Dose-Response for Reproductive Effects of ER Binders P. Schmieder R. Johnson

Dose-Response for Reproductive Effects of ER Binders

P. SchmiederR. Johnson

Acknowledgements:

In Vitro- J. Denny; R. Kolanczyk; Barb Sheedy; Mark TapperStudents: R. Maciewski; W. Backe; M. Dybvig; M. MerenessPost-Doc: H. Aladjov

In Vivo- K. Flynn; D. Hammermeister; D. Lothenbach; F. Whiteman; Students: J. Nagel; W. BackePost-Doc: M. Haasch

QSAR

Inert/Antimicrobial Chemical

ER Binding

AlteredProtein

Expression

Altered proteins,

hormones;Ova-testis

Sex reversal;Altered

behavior;Repro.

Estrogen Receptor (ER) Toxicity PathwayChemical interacts with Receptor at Molecular Level,

Initiates a Series of events, and leads to Adverse Outcome

Toxicological Understanding

Risk Assessment Relevance

In vivo Assays

MOLECULARInitiating Event

CELLULARResponse

TISSUE/ORGANINDIVIDUAL

Skewed Sex

Ratios;Yr Class

POPULATION

In vitro Assays

ER Prioritization System – Establishing Chemical Categories

Test chemicals within FI and AM inventories for potential to bind ER to prioritize which chemicals, of hundreds EPA is required to evaluate, should go on for further testing. Focus in on the chemical likely to have the activity.

Predictions are needed for chemicals with little of no data

EPA problem is low affinity chemicals

0.000001

0.00001

0.0001

0.001

0.01

0.1

0 1 2 3 4 5 6 7 8

LogKow

Lo

gR

BA

ChemicalUniverse

Contains CycleYes Contains 2 OH,

or OH and =0, at Spec. Dist

Possible High Affinity,“A-B”; “A-C”; or

“A-B-C” type binder

Contains some attenuating feature

steric?; other?

High Binding Affinity “A-B”;“A-C” or “A-B-C” type

No

Non binder Ex: ProgesteroneCorticossterone(RBA<0.00001)

Special Rule Classes

Log Kow <1.3

Low Affinity Binder“A-B”,“A-C” or “A-B-C” type

Assess strength of attenuation steric?;

other?

Some

Complete

Contains at least

one possible

H-bonding site

Type “A”Contains Phenol

Fragment

• Alkyl Anilines• Phthalates• Branched Phenones• Cyclo Phenones• p-subst Cyclohexanols• p-subst Cyclohexanones• ring subst (o-CH3, tri-CH3) Benzoates

V

Possible Low AffinityBinder

Type “B”Contains identif. Type B

Fragment

• N-alkyl Phenones• Non ring subst Benzoates

• Alkyl Phenols • Alkoxy Phenols• Parabens• Salicylates

Belongs to known Active

class

Potency Rules / Equations

No

Potency Rules / Equations

No

Yes No

No

II

Needs testing

Yes

No

No

Yes

Yes

Belongs to class with possible

Type B low affinity under investigation

Belongs to known Inactive

class

IIIBelongs to

known Inactive class

• Fully-hindered Alkyl Phenols

Belong to untested classwith possible

Type A low affinity

• ring subst p-CH3 Benzoates•Thiophosphate Esters• Mixed Organics• Cyclic Alcohols (not p-subst hexanols)• Cyclic Pentanones/Others• Br, I halobenzenes

No

No

Yes

Belongs to known Active

class

Belong to untested class

Needs testing

Needs testing

Non binder(RBA<0.00001)

• Mixed Phenols

No

No

No

Yes

Yes

YesIII


No

Belongs to known Inactive

class


• Alkylbenzsulfonic Acids• Sulfonic Acid Dyes• p-Alkyl Fluorobenzenes• Bis–anilines• Alkoxy Anilines• Imidazolidines• Isothiazolines• Alkyl Benzthiols• Pyrrolidiones

IV

Yes

• Oxazoles• Benzamide• Furans• Sorbitans• Triazines

Yes

Yes

No

Yes

• DDT-Like• Tamoxifen-Like• Multi-Cyclohydrocarbons• Alkyl Chlorobenzenes

0.000001

0.00001

0.0001

0.001

0.01

0.1

0 1 2 3 4 5 6 7 8

LogKow

Lo

gR

BA

0.000001

0.00001

0.0001

0.001

0.01

0.1

0 1 2 3 4 5 6 7 8

LogKow

Lo

gR

BA

Will a chemical with rtER binding affinity 0.001 in our assay have ER-mediated effects in vivo?

How do you compare dosimetry and effects across in vitro and in vivo assay systems?

Dosimetry: Chemical concentration – How much? Where?Total Chemical Conc; Bound vs Free – internal bioavailabilityChemical Solubility

Effects: Biological Response; Toxicity

How do you compare data across levels of biological organization?

Comparing Biological Response

Comparing Dosimetry

ER Toxicity Pathway

In vitroAssayEndpts

ER Binding

AlteredProtein

Expression

Chg 2ndry Sex Char

AlteredRepro.

Skewed Sex

Ratios,AlteredRepro.


CELLULARRespopse

TISSUE/ORGANResponse

INDIVIDUALResponse

POPULATIONResponse

Altered proteins (VTG),

Ova-testis

In vivoAssay Endpts

ER Pathway

ER Binding

AlteredProtein

Expression

Chg 2ndry Sex Char

AlteredRepro.

Skewed Sex



CELLULARRespopse


INDIVIDUALResponse

POPULATIONResponse

In vitroAssayEndpts

In vivoAssay Endpts

Troutcyto rtERBinding

MaleTrout Liver Slice Vtg Induction


Ova-testis

The in vitro Assays

rtER Binding – 3H-E2 displacementcytosolic fraction from M, F trout liverscytosol diluted in TEDG buffer (Tris, EDTA, dithiothreitol, glycerol);

avg protein = 4.6 mg/ml, ± 1.2, (range 3.4 - 8.8) N=97

rtER Liver Slice Vtg Induction precision cut trout liver slices incubated in 1.4ml of L-15 buffer (10% FBS);

~ 3 mg/ml protein

Alkyl Anilines rtER Binding

CTRL

0

10

20

30

40

50

60

70

80

90

100

110

120

-10 -9 -8 -7 -6 -5 -4 -3 -2

BA

E2

TBA

HALAAN

OA

ANL

EAPA

PT

Log Concentration (M)

[3H

]-E

2 B

indi

ng (

%)

Alkyl Anilines rtER Gene Expression

CTRL1.0×103

1.0×104

1.0×105

1.0×106

1.0×107

1.0×108

-10 -9 -8 -7 -6 -5 -4 -3 -2

TBABA

OA

AANHAL

PA

EA

PT

ANL

Log Concentration (M)

VT

G m

RN

A c

opie

s/40

0 ng

tot

al R

NA


NH2

CH3

4-n-amyl aniline

Comparison are made across the assay systems using one chemical RBA = 0.001

Concentration (logM)

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1

[3H

]-E

2 B

ou

nd

(%

)

0

20

40

60

80

100

VT

G m

RN

A c

op

ies

/ 400

ng

to

tal R

NA

105

106

107

108

rtER cytosolic receptor binding - blue

rtER liver slice VTG - red

4-n-amylaniline

Comparing Dosimetry

MED Analytical rt cytosol 4-n-amylaniline (AAN) [33228443]

-5.0

-4.0

-3.0

-2.7

-2.3 -2

1.28

5M

0.12

85M

0.01

285M

0.00

1285

M0

25

50

75

100

125

150

AAN2-0h

AAN2-20h

Cytosol nominal conc.

Per

cen

t o

f N

om

inal

Co

nce

ntr

atio

n

AAN [Slice] (nmoles/g liver)

0

200

400

600

800

1000

1200

1400

1600

1800

0 4 8 12 16 20 24 28 32 36 40 44 48

Time (hrs)

nm

ole

s/g

live

r

-4.3 logM (50 uM)

-4.0 logM (100uM)

Media + Slice

0

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48

Time (Hrs)

[AA

N]

uM

-4.3 logM (50uM)

-4 logM (100uM)

-3.92

-4.69

LogM

Total vs. free concentration of octylphenol in an estrogenicity reporter gene assay

using SPME to measure “free” chemical

-9 -8 -7 -6 -5 -40

100000

200000

300000

4000005% serum20% serum50% serum

log nominal conc. (M)

resp

on

se

-9 -8 -7 -6 -5 -40

100000

200000

300000

4000005% serum20% serum50% serum

log free conc. (M)

resp

on

se

Heringa M.B. et al. Environ. Sci. Technol. 2004, 38, 6263-6270Picture from J. Hermens, Utrecht University

Nominal / total concentration

Free aqueous concentration

Dose

[Effect] protein “free” Solubility Limit(LogMolar) (mg/ml) fraction (LogMolar)

Water 0.97 -3.43

Cyto rtER EC50 -3.7 7 0.27 -2.88

Slice Vtg LOEC -4.3 3.1 0.20 -2.76 MaxEC -4.0 3.1 0.20 -2.76

4- n amyl aniline in: in vitro Assay Media


-12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1

[3H

]-E

2 B

ou

nd

(%

)

0

20

40

60

80

100

VT

G m

RN

A c

op

ies

/ 400

ng

to

tal R

NA

105

106

107

108



4-n-amylaniline

Binding EC50[free]= -3.7

Vtg maxEC[free]= -4.7


In Vivo Endpoints

ER Pathway

ER Binding

AlteredProtein

Expression

Chg 2ndry Sex Char

AlteredRepro.

Skewed Sex



CELLULARRespopse


INDIVIDUALResponse

POPULATIONResponse

In vitroAssayEndpts

In vivoAssay Endpts




Ova-testis

Medaka Bioassay with p-n-amylaniline (AAN)

NH2

CH3

Generation

Weeks total 1 2 3 4 5 6 7 8 9 10

1 2 3F0

1 2 3 4 5 6 7 8F1

Development

Reproduction

Adults (F0) exposed for 21 d; Offspring (F1) exposed for 8 wks

Ctrl -7.18 -6.55 -6.16 -5.61 -5.06

21d-Lethality F0 MALE NOEC 50%

F0 FEMALE NOEC 25% 100%

Fecundity (eggs/female) F0 FEMALE NOEC LOEC:↓ ↓

Body Weight FO MALE LOEC:↓

F0 FEMALE NA

Liver Vitellogenin FO MALE LOEC

F0 FEMALE

Sex Reversal (M to F) FO MALE

Papil. Process

Body Weight F1 MALE NOEC LOEC:↓

F1 FEMALE NOEC LOEC:↓

Liver Vitellogenin F1 MALE NOEC LOEC:↑ ↑

F1 FEMALE NOEC LOEC: ↓ ↓

Sex Reversal (M to F) F1 MALE NOEC LOEC:↓ ↓

Papil. Process

Adults – 21d

Eggs from exposed adults hatched and raised thru 8 wk exposure – F1

ER mediated responses were observedSome genetic males had only ovarian tissue with no sign of testes

-4.87

96hr LC50Newly hatched

Lethality (reduced hatch) F1 M,F NOEC LOEC:↑

Pathology - FO MALE NOEC LOEC:↑

Spleen F0 FEMALE NOEC LOEC:↑

Pathology- FO MALE none

kidney hematopoesis F0 FEMALE none

Pathology - F1 MALE LOEC:↑

Spleen F1 FEMALE NOEC LOEC:↑

Pathology- F1 MALE NOEC LOEC LOEC

kidney hematopoesis F1 FEMALE NOEC

Additional pathology observed is indicative of aromatic amine toxicity (via metabolic activation)

ER Toxicity Pathway

In vitroAssayEndpts



ER Binding

AlteredProtein

Expression

Chg 2ndry Sex Char

AlteredRepro.

Skewed Sex



CELLULARRespopse


INDIVIDUALResponse

POPULATIONResponse


Ova-testis

In vivoAssay Endpts

Male MedakaLiver Vtg Induction

Female MedakaLiver Vtg decr.

Male MedakaChg in 2ndary sex characteristic; behavior?Papillary Processes

Male MedakaGonadComplete conversion to ovary

Female MedakaFecundityHatch

Sex ReversalGenetic Males to Phenotypic Females

ReproductiveImpairment, butis it EDC in F0??

Comparing Dosimetry

Acute Toxicity – 4-n-pentyl aniline

In Vivo

96h LC50 96h LC50Species (mg/L) LogMolar SourceFathead minnow 4.3 -4.58 ASTER-calcMedaka 2.2 -4.87 MED-msrdTrout 1.7 -4.99 ASTER-calc

Activity = 96h LC50 = -4.87 =0.038 Water Solubility -3.45

21d LC50Species LogMolar SourceMedaka ~-5.3 msrd

Activity = 21d LC50 = -5.3 =0.014 Water Solubility -3.45

How do we factor in time component in comparing effects?

Chronic ER-mediated Endpoint

Vitellogenin Induction

In vitro – Liver slice conc where Vtg induction was observed Media = -4.0 LogM

In vivo – Lowest conc where Vtg Induction was observedWater = -6.16 LogM


-12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1

[3H

]-E

2 B

ou

nd

(%

)

0

20

40

60

80

100

VT

G m

RN

A c

op

ies

/ 400

ng

to

tal R

NA

105

106

107

108



4-n-amylaniline

In vivo uptake of 4-n-amylaniline in Medaka Liver (7 d)

Water Conc = 0.117 ppm; -6.14 LogMolarBCF =160

Medaka VTG induction in Males, [Water Conc]= -6.16 Estimate Liver Conc as 160 times Water Conc = -3.95

Trout Liver Slice Media Conc for Vtg induction = -4.0

We don’t have a measured free concentration in Medaka blood (although it can be estimated from Log Kow), but we do have a measured free fraction of AAN in slice media = 0.20.

Correcting Vtg Induction conc for free fraction = -4.7

Activity = Effect conc (“free”) = -4.7 = 0.056 Water Solubility -3.45

Common Reference Pointfor comparing among in vitro assays

and from in vitro to in vivo?

AlkylAnilines

-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5 6 7 8Log Kow

Wat

erS

olu

bil

ity,

Bin

din

g E

C50

"fr

ee",

Sli

ce V

tg "

free

" (L

og

Mo

lar)

Water Solubility Binding EC50 (free) Liver Slice VTG FHM LC50 FHM MATC

AlkylPhenols

-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5 6 7 8Log Kow

Wat

erS

olu

bil

ity,

B

ind

ing

EC

50 "

free

", S

lice

VT

G L

OE

C

"fre

e"

[Lo

gM

ola

r]

Water Solubility Binding EC50 FHM LC50 FHM MATC Slice VTG LOEC

Common Reference Pointfor comparing among in vitro assays

and from in vitro to in vivo?

Couldn’t go from binding EC50 to in vivo Vtg,

But could go from in vitro Vtg to in vivo Vtg if you get on a comparable conc basis

Need to compare similar biological effect as well as get dosimetry on common scale

Documents

Dose-Response for Reproductive Effects of ER Binders P. Schmieder R. Johnson