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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