1. In Vitro Aromatase Assay: Prevalidation Studies
Susan Laws, Ph.D.
Endocrinology Branch
Reproductive Toxicology Division
NHEERL
Office of Research and Development
U.S. EPA

2. In Vitro Aromatase Assay:
A cytochrome P450 enzyme complex bound in endoplasmic reticulum
Catalyzes the conversion of androgens to estrogens
Androstendione Estrone
Testosterone Estradiol
Present in ovary, placenta, testis, brain, bone, vasculature and adipose tissue
Present in all vertebrates
Known to be inhibited by EDCs
Entire vertebrate phylum including mammals, birds, reptiles, amplhibians, teleost and elasmobranch fish , and agnatha (hagfish and lampreys).
In most vertebrates, aromatse occurs in gonads and brain. (sex-related behavior such as mating responses, frequently a marked sexually dimorphic difference.
Human and higher primates (more extensive tissue distribution….placenta and liver of developing fetus, adipose tissue of adult. Placenta = ungulate species such as cows, pigs, horses and sheep….note: not in placenta of rats.

3. In Vitro Aromatase Assay: Prevalidation Studies
Historical Perspective
EDSTAC recommended as alternative assay
EDSP Detailed Review Paper
Radiometric method
Human placental microsomes
Initial prevalidation studies
DRP protocol
Compared tissue sources for enzyme

4. Prevalidation Studies: Goals
Optimize protocols
Enzyme, substrate and cofactor concentrations
Linear time course response
Positive control
Performance Criteria
Intra- and inter-assay variation
Technician variation
Compare placental and recombinant microsomes (11 test chemicals)
Protocol for multi-laboratory studies

5. Androstenedione to Estrone
Reaction Mechanism:
Androstenedione to Estrone
Enzyme complex bound to endoplasmic reticulum of cell and comprised of 2 proteins
Cytochrome P450 (hemoporteins that converts C19 steroids (androgen) to C18 (estrogens) containing a phenolic A ring
NADPH-cytochrome P450 reductase, transfers the reducing equivalents to cytochrome P450 aromas.
3 moles of NADPH and 2 moles of Oxygen are used in the conversion of one mole of substrate into one mole of estrogen product.
Androstenedione (perferred substrate) proceed via 3 sucessive oxidation steps, with the first two being hydroxylations of the angular c-19 methyl group.
Final oxidation step, whose mechanism not fully understood, proceeds with the aromatization of the A ring and loss of the c-19 carbon atom as formic acid.
This is the basis for the classical 3H-water methods used for measuring aromatase acativity, quantifies the relase of tritium from the 1beta-position of androsteindion into the aqueous phase.
Km and Vmas in human placental microsomes was 68 nM and 57 pmol/min/mg protein, respectively.
Positive control: 2-hydroxyandrostendione (1 uM) inhibits reactions.
The heme protein is responsible for binding of the C19 androgenic steroid substrate and catalyzine the series of reactions leading to formation of the phenolic A ring characteristic of estrogen. )
(reducing equivalents are supp;ied from NADPH via a ubiquitosu microsomal flavoprotein, NADPH-cytochrome P450 reductase.
Humans: ovaries and testes, placenta and fetal (not adult) liver, adipose tissue, chondrocytes and osteoblasts of bone, vasculature smooth muscle, brain (hypothalamus, limbic system, cerebral cortex).
- Cytochrome P450arom and NADPH-cytochrome P450 reductase

6. In Vitro Aromatase Assay: Radiometric (3H20) Method

7. Indicators of Optimized Protocol
Small fraction (10-15%) substrate converted to estrone
Estrone production linear with time and enzyme concentration
Estrone production dependent upon presence of enzyme and NADPH
Estrone formation can be inhibited

8. Placental Microsomes: Product Formation versus Protein
(38%)
(18%)
(9%)
(%) conversion substrate to product
Inhibited: 4OH-androstenedione (100nM)
Intra-assay (triplicates) CV=3%; Inter-assay (3 exp.) CV=7.4%
Aromatase Optimization Supplementary Studies
(pages 11,28,29)

9. Placental Microsomes: Product Formation versus Time
2.7, 5.1, 10, 14, 17.5% Substrate conversion over time
Inhibited= 4OH-androstenedione (100 nM)
Estrogen Production: Human Placental Assay Results
Quick Response Task 2
(Table 1, pages 2-3)

10. Placental Microsomes: Inhibition of Aromatase Activity
Estrogen Production: Human Placental Assay Results
Quick Response Task 3
(Figure 3)

11. Placental Microsomes: Intra- and Inter-Assay Variance
Intra-assay (triplicates)
CV %
Inter-assay
(3 days)
CV 1.7 – 11.5%
Estrogen Production: Human Placental Assay Results
Quick Response Task 3 (Table 3, Figure 4)

12. Human Recombinant: Protocol Optimization Experiments
7, 12, 25, 33, 42% Substrate conversion over time
Inhibited= 4OH-androstenedione (100 nM)
Intra-assay (triplicates): CV = 1-3%
Inter-assay (2 days): CV = 5 – 20%
Estrogen Production: Human Placental Assay Results
Quick Response Task 4 and 5 (Tables 4, 5, Figures 5-7)

13. In Vitro Aromatase Assay: Optimized Assay Conditions
Assay Factor
Assay Type
Human Placenta
Human Recombinant
Protein (mg/mL)
0.0125
0.004
NADPH (mM)
0.3
[3H]ASDN (nM)
100
Incubation time (min) 15
Activity (nmol/mg/min)
/ (3)
/ (2)
Estrogen Production: Human Placental Assay Results
Quick Response Task 4

14. Optimized Protocols: Variability Between Assay Day and Technicians
Experiment design
Three technicians conducted each assay independently over 3 days
Triplicate assay tubes
Maximum aromatase activity determined
Comparison of coefficient of variations
Estrogen Production: Human Placental Assay Results
Quick Response Task 4
Tables 7 and 8

15. Coefficient of Variation: Intra-assay, Assay Day, and Technician Variability
Parameter
Placenta
Recombinant
Triplicates 4%
3.7%
Tech 1
22%
17%
Tech 2
49% (12%)*
50% (11%)*
Tech 3
12%
19%
Day 3
36%
Day 4
29%
30%
Day 5
47% (10%)*
53% (15%)*
(%)* CV after Tech 2, Day 5 data deleted
Estrogen Production: Human Placental Assay Results
Quick Response Task 4, Tables 7 and 8

16. In Vitro Aromatase Assay: Comparison of Test Chemicals
Experiment Design
Optimized protocols using placental and recombinant microsomes
Test chemicals (11, positives and negatives)
Complete concentration curve for each chemical ran on 4 separate days
Two technicians (one ran placental, the other recombinant)
Single set of test chemical concentrations shared by 2 tech. each day

17. Test Chemicals: Inhibitors Negative for Inhibition
4-OH-androstenedione
Chrysin
Ketoconazole
Aminoglutethimide
Econazole
Genistein (?)
Negative for Inhibition
Nonylphenol
Atrazine
Bis-(2-ethylhexyl)phthlate
Lindane
Dibenz(a,h)anthracene

18. In Vitro Aromatase Activity: Comparison of Inhibition
CV (20%)
CV (54%)
Figure 10-Placenta aromatase response curves
Figure 11-Recombinant aromatase response curves

19. In Vitro Aromatase Activity: Comparison of Inhibition
CV (47%)
CV (14%)
Figure 10-Placenta aromatase response curves
Figure 11-Recombinant aromatase response curves

20. In Vitro Aromatase Activity: Examples of Data
Figure 10-Placenta aromatase response curves
Figure 11-Recombinant aromatase response curves

21. Conclusions: Test Chemical Experiment
Variability between reps. is greater than expected for both assays
IC50s for inhibitors (CVs ranged 7 – 49%)
Technician error rather than inadequate protocol method is likely cause of variability
Despite variability, both protocols correctly identified inhibitors

22. In Vitro Aromatase Assay: Next Steps
Identify source of variability
Substrate concentration
Technician training
Conduct additional experiment to evaluate day-to-day and technician variability (e.g, better estimate of performance criteria)
2 Tech., 3 test chemicals (8-9 concentrations in triplicates), 4 days, both protocols

23. In Vitro Aromatase Assay: Next Steps
Rerun assays for test chemicals with incomplete curves
econazole, ketoconazole
Evaluate the usefulness of estrone measurement rather than 3H20 for recombinant protocol
Prepare updated protocols for validation
Broader concentration range for test chemicals
Guidelines for data analysis and interpretation

24. In Vitro Aromatase Assay: Summary
Protocols optimized for placenta and recombinant assays
Assays produce similar data
Assays differ in advantages/disadvantages
High throughput assays
KGN cell line
CYP19/Fluorescent substrate (HTP) kit available

25. Acknowledgements: Endocrinology Branch Battelle Memorial Institute
Columbus, OH
David Houchens
Paul Feder
Terri Pollock
Chemical and Life Sciences
Research Triangle Institute
RTP, NC
Sherry Black
RTI Technical Staff
James Mathews
Marcia Phillips
Rochelle Tyl
Endocrinology Branch
RTD, NHEERL, ORD
U.S. EPA
RTP, NC
Ralph Cooper
Earl Gray
Tammy Stoker
Vickie Wilson
Jerome Goldman
OSCP, U.S. EPA
Washington, DC
Gary Timm
Jim Kariya
Jane Scott-Smith