418539_a_at U35368 protein tyrosine phosphatase, receptor type, E 0.92 2.06E-18 Downloaded from 1420699_at NM_020008 C-type (calcium dependent, carbohydrate recognition domain) lectin, superfamily member 12 0.92 4.96E-18 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 0.92 8.71E-18 1455899_x_at BB241535 suppressor of cytokine signaling 3 0.91 1.10E-17 1421078_at NM_053085 transcription factor 23 0.91 2.01E-17

1460378_a_at BC010465 testis derived transcript 0.91 2.44E-17 jpet.aspetjournals.org 1422562_at NM_019662 Ras-related associated with diabetes 0.91 2.72E-17 1420589_at NM_008217 hyaluronan synthase 3 0.91 3.81E-17 1433530_at BI694945 RIKEN cDNA 2210411K19 gene 0.91 3.83E-17 1448257_at NM_007854 solute carrier family 29 (nucleoside transporters), member 2 0.91 4.78E-17 1421134_at NM_009704 amphiregulin 0.91 5.21E-17 at ASPET Journals on September 24, 2021 1450791_at NM_008726 natriuretic peptide precursor type B 0.91 7.40E-17 1437271_at BB825816 B-cell stimulating factor 3 0.91 7.48E-17 1456212_x_at BB831725 suppressor of cytokine signaling 3 0.91 7.96E-17 1431936_a_at AK009828 neuraminidase 2 0.90 9.19E-17 1418480_at NM_023785 chemokine (C-X-C motif) ligand 7 0.90 9.55E-17 1418612_at NM_011407 schlafen 1 0.90 1.01E-16 1423227_at AA798563 keratin complex 1, acidic, gene 17 0.90 1.05E-16 1451160_s_at BB049138 DNA segment, Chr 7, ERATO Doi 458, expressed 0.90 1.36E-16 1437270_a_at BB825816 B-cell stimulating factor 3 0.90 1.54E-16 1420664_s_at NM_011171 protein C receptor, endothelial 0.90 1.72E-16 1417406_at AF366401 SERTA domain containing 1 0.90 1.81E-16 1419647_a_at NM_133662 immediate early response 3 0.90 1.87E-16 1419569_a_at BC022751 interferon-stimulated protein 0.90 2.02E-16 1437226_x_at AV110584 MARCKS-like protein 0.90 2.17E-16 1451340_at BC027152 AT rich interactive domain 5A (Mrf1 like) 0.90 2.32E-16 1418616_at NM_010757 v-maf musculoaponeurotic fibrosarcoma oncogene family, protein K (avian) 0.90 2.81E-16 1436235_x_at BB750674 RIKEN cDNA 4732471D19 gene 0.90 3.09E-16 1449984_at NM_009140 chemokine (C-X-C motif) ligand 2 0.90 3.16E-16 1425837_a_at AF199491 CCR4 carbon catabolite repression 4-like (S. cerevisiae) 0.90 3.73E-16

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Table 2. Genes showing a negative correlation between increased ear weight and expression level (r≥0.90).

Probe ID Accession Gene Name r p-value (r) Number 1416855_at BB550400 growth arrest specific 1 -0.94 7.50E-21 1450645_at NM_008631 metallothionein 4 -0.94 7.72E-21 1426530_a_at BM120233 kelch-like 5 (Drosophila) -0.93 4.77E-20 1436528_at AI842353 expressed sequence AI842353 -0.93 1.47E-19 1451075_s_at BB294133 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 2 -0.93 1.73E-19 1454704_at BI106458 RIKEN cDNA 9330185J12 gene -0.93 2.11E-19 1423902_s_at AF467766 Rho guanine nucleotide exchange factor (GEF) 12 -0.93 3.54E-19 1448240_at NM_019709 membrane-bound transcription factor protease, site 1 -0.92 7.99E-19

1423994_at BQ175246 kinesin family member 1B -0.92 1.08E-18 Downloaded from 1418000_a_at NM_008410 integral 2B -0.92 1.31E-18 1452882_at BF322962 progesterone receptor membrane component 2 -0.92 1.90E-18 1417533_a_at NM_010580 integrin beta 5 -0.92 2.16E-18 1428187_at AK018679 CD47 antigen (Rh-related antigen, integrin-associated signal transducer) -0.92 3.04E-18 jpet.aspetjournals.org 1449815_a_at NM_024186 single-stranded DNA binding protein 2 -0.92 3.65E-18 1426401_at BQ173889 protein phosphatase 3, catalytic subunit, alpha isoform -0.92 4.13E-18 1434777_at BG064871 lung carcinoma myc related oncogene 1 -0.92 5.13E-18 1423513_at BG141609 neogenin -0.92 5.27E-18 1425981_a_at U47333 retinoblastoma-like 2 -0.92 5.81E-18

1460303_at NM_008173 nuclear receptor subfamily 3, group C, member 1 -0.92 6.84E-18 at ASPET Journals on September 24, 2021 1417639_at BC010590 solute carrier family 22 (organic cation transporter), member 4 -0.91 1.05E-17 1456195_x_at BB543979 integrin beta 5 -0.91 1.33E-17 1428714_at BF322962 progesterone receptor membrane component 2 -0.91 1.99E-17 1434010_at AV326938 RIKEN cDNA 2810425F24 gene -0.91 2.26E-17 1427243_at BI248354 expressed sequence AA536743 -0.91 2.43E-17 1427233_at AV291373 serologically defined colon cancer antigen 33 -0.91 3.22E-17 1434205_at BM247370 protein phosphatase 2, regulatory subunit B (B56), gamma isoform -0.91 4.69E-17 1433670_at BE571790 epithelial membrane protein 2 -0.91 5.60E-17 1418501_a_at AW548944 oxidation resistance 1 -0.91 5.64E-17 1448147_at NM_013869 tumor necrosis factor receptor superfamily, member 19 -0.91 7.34E-17 1422742_at NM_007772 human immunodeficiency virus type I enhancer binding protein 1 -0.90 9.20E-17 1425658_at AY083458 CD109 antigen -0.90 1.63E-16 1449070_x_at BB770932 cDNA sequence AB023957 -0.90 1.68E-16 1436038_a_at BF318238 Down syndrome critical region homolog 5 (human) -0.90 1.72E-16 1421841_at NM_008010 fibroblast growth factor receptor 3 -0.90 1.84E-16 1450420_at BQ174473 stromal antigen 1 -0.90 2.32E-16 1422852_at NM_019686 calcium and integrin binding family member 2 -0.90 2.33E-16 1455254_at AV250006 RIKEN cDNA 4833420G11 gene -0.90 2.33E-16 1451783_a_at D50366 kinesin-associated protein 3 -0.90 2.74E-16 1418925_at NM_009886 cadherin EGF LAG seven-pass G-type receptor 1 -0.900 3.37E-16 1460192_at NM_020573 oxysterol binding protein-like 1A -0.90 3.57E-16 1429063_s_at BG066903 kinesin family member 16B -0.90 3.86E-16

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1420350_at NM_028625 small proline rich-like 2 -0.90 4.17E-16 1416430_at NM_009804 catalase -0.90 4.21E-16 1427040_at U13371 kidney cell line derived transcript 1 -0.90 4.47E-16 1417061_at AF226613 solute carrier family 40 (iron-regulated transporter), member 1 -0.90 4.82E-16 1423679_at BC013800 RIKEN cDNA 2810432L12 gene -0.90 4.87E-16

Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

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Table 3. Molecular functions most highly correlated with ear weight.

Function Name Accession number Gene Name Unique Reference Total Unique Input Total P-Value cytokine activity 141 6 5.89626E-06 Probe Gene UniGene cluster This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 1421134_at NM_009704 amphiregulin Areg 8039

1437270_a_at BB825816 B-cell stimulating factor 3 Bsf3 347919 JPET FastForward.PublishedonDecember23,2005asDOI:10.1124/jpet.105.097014 1437270_a_at BB825816 B-cell stimulating factor 3 Bsf3 347919 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 Cxcl1 21013 1449984_at NM_009140 chemokine (C-X-C motif) ligand 2 Cxcl2 4979 1418480_at NM_023785 chemokine (C-X-C motif) ligand 7 Cxcl7 293614 chemokine activity 37 3 1.07037E-05 Probe Gene UniGene cluster 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 Cxcl1 21013 1449984_at NM_009140 chemokine (C-X-C motif) ligand 2 Cxcl2 4979 1418480_at NM_023785 chemokine (C-X-C motif) ligand 7 Cxcl7 293614 microtubule motor activity 52 3 4.31521E-05 Probe Gene UniGene cluster 1451783_a_at D50366 kinesin-associated protein 3 Kifap3 4651 1423994_at BQ175246 kinesin family member 1B Kif1b 252497 1429062_at BG066903 kinesin family member 16B Kif16b 251934 motor activity 101 2 0.000663603 Probe Gene UniGene cluster 1423994_at BQ175246 kinesin family member 1B Kif1b 252497 1429062_at BG066903 kinesin family member 16B Kif16b 251934 calmodulin binding 114 3 0.000783378 Probe Gene UniGene cluster 1437226_x_at AV110584 MARCKS-like protein Mlp 2769 1426401_at BQ173889 protein phosphatase 3, catalytic subunit, alpha isoform Ppp3ca 331389 1422562_at NM_019662 Ras-related associated with diabetes Rrad 29467 growth factor activity 132 3 0.001748999 Probe Gene UniGene cluster 1421134_at NM_009704 amphiregulin Areg 8039 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 Cxcl1 21013 1418349_at L07264 diphtheria toxin receptor Dtr 289681

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Function Name Accession number Gene Name Unique Reference Total Unique Input Total P-Value protein binding 1814 9 0.007787853 Probe Gene UniGene cluster 1417533_a_at NM_010580 integrin beta 5 Itgb5 6424

1456195_x_at BB543979 integrin beta 5 Itgb5 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 1423903_at BB049138 DNA segement, Chr7, ERATO Doi 458 expressed D7Ertd458e 227506

1426401_at BQ173889 protein phosphatase 3, catalytic subunit, alpha isoform Ppp3ca 331389 JPET FastForward.PublishedonDecember23,2005asDOI:10.1124/jpet.105.097014 1418616_at NM_010757 v-maf musculoaponeurotic fibrosarcoma oncogene family, protein K (avian) Mafk 157313 1416855_at BB550400 growth arrest specific 1 Gas1 1421866_at NM_008173 nuclear receptor subfamily 3, group C, member 1 Nr3c1 129481 1423902_s_at AF467766 Rho guanine nucleotide exchange factor (GEF) 12 2310014B11Rik 275266 1423513_at BG141609 neogenin Neo1 42249 structural constituent of cytoskeleton 113 2 0.007925622 Probe Gene UniGene cluster 1448169_at NM_010664 keratin complex 1, acidic, gene 18 Krt1-18 22479 1423227_at AA798563 keratin complex 1, acidic, gene 17 Krt1-17 14046 receptor activity 1099 7 0.008284202 Probe Gene UniGene cluster 1417533_a_at NM_010580 integrin beta 5 Itgb5 6424 1456195_x_at BB543979 integrin beta 5 Itgb5 1423903_at BB049138 DNA segement, Chr7, ERATO Doi 458 expressed D7Ertd458e 227506 1421866_at NM_008173 nuclear receptor subfamily 3, group C, member 1 Nr3c1 129481 1421841_at NM_008010 fibroblast growth factor receptor 3 Fgfr3 6904 1420664_s_at NM_011171 protein C receptor, endothelial Procr 3243 1415921_a_at NM_013869 tumor necrosis factor receptor superfamily, member 19 Tnfrsf19 281356

1All genes with an r≥0.90 (Tables 2 and 3) were uploaded into OntoExpress. Gene ontology categories containing less that two genes were excluded from this table.

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Table 4. Biological processes most highly correlated with ear weight.

Function Name Accession number Gene Name Unique Reference Total Unique Input Total P-Value regulation of cell cycle 215 3 1.82E-04 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Probe Gene UniGene cluster

1418616_at NM_010757 v-maf musculoaponeurotic fibrosarcoma oncogene family, protein K (avian) Mafk 157313 JPET FastForward.PublishedonDecember23,2005asDOI:10.1124/jpet.105.097014 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 Cxcl1 21013 1434777_at BG064871 lung carcinoma myc related oncogene 1 Lmyc1 318431 cell cycle arrest 31 2 2.02E-04 Probe Gene UniGene cluster 1416855_at BB550400 growth arrest specific 1 Gas1 1418612_at NM_011407 schlafen 1 Slfn1 10948 signal transduction 451 5 2.33E-04 Probe Gene UniGene cluster 1455899_x_at BB241535 suppressor of cytokine signaling 3 Socs3 1456212_x_at BB831725 suppressor of cytokine signaling 3 Socs3 1449984_at NM_009140 chemokine (C-X-C motif) ligand 2 Cxcl2 4979 1421841_at NM_008010 fibroblast growth factor receptor 3 Fgfr3 6904 1434205_at BM247370 protein phosphatase 2, regulatory subunit B (B56), gamma isoform Ppp2r5c 240396 cytoskeleton organization and biogenesis 119 2 3.46E-04 Probe Gene UniGene cluster 1448169_at NM_010664 keratin complex 1, acidic, gene 18 Krt1-18 22479 1423227_at AA798563 keratin complex 1, acidic, gene 17 Krt1-17 14046

G1/S transition of mitotic cell cycle 28 2 3.94E-04 Probe Gene UniGene cluster 1426401_at BQ173889 protein phosphatase 3, catalytic subunit, alpha isoform Ppp3ca 331389 1418612_at NM_011407 schlafen 1 Slfn1 10948 cell cycle 208 2 5.04E-04 Probe Gene UniGene cluster 1425981_a_at U47333 retinoblastoma-like 2 Rbl2 235580 1421939_a_at BQ174473 Stromal Antigen 1 Stag1 42135 immune response 205 3 1.01E-03 Probe Gene UniGene cluster 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 Cxcl1 21013

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Function Name Accession number Gene Name Unique Reference Total Unique Input Total P-Value 1449984_at NM_009140 chemokine (C-X-C motif) ligand 2 Cxcl2 4979 1418480_at NM_023785 chemokine (C-X-C motif) ligand 7 Cxcl7 293614

microtubule-based process 69 3 1.42E-03 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Probe Gene UniGene cluster

1451783_a_at D50366 kinesin-associated protein 3 Kifap3 4651 JPET FastForward.PublishedonDecember23,2005asDOI:10.1124/jpet.105.097014 1423994_at BQ175246 kinesin family member 1B Kif1b 252497 1429062_at BG066903 kinesin family member 16B Kif16b 251934 cell growth and/or maintenance 231 3 1.47E-03 Probe Gene UniGene cluster 1418616_at NM_010757 v-maf musculoaponeurotic fibrosarcoma oncogene family, protein K (avian) Mafk 157313 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 Cxcl1 21013 1434777_at BG064871 lung carcinoma myc related oncogene 1 Lmyc1 318431 cell adhesion 371 4 3.75E-03 Probe Gene UniGene cluster 1417533_a_at NM_010580 integrin beta 5 Itgb5 6424 1456195_x_at BB543979 integrin beta 5 Itgb5 1423513_at BG141609 neogenin Neo1 42249 1418925_at NM_009886 Celsr1 22680 intracellular signaling cascade 357 3 5.38E-03 Probe Gene UniGene cluster 1423902_s_at AF467766 cadherin EGF LAG seven-pass G-type receptor 1 2310014B11Rik 275266 1455899_x_at BB241535 suppressor of cytokine signaling 3 Socs3 1456212_x_at BB831725 suppressor of cytokine signaling 3 Socs3 inflammatory response 85 2 7.37E-03 Probe Gene UniGene cluster 1419209_at NM_008176 chemokine (C-X-C motif) ligand 1 Cxcl1 21013 1449984_at NM_009140 chemokine (C-X-C motif) ligand 2 Cxcl2 4979 cell surface receptor linked signal transduction 86 3 7.61E-03 Probe Gene UniGene cluster 1437270_a_at BB825816 B-cell stimulating factor 3 Bsf3 347919 1437270_a_at BB825816 B-cell stimulating factor 3 Bsf3 347919 C-type (calcium dependent, carbohydrate recognition domain) lectin, superfamily 1420699_at NM_020008 member 12 Clecsf12 239516

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Function Name Accession number Gene Name Unique Reference Total Unique Input Total P-Value cell-cell adhesion 26 2 9.79E-03 Probe Gene UniGene cluster 1423903_at BB049138 DNA segement, Chr7, ERATO Doi 458 expressed D7Ertd458e 227506 C-type (calcium dependent, carbohydrate recognition domain) lectin, superfamily This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 1420699_at NM_020008 member 12 Clecsf12 239516

JPET FastForward.PublishedonDecember23,2005asDOI:10.1124/jpet.105.097014 1All genes with an r≥0.90 (Tables 2 and 3) were uploaded into OntoExpress. Gene ontology categories containing less that two genes were excluded from this table

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Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 24, 2021 24, September on Journals ASPET at JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on December 23, 2005 as DOI: 10.1124/jpet.105.097014 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021