In Vitro Aromatase Assay:Prevalidation Studies

Susan Laws, Ph.D.

Endocrinology Branch

Reproductive Toxicology Division

NHEERL

Office of Research and Development

U.S. EPA

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

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

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

HO

O

O

O

androstenedione

O

O

estrone

HOO2

NADPH

O

O

HOO2

NADPH

HO

- H2O

O

O

O

19-hydroxyandrostenedione 19,19-dihydroxyandrostenedione

19-oxoandrostenedione

O2

NADPHO

O

HOO

O+3Fe

- H2O

- HCOOH

peroxy enzyme intermediate

Reaction Mechanism: Androstenedione to Estrone

- Cytochrome P450arom and NADPH-cytochrome P450 reductase

In Vitro Aromatase Assay:Radiometric (3H20) Method

Microsomal(Arom(CYP19))

Complex

3H-Androstenedioneand NADPH

3H2O

Estrone

+

Postitive Control: 4-OH-androstenedione (100 nM)

Test Chemical(0.1 nM - 1.0 mM)

+

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

Placental Microsomes: Product Formation versus Protein

Figure 1: Product formation vs. [protein]

0.0000

0.0010

0.0020

0.0030

0.0040

0.0050

0.0060

0 0.01 0.02 0.03 0.04 0.05 0.06

[protein] mg/mL

nm

ol e

str

on

e f

orm

ed

/min

Complete

Inhibited

(9%)

(18%)

(38%)

(%) conversion substrate to productInhibited: 4OH-androstenedione (100nM)

Intra-assay (triplicates) CV=3%; Inter-assay (3 exp.) CV=7.4%Aromatase Optimization Supplementary Studies

(pages 11,28,29)

Placental Microsomes: Product Formation versus Time

Figure 1: Activity vs. Time

R2 = 0.96

R2 = 0.78

0.0000

0.5000

1.0000

1.5000

2.0000

0 10 20 30 40

Time (min)

Pro

du

ct

form

ati

on

rate

(n

mo

l/m

g)

Complete

Inhibited

2.7, 5.1, 10, 14, 17.5% Substrate conversion over timeInhibited= 4OH-androstenedione (100 nM)

Estrogen Production: Human Placental Assay ResultsQuick Response Task 2

(Table 1, pages 2-3)

Placental Microsomes: Inhibition of Aromatase Activity

Estrogen Production: Human Placental Assay ResultsQuick Response Task 3

(Figure 3)

Placental Microsomes: Inhibition ofAromatase Activity with 4-OH-Androstenedione

-10 -9 -8 -7 -6 -50

25

50

75

100

IC50=42 nM

log[4-OH-ASDN]

Pe

rce

nt

of

co

ntr

ol a

cti

vity

Placental Microsomes:Intra- and Inter-Assay Variance

Intra-assay (triplicates)

CV 1.4-7.5% Inter-assay

(3 days)

CV 1.7 – 11.5%

Comparison of QR1, QR2 and QR3 Aromatase Activities (0.0125 mg/mL protein and 15 min)

0.0000

0.0100

0.0200

0.0300

0.0400

0.0500

0.0600

Total activity Inhibited

QR1

QR2

QR3

Estrogen Production: Human Placental Assay Results

Quick Response Task 3 (Table 3, Figure 4)

Human Recombinant: Protocol Optimization Experiments

Activity vs. Time

R2 = 0.96

R2 = 0.84

0.0000

2.0000

4.0000

6.0000

8.0000

10.0000

0 10 20 30 40

Time (min)

Pro

duct

form

atio

n ra

te (n

mol

/mg)

Complete

Inhibited

Recombinant: Inhibition of AromataseActivity with 4-OH-Androstenedione

-10 -9 -8 -7 -6 -50

25

50

75

100

IC50=25 nM

log[4-OH-ASDN]

Pe

rce

nt

of

Co

ntr

ol

Ac

tiv

ity

Estrogen Production: Human Placental Assay Results

Quick Response Task 4 and 5 (Tables 4, 5, Figures 5-7)

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%

Comparison of QR4 and QR5 Aromatase Activities

0.0000

0.0500

0.1000

0.1500

0.2000

0.2500

0.3000

0.3500

0.4000

0.4500

0.5000

Total activity Inhibited

QR4

QR5

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 0.3

[3H]ASDN (nM) 100 100

Incubation time (min)

15 15

Activity (nmol/mg/min)

0.053 +/-0.001 (3) 0.283 +/- 0.0005 (2)

Estrogen Production: Human Placental Assay ResultsQuick Response Task 4

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

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% 17%

Day 4 29% 30%

Day 5 47% (10%)* 53% (15%)*

(%)* CV after Tech 2, Day 5 data deletedEstrogen Production: Human Placental Assay Results

Quick Response Task 4, Tables 7 and 8

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

Test Chemicals:

Inhibitors 4-OH-androstenedione Chrysin Ketoconazole Aminoglutethimide Econazole Genistein (?)

Negative for Inhibition Nonylphenol Atrazine Bis-(2-ethylhexyl)phthlate Lindane Dibenz(a,h)anthracene

In Vitro Aromatase Activity: Comparison of Inhibition

Recombinant Microsomes:4-OH-androstenedione

-10 -9 -8 -7 -6 -5 -4 -3 -20

25

50

75

100 Day 1Day 2Day 3Day 4

Log (4-OH-androstenedione)

Pe

rce

nt

of

Co

ntr

ol A

cti

vity

Human Placenta:4-OH- Androstenedione

-10 -9 -8 -7 -6 -5 -4 -3 -20

25

50

75

100 Day 1Day 2Day 3Day 4

Log (4-OH-androstenedione)

Pe

rce

nt

of

Co

ntr

ol A

cti

vity

CV (20%) CV (54%)

Figure 10-Placenta aromatase response curvesFigure 11-Recombinant aromatase response curves

In Vitro Aromatase Activity: Comparison of Inhibition

Recombinant Microsomes:Aminoglutethimide

-10 -9 -8 -7 -6 -5 -4 -3 -20

25

50

75

100 Day 1Day 2

Day 3

Day 4

Log (Aminoglutethimide)

Pe

rce

nt

of

Co

ntr

ol A

cti

vity

Human Placenta: Aminoglutethimide

-10 -9 -8 -7 -6 -5 -4 -3 -20

25

50

75

100Day 1

Day 2Day 3Day4

log (Aminoglutethimide)

Pe

rce

nt

of

Co

ntr

ol A

cti

vity

CV (47%) CV (14%)

Figure 10-Placenta aromatase response curvesFigure 11-Recombinant aromatase response curves

In Vitro Aromatase Activity:Examples of Data

Human PlacentaGenistein

-10 -9 -8 -7 -6 -5 -4 -3 -20

25

50

75

100

4-OH-Androstenedione

Log (genistein)P

erc

en

t o

f C

on

tro

l Ac

tivi

ty

Placental MicrosomesEconazole and Atrazine

-10 -9 -8 -7 -6 -5 -4 -3 -20

25

50

75

100

4-OH-androstenedione

AtrazineEconazole

Log (Test Chemicals)

Pe

rce

nt

of

Co

ntr

ol A

cti

vity

Figure 10-Placenta aromatase response curvesFigure 11-Recombinant aromatase response curves

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

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

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

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

Acknowledgements:Battelle Memorial InstituteColumbus, OH• David Houchens• Paul Feder• Terri Pollock

Chemical and Life SciencesResearch Triangle InstituteRTP, NC• Sherry Black• RTI Technical Staff • James Mathews• Marcia Phillips• Rochelle Tyl

Endocrinology BranchRTD, NHEERL, ORDU.S. EPARTP, NC• Ralph Cooper• Earl Gray• Tammy Stoker• Vickie Wilson• Jerome Goldman

OSCP, U.S. EPAWashington, DC• Gary Timm• Jim Kariya• Jane Scott-Smith