# Integration of Toxicogenomic Technology to Inform Risk 

# Assessment A Case Study: Propiconazole Stephen Nesnow, Ph.D. Senior Scientist National Health and Environmental Effects Research Laboratory, ORD, US EPA, Research Triangle Park, NC

5/24/2011

# Overview of presentation

• Goals of the project • Omics overview • Recap of propiconazole toxicological effects and 

## description of MOA • Summary of incorporation of data from omics and other technologies into MOA 

## • Selected MOA key events in detail • Summary of how omics could be incorporated into risk assessment and human relevance frameworks 

## • Summary of other advantages/uses of omics 2

# Goals of the project



# • To apply omics and other emerging technologies into the development of an 

# MOA for conazoles using propiconazole as a model.

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# Omics characteristics

• Powerful research tools that measure global changes in organs, tissues, cells and biological fluids. 

### • Captures static views of dynamic molecular events. • Strong biological inferences can be made. • Provides discovery - driven rather than hypothesis - driven views. • Serves to identify molecular components or patterns of components which can support of modes of action. • Can be integrated with toxicological alterations /apical endpoints (e.g. toxicogenomics). 

### • Omics can enhance the scientific plausibility of risk assessment. • Interpretation of omics data within a toxicology context require knowledge of toxicity mechanisms.

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# Description of omics

Metabolomics Proteomics Transcriptomics Field of Study Collection of components Macromolecule6 Microarray chip 44K genes

Microarray process flow Heatmap of differentially expressed genes Transcriptomic analysis pathway software and knowledge databases



# Transcriptomics workflow
mRNA from tissues or cells

# Proteomics workflow

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# Metabolomics workflow Tissue, cell or fluid sample extract Ultra -HP/LC Mass

Spectrometer HPLC chromatogram

Identification and quantitation of individual HPLC chromatogram peaks Table of endogenous metabolites and fold values Source: Metabolon, Inc .9



# Propiconazole

Induces malignant and benign liver tumors in mice. Induces hepatic hypertrophy. Increases hepatic cell proliferation. Reduces serum cholesterol levels. Increases hepatic Cyp levels: Cyp1a2, Cyp2b10, Cyp3a11; and activities: MROD, PROD, BROD, testosterone 16 β - hydroxylase. 

### Toxicology in mice General information One member of large group of triazole or imidazole - containing fungicides used in agriculture to prevent fungal growth and used in humans to treat local and systemic fungal infections. Designed to inhibit CYP51 (lanosterol 14 - α - demethylase), a critical step in the biosynthesis of ergosterol, required for the formation of the fungal cell wall. In mammals they can affect cholesterol levels. In mammals, propiconazole can both induce and inhibit hepatic CYPs. Propiconazole is considered to be non genotoxic (non - DNA reactive) in standardized tests.

## Apical based MOA for propiconazole

Increase Liver Tumors Increased Liver/body weight Induce Cyp2b protein and increased PROD activity

Increase cell proliferation Hypertrophy

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## Proposed MOA for propiconazole derived from 

## toxicological, omic, molecular, and biochemical data

Increase Liver Tumors Activate CAR, PXR Induce high activities of Cyp2b, Cyp3a Increase atRA metabolism/ decrease atRA levels Increase cell proliferation/alter cell signaling Oxidative stress Dysregulate cholesterol biosynthesis/ metabolism Mutation in vivo 

## Propiconazole parent

Hypertrophy
Decrease apoptosis Increase mevalonic acid levels

1112 Data used to derive key events in the propiconazole MOA: in vivo data from studies conducted under the same conditions used in the cancer bioassay13



# Propiconazole is cytotoxic, 

# however its metabolites are less cytotoxic in hepatic cells in vitro

Chen et al., 2008 Propiconazole parent 

## Key Event

## Key Event

Ratio: # genes/ total # genes in pathway Activate CAR, PXR


# Ingenuity TM Tox List

Nesnow et al., 2009 1415 

## Key Event Increase atRA metabolism/ decrease atRA levels

Thermometer symbols represent gene expression changes 1=4d, 2=30d, 3=90d Z=catalysis 

## Effects of propiconazole treatment on retinoic

acid metabolism pathway: transcriptomic map

Ward et al., 2006 Nesnow et al., 200916 

## Key Event Increase atRA metabolism/ decrease atRA levels 

## Conazoles increase hepatic 

## microsomal atRA metabolism after 4

days of treatment

Chen et al., 2009 atRA 4-hydroxy -atRA 4-oxo -atRA

# * * * Key Event Increase atRA metabolism/ decrease atRA levels 

## Propiconazole decreases hepatic



## atRA levels after 4- days of treatment

Chen et al., 2009 

### ** Retinoids can inhibit cell proliferation in a large variety of normal and neoplastic cell types in vitro and tumors in vivo. Conversely, reduced cellular atRA levels increase cell proliferation. 17

# * * 10- fold 5- fold 

## Conazoles induce cell proliferation measured by PCNA immunohistochemistry at 4 days Key Event

Allen et al, 2006 Increase cell proliferation/alter cell signaling

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## Pro

Dnajb4 Pcaf H2afx Kif5b Kpna2 Nr3c1 Stat5b Tmpo Adh4 C6 Dnajb1 Gcsh Got1 Itpr1 Ptp4a1 Rnps1 Sfpq Slc17a2 Stch Ywhah Bag3 Eif4e Hspa8 Kpnb1 Mrpl19 Nfia Psen1 Rpl13a Sgk Capn7

Dnajb9 Dnajc3 Ssr3 G1/S S G2 G2/M M/G1 

## Key Event

Nesnow et al, 2009 Altered cell cycle genes at two time points 

## Pro

Rnps1 Slc17a2 Stch YwhahCrk Kmo Kpnb1 Ppp3ca Prkar1a Ran Stag1

Psmd11 Rock1 Stat5b Tmpo H1f0

Rbbp8 Nktr Sdc1 Ssr3

Eif4ebp2 Esd Ubqln2

Fkbp1a Treatment: 4 days Treatment: 30 days Transcriptomics Increase cell proliferation/alter cell signaling

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## Key Event 

# Signaling pathways altered



# by propiconazole

30 days 

### 4 days

Nesnow et al, 2009 

## Ingenuity 

## Pathway Analysis Tox List or Canonical Pathway TM Transcriptomics Increase cell proliferation/alter cell signaling

2021 

## The cholesterol biosynthesis 

## pathway is altered by propiconazole Thermometer symbols represent significant gene expression changes at 4d Ward et al., 2006 Nesnow et al., 2009 

## Key Event Dysregulate cholesterol biosynthesis/ metabolism22 

## Propiconazole dysregulates cholesterol/bile acid 

## biosynthesis -metabolomics results
cholesterol 7 - α - hydroxycholesterol 7 - β - hydroxycholesterol chenodeoxycholate cholate taurochenodeoxycholate taurocholate

Cholesterol Metabolism/Bile Acid Biosynthesis

1.8 - fold Cyp7a

Nesnow et al., 201123 Sc4mol Liver cholesterol Propiconazole Cyp51 Serum cholesterol

Bile acids Acat1 Cyp51

Cyp7a 7 α , β - hydroxy cholesterols

Sqle HMG- CoA

Dhcr7 

## Key Event Dysregulate cholesterol biosynthesis/ metabolism Akr1d7 Aldh1a7 

### Propiconazole increases cholesterol biosynthesis and metabolism through a negative feedback loop

Transcriptomics, proteomics and metabolomics24 Acetyl CoA HMG - CoA Mevalonate Farnesyl - PP Squalene Lanosterol Demethylated lanosterol Cholesterol Bile acids 

### The increased cholesterol biosynthesis and metabolism increases cell proliferation through increased ras farnesylation

Farnesylation of Ras allows it to be transported to plasma membrane where it initiates cell proliferation. Increase mevalonic acid levels 

## Key Event
Mevalonate promotes the growth of human tumor cells in mice and increases cell proliferation in human melanoma tumor cells in culture (Duncan et al, 2004). Elevated mevalonic acid synthesis has been reported in human hepatocarcinoma samples (Kawata et al, 1990). p-Rb (cell proliferation) Murphy et al., 2011 MEK1/2 p-Erk Raf activation
Erk Rb Ras activation25 Decrease apoptosis 

## Key Event Propiconazole generally increases the



## expression of anti

-apoptotic genes and suppresses the expression of pro -apoptotic genes over time

The Hallmarks of Cancer (Hanahan and Weinberg, 2011) 4 day 30 day 90 day26 Oxidative stress 

## Key Event

Propiconazole induces both lipid oxidation, decreases the GSH/GSSH ratio and induces GST enzyme activity. Transcriptomics

Metabolomics

Ward et al., 2006; Bruno et al., 2009; Ortiz et al., 2010; Nesnow et al., 2011 Biochemical measurements Proteomics Propiconazole induces protein oxidation.

## Big Blue ® 

## transgenic mouse mutation assay

• Big Blue ® 

### transgenic mice were fed propiconazole at the 

### tumorigenic dose and mutations scored in the liver after 4 days. • Transgenic rodent model to detect mutations in the LacI gene from the E. coli lactose operon. • Each cell of each tissue of the rodent has multiple copies of the transgene integrated into the chromosome. • The transgene is not functional in the rodent, but may be easily recovered and analyzed for induction of mutations. 

### • Model responds to a wide variety of mutagens. • Forward mutation assay, detects many different types of mutations.

Ross et al., 2009, 2010 27Mutation in vivo 

### Key Event

Ross et al. , 2009, 2010 

### Propiconazole increases mutations in mouse 

### liver presumably by an increase in oxidative stress -induced DNA damage Mutant frequency/mean control mutant frequency

Preliminary data show that endogenous DNA adduct patterns were the same in treated and untreated mice, while propiconazole significantly increased the levels of a group of endogenous DNA adducts compared to concurrent controls (Nelson et al., 2011). Propiconazole Control Transcriptomics A series of DNA damage pathways were altered by propiconazole. Endogenous DNA adduct analysis

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# MOA framework for risk assessment

• Postulate a mode of action. • Identify key events. • Detail dose response relationships. 

## • Identify temporal relationships. • Discuss strength, consistency and specificity of association of tumor response with key events. • Discuss biological plausibility. • Discuss other modes of action. • Assess the postulated mode of action - confidence level. 

## • Discuss uncertainties, inconsistencies and data gaps. 29 

# Procedures that omics and related technologies could address X X X X X X X X X

## Current practice Toxicological alterations/apical events Cytotoxicity Cell proliferation Apoptosis 

### MODE OF ACTION Key Event 1 Key Event 2 Key Event 3 Tumors 

## Proposed practice Toxicological alterations/apical events Omic alterations Transcription Protein Metabolite Other 

# Omics and related technologies 

# in MOA assessment

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# Potential uses of omics and related 

# technologies in risk assessment activities

• Identify potential key events that could be linked to adverse outcomes. • Link a continuum of precursor key events that lead to apical responses. • Identify cellular pathways and networks associated with key events for both linkage and coherence by enhancing information contained in apical key events. • Infer key events not measured through apical approaches. • Extend apical dose response relationships to lower doses. • Link key events observed in animal models to humans thus enhancing human relevance. • Support or refute other MOAs. • Develop (Q)SAR databases/methods to predict biological responses and linkage to AOPs.
3132 

## Extended apical dose response relationships to lower doses

Ward et al., 2006 100 500 2500 

### Propiconazole, ppm 

## *
* * *

100 500 2500 

### Propiconazole, ppm Gene expression Enzyme activity 

### Cyp2b10 over - expression in mouse liver can be detected at 100 ppm treatment while PROD activity is not statistically increased33



# Support or refute other MOAs Phenobarbital (PB) induces mouse liver cancer and exhibits many, but not all, of the key events induced by propiconazole (Pro). Transcriptomics data show that the two agents are different based on global and gene pathway analyses.

PCA plots of all genes from microarray data from individual animals after Pro or PB treatment. Alterations of cell cycle genes within the phases of the cell cycle by PB and Pro are different based on differentially expressed genes. Genes in red are overexpressed, whereas genes in green are underexpressed. Nesnow et al., 2009

# MOA human relevance framework:

• Question 1: Is the weight of evidence sufficient to establish the MOA in animals? • Question 2: Are key events in the animal MOA plausible in humans? • Question 3: Taking into account kinetic and dynamic factors, is the animal MOA plausible in humans? 

## • Question 4: Statement of confidence; analysis; implications.

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# Questions that omics and related technologies could address X X35 

# Integrated approaches to knowledge-based 

# data models for the prediction of toxicity

• ( 

### Q)SAR combined with omic pathway, and toxicity endpoint data can provide enhanced predictive models.

PLoS ONE 5(8): e12385.36 

# In vitro approaches combined with omics and related technologies can reduce animal use

• Omic studies use reduced numbers of animals compared to classical toxicity studies. • Omics can be used to develop biosensors/bioindicators for key events/AOPs using in vitro cell systems. 

### • Cell based systems include primary, immortalized and tumorigenic cell lines. • These cells are available from most major organs and or tissues from both experimental animals and humans.

## • The EPA Conazole Project Research Team • The Office of Pesticide Programs 

## • The US Triazole Task Force 

# Acknowledgements

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