US Army Corps of EngineersBUILDING STRONG®

Systems Biology Investigation to Explore the Computational Toxicology Tool, GO-Modeler

Kurt A. GustBindu NanduriArun RawatMitchell S. WilbanksMichael Quinn Jr.Jeff ChenShane BurgessEdward J. Perkins

Authors

K.A. Gust1, Bindu Nanduri2, Arun Rawat3, Mitchell S. Wilbanks1, Michael Quinn Jr.4, Jeff Chen1, Shane Burgess2, Edward J. Perkins1

1US Army, Engineer Research and Development Center2Mississippi State University3University of Southern Mississippi4US Army, Public Health Command

"Today’s challenge is to realize greater knowledge and understanding from the data-rich opportunities provided by modern high-throughput genomic technology."

Professor Andrew Cossins,

Consortium for Post-Genome Science, Chairman.

What is the Gene Ontology?“a controlled vocabulary that can be applied to all organisms even as knowledge of gene and protein roles in cells is accumulating and changing” the de facto standard for functional annotationassign functions to gene products at different levels, depending on how much is known about a gene product is used for a diverse range of species structured to be queried at different levels, eg:

► find all the chicken gene products in the genome that are involved in signal transduction

► zoom in on all the receptor tyrosine kinases human readable GO function has a digital tag to allow computational analysis of large datasets

COMPUTATIONALLY-AMENIABLE ENCYCLOPEDIA OF GENE FUNCTIONS

Use GO for……. Determining which classes of gene products

are over-represented or under-represented. Grouping gene products by biological

function. Relating a protein’s location to its function. Focusing on particular biological pathways

and functions Hypothesis-testing: GO Modeler.

“GO-slim”

In contrast, GO-Modeler uses the deep granular information rich data suitable for hypothesis-testing

Many people use “GO Slims” which capture only high-level terms which are more often then not extremely poorly informative and not suitable for hypothesis-testing.

Step I. GO-based Phenotype Scoring.

Gene product Th1 Th2 Treg Inflammation

IL-2 1.58 1.58 -1.58

IL-4 0.00 0.00 0.00 0.00

IL-6 0.00 -1.20 1.20 -1.20

IL-8 0.00 0.00 1.18 1.18

IL-10 0.00 0.00 0.00 0.00

IL-12 0.00 0.00 0.00 0.00

IL-13 1.51 -1.51 0.00 0.00

IL-18 0.91 0.91 0.91 0.91

IFN-g 0.00 0.00 0.00 0.00

TGF-b -1.71 0.00 1.71 -1.71

CTLA-4 -1.89 -1.89 1.89 -1.89

GPR-83 -1.69 -1.69 1.69 -1.69

SMAD-7 0.00 0.00 0.00 0.00

Net Effect -1.29 -5.38 10.15 -5.98

Step III. Inclusion of quantitative data to the phenotype scoring table and calculation of net affect.

Underlying GO-Modeler

1-111SMAD-7

-11-1-1GPR-83

-11-1-1CTLA-4

-110-1TGF-b

11-11IFN-g

1111IL-18

NDND1-1IL-13

NDND-11IL-12

011-1IL-10

11NDNDIL-8

1-11IL-6

ND11-1IL-4

-11ND1IL-2

InflammationTregTh2Th1Gene product

ND = No data

Step II. Multiply by quantitative data for each gene product.

GO Modeler

http://agbase.msstate.edu/cgi-bin/tools/GOModeler.pl

Problem Identification

Daphnia magna

Northern bobwhite

Earthworm

Fathead minnow

Rat

Animals may be exposed to soils, water and/or food contaminated with energetic compounds on Army ranges.

Song Birds

Western Fence Lizard

Contamination

Problem Identification

Toxicology (Parent Compound & Metabolites):

Aberrant Neuromuscular Effects Anemia and various impacts on Blood Chemistry Gastrointestinal Impacts / General Impacts on Viscera. Mortality at High Doses

Result: Increased regulatory concern over RDX, TNT & their breakdown products.

2,4,6-trinitrotoluene (TNT)

CH3

NO2

NO2

NO2

2-Amino-4,6-dinitrotoluene (2A-DNT)

CH3

NH2

NO2

NO2

Systems Biol. - Transcriptomics + ProteomicsProteomics

TranscriptomicsBiological Networks?

Northern Bobwhite

2A-DNT

2A-DNT Exposures with Northern BobwhiteSub-Acute Exposure – Birds dosed with 2A-DNT for 14d at 0, 50, 125, 225, 550, or 1000 mg/kg/day via oral gavage.

Sub-Chronic Exposure -Birds dosed with 2A-DNT for 60d at 0, 0.5, 3, 14 or 30 mg/kg/day via oral gavage, 12 biol reps for each sex.

Transcriptomics and Proteomics – Leveraging Sub-Chronic Exp.•Proteomics investigated in Liver and Kidney tissues. Four biological replicates were investigated for the 0 and 30 mg/kg/d treatments. Liver tissue was investigated in males and females, and kidney tissue was examined in males only.

•Transcriptomics investigated in Liver and Kidney tissues. Four biological replicates for both males and females were examined for the 0, 3, 14 and 30 mg/kg/d treatments for each tissue.

2A-DNT Toxicology

Figure 1. The number of days survived by northern bobwhite (Colinus virginianus) exposed daily to oral gavages of 2A-DNT (2-amino-2,6-dinitrotoluene; mg/kg-d) for a total of 14 days.

Quinn et al 2010, Ecotoxicology

Sub-Acute 14d Exposure

2A-DNT ToxicologySub-Chronic 60d Exposure

Increased Liver weights (Brain-normalized) at the highest 2A-DNT dose (30 mg/kg/d) in both males and females.

significant reduction in white blood cell counts at the 30 mg/kg/d dose in females

Out of 15 blood chemistry investigations:

Alanine aminotransferase (ALT) significantly decreased and Triglycerides (TRIG) were significantly increased respectively at intermediate 2A-DNT concentrations (0.5 – 3 mg/kg/d) in males only.

Quinn et al 2010, Ecotoxicology

Hypotheses Based on Phenotypes 1. Daily oral dosing of 2A-DNT had no effect on genes and molecular pathways

involved in Lipid metabolism in liver tissues of Northern bobwhite.

2. Daily oral dosing of 2A-DNT had no effect on peroxisome proliferator-activated receptor (PPAR)-controlled pathways in Northern bobwhite.

3. Daily oral dosing of 2A-DNT had no effect on genes and molecular pathways involved in energy metabolism in Northern bobwhite.

4. Daily oral dosing of 2A-DNT had no effect on genes and molecular pathways involved in immune function in Northern bobwhite.

5. Daily oral dosing of 2A-DNT had no effect on genes and molecular pathways involved in xenobiotic metabolism in Northern bobwhite.

GO Modeler – Hypothesis Statements1. lipid metabolismGO:0006629 lipid metabolic process 

2. PPAR controlled pathwaysGO:0006629 lipid metabolic process (includes GO:0006631 fatty acid metabolic process)GO:0006699 bile acid biosynthetic process GO:0046950 cellular ketone body metabolic processGO:0045444 fat cell differentiation, GO:0002024 diet induced thermogenesis AND/OR GO:0050873 brown fat cell differentiation, GO:0060548 negative regulation of cell death AND/OR GO:0019725 cellular homeostasis, GO:0016567 protein ubiquitination AND/OR GO:0016574 histone ubiquitination, GO:0006094 gluconeogenesis 

3. energy metabolismGO:0015975 energy derivation by oxidation of reduced inorganic compounds GO:0015980 energy derivation by oxidation of organic compoundsGO:0006119 oxidative phosphorylation 

4. immune functionGO:0006955 immune response 

5. xenobiotic metabolismGO:0006805 xenobiotic metabolic processGO:0009410 response to xenobiotic stimulus 

6. liver weightGO:0035265 organ growthGO:0031100 organ regeneration

7. alanine transferaseGO:0004021 L-alanine:2-oxoglutarate aminotransferase activity GO:0047810 D-alanine:2-oxoglutarate aminotransferase activity 

8. triglycerideGO:0006641 triglyceride metabolic processGO:0034197 triglyceride transport

2nd Generation Multi-tissue Microarray• Agilent G2 one-color platform• 8 x 15K spot, high-density

oligonucleotide• Source cDNA library

developed using Next Gen Seq • Fully Annotated, Open Source

Knowledgebase www.quailgenomics.info

Northern Bobwhite Genome Tools

Rawat et al 2010 BMC Bioinformatics

TreatmentIncreased

ExpressionDecreased Expression Total

Female, Liver 3 mg/kg/day 63 69 132Female, Liver 14 mg/kg/day 139 130 269Female, Liver 30 mg/kg/day 69 110 179Male, Liver 3 mg/kg/day 139 206 345Male, Liver 14 mg/kg/day 108 170 278Male, Liver 30 mg/kg/day 128 141 269

2A-DNT TranscriptomicsResults Overview

•Total Differentially Expressed Transcripts (DET)•Even Distribution of Increased and Decreased Expr.

2A-DNT TranscriptomicsResults Overview

L = Liver Tissue3 = 3 mg/kg/d14 = 14 mg/kg/d30 = 30 mg/kg/d

132 269

179

345 278

270

•Total Differentially Expressed Transcripts (DET)•Commonality in DET among Doses

2A-DNT TranscriptomicsResults Overview

L = Liver Tissue3 = 3 mg/kg/d14 = 14 mg/kg/d30 = 30 mg/kg/d

•Commonality in DET among Sexes was Limited

Methods: Comparative shotgun proteomics Male and Female liver tissue of Northern

bobwhite, 2-ADNT 30mg/kg/d vs Controls. Pressure cycling technology sample

preparation & trypsin digested proteins Analysis: 1 dimensional liquid

chromatography nano-spray tandem mass spectrometry

2A-DNT Proteomics

Results Overview: 2,672 proteins identified Total Differentially Expressed:

2A-DNT Proteomics

TreatmentIncreased

ExpressionDecreased Expression Total

Male, Liver 30 mg/kg/day 77 215 292Female, Liver 30 mg/kg/day 99 59 158

Transcriptiomics vs ProteomicsTarget by Target Comparison

•Limited Overlap of differentially expressed targets.

•HOWEVER, Syntax differences among annotations still need to be addressed. Commonality is likely UNDER-Represented.

GO-Modeler Hypothesis Tests

•“( )” Reject Null Hypothesis•Expression in liver was sex specific•Lipid metabolism impacted, but not strongly at transcript level.•PPAR controlled pathways were impacted by 2A-DNT Exposure.

Transcriptomics Data

Female, Liver 30 mg/kg/day

Male, Liver 30 mg/kg/day

Increased Decreased

0 1

1 8

0 0

1 3

0 0

1 0

0 0

0 0

GO-Modeler Hypothesis Tests

PPAR controlled pathways

•Expression in liver was largely sex specific.

•Marginal Impacts on xenobiotic metabolism and Immune response – Do not Reject Null Hypothesis•Significant Impacts: Energy Metabolism, PPAR controlled pathways and Lipid Metabolism – Reject Null Hypothesis•Some Parallelism with Transcriptomics Results

Proteomics Data

Related Work Investigating 2,6-DNT

Impacts on Lipid Metabolism

•Decreased Expression of PPAR Signaling Pathway

•Decreased Lipid Metabolism Lipid Inundation in Liver Wintz et al 2006 Toxicol Sci

Rawat et al 2010 Physiol Genomics

Female, Liver Tissue, 30 mg/kg/day

Netw ork Function

ScoreOrganismal Functions, Cell Death, Carbohydrate Metabolism 19Lipid Metabolism, Small Molecule Biochemistry, Vitamin and Mineral Metabolism 18Cellular Grow th and Proliferation, Skeletal and Muscular System Development and Function, Cellular Function and Maintenance 16Lipid Metabolism, Small Molecule Biochemistry, Vitamin and Mineral Metabolism 16Cellular Movement, Nervous System Development and Function, Organ Development 16

Male, Liver Tissue, 30 mg/kg/dayDNA Replication, Recombination, and Repair, Protein Degradation, Protein Synthesis 20Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry 18Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry 17Cell Cycle, Cellular Development, Cellular Assembly and Organization 17Cell Cycle, Hair and Skin Development and Function, Cellular Development 15

Female, Liver Tissue, 14 mg/kg/dayLipid Metabolism, Small Molecule Biochemistry, Cellular Development 61Cell Death, Cellular Development, Tissue Development 21Organismal Injury and Abnormalities, Antigen Presentation, Humoral Immune Response 19Cancer, Dermatological Diseases and Conditions, Cellular Development 18Cell Death, Organ Morphology, Lipid Metabolism 15

Male, Liver Tissue, 14 mg/kg/dayLipid Metabolism, Small Molecule Biochemistry, Cellular Development 21Lipid Metabolism, Small Molecule Biochemistry, Cellular Movement 19Lipid Metabolism, Small Molecule Biochemistry, Vitamin and Mineral Metabolism 18Lipid Metabolism, Small Molecule Biochemistry, Vitamin and Mineral Metabolism 16Cell-To-Cell Signaling and Interaction, Tissue Development, Cellular Movement 16

Female, Liver Tissue, 3 mg/kg/dayCarbohydrate Metabolism, Behavior, Genetic Disorder 21Cellular Grow th and Proliferation, Cell Death, DNA Replication, Recombination, and Repair 19Cellular Grow th and Proliferation, Cellular Movement, Cancer 17Cellular Grow th and Proliferation, Hematological System Development and Function, Inflammatory Response 15Cell-To-Cell Signaling and Interaction, Hair and Skin Development and Function, Tissue Development 10

Male, Liver Tissue, 3 mg/kg/dayAntigen Presentation, Cell-To-Cell Signaling and Interaction, Hematological System Development and Function 23Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry 20Cellular Development, Cell Cycle, DNA Replication, Recombination, and Repair 15Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry 15Cellular Grow th and Proliferation, Small Molecule Biochemistry, Cancer 15

Table 1. Top netw orks based on transcriptomics data using Ingenutity Pathw ay Analysis.

2A-DNT TranscriptomicsIngenuity Pathway Analysis

•Many of Top 5 Networks involved in Lipid Metabolism

•Results Similar to impacts of 2,6-DNT (Rawat et al 2010, Physiol Genomics)

2A-DNT ProteomicsIngenuity Pathway Analysis

•Many of Top 5 Networks involved in Lipid Metabolism

•Parallels Results of Transcriptomics Analysis

Female, Liver 30 mg/kg/d

Network Functions

Score1 Lipid Metabolism, Small Molecule Biochemistry, Molecular Transport 92 Hepatic System Disease, Lipid Metabolism, Small Molecule Biochemistry 73 Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry 74 Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry 75 Lipid Metabolism, Small Molecule Biochemistry, Molecular Transport 6

Male, Liver 30 mg/kg/d1 Lipid Metabolism, Small Molecule Biochemistry, Metabolic Disease 92 Lipid Metabolism, Small Molecule Biochemistry, Molecular Transport 83 Hepatic System Disease, Lipid Metabolism, Molecular Transport 74 Hepatic System Disease, Cell Death, Lipid Metabolism 75 Carbohydrate Metabolism, Small Molecule Biochemistry, Molecular Transport 6

Table 2. Top netw orks based on transcriptomics data using Ingenutity Pathw ay Analysis.

Top Network: Male, Liver 30 mg/kg/day2A-DNT Transcriptomics

Top Network: Female, Liver 30 mg/kg/day2A-DNT Transcriptomics

Summary and Conclusions•GO Modeler provides a novel a priori hypothesis testing mechanism utilizing functional annotation.•GO Modeler successfully identified impacts related toxicological phenotypes.•Provides function-based down-selection of targets focusing on most relevant bio-molecules.•Results parallel enrichment identified by Ingenuity Pathway Analysis.•Improvements Needed: More Objective Hypothesis Test, Increased Automation, Robust Validation. http://agbase.msstate.edu/cgi-bin/tools/GOModeler.pl