A

new

highly

specific

and

robust

yeast

androgen

bioassay

for

the

detection

of

agonists

and

antagonists

Public

concern

about

the

presence

of

natural

and

anthropogenic

compounds

which

affect

human

health

by

modulating

normal

endocrine

functions

is

continuously

growing

Fast

and

simple

high-throughput

screening

methods

for

the

detection

of

hormone

activities

are

thus

indispensable

During

the

last

two

decades

a

panel

of

different

in

vitro

assays

has

been

developed

mainly

for

compounds

with

an

estrogenic

mode

of

action

Here

we

describe

the

development

of

an

androgen

transcription

activation

assay

that

is

easy

to

use

in

routine

screening

Recombinant

yeast

cells

were

constructed

that

express

the

human

androgen

receptor

and

yeast

enhanced

green

fluorescent

protein

-LRB-

yEGFP

-RRB-

the

latter

in

response

to

androgens

Compared

with

other

reporters

the

yEGFP

reporter

protein

is

very

convenient

because

it

is

directly

measurable

in

intact

living

cells

i.e.

cell

wall

disruption

and

the

addition

of

a

substrate

are

not

needed

When

yeast

was

exposed

to

the

concentration

where

half-maximal

activation

is

reached

-LRB-

-RRB-

was

nM

The

relative

androgenic

potencies

defined

as

the

ratio

between

the

of

and

the

of

the

compound

of

methyltrienolone

and

are

and

respectively

The

results

presented

in

this

paper

demonstrate

that

this

new

yeast

androgen

bioassay

is

fast

sensitive

and

very

specific

and

also

suited

to

detect

compounds

that

have

an

antiandrogenic

mode

of

action

Introduction

There

is

concern

that

chemicals

in

our

food

water

and

environment

affect

human

health

by

disrupting

normal

endocrine

function

possibly

leading

to

reproductive

failure

in

humans

and

tumors

in

sensitive

tissues

-LSB-

-RSB-

This

relates

to

chemicals

with

previously

unknown

hormonal

properties

like

certain

pesticides

and

plasticizers

but

also

to

compounds

used

in

pharmaceutical

preparations

eg.

oral

contraceptives

and

tablets

for

hormone-replacement

therapy

the

endogenous

steroids

excreted

in

urine

of

man

and

domestic

animals

and

potentially

also

compounds

used

for

their

growth-promoting

properties

in

animals

Of

all

endocrine

disruptors

environmental

estrogens

are

the

most

studied

-LSB-

-RSB-

However

recent

studies

show

a

crucial

involvement

of

the

androgen

receptor

in

abnormal

sex

development

The

presence

of

pollutants

with

adverse

effects

on

human

androgen

receptor

-LRB-

hAR

-RRB-

has

been

reported

from

paper-mill

effluents

and

as

a

result

of

intensive

farming

-LSB-

-RSB-

Xenoandrogenic

exposure-related

disorders

include

testicular

cancer

hypospadias

cryptorchidism

and

poor

sperm

and

very

recently

prepubertal

gynecomastia

was

linked

to

both

estrogenic

and

antiandrogenic

effects

of

lavender

and

tea

tree

oil

-LSB-

-RSB-

Chemical

and

immunological

methods

are

commonly

used

to

detect

steroid

hormones

in

food

clinical

practice

environmental

samples

or

doping

control

Owing

to

the

great

variety

of

chemicals

with

hormonelike

activity

these

methods

have

the

drawback

that

they

only

quantify

the

compound

of

interest

and

are

not

able

to

determine

biological

activity

of

unknown

compounds

and

their

metabolites

this

in

contrast

to

biological

assays

Receptor-based

transcription

activation

assays

can

be

used

to

detect

all

compounds

having

affinity

for

a

given

receptor

-LSB-

-RSB-

In

contrast

to

receptor

binding

assays

receptor

gene

bioassays

also

include

the

transactivation

step

and

can

distinguish

between

receptor

agonists

and

receptor

antagonists

-LSB-

-RSB-

This

feature

is

very

helpful

in

detecting

both

known

and

unknown

compounds

Several

assays

have

been

developed

for

this

purpose

using

both

mammalian

and

yeast

cells

In

general

transcription

activation

assays

based

on

mammalian

or

more

particular

human

cell

lines

have

been

shown

to

be

more

sensitive

than

yeast-based

assays

and

may

be

able

to

identify

compounds

that

require

human

metabolism

for

activation

into

their

active

state

Metabolic

conversion

can

either

activate

or

inactivate

some

compounds

-LSB-

-RSB-

whereas

the

relatively

low

metabolic

capacity

of

yeast

ensures

that

the

test

reflects

the

activity

of

the

original

compound

In

addition

yeast-based

assays

have

several

other

advantages

These

include

low

costs

easy

handling

lack

of

known

endogenous

receptors

that

may

compete

with

the

receptor

activity

under

investigation

-LRB-

no

crosstalk

-RRB-

and

the

use

of

media

that

are

devoid

of

steroids

-LSB-

-RSB-

Furthermore

yeast

cell

assays

are

extremely

robust

and

survive

extracts

from

dirty

sample

matrices

such

as

sediments

urine

and

feed

-LSB-

-RSB-

Especially

in

the

case

of

androgens

the

lack

of

known

endogenous

receptors

in

yeast

is

a

great

advantage

compared

with

mammalian

cell

lines

as

androgen

responsive

elements

-LRB-

AREs

-RRB-

can

also

be

activated

by

the

progesterone

receptor

and

the

glucocorticoid

receptor

To

avoid

potential

crosstalk

in

mammalian

cell

lines

a

lot

of

efforts

was

expended

to

construct

an

ARE

that

is

specific

and

no

longer

inducible

by

the

progesterone

and

glucocorticoid

receptor

-LSB-

-RSB-

However

up

till

now

such

an

ARE

does

not

exist

and

it

is

doubtful

whether

it

will

be

found

as

the

consensus

progesterone

responsive

element/glucocorticoid

responsive

element

is

equal

to

the

consensus

ARE

Moreover

the

glucocorticoid

receptor

is

normally

expressed

in

all

mammalian

cell

types

So

far

this

has

resulted

in

cell

lines

that

are

not

specific

for

androgens

and

that

also

respond

to

gestagens

or

glucocorticoids

-LSB-

-RSB-

This

paper

reports

the

development

of

a

new

yeast

androgen

bioassay

by

creating

a

stably

transfected

yeast

strain

that

expresses

yeast

enhanced

green

fluorescent

protein

-LRB-

yEGFP

-RRB-

as

a

measurable

reporter

protein

in

response

to

androgens

The

lack

of

known

endogenous

receptors

in

yeast

enabled

us

to

use

the

strong

nonspecific

consensus

ARE

sequence

which

is

actually

a

common

hormone

responsive

element

that

is

recognized

by

the

androgen

progesterone

and

glucocorticoid

receptors

and

can

therefore

not

be

used

in

mammalian

cell

lines

expressing

more

than

one

of

these

receptors

Exposures

to

progesterone

dexamethasone

and

other

compounds

were

performed

in

plates

in

order

to

demonstrate

the

suitability

and

specificity

of

this

new

yeast

androgen

bioassay

Additionally

flutamide

and

several

brominated

flame

retardants

were

tested

for

their

antagonistic

mode

of

action

and

the

results

were

compared

with

a

yeast

androgen

bioassay

expressing

β-galactosidase

as

a

reporter

protein

Materials

and

methods

Chemicals

Chemicals

and

methods

to

prepare

the

growth

media

to

perform

PCR

to

isolate

DNA

and

to

transform

bacteria

and

yeast

were

as

described

earlier

-LSB-

-RSB-

Corticosterone

dexamethasone

estrone

flutamide

medroxyprogesterone

and

progesterone

were

obtained

from

Sigma

-LRB-

St.

Louis

MO

USA

-RRB-

The

following

compounds

were

obtained

from

Steraloids

-LRB-

Newport

RI

USA

-RRB-

diethylstilbestrol

and

Tetrahydrogestrinone

-LRB-

THG

-RRB-

was

a

gift

from

M.

Thevis

-LRB-

DSHS

Cologne

Germany

-RRB-

Copper

sulfate

and

dimethyl

sulfoxide

-LRB-

DMSO

-RRB-

were

obtained

from

Merck

-LRB-

Darmstadt

Germany

-RRB-

and

methyltrienolone

was

obtained

from

PerkinElmer

-LRB-

USA

-RRB-

All

restriction

endonucleases

and

corresponding

buffers

were

obtained

from

New

England

Biolabs

-LRB-

Hitchin

UK

-RRB-

and

the

yeast

β-galactosidase

assay

kit

was

from

Pierce

Biotechnology

-LRB-

Rockford

IL

USA

-RRB-

-LRB-

TBP

-RRB-

and

the

hydroxyl

derivative

were

synthesized

at

the

Wallenberg

Laboratory

-LRB-

Stockholm

University

Sweden

-RRB-

Yeast

strains

The

yeast

Saccharomyces

cerevisiae

-LRB-

CEN.PK

LEU

-RRB-

host

strain

was

a

gift

from

H.

Silljé

-LRB-

University

of

Utrecht

The

Netherlands

-RRB-

The

yeast

androgen

bioassay

with

β-galactosidase

as

a

marker

was

kindly

provided

by

D.P.

McDonnell

-LRB-

Duke

University

USA

-RRB-

Plasmids

The

and

yeast

expression

vectors

were

obtained

from

the

American

Type

Culture

Collection

-LRB-

ATCC

Rockville

Maryland

USA

-RRB-

The

plasmid

was

a

gift

from

A.J.

Brown

-LRB-

Stanford

University

USA

-RRB-

Construction

of

the

expression

vector

The

yeast

cells

provided

by

McDonnell

were

grown

overnight

and

chromosomal

DNA

was

isolated

This

DNA

was

used

to

serve

as

a

template

for

the

PCR

to

obtain

the

complementary

DNA

-LRB-

cDNA

-RRB-

of

hAR

Full-length

hAR

cDNA

was

obtained

using

the

Expand

High

Fidelity

PCR

system

-LRB-

Boehringer

Mannheim

-RRB-

and

an

Eppendorf

Mastercycler

gradient

The

sequence

of

the

primer

was

containing

a

restriction

site

for

XbaI

just

before

the

ATG

start

codon

The

sequence

of

the

primer

was

containing

a

restriction

site

for

BamHI

just

after

the

TGA

stop

codon

This

PCR

generated

a

full-length

double-stranded

-LRB-

ds

-RRB-

cDNA

of

bp

of

the

hAR

gene

with

a

XbaI

and

a

BamHI

restriction

site

The

full-length

hAR

PCR

product

was

isolated

from

a

low-melt

agarose

gel

cleaved

with

XbaI

and

BamHI

and

ligated

into

the

corresponding

site

of

the

yeast

vector

Plasmid

digestion

and

PCR

controls

revealed

several

good

clones

Construction

of

the

reporter

vector

A

set

of

complementary

oligonucleotides

-LRB-

a

and

b

-RRB-

each

with

two

consensus

ARE

sequences

-LRB-

in

bold

-RRB-

were

synthesized

A

solution

with

both

cDNA

oligonucleotides

μM

of

each

was

heated

at

°C

and

cooled

down

to

room

temperature

in

h

This

set

gave

a

ds

DNA

with

a

SacI

sticky

end

and

a

MscI

blunt

end.Sa

AAAGTCAGAACAGCATGTTCTGATCAAATCTAGAAGATCCAAAGTCAGAACAGCATGTTCTGATCAAACTCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGG-3

Sb

CCATCCACGCTATATATACACGCCTGGCGGATCTGCTCGAGTTTGATCAGAACATGCTGTTCTGACTTTGGATCTTCTAGATTTGATCAGAACATGCTGTTCTGACTTTAGCT-3

This

ds

DNA

was

cloned

into

the

corresponding

site

of

the

vector

Subsequently

yEGFP

-LSB-

-RSB-

obtained

from

a

HindIII/SalI

double

digestion

of

pyEGFP

was

cloned

in

the

corresponding

HindIII/SalI

sites

of

the

reporter

construct

Plasmid

digestion

and

PCR

controls

revealed

several

good

clones

Transformation

of

yeast

cells

Transformation

of

yeast

host

strain

-LRB-

Ura

His

and

Leu

-RRB-

was

performed

by

the

lithium

acetate

protocol

as

described

earlier

-LSB-

-RSB-

First

the

yeast

was

transformed

with

the

reporter

vector

integrated

at

the

chromosomal

location

of

the

uracil

gene

via

homologous

recombination

Therefore

prior

to

transformation

the

reporter

vector

was

linearized

by

cutting

with

StuI

which

has

a

unique

restriction

site

in

the

marker

gene

Transformants

were

grown

on

minimal

medium

plates

containing

l-leucine

and

l-histidine

-LRB-

MM/L

plates

-RRB-

This

yeast

reporter

strain

was

then

transformed

with

the

expression

vector

which

was

linearized

by

cleavage

with

NsiI

which

has

a

unique

restriction

site

in

the

marker

gene

-LRB-

histidine

-RRB-

Transformants

were

grown

on

MM/L

plates

and

PCR

controls

were

used

to

select

clones

that

contain

the

reporter

and

the

expression

construct

PCR

controls

PCR

controls

were

performed

on

the

reporter-receptor

transformants

Yeast

chromosomal

DNA

of

transformants

was

isolated

and

PCR

controls

were

performed

PCR

I

was

performed

with

a

primer

on

the

backbone

of

the

reporter

plasmid

and

a

primer

on

the

sequence

The

sequence

of

the

primer

was

and

the

sequence

of

the

primer

was

PCR

II

was

performed

with

a

primer

on

the

-LRB-

cytochrome

c

oxidase

-RRB-

promoter

of

the

reporter

plasmid

and

a

primer

on

the

terminator

The

sequence

of

the

primer

was

and

the

sequence

of

the

primer

was

PCR

III

was

performed

with

the

primers

that

were

also

used

to

obtain

the

full

length

cDNA

of

the

hAR

-LRB-

see

Construction

of

the

expression

vector

-RRB-

Streamlined

yEGFP

assay

with

the

yeast

androgen

bioassay

The

day

before

running

the

assay

a

single

colony

from

a

MM/L

agar

plate

was

used

to

inoculate

mL

of

the

selective

MM/L

medium

This

culture

was

grown

overnight

at

°C

with

vigorous

orbital

shaking

At

the

late

log

phase

the

yeast

androgen

receptor

biosensor

was

diluted

in

the

selective

MM/L

medium

to

an

optical

density

-LRB-

OD

-RRB-

at

nm

between

and

For

exposure

aliquots

of

μL

of

this

diluted

yeast

culture

were

pipetted

into

each

well

of

a

plate

and

μL

of

a

or

other

stock

solution

in

DMSO

was

added

DMSO-only

controls

were

included

in

each

experiment

and

each

sample

concentration

was

assayed

in

triplicate

Exposure

was

performed

for

h

at

°C

and

orbital

shaking

at

rpm

Fluorescence

was

measured

at

and

h

directly

in

a

CytoFluor

multiwell

plate

reader

-LRB-

Series

PerSeptive

Biosystems

-RRB-

using

excitation

at

nm

and

measuring

emission

at

nm

The

fluorescence

signal

was

corrected

with

the

signals

obtained

with

MM/L

containing

DMSO

solvent

only

Densities

of

the

yeast

culture

were

determined

by

measuring

the

OD

at

nm

but

this

was

only

done

to

check

whether

a

sample

was

toxic

for

the

yeast

cells

For

the

calculation

of

the

relative

androgenic

potency

-LRB-

RAP

-RRB-

of

the

compounds

in

the

yeast

androgen

bioassay

the

data

of

a

complete

dose

response

curve

were

fitted

using

the

equation

-LRB-

Slide

write

Plus

version

-RRB-

This

is

equivalent

to

where

response

is

the

measured

fluorescence

signal

-LSB-

agonist

-RSB-

is

the

concentration

of

the

test

compound

and

is

the

concentration

of

the

test

compound

giving

half-maximum

response

lacZ-based

yeast

androgen

bioassay

β-galactosidase

assay

An

agar

plate

containing

the

selective

growth

medium

consisting

of

a

yeast

nitrogen

base

with

dextrose

-LRB-

-RRB-

lysine

-LRB-

mg/L

-RRB-

tryptophan

-LRB-

mg/L

-RRB-

uracil

-LRB-

mg/L

-RRB-

and

adenine

-LRB-

mg/L

-RRB-

was

inoculated

with

the

yeast

androgen

receptor

cytosensor

from

a

frozen

°C

stock

-LRB-

glycerol

v/v

-RRB-

The

plate

was

incubated

at

°C

for

h

and

then

stored

at

°C

The

day

before

running

the

assay

a

single

colony

of

the

yeast

was

used

to

inoculate

mL

of

the

selective

growth

medium

This

culture

was

grown

overnight

at

°C

with

vigorous

orbital

shaking

at

rpm

At

the

late

log

phase

the

yeast

androgen

receptor

cytosensor

was

diluted

in

growth

medium

to

an

OD

of

at

nm

and

-LRB-

mM

-RRB-

was

added

to

induce

the

expression

of

the

hAR

For

exposure

in

plates

aliquots

of

μL

of

this

diluted

yeast

culture

were

pipetted

into

each

well

and

μL

of

stock

solutions

in

DMSO

was

added

Exposure

was

performed

for

h

at

°C

and

rpm

and

the

β-galactosidase

activity

was

measured

with

a

commercial

yeast

β-galactosidase

assay

kit

from

Pierce

-LRB-

Rockford

IL

USA

-RRB-

This

kit

uses

o-nitrophenyl

β-d-galactopyranoside

as

a

substrate

and

the

solution

turns

yellow

upon

hydrolysis

of

β-d-galactopyranoside

to

o-nitrophenol

and

galactose

The

yellow

o-nitrophenol

is

measured

in

a

Biotek

-LRB-

Winooski

VT

USA

-RRB-

model

ELx

series

ultra

microplate

reader

at

nm

Densities

of

the

yeast

culture

were

determined

by

measuring

the

OD

at

nm

The

measured

response

at

nm

was

corrected

for

the

OD

at

nm

Results

and

discussion

A

recombinant

yeast

cell

was

constructed

that

expresses

the

hAR

and

yEGFP

as

a

reporter

protein

in

response

to

androgens

Both

the

receptor

construct

as

well

as

the

reporter

construct

were

stably

integrated

into

the

yeast

genome

by

the

use

of

yeast-integrating

plasmids

For

the

construction

of

the

reporter

vector

the

plasmid

containing

the

marker

gene

was

used

Two

consensus

AREs

were

placed

in

the

SacI/MscI

site

of

the

truncated

promoter

in

a

way

that

the

to

XhoI-SphI

part

of

the

promoter

was

restored

-LSB-

-RSB-

High

expression

levels

of

the

androgen

receptor

were

obtained

by

placing

the

cDNA

of

the

hAR

gene

behind

the

strong

constitutive

yeast

dehydrogenase

-LRB-

GPD

-RRB-

promoter

in

the

plasmid

This

plasmid

contains

the

marker

gene

Transfected

strains

were

checked

with

PCR

The

correct

and

specific

functioning

of

the

yeast

androgen

bioassay

was

studied

by

exposures

to

and

other

compounds

and

the

results

were

compared

with

results

obtained

with

the

lacZ-based

yeast

androgen

bioassay

provided

by

McDonnell

In

addition

the

antiandrogenic

properties

of

several

brominated

flame

retardants

were

investigated

PCR

controls

A

number

of

different

PCR

controls

were

carried

out

to

check

the

integration

of

the

vectors

into

the

yeast

genome

Figure

shows

the

gel

electrophoresis

results

of

these

PCR

controls

PCR

I

-LRB-

Fig.

-RRB-

was

performed

with

primers

on

the

backbone

of

the

plasmid

and

on

the

sequence

As

expected

it

gave

the

specific

band

with

the

reporter

vector

and

the

DNA

that

was

isolated

from

the

yeast

androgen

biosensor

The

negative

controls

performed

with

the

empty

plasmid

and

with

the

DNA

that

was

isolated

from

the

empty

yeast

host

-LRB-

the

nontransfected

yeast

cell

-RRB-

showed

no

PCR

bands

PCR

II

-LRB-

Fig.

-RRB-

was

performed

with

primers

on

the

promoter

and

the

terminator

As

expected

it

gave

the

specific

band

with

the

reporter

vector

and

the

DNA

that

was

isolated

from

the

biosensor

because

both

contain

the

reporter

construct

with

the

yEGFP

that

was

ligated

between

the

promoter

and

terminator

The

negative

controls

performed

with

the

empty

plasmid

and

with

the

DNA

that

was

isolated

from

the

empty

yeast

host

did

not

show

the

reporter-specific

band

However

this

PCR

generated

a

band

with

the

DNA

of

the

empty

yeast

host

and

the

biosensor

This

band

corresponds

to

the

CYC

gene

of

the

yeast

host

itself

and

is

therefore

also

a

specific

band

PCR

III

-LRB-

Fig.

-RRB-

was

performed

with

the

primers

on

the

hAR

gene

As

expected

it

gave

the

specific

band

with

the

expression

vector

and

the

DNA

that

was

isolated

from

the

biosensor

In

the

negative

control

performed

with

the

DNA

that

was

isolated

from

the

yeast

host

the

receptor-specific

band

was

not

present

These

PCR

controls

demonstrate

that

all

specific

PCR

bands

can

be

seen

thus

demonstrating

that

our

yeast

androgen

bioassay

contains

the

expression

vector

and

the

reporter

vector

both

stably

integrated

in

the

yeast

genome

Fig.

controls

The

PCR

controls

were

performed

as

described

in

PCR

controls

a

Lanes

and

contain

a

ladder

PCR

I

was

performed

with

primers

on

the

backbone

of

the

plasmid

and

on

the

sequence

Lanes

are

PCR

I

on

the

reporter

vector

the

DNA

that

was

isolated

from

the

yeast

transformant

the

empty

plasmid

and

the

DNA

that

was

isolated

from

the

empty

yeast

host

-LRB-

the

nontransfected

yeast

cells

-RRB-

respectively

PCR

II

was

performed

with

primers

on

the

promoter

and

the

terminator

Lanes

are

PCR

II

on

the

reporter

vector

the

DNA

from

the

yeast

transformant

the

empty

plasmid

and

the

DNA

from

the

empty

yeast

host

respectively

b

Lane

contains

a

ladder

PCR

III

was

performed

with

the

primers

on

the

human

androgen

receptor

gene

Lanes

are

PCR

III

on

the

expression

vector

the

DNA

from

the

yeast

transformant

and

the

DNA

from

the

empty

yeast

host

respectively

yEGFP

yeast

enhanced

green

fluorescent

protein

Dose

response

curves

obtained

with

the

new

yeast

androgen

bioassay

The

dose

response

curves

for

several

natural

and

synthetic

androgens

are

shown

in

Fig.

and

caused

a

dose-related

increase

in

the

production

of

yEGFP

demonstrating

that

these

compounds

are

potent

androgens

The

bioassay

showed

a

limit

of

detection

of

nM

for

with

a

dynamic

range

from

to

nM

and

very

low

standard

deviations

-LRB-

les

than

-RRB-

The

figure

also

shows

that

and

progesterone

give

a

response

The

female

hormone

gives

a

full

dose

response

curve

but

the

maximum

of

the

curve

is

reached

at

a

times

higher

concentration

than

that

of

and

is

less

steep

Progesterone

gives

a

response

but

the

maximum

response

is

only

about

of

that

of

and

is

reached

at

a

times

higher

concentration

Both

and

progesterone

are

known

to

possess

androgenic

properties

Progesterone

displays

low

binding

to

the

androgen

receptor

-LSB-

-RSB-

and

shows

androgenic

effects

in

vivo

-LSB-

-RSB-

According

to

-LSB-

-RSB-

and

progesterone

showed

androgenic

activity

in

ten

out

of

and

seven

out

of

nine

mammalian

cell

reporter

gene

-LRB-

MCRG

-RRB-

systems

respectively

The

corticosteroids

corticosterone

and

dexamethasone

showed

no

response

in

our

assay

Fig.

of

the

yeast

androgen

biosensor

to

different

substances

Exposure

to

progesterone

dexamethasone

and

was

started

by

adding

to

μL

of

a

yeast

culture

a

aliquot

of

a

stock

solution

of

the

compound

in

dimethyl

sulfoxide

-LRB-

DMSO

-RRB-

Fluorescence

was

determined

after

h

as

described

in

Streamlined

yEGFP

assay

with

the

yeast

androgen

bioassay

Fluorescence

signals

are

the

mean

of

a

triplicate

with

the

standard

deviation

-LRB-

SD

-RRB-

DHT

Prog

progesterone

Dex

dexamethasone

Bold

Table

shows

the

calculated

ie.

the

concentration

giving

a

half-maximum

response

and

the

RAP

defined

as

the

ratio

between

the

of

and

the

of

the

compound

for

several

compounds

The

yeast

androgen

bioassay

showed

good

sensitivity

towards

all

androgens

tested

with

the

following

range

of

potencies

methyltrienolone

tetrahydrogestrinone

medroxyprogesterone

acetate

progesterone

Steroids

representative

for

other

hormone

receptors

like

estrone

and

diethylstilbestrol

for

the

estrogen

receptor

and

corticosterone

and

dexamethasone

for

the

glucocorticoid

receptor

showed

no

agonistic

response

Only

progesterone

and

medroxyprogesterone

acetate

gave

a

clear

agonistic

response

However

these

compounds

are

known

to

exert

androgenic

effects

Table

concentrations

and

relative

androgenic

potencies

-LRB-

RAP

-RRB-

of

compounds

in

the

yeast

androgen

biosensor

expressing

yeast

enhanced

green

fluorescent

protein

in

response

to

androgensCompoundQualitative

response

for

AR

-LRB-

nM

-RRB-

in

the

yeast

androgen

Positive

-LRB-

-RRB-

Strong

AR

agonist761.05α-DihydrotestosteronePositive

-LRB-

-RRB-

Strong

AR

agonist

weak

ER

β-TrenbolonePositiveBinds

strongly

to

MethyltrienolonePositive

-LRB-

-RRB-

AR

TetrahydrogestrinoneAR

-LRB-

-RRB-

AR

agonist

and

antagonist

strong

ER

EstronePositive

-LRB-

-RRB-

AR

agonist

strong

ER

-LRB-

-RRB-

ER

-LRB-

-RRB-

Strong

ER

agonistNRNRDiethylstilbestrolNegative

-LRB-

-RRB-

Strong

ER

-LRB-

-RRB-

ER

antagonistNRNRProgesteronefPositive

-LRB-

-RRB-

Medroxyprogesterone

acetate

Positive

-LRB-

-RRB-

Weak

AR

CorticosteroneNegative

-LRB-

-RRB-

Binds

weakly

to

ARNRNRDexamethasonePositive

-LRB-

-RRB-

AR

agonistNRNRFlutamideNegative

-LRB-

-RRB-

AR

androgen

receptor

ER

estrogen

receptor

NR

no

responseaQualitative

response

for

AR

agonism

across

all

mammalian

cell

reporter

gene

studies

-LRB-

data

obtained

from

-LSB-

-RSB-

-RRB-

bComments

obtained

from

-LSB-

-RSB-

cThe

is

the

concentration

giving

half-maximum

response.dThe

RAP

is

defined

as

the

ratio

between

the

of

and

the

of

the

compound.eThis

compound

was

toxic

to

yeast

above

μM.fThese

compounds

reach

a

maximum

response

that

is

lower

than

of

the

maximum

response

obtained

with

The

maxima

obtained

with

and

progesterone

are

about

and

respectively

Compared

with

the

NIH

publication

-LSB-

-RSB-

there

are

a

few

discrepancies

According

to

-LSB-

-RSB-

estrone

is

an

androgen

receptor

agonist

that

showed

androgenic

activity

in

two

out

of

two

MCRG

systems

However

some

mammalian

cells

are

able

to

convert

estrone

into

and

vice

versa

This

conversion

is

ascribed

to

dehydrogenase

and

this

enzyme

is

responsible

for

the

high

relative

estrogenic

potency

-LRB-

REP

-RRB-

of

estrone

in

the

estrogen

bioassay

with

the

breast

cancer

cells

-LRB-

ER-CALUX

test

-RRB-

In

that

test

the

estrogenic

potency

of

estrone

was

equal

to

that

of

and

a

REP

of

was

reported

for

estrone

-LSB-

-RSB-

This

probably

explains

why

estrone

gave

a

positive

result

for

androgenic

activity

in

two

MCRG

systems

but

gave

a

negative

result

in

our

yeast

androgen

bioassay

Yeast

is

obviously

not

able

to

convert

estrone

into

The

reported

REP

of

in

our

yeast

estrogen

biosensor

corresponds

nicely

with

the

in

vivo

potency

of

this

compound

-LSB-

-RSB-

Dexamethasone

was

also

described

as

an

androgen

receptor

agonist

in

the

NIH

publication

showing

androgenic

activity

in

three

out

of

four

mammalian

assays

but

gave

a

negative

result

in

our

yeast

androgen

bioassay

However

these

MCRG

systems

use

an

MMTV-Luc

reporter

construct

or

an

ARE-Luc

reporter

construct

Both

the

MMTV

and

the

ARE

sequence

are

recognized

by

the

glucocorticoid

receptor

and

this

means

that

the

response

found

in

these

MCRG

systems

is

probably

due

to

crosstalk

as

the

glucocorticoid

receptor

is

normally

expressed

in

all

cell

types

Dose

response

curves

obtained

with

the

lacZ-based

yeast

androgen

bioassay

Figure

shows

the

dose

response

curves

for

several

natural

and

synthetic

androgens

obtained

with

the

lacZ-based

yeast

androgen

bioassay

The

assay

was

simplified

in

our

laboratory

by

scaling

it

down

to

a

format

and

the

use

of

a

β-galactosidase

assay

kit

With

increasing

β-galactosidase

activity

the

density

of

the

yeast

culture

measured

at

nm

dropped

to

about

-LRB-

data

not

shown

-RRB-

Therefore

the

measured

â-galactosidase

activity

was

corrected

for

the

OD

of

the

yeast

culture

Table

shows

the

calculated

and

the

corresponding

RAP

values

The

data

demonstrate

that

there

is

a

good

correlation

between

the

values

for

androgens

determined

in

our

laboratory

and

those

reported

by

Gaido

et

al.

-LSB-

-RSB-

in

It

seems

that

the

lacZ-based

yeast

androgen

bioassay

in

terms

of

values

is

times

more

sensitive

than

our

new

bioassay

However

there

was

almost

no

difference

in

the

limit

of

detection

Although

the

curves

in

the

β-galactosidase

assay

go

up

at

around

nM

and

in

the

yEGFP

assay

at

around

nM

the

limit

of

detection

is

about

nM

for

in

both

assay

types

This

is

mainly

because

the

standard

deviations

are

much

higher

in

the

β-galactosidase

assay

However

the

curves

in

the

β-galactosidase

assay

are

much

steeper

and

are

thus

responsible

for

the

lower

values

The

steeper

curves

are

probably

due

to

using

an

enzyme

as

a

marker

β-galactosidase

compared

with

a

yEGFP

marker

protein

and

the

expression

of

the

cofactor

that

enhances

transcription

activation

in

the

lacZ-based

yeast

androgen

bioassay

The

dynamic

range

for

in

the

McDonnell

assay

was

from

to

nM

and

is

slightly

smaller

than

the

range

of

the

new

bioassay

-LRB-

nM

-RRB-

There

were

also

no

great

differences

in

the

RAP

values

determined

although

methyltrienolone

was

more

potent

in

the

lacZ-based

bioassay

However

the

new

yEGFP

bioassay

is

less

sensitive

for

and

progesterone

RAPs

of

and

respectively

and

for

the

latter

no

full

dose

response

curve

compared

with

RAPs

of

and

and

full

dose

response

curves

in

the

lacZ-based

bioassay

This

means

that

our

new

bioassay

is

more

specific

for

detecting

compounds

with

a

pure

androgenic

mode

of

action

The

main

difference

between

the

lacZ-based

and

our

yEGFP

biosassay

is

that

protein

which

is

a

counterpart

of

the

mammalian

is

overexpressed

in

the

lacZ-based

bioassay

in

order

to

enhance

transcriptional

efficacy

However

this

can

not

explain

the

observed

difference

with

methyltrienolone

as

according

to

Imhof

and

McDonnell

-LSB-

-RSB-

this

did

not

alter

the

potency

or

specificity

of

the

assay

In

addition

and

in

contrast

to

the

lacZ-based

yeast

androgen

bioassay

there

were

no

differences

in

the

density

of

the

yeast

culture

measured

at

nm

upon

exposure

to

different

compounds

that

induced

yEGFP

expression

which

indicates

a

decreased

growth

of

yeast

cells

exposed

to

androgens

in

the

lacZ-based

yeast

androgen

bioassay

The

only

other

known

yeast

androgen

bioassay

one

that

uses

luciferase

as

a

reporter

protein

displays

similar

characteristics

in

terms

of

specificity

and

was

times

more

sensitive

in

terms

of

values

-LSB-

-RSB-

However

this

assay

needs

the

correction

of

the

same

yeast

strain

that

only

and

stably

expressed

luciferase

as

an

external

control

to

correct

for

and

normalize

the

aspecific

responses

caused

by

variation

in

cell

vitality

due

to

matrix

and

analyte

toxicity

Unorrected

dose

response

curves

for

displayed

more

than

times

higher

values

An

earlier

assay

described

by

Lee

et

al.

-LSB-

-RSB-

in

uses

β-galactosidase

but

only

expresses

the

hinge-ligand

binding

domain

of

the

androgen

receptor

Fig.

of

the

McDonnell

yeast

androgen

bioassay

to

different

substances

Exposure

to

progesterone

dexamethasone

and

was

started

by

adding

to

μL

of

a

yeast

culture

a

aliquot

of

a

stock

solution

of

the

compound

in

DMSO

The

β-galactosidase

activity

was

determined

after

h

and

corrected

for

the

optical

density

at

nm

as

described

in

lacZ-based

yeast

androgen

bioassay

β-galactosidase

assay

Signals

are

the

mean

of

a

triplicate

with

the

SDTable

concentrations

and

RAPs

of

compounds

in

the

yeast

androgen

bioassay

expressing

β-galactosidase

in

response

to

-LRB-

nM

-RRB-

a

by

Gaido

et

al.

-LSB-

-RSB-

-LRB-

nM

-RRB-

b

in

our

MethyltrienoloneND3.73.1TetrahydrogestrinoneND11.51.017β-Estradiol

86.1950.117α-EstradiolNDNRNRProgesterone89.31700

CorticosteroneNDNRNRDexamethasoneNDNRNRND

not

determinedaValues

obtained

by

Gaido

et

al.

-LSB-

-RSB-

using

the

McDonnell

yeast

androgen

bioassaybValues

determined

in

our

laboratory

using

the

McDonnell

yeast

androgen

bioassay

-LRB-

see

Dose

response

curves

obtained

with

the

lacZ-based

yeast

androgen

bioassay

-RRB-

Antiandrogenic

activity

The

specificity

of

the

new

yeast

androgen

bioassay

was

further

demonstrated

by

the

ability

of

antiandrogens

to

suppress

the

induction

of

yEGFP

Figure

shows

the

antiandrogenic

activity

of

the

known

antagonist

flutamide

and

three

brominated

flame

retardants

TBP

and

The

antagonistic

properties

were

examined

by

coexposure

with

a

concentration

of

that

induced

a

submaximal

response

-LRB-

nM

-RRB-

of

these

four

compounds

were

able

to

show

an

agonistic

response

-LRB-

Table

-RRB-

but

Fig.

clearly

shows

that

all

three

were

able

to

inhibit

the

response

induced

by

The

value

was

about

μM

for

flutamide

and

TBP

and

were

about

as

potent

while

was

clearly

less

antiandrogenic

Similar

results

were

obtained

with

a

human

cell

line

Only

TBP

was

less

potent

in

that

test

but

cytotoxicity

of

TBP

could

not

be

excluded

-LSB-

-RSB-

Fig.

of

a

submaximal

response

obtained

by

with

flutamide

and

three

brominated

flame

retardants

-LRB-

BFRs

-RRB-

Coexposure

to

a

concentration

of

that

induced

a

submaximal

response

was

started

by

adding

to

μL

of

a

yeast

culture

μL

of

a

and

μL

of

the

BFR

stock

solution

in

DMSO

Fluorescence

was

determined

after

h

as

described

in

Streamlined

yEGFP

assay

with

the

yeast

androgen

bioassay

Fluorescence

signals

are

the

mean

of

a

triplicate

with

the

SD

TBP

Conclusions

A

recombinant

yeast

cell

was

constructed

that

expresses

the

hAR

and

yEGFP

as

a

reporter

protein

in

response

to

androgens

Compared

with

other

yeast

androgen

bioassays

this

new

biosassay

showed

a

similar

limit

of

detection

and

dynamic

range

However

the

measurement

of

the

fluorescence

-LRB-

yEGFP

-RRB-

can

be

followed

as

a

function

of

incubation

time

and

is

easier

quicker

and

cheaper

than

the

measurement

of

the

β-galactosidase

or

the

luciferase

activity

which

needs

cell

wall

disruption

and/or

the

addition

of

expensive

substrates

Owing

to

the

ease

of

the

yEGFP

measurement

standard

deviations

are

generally

less

than

Moreover

the

assay

seems

to

be

more

robust

and

more

specific

for

detecting

compounds

with

a

pure

androgenic

mode

of

action

Brominated

flame

retardants

with

suspected

antiandrogenic

properties

were

able

to

inhibit

the

response

obtained

with

the

most

potent

endogenous

androgen

demonstrating

that

this

yeast

androgen

bioassay

is

suited

to

detect

compounds

with

both

agonistic

and

antagonistic

characteristics

As

all

compounds

tested

were

able

to

show

either

their

agonistic

or

their

antagonistic

properties

neither

the

cell

wall

nor

the

cell

membrane

seemed

to

be

an

obstacle

As

for

the

yeast

estrogen

bioassay

we

validated

this

new

yeast

androgen

bioassay

according

to

international

criteria

eg.

the

determination

of

the

decision

limit

-LRB-

CCα

-RRB-

and

the

detection

capability

-LRB-

CCβ

-RRB-

The

decision

limit

is

then

used

to

distinguish

negative

and

suspect

samples

-LSB-

-RSB-

-LRB-

unpublished

results

-RRB-

The

assay

was

proven

to

be

useful

to

detect

the

new

designer

steroid

tetrahydrogestrinone

in

human

urine

-LSB-

-RSB-

while

prohormones

with

an

androgenic

mode

of

action

e.g.

dehydroepiandrosterone

are

not

active

in

yeast-based

bioassays

and

need

metabolic

activation

before

they

can

be

detected

-LSB-

-RSB-

Future

work

will

include

the

validation

of

the

assay

for

urine

and

feed

and

the

screening

of

prohormones

with

and

without

metabolic

activation

To

mimic

the

in

vivo

metabolic

activation

liver

slices

liver

cell

lines

liver

enzymes

and

pure

enzymes

will

be

used

O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O B O O O O O O O O O O O B O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

antagonists
brominated flame retardants
crosstalk
metabolism
receptor
saccharomyces cerevisiae