jpk ¦¦¦ a/k United States
£2 Environmental Protection
\0 |jP% Agency
Prevention, Pesticides EPA 740-C-09-004
and Toxic Substances October 2009
(7101)
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1200:
Aromatase (Human
Recombinant)
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NOTICE
This guideline is one of a series of test guidelines established by the Office of
Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection
Agency for use in testing pesticides and chemical substances to develop data for submission to
the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section
408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
The OPPTS test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test,
and is also used by EPA, the public, and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these
guidelines are not binding on either EPA or any outside parties, and the EPA may depart from
the guidelines where circumstances warrant and without prior notice. The procedures contained
in this guideline are strongly recommended for generating the data that are the subject of the
guideline, but EPA recognizes that departures may be appropriate in specific situations. You
may propose alternatives to the recommendations described in these guidelines, and the
Agency will assess them for appropriateness on a case-by-case basis.
For additional information about OPPTS harmonized test guidelines and to access the
guidelines electronically, please go to http://www.epa.gov/oppts and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
httpJ/www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150
through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OPPTS 890.1200: Aromatase (Human Recombinant)
(a) Scope.
(1) (1) Applicability. This guideline is intended to meet testing
requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C.
2601, etseq.), the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) (7 U.S.C. 136, et seq.), and the Federal Food, Drug, and
Cosmetic Act (FFDCA) (21 U.S.C. 346a).
(2) Background. The Endocrine Disruptor Screening Program (EDSP)
reflects a two-tiered approach to implement the statutory testing
requirements of FFDCA section 408(p) (21 U.S.C. 346a). In general, EPA
intends to use the data collected under the EDSP, along with other
information, to determine if a pesticide chemical, or other substances, may
pose a risk to human health or the environment due to disruption of the
endocrine system.
This test guideline is intended to be used in conjunction with other
guidelines in the OPPTS 890 series that make up the full screening
battery under the EDSP to identify substances that have the potential to
interact with the estrogen, androgen, or thyroid hormone (Tier 1
"screening"). The determination will be made on a weight-of-evidence
basis taking into account data from the Tier 1 assays and other
scientifically relevant information available. The fact that a substance may
interact with a hormone system, however, does not mean that when the
substance is used, it will cause adverse effects in humans or ecological
systems.
Chemicals that go through Tier 1 screening and are found to have the
potential to interact with the estrogen, androgen, or thyroid hormone
systems will proceed to the next stage of the EDSP where EPA will
determine which, if any, of the Tier 2 tests are necessary based on the
available data. Tier 2 testing is designed to identify any adverse
endocrine-related effects caused by the substance, and establish a
quantitative relationship between the dose and that endocrine effect.
(b) Purpose. The Aromatase (Human Recombinant) Assay is a screening assay
intended to identify chemicals that may affect the endocrine system (e.g.,
steroidogenesis) by inhibiting catalytic activity of aromatase, the enzyme
responsible for the conversion of androgens to estrogens.
(c) Introduction. The objective of this protocol is to describe procedures for
conducting the Aromatase (CYP19) Assay for use as a screening tool in EPA's
EDSP Tier I Screening Battery. The assay is based upon the 3H20- aromatase
assay, an in vitro method that has been used extensively for the determination of
the presence of aromatase in multiple target tissues in all vertebrates, and has
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long been used in pharmaceutical research to identify chemicals that can inhibit
the catalytic activity of aromatase through an interaction with the substrate
binding site on the enzyme. The assay measures the conversion of androgen to
estrogen in microsomes isolated from various target tissues or cell lines
containing aromatase, or recombinant aromatase respectively, and cytochrome
P450 reductase. In brief, radioactive substrate (3H-androstenedione) and
NADPH are added to microsomes containing the aromatase (CYP19) and
reductase complex. 3H20 is released during the conversion of androstenedione
to estrone, and can be quantified as a direct measurement of aromatase activity
per unit reaction time. Competitive inhibition of aromatase activity by test
chemicals can be detected by serial reaction tubes containing increasing
concentrations of the chemical of interest.
This protocol has been optimized for use with recombinant microsomes
containing human aromatase (CYP19) and cytochrome P450 reductase. This
protocol uses relatively large volumes. The assay may be conducted using
smaller volumes than stated in this protocol as long as the relative concentrations
of each component of the assay remain the same as indicated in Table 2 and
results adhere to the performance criteria, as described in this protocol.
Terms.
HPLC High Performance Liquid Chromatography
ADSN androstenedione (4-Androstene-3,17-dione)
[3H]ASDN tritiated androstenedione
LSS liquid scintillation spectrometry
DMSO dimethylsulfoxide
NADPH p-nicotinamide adenine dinucleotide phosphate
4-OH ASDN 4-hydroxy-androstenedione
Materials Receipt and/or Preparation.
(1) Equipment.
~ HPLC (pumps, injector, reversed phase column, uv/vis and
radiochemical detectors, data collection/analysis system)
~ HPLC column (suggested: Zorbax SB-C18, 4.6 x 250 mm) with a
mobile phase of 55:15:30 (v:v:v) and a flow rate of 1 mL/min
~ UV spectrophotometer that can read absorbance at 240 nm
~ Flow-through radiochemical detector
~ Liquid scintillation counter
~ Analytical balance
~ pH meter
~ Shaking water bath capable of maintaining 37°C
~ Multi-tube vortex
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~ Lab timer
~ Low speed refrigerated centrifuge
~ Micropipettes (various volumes, 1 |jl—5 mL)
~ Glassware free of detergent residue
~ Disposable pipet tips
~ Disposable test tubes (13 x 100 mm, glass)
~ Scintillation vials
~ Refrigerated centrifuges
Reagents
~
Distilled, deionized water
~
Tetrahydrofuran, CAS No. 109-99-9 (HPLC grade)
~
Absolute methanol (HPLC grade)
~
Absolute ethanol
~
Dimethylsulfoxide (DMSO)
~
4-hydroxyandrostenedione (4-OH ASDN), CAS# 566-48-3, mw
302.4
~
[1 -(3[3H(N)]-Androst-4-ene-3,17-dione, ([3H]ASDN)
~
Androstenedione (CAS# # 63-05-8, mw 286.45)
~
NADPH (p-nicotinamide adenine dinucleotide phosphate, reduced
form, tetrasodium salt, CAS# 53-59-8)
~
Propylene glycol (CAS# 57-55-6)
~
Methylene chloride (CAS# 75-009-2)
~
Liquid scintillation cocktail
~
Reference chemical formulations
~
Protein Assay Kit
~
Bovine serum albumin
~
Microsomes containing human CYP19 + P450 reductase
Substrate.
(i) Substrate Names and Purity. The substrate for the aromatase
assay is androstenedione (ASDN). Non-radiolabeled and
radiolabeled androstenedione ([1 p-3H]- Androst-4-ene-3,17-dione),
[3H]ASDN) are be used in this assay. Obtain a non-radiolabeled
ASDN that is > 98% pure. Include all applicable information
regarding supplier, lot numbers and reported/measured purity for
the substrate in study reports.
(ii) Radiochemical Purity. The radiochemical purity of the [3H]ASDN
is recommended to be greater than 95 percent. This is usually
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supplied at a specific activity of 20-30 Ci/mmol as specified by the
vendor. Determine the radiochemical purity of the [3H]ASDN using
high performance liquid chromatography (HPLC) and liquid
scintillation counting.
The HPLC method uses a 4.6 x 250 mm column with a mobile
phase of 55:15:30 (v:v:v) distilled, deionized water: tetrahydrofuran:
methanol and a flow rate of 1 mL/min. The eluant will be monitored
by UV absorbance at 240 nm and by a flow-through radiochemical
detector. Collect eluant fractions manually into vials and assay for
radiochemical content by liquid scintillation spectrometry (LSS).
Include a reference standard of nonradiolabeled ASDN, analyzed
by the same method, to confirm coelution of the nonradiolabeled
and radiolabeled ASDN.
If the radiochemical purity is less than 95 percent, then it suggested
that a new batch of radiochemical be obtained.
Preparation of Substrate Solution for use in Aromatase Assay.
Since the specific activity of the stock [3H]ASDN is too high for use
directly in the assay, a solution containing a mixture of
nonradiolabeled and radiolabeled [3H]ASDN is prepared such that
the final concentration of ASDN in the assay is 100 nM, and the
amount of tritium added to each incubation tube is about 0.1 jjCi.
The following example illustrates the preparation of a substrate
solution using a stock of [3H]ASDN with a specific activity of 25.3
Ci/mmol and a concentration of 1 mCi/mL. Calculations may
change based on the specific activity and concentration of the stock
purchased. The following procedure will reduce the specific activity
to 0.3-0.5 Ci/mmol.
~ Prepare a 1:100 dilution of the radiolabeled stock in buffer
(final concentration = 10 jjCi/mL) (see 2.5.1).
~ Prepare a 1 mg/mL solution of ASDN (MW 286.45 g/mol) in
ethanol and then prepare dilutions in buffer to a final
concentration of 1 |jg/ml_.
~ Combine 4.6 mL of the 1 |jg/ml_ solution of ASDN, 800 |jl_ of
the [3H]ASDN dilution and 2.7 mL buffer to make 8 mL of
substrate solution (enough for approximate 70-80 tubes).
• This substrate solution will have a concentration of 2 |jM
ASDN with a radiochemical content of about 1 jjCi/mL.
~ After mixing the solution well, confirm radiochemical content
by adding 20 |jL to scintillation cocktail and determining total
dpms for radiochemical content analysis.
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~ Add 100 |jl_ of the substrate solution to each 2 mL assay
volume to yield a final ASDN concentration of 100 nM and a
final [3H]ASDN content of 0.1 jjCi/tube.
~ Calculate the lower specific activity for each preparation of
substrate solution based upon the original specific activity
and appropriate decay of radioactivity on the day the assay
is performed. The agency intends to provide a suggested
data entry template, which will contain a calculation check
for specific activity on the day of the assay, with the posting
of this guideline on the Agency's Web site (Ref. 1).
Test Chemicals. Please provide the information listed below for each test
chemical:
• CAS Number
• Molecular Formula/Weight
• Supplier/source
• Lot Number
• Purity
• Storage Conditions
• Solvent
• Solubility Limit (if insoluble at any concentrations)
• Highest Concentration Tested
• Description of how stock solution was prepared
Prepare test chemical stock solutions and analyze as required by good
laboratory practice (GLP) standards described in 40 CFR part 160 (Ref.
2). Make the test chemicals in buffer, absolute ethanol or
dimethylsulfoxide (DMSO). Selection of the solvent is based upon the
physical properties of each test chemical (e.g., partition coefficient,
hydrophobicity, solubility, etc.), as well as indicators that a chemical is
precipitating out of solution after being added to the assay tube/buffer
(e.g., U-shaped inhibition curves with increasing chemical concentrations,
solubility test using light scattering techniques, etc.). In general, it is
expected that either ethanol or DMSO will be used as the primary solvents
to facilitate solubility of the test chemical at the higher dose concentrations
for the assay.
It is recommended that the total volume of test chemical formulation used
in each assay be no more than 1 percent of the total assay volume (i.e.,
20 |jL in a 2 mL assay) in order to minimize the potential of the solvent to
inhibit the enzyme. Fresh serial dilutions of the stock solution will be
prepared in the same solvent as the stock solution on the day of use such
that the target concentration of test chemical can be achieved by the
addition of 20 |jL of the dilution to a 2 mL assay volume. Information on
storage conditions and stability of stock solutions will be reported.
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(5) Positive Control. The known aromatase inhibitor, 4-
hydroxyandrostendione (4-OH ASDN), is used as the positive control.
Table 1 contains identity and property information for 4-OH ASDN.
Table 1. Positive Control Substance.
Test Substance
CAS Number
Molecular Formula
Molecular Weight (g/mol)
4-OH ASDN
566-48-3
C19H26O3
302.4
Prepare a stock solution and report storage conditions for this positive
control substance stock. Conduct and report methods for purity analysis
according to GLP standards. The 4-OH ASDN is formulated in either
absolute ethanol or DMSO, depending upon which solvent is being used
for the test chemicals on a given day. It is recommended that the total
volume of control substance formulation used in each assay be no more
than 1 percent of the total assay volume (i.e., 20 |jL in a 2 mL assay) in
order to minimize the potential of the solvent to inhibit the enzyme. Fresh
dilutions of the stock solution will be prepared in the same solvent as the
stock solution on the day of use. Dilutions will be prepared such that the
target concentrations of the positive control substance (0.01-10,000 nM;
Table 4) can be achieved by the addition of 20 |jl_ of the dilution to a 2 mL
assay volume.
(6) Microsomes. Microsomes can be denatured by detergents. Therefore, it
is important to ensure that all glassware and other equipment used for
microsome preparations be free of detergent residue. New disposable
test tubes, bottles, vials, pipettes and pipette tips may be used directly in
the assay. Rinse durable labware that may have been exposed to
detergents with water and/or buffer prior to use in the assay.
(i) Source of the Human Recombinant Microsomes. Any source of
human recombinant microsomes containing CYP19 + reductase
can be used in this assay provided the performance criteria
described in this test guideline can be met. Microsomes should be
soted at -70°C but should not be stored longer than 12 months.
Human recombinant microsomes are commercially available from
Gentest™ (Woburn, MA; www.qentest.com'). If a commercial
source of microsomes is used, supplier-provided values for protein
concentration, cytochrome c reductase activity, and aromatase
activity, found on the data sheet accompanying each shipment,
should be reported for each aromatase assay performed.
(ii) Human Recombinant Microsome Preparation. To minimize
freeze-thaw cycles, aliquot the human recombinant microsomes
into individual vials based on the protein content of each batch.
Quickly thaw the microsomes in a 37 ± 1 °C water bath, place them
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in an ice bath, and aliquot into individual vials in an amount
sufficient for use in a single assay (typically, 0.4 mg will be
sufficient for an 80-90 tube assay). The assay uses approximately
0.004 mg/ml (final concentration) of microsomal protein/assay tube.
After aliquoting the microsomes into individual vials, flash freeze
the vials in liquid nitrogen and then returned to the -70°C freezer for
storage for no longer than 6 months.
Note: Aliquot and flash freeze microsomes that were thawed and
kept on ice as quickly as possible to maintain enzyme activity as
best as possible.
(7) Other Assay Components.
(i) Buffer. The assay buffer is 0.1 M sodium phosphate buffer, pH
7.4. Sodium phosphate monobasic (e.g., JT Baker, cat # 4011-01,
137.99 g/mol) and sodium phosphate dibasic (e.g., JT Baker, cat#
4062-01, 141.96 g/mol) are used in the preparation of the buffer.
Solutions of each reagent at 0.1 M are prepared in distilled,
deionized water and then the solutions are combined in equal
volumes, resulting in a 0.1 M solution with a final pH of 7.4. The
assay buffer may be stored for up to one month in the refrigerator
(2-8°C).
(ii) Propylene Glycol. Propylene glycol (e.g., JT Baker, cat # 9402-
01, 76.1 g/mol) is added to the assay directly as described below.
Propylene glycol is necessary for optimal performance of this
assay.
(iii) NADPH. NADPH (p-nicotinamide adenine dinucleotide phosphate,
reduced form, tetrasodium salt, e.g., Sigma, cat # 1630, 833.4
g/mol) is the required co-factor for aromatase (CYP19). The final
concentration in the assay is 0.3 mM. Typically, a 6 mM stock
solution is prepared in assay buffer and then 100 |jl_ of the stock is
added to the 2 mL assay volume. Prepare NADPH fresh each day
and keep on ice.
Determine Suitability of Microsome Preparations.
(1) Protein Assay. Determine the protein concentration of the microsome
preparation on each day of use in the aromatase assay. A 6-point
standard curve is prepared using bovine serum albumin (BSA), ranging
from 0.13 to 1.5 mg protein/mL. Protein concentration is determined by
using a standard protein assay kit such as the DC Protein Assay kit (Bio-
Rad, Hercules, CA) or BCA Protein Assay (Pierce, Rockford, IL) as
directed in the manufacturer's instructions.
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(2) Cytochrome P450 (CYP19) Aromatase Activity. Measure aromatase
activity in each new lot of recombinant microsomes to demonstrate
sufficient activity for use with the test chemicals. The minimum acceptable
aromatase activity in human recombinant microsomes is 0.1 nmol/mg-
protein/min. If the aromatase activity for the microsomal preparation is
below the minimum level, it cannot be used in the assay. In this test,
tubes to determine full activity control and background activity will be run
in triplicate using the optimized conditions presented in Table 2.
Table 2. Tubes Needed for Determination of CYP19 Aromatase Activity.
Sample Type
Repetitions
(tubes)
Description
Full Activity Control
3
All test components3 plus solvent vehicle
Background Activity Control
3
Same as full activity control, but no NADPH
aThe complete assay ("all test components") contains buffer, propylene glycol, microsomal protein, fH]
ASDN, and NADPH.
(g) Assay Controls. A run is defined as an independent experiment. Each run
contains quadruple tubes (as shown in Table 5) for the full activity control and
background activity control and a full concentration curve run in triplicate (i.e., 3
replicates for each concentration) for the positive control.
(1) Definitions of Controls.
(i) Full Enzyme Activity Control. The full enzyme activity control is
used to determine the maximum aromatase activity as measured
by the production of 3H20 during the conversion of the substrate
([3H]ASDN) to the product (estrone). Assay tubes for the full
enzyme activity control contain the following components: buffer,
propylene glycol, microsomal protein, [3H]ASDN, NADPH, and the
vehicle solvent that will be used for any test chemicals.
Note: These tubes do not contain any test chemical or positive
control chemical.
(ii) Background Activity Control. The background activity control is
used to determine non-specific radioactivity when aromatase is not
activated by the co-factor NADPH. Assay tubes for the background
activity control consists of buffer, propylene glycol, microsomal
protein, [3H]ASDN, and vehicle solvent that will be used for any test
chemicals.
Note: These tubes do not contain NADPH, test chemicals or the
positive control chemical. In a properly functioning assay system
the average DPMs for the background activity control are generally
no greater than 10-15% of those observed for the full enzyme
activity control.
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(iii) Positive Control. The positive control is used to demonstrate that
the assay is being conducted properly for each run, by detecting a
known inhibitor of aromatase activity, 4-hydroxy-androstenedione
(4-OH ASDN), and producing a sigmoidal inhibition curve from 0.1
- 10,000 nM. Performance criteria (Table 3) are included for the
positive control. The assay components used in these assay tubes
consist of: buffer, propylene glycol, microsomal protein, [3H]ASDN,
NADPH, and varying concentrations of 4-OH ASDN.
Table 3. Positive Control Study Design.
Sample Type
Repetitions
(tubes)
Description
4-OH ASDN(M)
Full Activity Control
4
All test components3 plus
solvent vehicle
N/A
Background Activity Control
4
Same as full activity control,
but no NADPH
N/A
4-OH ASDN Cone 1
3
All test components plus 4-
OH ASDN (positive control)
1X10"5
4-OH ASDN Cone 2
3
All test components plus 4-
OH ASDN (positive control)
1X10"6
4-OH ASDN Cone 3
3
All test components plus 4-
OH ASDN (positive control)
1X10"65
4-OH ASDN Cone 4
3
All test components plus 4-
OH ASDN (positive control)
1X10"7
4-OH ASDN Cone 5
3
All test components plus 4-
OH ASDN (positive control)
1X10"75
4-OH ASDN Cone 6
3
All test components plus 4-
OH ASDN (positive control)
1X10"8
4-OH ASDN Cone 7
3
All test components plus 4-
OH ASDN (positive control)
1X10"9
4-OH ASDN Cone 8
3
All test components plus 4-
OH ASDN (positive control)
1X10"10
aThe complete assay ("all test components") contains buffer, propylene glycol, microsomal protein, [3H]
ASDN, and NADPH.
(h) Aromatase Assay Method. The aromatase assay is performed using 13x100
mm test tubes maintained at 37 +1 °C in a shaking water bath. Each set of test
chemical tubes is run in triplicate using the optimized conditions (Table 2) with a
final assay volume of 2mL/tube.
Follow the steps below to set up the assay:
~ Label each test tube.
~ Pipet the following assay components into each test tube (total volume, 1
mL):
• 100 uL propylene glycol
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• 100 |J|_3H-ASDN (200 nM)
• 100 |jL NADPH (Note: substitute assay buffer in tubes designated as
background control)
• 20 |jl_ test chemical (Note: use vehicle solvent for full activity and
background controls)
• 680 |jl_ buffer (0.1 M sodium phosphate, pH 7.4)
~ Place test tubes in water bath (37 +1 °C) for 5 min. prior to initiation of
assay.
~ Prepare microsomal protein for the assay by diluting in buffer to 0.008
mg/mL and warming in water bath (37 +1 °C) for 5 min. prior to initiation of
assay.
~ Initiate the assay by adding 1 mL recombinant microsomal preparation to
each assay tube and vortex gently using a multi-tube vortex.
~ Incubate all test tubes for 15 min. in water bath (37 +1 °C).
~ Stop the assay by the adding methylene chloride (2 mL).
~ Vortex-mix the tubes for ca. 5 sec. and placed on ice for 5 min.
~ Vortex-mixed the tubes an additional 20-25 sec.
~ Centrifuged the tubes at 200 x g for 10 min., 4°C.
~ Remove the methylene chloride (bottom layer) with Pasteur pipet and
discard.
~ Extract the aqueous layers again with methylene chloride (2 mL),
centrifuge for 10 min. at 1000 rpm, and again discard the methylene
chloride layer (bottom).
~ Perform this extraction procedure one additional time, again discarding the
bottom methylene chloride layer.
~ Transfer and separate the aqueous layer from each tube into two 20 mL
liquid scintillation counting vials as duplicate aliquots (0.5 mL each).
~ Add liquid scintillation cocktail (10 mL) to each counting vial, cap, and
vortex. The radiochemical content of each aliquot will be determined as
described below.
Table 4. Optimized Conditions for the Aromatase Assay.
Assay factor (units)
Final Concentration
Microsomal Protein (mg/mL)a
0.004°
NADPH (mM)a
0.3
[3H]ASDN (nM)a
100
Propylene glycol
5%
Incubation Time (min)
15
aThe complete assay contains buffer, propylene glycol, microsomal protein, fH] ASDN, and NADPH.
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bThe concentration of microsomal protein has been optimized when using microsomes that produce
approximately 1200 pmol product/(min x mg protein), 5 pmol product/pmol P450/min.
The amount of 3H20 in the aqueous fraction is quantified for each assay tube using
liquid scintillation spectrometry (LSS), and each mole of 3H20 represents the
conversion of one mole of substrate ([3H]- ASDN) to estrone. Aromatase activity
is reported as nmol3H20/mg microsomal protein/min and is calculated as
described below. Note: The assay may be conducted using smaller volumes as
long as the relative concentrations of each component of the assay remain the
same as indicated in Table 4, and all the performance criteria for the assay are
met (Table 5).
(i) Demonstration of Proficiency.
(1) Positive Control Chemical. Prior to conducting the assay for evaluation
of test chemicals, it is recommended that each technician conduct at least
one single run of the positive control experiment as outlined in Table 3 to
demonstrate assay proficiency. Evaluate these data against the following
criteria:
~ The suggested mean for aromatase activity (see above) in the
absence of an inhibitor is at least 0.1 nmol/mg-protein/min.
~ The recommended mean background control activity is < 10% of
the full activity control.
~ The suggested coefficient of variation (CV) for replicates within
each sample type and concentration of 4-OH ASDN is less than
15%.
~ Performance criteria are provided (Table 5) to serve as guidance in
identifying runs that provide parameters in the preferred ranges.
When data are analyzed as described later in this protocol,
preferential concentration response curve(s) generated for 4-OH
ASDN, can be identified by comparison with these performance
criteria listed (Table 5).
Table 5. Performance Criteria for the Positive Control.
Parameter
Lower limit
Upper Limit
Positive Control
Slope
-1.2
-0.8
Top (%)
90
110
Bottom (%)
-5
+6
Log IC50
-7.3
-7.0
(2) Proficiency Chemicals. After successfully conducting the positive
control run in below, each new technician is expected to conduct full scale
tests (3 runs) with the chemicals in Table 6. These chemicals were
selected to span the range of responses expected in the assay based on
the validation studies conducted by participating laboratories and are
described in the Integrated Summary Report for Aromatase (Ref. 3).
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Table 6. List of Proficiency Chemicals.
Compound
CAS No.
Class
Econazole
24169-02-6
Inhibitor
Fenarimol
60168-88-9
Inhibitor
Nitrofen
1836-75-5
Inhibitor
Atrazine
1912-24-9
Non-inhibitor
(j) Determination of the Response of Aromatase Activity to TEST Chemicals.
A run is an independent experiment. Each run contains replicate tubes for full
activity control, background activity control, positive control, and test chemical as
shown in Table 7 and described below.
Each run tests the response of aromatase activity to the presence of eight
concentrations of a test chemical run in triplicate (i.e., there are three tubes of
each test chemical concentration per run of the assay). Test each chemical in
three independent runs. Conduct each run for a given test chemical entirely
independently of the other runs for that test chemical. There are three (triplicate)
repetitions for each concentration of a test chemical. A single run of a given test
chemical is described in Table 7.
Three types of control samples will be included for each run. These include:
1. Full enzyme (aromatase) activity controls (substrate, NADPH, propylene
glycol, buffer, vehicle [used for preparation of test substance solutions]
and microsomes);
2. Background activity controls (all components that are in the full aromatase
activity controls, except NADPH); and
3. Positive controls (4-OH ASDN run at eight concentrations in the same
manner as test chemicals).
Four test tubes of the full enzyme activity control and background activity controls
are included with each run. The full enzyme and background activity controls sets
are split so that two tubes (of each control type) are run at the beginning and two
at the end of each run. The positive control is tested at eight concentrations in
each run as indicated in Table 7. All controls are treated the same as the other
samples.
The aromatase assay is conducted as described in Section (h).
After completion of the first run, the data are reviewed and, if necessary, the
concentration of test chemical used in the second and third runs is adjusted. The
decision on how to adjust test chemical concentration is based on the results
from the first run with the following guidelines in mind:
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~ If insolubility (cloudiness or a precipitate) is observed at the highest
concentration (103 M), then set the highest concentration for the second
and third runs at the highest concentration that appeared to be soluble
using mid-log concentrations; i.e., try 103 5 M if the test chemical is
insoluble at 103 M as it is important to define the lower portion of the
curve. Do not use a concentration lower than 10~5 M for the highest
concentration tested.
~ If the highest concentration to be tested is lowered to 10~4 or 10~5 M, then
add mid-log concentration(s) near the lower end of the curve (higher
concentrations) and around the estimated IC50 based on the results of the
first run in order to keep eight concentrations in the test set.
~ The lowest concentration to be tested is generally 10~10 M, but lower
concentrations may be required to obtain the "top of the curve". That is,
obtain the full enzymatic activity at the two lowest concentrations of the
test chemical in order to define the top of the concentration-response
curve.
Table 7. Test Chemical Study Design.
Sample Type
Repetitions
(tubes)
Description
Reference or
Chemical
(M)
Full Activity Control
4
All test components3 plus solvent vehicle+
N/A
Background Activity
Control
4
Same as full activity control, but no
NADPH
N/A
4-0H ASDN Cone 1
2
All test components plus 4-OH ASDN
1X10"5
4-0H ASDN Cone 2
2
All test components plus 4-OH ASDN
1X10"6
4-0H ASDN Cone 3
2
All test components plus 4-OH ASDN
1X10"65
4-0H ASDN Cone 4
2
All test components plus 4-OH ASDN
1X10"7
4-0H ASDN Cone 5
2
All test components plus 4-OH ASDN
1X10"75
4-0H ASDN Cone 6
2
All test components plus 4-OH ASDN
1X10"8
4-0H ASDN Cone 7
2
All test components plus 4-OH ASDN
1X10"9
4-0H ASDN Cone 8
2
All test components plus 4-OH ASDN
1X10"10
Test Chem. Cone 1
3
All test components3 plus test chemical
1X10"3
Test Chem. Cone 2
3
All test components3 plus test chemical
1X10"4
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Sample Type
Repetitions
(tubes)
Description
Reference or
Chemical
(M)
Test Chem. Cone 3
3
All test components3 plus test chemical
1X10"5
Test Chem. Cone 4
3
All test components3 plus test chemical
1X10"6
Test Chem. Cone 5
3
All test components3 plus test chemical
1X10"7
Test Chem. Cone 6
3
All test components3 plus test chemical
1X10"8
Test Chem. Cone 7
3
All test components3 plus test chemical
1X10"9
Test Chem. Cone 8
3
All test components3 plus test chemical
1X10"10
a The complete assay ("all test components") contains buffer, propylene glycol, microsomal protein,
fHJASDN and NADPH.
(k) Data Analysis.
(1) Aromatase Activity and Percent of Control Calculations. Convert raw
data to aromatase activity (nmol/mg protein/min) and percent control.
Report data in electronic format (spreadsheet or comma-separate values),
being sure to provide all formatting information that is necessary to read
the data. The Agency intends to provide a suggested template on the
Agency's Web site (Ref. 1). The following data are requested. Submit all
raw data and calculated endpoints with the report including:
~ DPM/mL for each aliquot of extracted aqueous incubation mixture.
~ Average DPM/mL for each aqueous portion (after extraction).
~ Total DPM for each aqueous portion (after extraction).
~ The total DPM present in the assay tube at initiation. The volume
(mL) of substrate solution added to the incubation multiplied by the
substrate solution's specific activity (DPM/mL) yields the total DPM
present in the assay tube at initiation.
~ Percent of substrate converted to product. The total DPM remaining
in the aqueous portion after extraction divided by the total DPM
present in the assay tube at initiation times 100 yields the percent
of the substrate that was converted to product.
~ Total DPM after extraction correct for background. The total DPM
remaining in the aqueous portion after extraction is corrected for
background by subtracting the average DPM present in the
aqueous portion of the background tubes. This corrected DPM is
then converted to nmol product formed by dividing by the substrate
specific activity (DPM/nmol).
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~ Aromatase activity. The activity of the enzyme reaction is
expressed in nmol (mg protein)"1 rnin1 and is calculated by dividing
the amount of 3H20 (nmol) produced by the product of mg
microsomal protein used times the incubation time (15 minutes).
~ Average activity in the full activity control tubes.
~ Percent of control activity remaining in the presence of various
inhibitor concentrations, including the positive control. This value is
calculated by dividing the aromatase activity at a given
concentration by the average full activity control and multiplying by
100.
Note: Nominally, one might expect the percent of control activity values for an
inhibitor to vary between 0 percent near the high inhibition concentrations and
100 percent near the low inhibition concentrations. However, due to
experimental variation, individual observed percent of control values will
sometimes extend slightly below 0 percent or above 100 percent as noted in the
performance criteria.
Model Fitting. The response curve is fitted by weighted least squares
nonlinear regression analysis with weights equal to 1/Y. Model fits are to
be carried out using a non-linear regression program such as Prism
software (Version 3 or higher). Concentration response trend curves are
fitted to the percent of control activity values within each of the repeat
tubes at each test chemical concentration. Concentration is expressed on
the log scale. In agreement with past convention, common logarithms
(i.e., base 10) are used. The variables in the response curve are defined
as follows:
Y = percent of control activity in the inhibitor tube
X = logarithm (base 10) of the concentration
T = average DPMs across the repeat tubes with the same test chemical
concentration that define the Top of the curve
B = average DPMs across the repeat tubes with the same test chemical
concentration that define the Bottom of the curve
(3 = slope of the concentration response curve ((3 will be negative)
|j = log10IC50 (IC50 is the concentration corresponding to percent of control activity
equal to 50%)
The following concentration-response curve is fitted to relate percent of
control activity to logarithm of concentration within each run:
Y = B + (T-B)
1+10( L°g ic50-x)P+iog[(T-Byso-B)-i]
A concentration-response model is fitted for each test run for each test
chemical.
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Graphical and Analysis of Variance Comparisons Among
Concentration Response Curve Fits. For each run the individual
percent of control values are plotted versus logarithm of the test chemical
concentration. The fitted concentration response curve is superimposed
on the plot. Is it suggested that individual plots be prepared for each run.
EPA requests that additional plots be prepared to compare the percent of
control activity values across runs through the following steps:
~ For each run, plot the average percent of control values versus
logarithm of test chemical concentration, and place all of the runs
for one chemical on the same graph.
~ Use different symbols to distinguish among runs.
~ Superimpose the fitted concentration response curves for each run
on the graph.
~ On a separate graph, plot the average percent of control values for
each run versus the logarithm of test chemical concentration.
~ Superimpose the average concentration response curve across
runs on the same plot.
For each run treat ((3, |x) as a random variables with mean (pavg, M-avg)- Let
X and Y (0< Y <100) denote logarithm of concentration and percent of
control, as defined above. The average response curve is
Y = B + (T-Bl + £
"I + "I 0* |javg-X)pavg+log[ (T-B/50-B)-1]
Compare slope ((3) and log10IC50 (jj) across runs based on one-way
random effects analysis of variance, treating the runs as random effects.
Prepare graphs that display the parameters within each run with
associated 95 percent confidence intervals based on the within-run
standard error and the average across-run standard error with the
associated 95 percent confidence interval incorporating run-to-run
variation.
Quality Control-Assay Drift Monitored using the Full Enzyme Activity
Control and Background Activity Control. Within each run of each test
chemical quadruplicate repetitions are made of the full enzyme activity
control and background activity control tubes. Half the repetitions will be
carried out at the beginning of the run and half at the end. If the
conditions are consistent throughout the test, the control tubes at the
beginning will be equivalent to those at the end.
To assess whether this is the case, the control responses are adjusted for
background DPMs, divided by the average of the (background adjusted)
full enzyme activity control values, and expressed as percent of control. It
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is recommended that the average of the four background activity controls
within a run fall around 0 percent (with an acceptable range of-5 - +6%)
and the average of the four full enzyme activity controls within a run fall
around 100 percent (with an acceptable range of 90 -110%).
(5) Data Interpretation. Data from the assay will be used to classify
chemicals according to their ability to inhibit aromatase. To be classed as
an inhibitor, the data fit the 4-parameter regression model to yield an
inhibition curve and result in greater than 50% inhibition at the highest
concentration. Average the value of the inhibition curve at each of three
runs at the highest concentration and compared with the following criteria:
Table 8. Data Interpretation Criteria.
Criteria
Classification
Data fit 4-parameter nonlinear
regression model
Average curve across runs crosses 50%*
Inhibitor
Average lowest portion of curves across runs is
between 50% and 75% activity **
Equivocal
Average lowest portion of curves across runs is
greater than 75% activity**
Non-Inhibitor
Data do not fit the model
...
* Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near-1. Unusually
steep curves may be a sign that the protein is being denatured or that solubility problems are being
encountered. If the slope of the curve is steeper than -2.0 label the result equivocal.
**lfthe test compound is not soluble above 10'6 M and the inhibition curve does not cross 50%, the
chemical is judged to be un-testable.
(6) Statistical Software. Concentration response curves will be fitted to the
data using the non-linear regression analysis features in a commercial
software package such as PRISM statistical analysis package, Version 3
or higher (GraphPad Software, Inc.). Supplemental statistical analyses
and displays such as summary tables, graphical displays, analysis of
variance, and multiple comparisons can be carried out using PRISM, the
SAS statistical analysis system (Version 8 or higher), or other general
purpose statistical packages (e.g., SPSS).
(7) Retention of Records. Retain records as required by GLP (Ref. 2).
(I) Data Reporting.
The data to be reported in the interim data summaries will include (but is not
limited to) for example: assay date and run number, technician code, chemical
code and log chemical concentration, background corrected aromatase activity
(for each control and test chemical repetition), percent of control activity, IC50,
slope, and graphs of activity versus log chemical concentration.
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In addition, draft and final reports will contain tables and graphs, as appropriate,
containing the results of the statistical analyses described in Section (k) of this
document.
Please include the following information in the test report, as well as any
additional relevant information:
(1) Test Compound(s).
~ Name, chemical structure, and CAS RN (Chemical Abstract Service
Registry Number, CAS#), if known.
~ Physical nature (solid or liquid), and purity, if known.
~ Physicochemical properties relevant to the study (e.g., solubility,
stability, volatility).
(2) Solvent.
~ Justification for choice of solvent/vehicle if other than ethanol.
~ Maximum concentration of solvent in assay wells. (Show
calculations.)
~ Information to demonstrate that the solvent/vehicle, if other than an
established solvent, does not bind to, or otherwise affect, the
components of the assay (e.g., the aromatase enzyme).
(3) Radiolabeled Androstenedione ([1P-3H]- Androst-4-ene-3,17-dione).
~ Name, including number and position of tritium atom(s).
~ Supplier, catalogue number, and batch number.
~ Specific Activity (SA) and date for which that SA was certified by
supplier.
~ Concentration as received from supplier.
~ Lower SA following addition of unlabeled ASDN.
~ Concentration (dpm/ml, nM) used in assay tubes.
~ Calculations made to obtain the appropriate concentrations.
(4) Unlabeled Androstenedione.
~ Supplier, batch, and catalog number.
~ CAS number.
~ Purity.
(5) Microsomes of Recombinant Human Aromatase.
~ Type and source of aromatase microsomes. If from a commercial
source, identify the supplier.
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~ Method and conditions of transport and storage.
Test Conditions.
~ Demonstration of aromatase activity for each batch of recombinant
microsomes used:
• Raw data for full and background activity controls.
~ Concentration range and spacing of the unlabeled androstenedione
positive control.
~ Composition of buffer(s) used.
~ Concentration range and spacing of test compound, with
justification if deviating from recommended range and spacing.
~ Volume of vehicle used to dissolve test compound and volume of
test compound added.
~ Total volume per assay tube.
~ Incubation time and temperature.
~ Concentration range and spacing of full activity and background
activity controls.
~ Notes on any precipitation of test compound while making stock
solutions or while adding test dilutions to the assay tubes.
~ Notes on any abnormalities during separation aqueous layer during
the methylene chloride extraction.
~ Notes on any problems in analysis of aromatase activity.
~ Notes on reasons for repeating a run, if a repeat was necessary.
~ Methods used to determine IC5o values (software used, formulas,
etc.).
~ Statistical methods used, if any.
Results. Report the following for each run. Be sure to include the run
number on each product.
~ Date of run.
~ Results (e.g., the dpm counts for each tube), inserted into a data
worksheet.
• Report data in electronic format (spreadsheet or comma-
separate values), being sure to provide all formatting
information that is necessary to read the data.
• The Agency intends to provide a suggested template with the
posting of this guideline on the Agency's Web site (Ref. 1).
• Adjust the data reported as necessary to accommodate the
actual concentrations, volumes, etc. used in the assay.
• Please provide one worksheet per run.
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~ Extent of precipitation of test compound (i.e., in stock solution,
assay well, or not visible by eye).
~ Values for the Full Activity and Background Activity controls tested
to determine aromatase activity of each batch of recombinant
microsomes used.
~ Comparison of Full Activity and Background Activity controls
positioned at the beginning and at the end of the assay.
~ DPM/ml for each aliquot of extracted aqueous incubation mixture.
~ Average DPM/mL for each aqueous portion (after extraction).
~ Total DPM for each aqueous portion (after extraction).
~ The total DPM present in the assay tube at initiation. The volume
(mL) of substrate solution added to the incubation multiplied by the
substrate solution's specific activity (DPM/mL) yields the total DPM
present in the assay tube at initiation.
~ Percent of substrate converted to product. The total DPM remaining
in the aqueous portion after extraction divided by the total DPM
present in the assay tube at initiation times 100 yields the percent
of the substrate that was converted to product.
~ Total DPM after extraction correct for background. The total DPM
remaining in the aqueous portion after extraction is corrected for
background by subtracting the average DPM present in the
aqueous portion of the background tubes. This corrected DPM is
then converted to nmol product formed by dividing by the substrate
specific activity (DPM/nmol).
~ Enzyme activity. The activity of the enzyme reaction is expressed
in nmol (mg product'"1 min"1 and is calculated by dividing the
amount of androstenedione converted to 3H20 (nmol) by the
product of mg microsomal protein used times the incubation time
(15 minutes).
~ Average activity in the full activity control tubes.
~ Percent of control activity remaining in the presence of various
inhibitor concentrations, including the positive control. This value is
calculated by dividing the aromatase activity at a given
concentration by the average full activity control and multiplying by
100.
~ % Activity data for each replicate at each concentration level for all
substances.
~ Plot of each data point, along with the unconstrained curve fitted to
the Hill equation, which demonstrates the performance of the test
compound in repeat runs.
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• Plot the data points and curves for the positive control chemical
on the same graph as that of the compound(s) tested in that
same run.
• Differentiate data and curves by run (i.e., distinguish the
reference chemical and test compound data points and fitted
curve from Run A from those of Run B and Run C, and those of
Run B from Run C).
~ log(IC50) values for androstenedione, and the test compound.
~ Keep a record of all protocol deviations or problems encountered
and include in the final report.
• Use this record to improve subsequent runs.
(8) Discussion.
~ log(IC50) values for positive control, including ranges, means, and
standard deviations.
~ Reproducibility of the log(IC50) values of the positive control.
(9) Conclusion.
~ Classification of test compound activity: inhibitor, equivocal, or no
inhibition.
(10) Replicate Studies.
~ Generally, replicate studies are not mandated for screening assays.
However, in situations where questionable data are obtained (i.e.,
the IC50 value is not well defined), replicate tests to clarify the
results of the initial study would be prudent.
(m) Study Records to be Maintained. All records that document the conduct of the
laboratory experiments and results obtained, as well as the equipment and
chemicals used.
(1) Protocol and any Amendments. *
(2) List of any Protocol Deviations.
(3) List of Standard Operating Procedures. '
(4) QAPP and any Amendments.
(5) List of any QAPP Deviations.
(n) References.
1. EPA. OPPTS Harmonized Test Guidelines. Available on-line at:
http://www.epa.gov/oppts (select "Test Methods & Guidelines" on the left side
navigation menu). You may also access the guidelines in
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http://www.reQulations.Qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-
2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
2. EPA. Good Laboratory Practice (GLP) Standards. 40 CFR part 160.
3. EPA. Integrated Summary Report on Aromatase. December 11, 2007. Available
electronically at: http://www.epa.gov/endo/pubs/aromatase isr.pdf.
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