# Presentation: 056429

## Toxicogenomic and Traditional Approaches Applied to Identifying the 

**Research Goals**

**Methods/Approach**

**Impact and Outcomes**

**Conclusions**

**Future Directions**

**Science Question**

**Acknowledgements**

**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

- 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?

- 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:

_**Endpoints**_

**Histology**

**Hormone Analysis**

- Serum levels.

**Enzyme activity**

- O-dealkylations of alkoxyresorufins for measuring different isoforms of cytochrome P450.

- EthoxyResorufin (EROD) highly sensitive and selective for 1A1.
- PentoxyResorufin (PROD)  selective for 2B1 (some 1A1, 1A2).
- MethoxyResorufin (MROD)  selective for 1A2 (some 1A1, 2C6).

**Transcriptional alterations**

- Quantitative rt-PCR to assess selected hepatic CYP gene expression.

-   Quantitative rt-PCR to assess selected thyroid hormone.
-    control gene of expression in hypothalamus, pituitary, thyroid, liver.

- Microarray analysis of gene expression in liver.

**Protein analysis**

- Western analysis of selected proteins in liver, other tissues.

_**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     **

- Begin dosing
-     (2/04)
-    Harvest 17 CD-1 mice/dose group

**     ****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.

**Comparative phenobarbital-like**** ****effects **                                                                                                                                                                                                                                                                        Gene expression patterns / 8-hydroxy-dG levels.

**     ****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.

-       Mitogenic stimulation
-         of hepatocytes

- Increased likelihood of
- initiated cells and subsequent
- proliferation of initiated cells

- Continuous exposure would
- result in tumor development

- 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.

-  Several biochemical, molecular, and tissue responses will be                                             examined and used for comparison across conazoles and species.

- Quantitative assessment of thyroid and liver histopathology.
- Cell proliferation.

**     ****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.

|  |
| --- |

_**All 3 Conazoles Induced Changes in Gene Expression **_
_**Some Patterns In Common and Some Unique **_

**Results**

-          Time dependence of PROD activity

**Days of treatment**

- 0

- 20

- 40

- 60

- 80

- 100

**pmol resorufin/min/mg**

- 0

- 100

- 200

- 300

- 400

- 500

- Myclobutanil

- Propiconazole

- Triadimefon

_**All 3 Conazoles Induced Liver P450 Enzymes**_
_** **__**(PROD>EROD/MROD)  **_

-      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.

- Unique: triadimefon-induced functional gene categories

- In common: propiconazole and triadimefon, 2-tier dose responses

-                 P450 induction

- Major pathways in triadimefon toxicity

**9**

**8**

**7**

**7**

**6**

**5**

**4**

**3**

**2**

**1**

**Cyp51**

- 1,2

- 3

- 4

- 5

- 6

- 7

- 8

- 9

|      Conazole  | Low dose ppm | Mid dose ppm | High dose ppm |
| --- | --- | --- | --- |
|  Propiconazole | 100  | 500  | 2500*  |
|  Triadimefon | 100 | 500 | 1800* |
|  Myclobutanil | 100 | 500 | 2000^ |
|  Control | --- | --- | --- |
|  Treated feeds from the USTTF:  *tumorigenic dose,  ^ non-tumorigenic dose |  |  |  |

| Cholesterol Biosynthesis  |  |  |  |
| --- | --- | --- | --- |
|     
                     Gene |       Fold-Change
 Tri     Myc    Pro |  |  |
| acetoacety-CoA  synthetase |   1.7 | 1.2 |  1.0 |
| HMG-Coenzyme A synthase1 |   1.5 |  1.1 |  1.1 |
| HMG-Coenzyme A reductase |   1.9 |  1.0 |  1.4 |
| mevalonate kinase |   1.4 |  1.2 |  0.9 |
| mevalonate decarboxylase |   2.1 |  1.7 |  1.3 |
| IPP isomerase |   2.0 |  1.3 |  1.3 |
| farnesyl phosphatetransferase1 |   1.5 |  1.1 |  1.1 |
| squalene epoxidase |   2.9 |  2.1 |  2.5 |
| Cyp51 |   1.9 |  1.3 |  1.6 |
|                     Average |   1.9 |  1.3 |  1.4 |

_** **__**PROD Does Not Correlate With Tumorigenicity**_

_**Triadimefon Alters Most Genes in Cholesterol Biosynthesis**_** **