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.

3
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.

4
5

Description of omics

Metabolomics

Proteomics

Transcriptomics

Field of Study Collection of components Macromolecule 
6

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

7
8

Metabolomics workflow

Tissue, 

cell or 

fluid 

sample 

extract

Ultra-HP/LC Mass 

Spectrometer

HPLC chromatogram

Identification and quantitation of 

individual HPLC chromatogram peaks

Source: Metabolon, Inc. Table of endogenous metabolites and fold values
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 16β-hydroxylase.

Toxicology in mice

General information 

One member of large group of triazoleor 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 (lanosterol14-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 nongenotoxic (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

10
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 

11
12

Data used to derive key events in the propiconazole MOA: in vivo data from 

studies conducted under the same conditions used in the cancer bioassay

 In vivo In 

vitro 

In vivo and/or In 

vitro 

Key Event Apical Transcriptomic Proteomic Metabolomic Other Biochem/Molecular 

Propiconazole

parent

X

CAR/PXR activation X

Cyp2b &Cyp3a 

induction/activity

X X X

Increase 

hypertropy

X

Increase oxidative 

stress

X X X X

Induce 

mutations

X X

Increase hepatic atRA

metabolism/ Decrease 

hepatic atRA levels

X X

Dysregulate 

cholesterol 

biosynthesis and 

metabolism

X X X X X

Increase mevalonic 

acid levels

X X X

Increase cell 

proliferation

X X X

Decrease

apoptosis

X

Increase 

tumors

X
13

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

IngenuityTM

Tox List

Nesnow et al., 2009 14
15

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., 2009
16

Key Event

Increase atRA 

metabolism/ 

decrease atRA 

levels

Conazoles increase hepatic 

microsomal atRA metabolism after 4 

days of treatment 

Chen et al., 2009

Control Propiconazole [-]NADPH

atRA metabolism, pmol/min/mg protein

0

50

100

150

200

250

300

4-hydroxy-atRA 

4-oxo-atRA 

Total metabolism 

*

*

*

atRA 4-hydroxy-atRA 4-oxo-atRA
Control Myclobutanil Propiconazole Triadimefon

Hepatic RA levels (% of Control)

0

20

40

60

80

100

120

140

* 

*

*

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
The effects of conazoles on cell proliferation 

Control MyclobutanilPropiconazole Triadimefon

Labeling index, % 0

1

2

3

4

5

*

*

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 

18
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

Ywhah

Crk

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 

19
Key Event

 Aryl Hydrocarbon Receptor Signaling Can, Tox S

 EGF Signaling Can S

 Glucocorticoid Receptor Signaling Can S

 IL-4 Signaling Can S

 Insulin Receptor Signaling Can S

 LPS/IL-1 Mediated Inhibition of RXR Function Can, Tox S

 Neuregulin Signaling Can S

 PI3K/AKT Signaling Can S

 PTEN Signaling Can S

 Xenobiotic Metabolism Signaling Can S

 14-3-3-mediated Signaling Can S

 Acute Phase Response Signaling Can S

 Apoptosis Signaling Can S

 Aryl Hydrocarbon Receptor Signaling Can, Tox S

 Ceramide Signaling Can S

 EGF Signaling Can S

 Estrogen Receptor Signaling Can S

 GM-CSF Signaling Can S

 Hypoxia Signaling in the Cardiovascular System Can S

 Hypoxia-Inducible Factor Signaling Tox S

 IGF-1 Signaling Can S

 IL-2 Signaling Can S

 Insulin Receptor Signaling Can S

 JAK/Stat Signaling Can S

 p38 MAPK Signaling Can S

 PDGF Signaling Can S

 SAPK/JNK Signaling Can S

 TGF-β Signaling Can, Tox S

 Toll-like Receptor Signaling Can S

 Xenobiotic Metabolism Signaling Can S

Glucocorticoid Receptor Signaling Can S

Signaling pathways altered 

by propiconazole

30 days

4 days

Nesnow et al, 2009

Ingenuity 

Pathway Analysis 

Tox List or 

Canonical 

PathwayTM

Transcriptomics

Increase cell 

proliferation/alter 

cell signaling 

20
21

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/

metabolism 
22

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

7-Hydroxycholesterol

500 ppm

1250 ppm2500 ppm

Fold

1.0

1.5

2.0

2.5

3.0

7-Hydroxycholesterol

500 ppm

1250 ppm2500 ppm

Fold

1.0

1.2

1.4

1.6

1.8

2.0

Chenodeoxycholate

500 ppm

1250 ppm2500 ppm

Fold

0.0

0.5

1.0

1.5

2.0

2.5

Taurochenodeoxycholate

500 ppm

1250 ppm2500 ppm

Fold

0

1

2

3

4

5

Cholate

500 ppm

1250 ppm2500 ppm

Fold

-3.0

-2.5

-2.0

-1.5

-1.0

Nesnow et 

al., 2011
23

Sc4mol

Liver 

cholesterol

Propiconazole Cyp51

Serum 

cholesterol

Bile 

acids

Acat1 

Cyp51

Cyp7a

7α,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 

metabolomics
24

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 activation
25

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 day
26

Oxidative 

stress 

Key Event

Ascorbate

500 ppm

1250 ppm2500 ppm

Fold

0

1

2

3

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

Glutathione reduced, GSH

500 ppm

1250 ppm2500 ppm

Fold

-2.0

-1.5

-1.0

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 27
Mutation 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). 

Control Propiconazole

Transcriptomics

A series of DNA damage 

pathways were altered by 

propiconazole.

Endogenous DNA 

adduct analysis 

28
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

30
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.

31
Propiconazole Mouse 30-days

Control Low Mid High

pmol resorufin/min/mg

0

100

200

300

400

500

EROD 

MROD 

PROD 

32

Extended apical dose response relationships to lower doses 

Ward et al., 2006

100 500 2500

Propiconazole, ppm 

*

* *

*

100 500 2500

Propiconazole, ppm 

Enzyme activity Gene expression

Cyp2b10 over-expression in mouse liver can be detected at 100 ppm 

treatment while PROD activity is not statistically increased
33

-4

-2

0

2

4

6

-16.5

-16.0

-15.5

-15.0

-14.5

-14.0

-13.5

-13.0

4 2 0 -2 -4 -6

PCA #3

PCA #1

PCA #2

Phenobarbital

Propiconazole

Triadimefon

PB

Tri

Pro

-4

-2

0

2

4

6

-16.5

-16.0

-15.5

-15.0

-14.5

-14.0

-13.5

-13.0

4 2 0 -2 -4 -6

PCA #3

PCA #1

PCA #2

Phenobarbital

Propiconazole

Triadimefon

-4

-2

0

2

4

6

-16.5

-16.0

-15.5

-15.0

-14.5

-14.0

-13.5

-13.0

4 2 0 -2 -4 -6

PCA #3

PCA #1

PCA #2

Phenobarbital

Propiconazole

Triadimefon

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.

34

Questions that omics and related 

technologies could address

X

X
35

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

37