# Presentation: 005474

## Pre-Validation of the Aromatase Assay using Human and Bovine Placental, and Human Recombinant MicrosomesEndocrine Disruptor Methods Validation Subcommittee (EDMVS) Plenary SessionJune 5, 2003

**Pre-Validation of the Aromatase Assay using Human and Bovine Placental, and Human Recombinant Microsomes*****Endocrine Disruptor Methods Validation Subcommittee (EDMVS) Plenary Session*****June 5, 2003**

- Research Triangle Park,  North Carolina

*RTI International is a tradename of Research Triangle Institute.*

## Overview

- Background:  Aromatase (CYP19)
- Study Goals
- Substrate characterization
- Placenta tissues – human, bovine, porcine
- Methods
- Results
-                Protein yield
-                P450 Spectra
-                Aromatase activities
- Conclusions
- Future studies

## Aromatase

- Cytochrome P450 enzyme – CYP19
- Present in the gonads and placenta
- Responsible for the biosynthesis of estrogen steroid hormones
- Can be inhibited at the level of gene expression (e.g., ethylhexylphthalate), or directly at enzyme (e.g. azoles)

## Steroid Hormone Biosynthesis by Aromatase

*Steroid Hormone Biosynthesis by Aromatase*

## Steroid Hormone Biosynthesis by Aromatase (cont’d)

*Steroid Hormone Biosynthesis by Aromatase (cont’d)*

## Study Goals

**	****The goal of this work is to identify the optimal factors and conditions for the assay of aromatase, including:**
- Characterization of radiolabeled substrate (androstenedione)
- Selection of mammalian placenta allowing sufficient yield of catalytically-active microsomal protein, and assessment of human recombinant CYP19
- Optimization of assay with respect to concentration of protein, cofactors, substrate, and incubation time using a factorial design
- Using this optimized assay, determine the effect of selected substances on aromatase activity

## Substrate Characterization

- Sources
- Nonradiolabeled 4-androstene-3,17-dione (99%): Sigma Chemical Co.
- Radiolabeled [1-3H(N)]androst-4-ene-3,17-dione:  Perkin-Elmer Life Science
- Specific activity: 25.3 Ci/mmol
- Radiochemical purity: 98%

## HPLC Radiochromatogram for[3H]Androstenedione

## Placental Tissues 

- http://www.vivo.colostate.edu/hbooks/pathphys/reprod/placenta/structure.html

## Placental Tissues (con’t)

- http://www.vivo.colostate.edu/hbooks/pathphys/reprod/placenta/ruminants.html

## Placenta Tissues (con’t)

## Methods 

- _Preparation of microsomes from tissues__ _
- Iced-down (not frozen!) within 10 min of delivery_ _
- Soft tissue harvested, homogenized in cold buffer
- Homogenate centrifuged @ 10,000*g* for 30 min, 4C
- Supernatant centrifuged @ 100,000*g* for 60 min, 4C
- Pellet resuspended in buffer, centrifuged @ 100,000*g* for 60 min, 4C
- Pellet “washed” by repeating above
- Resuspended in buffer, protein concentration determined
- _Spectral P450 content determination_: difference spectrum, 400 – 500 nm, of CO vs. CO/dithionite reduced microsomes, quantitation using extinction coefficient for the450 nm absorbance of 100 mM-1 cm-1

## Methods (cont’d) 

- 0.1 mL	propylene glycol
- 50.0 nM	3H-androstenedione (0.1 μCi)
- 1.7 mM	NADP+
- 2.8 mM	glucose-6-phosphate
- 1.0 u	glucose-6-phosphate dehydrogenase
- Dilute to 1.0 mL volume with phosphate buffer, pH 7.4, and warm to 37 °C

- Preparation of placental or recombinant microsomal pellet

- Suspension of placental or recombinant microsomes and dilution to
- ~0.1 mg/mL in phosphate buffer

- Warm microsomes to 37 °C

- Add 1.0 mL placental microsomal suspension to each test tube

- Incubate at 37 °C for 30 minutes in shaking water bath

*Methods (cont’d) *

## Methods (cont’d) 

- Transfer organic layer to a capped vial.
- Add 2.0 mL of CH2Cl2 to test tube containing aqueous layer; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

- Transfer organic layer to a capped vial.
- Add 2.0 mL of CH2Cl2 to test tube containing aqueous layer; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

- Transfer organic layer to a capped vial.  Prepare aliquots of each organic extract for analysis by LSC.
- Transfer aqueous layer to a capped vial; transfer 0.5 mL to
- a LSC vial; add 10.0 mL scintillation cocktail; count in LSC.

*Methods (cont’d) *

## Tissue Procurement Issues

- Human placenta
- Caesarian section allows for 1) timed delivery and optimal collection conditions, and 2) less chance of disease transmission
- Bovine and porcine
- Requires farms close to laboratories, assistance of farm staff
- Deliveries seasonal, and any time of day

## P450 Difference Spectrum

- Human Recombinant CYP19

## Results (cont’d)

## Conclusions

**Collection conditions used for placentas are crucial to activity**
- Human Placentas
- easiest to collect under optimal conditions
- well-defined morphology and good yield of microsomal protein
- high activity
- Human recombinant, comparable activity with best placental preparations
- However,
- SOPs must be in place for handing potentially infectious materials
- Although Caesarian vs. birth canal delivery minimizes infection of the placental tissue, information regarding screening for HIV, hepatitis, etc. should be obtained if available.

## Future Studies

- Optimization of conditions using a factorial design

## Future Studies

- Determination of variance of the optimized assay
- Using the optimized conditions determined for each preparation, three technicians independently conduct the assay on three separate days
- The results are assessed for technician-to-technician and day-to-day variance.

## Future Studies

- Determination of IC50 for:
- aminoglutethimide  (non-steroidal aromatase Inhibitor)
- 4-hydroxyandrostenedione (potent steroidal aromatase inhibitor
- chrysin (potent flavonoid)
- genistein (weak isoflavonoid)
- ketoconazole (weak imidazole anti-fungal)
- econazole (potent imidazole anti-fungal)
- atrazine (affects aromatase gene expression; no aromatase inhibition)
- bis-(2-ethylhexyl)phthlate (affects aromatase gene expression; no aromatase inhibition)
- nonylphenol (affects AR/ER; no aromatase inhibition)
- lindane (affects StAR and cholesterol metabolism; no aromatase inhibition)
- dibenz(a,h)anthracene

## Acknowledgements

_**Study Plans**_
- Dr. Robert Brueggemeier
- (Ohio State University)
- Dr. Jerry Johnson (Battelle)

_**Placental Tissues**_
- Dr. Amy Murtha (Duke University)
- Mr. Rick Kern (North Carolina State University)
- Mr. Curtis Powell (North Carolina State University)

_**RTI International**_
- Ms. Sherry Black
- Ms. Purvi Patel