Research Goals

1
Toxicogenomic and Traditional Approaches Applied
to Identifying the Mode(s) of Action of
Conazole-Induced Mouse Liver Cancer 1James Allen,
1Larry Claxton, 1Don Delker, 1Susan Hester, 1Leon
King, 1Stephen Nesnow, 1Jeffrey Ross, 1Sheau-Fung
Thai, 1Doug Wolf, 2Vicki Dellarco, 2Karl
Baetcke 1US Environmental Protection Agency,
Office of Research and Development 2Office of
Pesticide Programs

Conclusions
Results
Methods/Approach
All 3 Conazoles Induced Changes in Gene
Expression Some Patterns In Common and Some
Unique
The results from these studies do not
provide support for the current hypothesis that
differential induction of xenobiotic metabolizing
P450 enzyme activity is a key determinant of
conazole tumorigenicity. Overall, the three
conazoles produced similar results for endoints
commonly used in chemical toxicity/carcinogenesis
assessment. The traditional assays used did not
distinguish the two tumorigenic conazoles,
triadimefon and propiconazole, from the
non-tumorigenic conazole, myclobutanil. The
results from genomic analyses provide new leads
toward understanding the mode(s) of action
responsible for conazole tumorigenesis. Some
gene expression patterns were common among
different combinations of conazoles, while others
appeared to be unique to a specific agent.
Propiconazole exhibited an especially large
number of unique genes, including some
functioning in cell cycling and DNA replication,
suggesting that the mechanism of carcinogenic
action may be different from that of triadimefon
which showed high activity for altering genes
functioning in cholesterol biosynthesis. The
overexpression of cholesterol biosynthetic
enzymes is consistent with the inhibition of
CYP51 by triadimefon. This suggests depressed
circulating levels of cholesterol and Vitamin D3
which have been associated with cytogenetic
alterations and increased cell proliferation,
both hallmarks of cancer. These analyses reveal
functional categories of chemical response genes
that indicate mechanisms not previously
considered, and provide guidance for further
research.
Science Question
In common propiconazole and triadimefon, 2-tier
dose responses
Study Design 3 Test Conazoles Myclobutanil -
nontumorigenic in mouse and rat. Propiconazole -
tumorigenic for mouse liver only. Triadimefon -
tumorigenic for mouse liver and rat thyroid.
4 days 30 days
90 days

Conazoles are antifungal agents used as
pesticides and pharmaceuticals. Some of these
fungicides cause liver tumors in mice. It is
hypothesized that conazoles are hepatotoxic and
hepatotumorigenic through their specific
induction of cytochrome P450 enzyme activities
which lead to oxidative stress, mitogenesis and
altered foci development. This pathway is similar
to that for phenobarbital which is tumorigenic in
mice but apparently not in humans. Key
scientific questions in the present work are
-What are the mode(s) of action by which some
conazoles induce liver


toxicity and
tumors in mice? -Are these modes of action
similar to those of phenobarbital? -Could these
modes of action also occur in humans and are they
predictive of human health risks?
Begin dosing (2/04)
Harvest 17 CD-1 mice/dose group
Research Goals
Impact and Outcomes
Primary research goals are to identify modes of
action for hepatotumorigenic conazoles, and
determine their relevance for assessing human
risks. Traditional and toxicogenomic approaches
are used to test the proffered hypothesis that
liver cancer arises from P450 enzyme induction as
an early key event, and to identify patterns of
altered gene expression which may signify
tumorigenic pathways.
Hypothesis

This research is central to Agency efforts
in determining how mode of action information can
be used in conazole chemical risk assessment,
particularly as it relates to quantitation and
human extrapolation. -A model approach is
provided for combining genomic technologies with
traditional toxicological methods to assess
cross-species/tissues commonality in modes of
action for conazole induction of tumors and other
toxicities, and should improve assessments of
human health risks. -The results from this study
will specifically strengthen risk assessments
using mouse liver tumor models for evaluating the
20 to 30 conazoles in commerce by affording
important new information relevant to hazard
identification, dose-response and
cross-species/tissues extrapolation. These
studies should inform and improve the process for
assessing and predicting human health risk from
chemical exposure. -The toxicity-anchored gene
profiling approach taken should enhance abilities
to identify and classify new environmental agents
according to mode of action. -Associated
computational toxicological approaches can be
developed to prioritize the testing of new
environmental agents through improved prediction
of chemical toxic effects.
Unique triadimefon-induced functional gene
categories
Endpoints
Several biochemical, molecular, and tissue
responses will be
examined and used for comparison
across conazoles and species.


All 3 Conazoles Induced Liver P450 Enzymes
(PRODgtEROD/MROD)
Histology
Quantitative assessment of thyroid and liver
histopathology. Cell proliferation.
P450 induction
Mitogenic stimulation of
hepatocytes
Hormone Analysis
Serum levels.
Enzyme activity
O-dealkylations of alkoxyresorufins for measuring
different isoforms of cytochrome P450.

Increased likelihood of initiated cells and
subsequent proliferation of initiated cells
Future Directions
EthoxyResorufin (EROD) highly sensitive and
selective for 1A1. PentoxyResorufin (PROD)
selective for 2B1 (some 1A1, 1A2). MethoxyResorufi
n (MROD) selective for 1A2 (some 1A1, 2C6).
Major pathways in triadimefon toxicity
Triadimefon Alters Most Genes in Cholesterol
Biosynthesis

• Genomic studies will be expanded to further
  explore the key pathways involved in conazole
  mode of action related to hepatotumorigenesis and
  other toxicities.
• As part of interspecies comparison and
  harmonization studies, genomic analyses will be
  compared among mouse and rat experimental
  systems.
• Studies will be aimed at determining whether
  modes of action in rodents are concordant with
  processes that could occur in humans and be
  predictive of human health risks.
• Parallelogram gene expression studies will be
  conducted to assess extrapolation to humans of
  conazole responses in mice.
• Studies will be conducted to determine
  similarities in mode(s) of action between
  phenobarbital and conazoles.

Transcriptional alterations
1
Quantitative rt-PCR to assess selected hepatic
CYP gene expression.
Continuous exposure would result in tumor
development
2
Quantitative rt-PCR to assess selected thyroid
hormone. control gene of expression in
hypothalamus, pituitary, thyroid, liver.
3
PROD Does Not Correlate With Tumorigenicity
3
4
6
5
Time dependence of PROD activity
1,2
4
Microarray analysis of gene expression in liver.
500
5
Specific study aims include -determination of
specific conazole effects on P450
activities/related toxicities. -determination of
specific conazole effects on liver cell gene
expression. -comparison of results across species
and tissues. -assessment of human relevance
through parallelogram studies.
Protein analysis
6
Western analysis of selected proteins in liver,
other tissues.
7
400
8
7
Comparative phenobarbital-like effects





Gene expression patterns / 8-hydroxy-dG
levels.
300
Myclobutanil
7
9
pmol resorufin/min/mg
Propiconazole
Triadimefon
Acknowledgements
200
8
100
The authors gratefully acknowledge the
assistance of all ECD conazole project team
members. Special thanks are given to Drs. Guobin
Sun and William Ward, Michael George, and the US
Triazole Task Force, for their key contributions
to this work.
9
0
Cyp51
100
0
20
40
60
80
Days of treatment