We
are
working
in
Tier
I
with
mechanisms
Are
mechanisms
similar
or
dissimilar
across
phyla,

species,
classes,
etc.?
In
other
words
–
What
is
the
phylogeny
of
the
various
mechanisms?
Attempt
at
`Problem
Statement'
In
particular,
what
is
the
phylogeny
of:
1.

Nuclear
receptors
–
as
they
bind
the
the
ligands
and
activate
particular
genes
and
pathways
2.

P450s
–
as
they
synthesize
and
metabolize
most
NR
ligands
Regrettably,
will
emphasis
more
of
what
we
don't
know,
than
what
we
know
Nuclear
Receptors
Metazoan
signaling
pathways
5
Basic
evolutionary
groups:
­

based
on
DNA
element
structure
(a
repeated
sequence,
some
serial
repeats,
some
mirrored,
and
with
n
nucleotides
spacer)
­

based
on
gene
structure
–
intron/
extron
point
in
DNA
binding
region
of
protein
­

based
on
amino
acid
sequence
homology
At
this
time,

steroid
family
(E,

A,

G,

P,

M)

appears
to
be
unique
to
vertebrates.
Echinoderms
Hemichordates
Urochordates
Cephalochordates
Vertebrates
Poriferans
Cnidarians
Ctenophorans
Gastrotrichs
Nematodes
Priapulids
Kinorhynchs
Onychophorans
Tardigrades
Arthropods
Bryozoans
Entoprocts
Platyhelminthes
Pogonophorans
Brachiopods
Phoronids
Nemerteans
Annelids
Echiurans
Molluscs
Sipunculans
Gnathostomulids
Rotifers
Metazoans
Bilateria
Deuterostomes
Protostomes
Lophotrochozoans
Ecdysozoans
Gastrotrichs
Nematodes
Priapulids
Kinorhynchs
Onychophorans
Tardigrades
Arthropods
Bryozoans
Entoprocts
Platyhelminthes
Pogonophorans
Brachiopods
Phoronids
Nemerteans
Annelids
Echiurans
Molluscs
Sipunculans
Gnathostomulids
Rotifers
Metazoans
&

Nuclear
Receptors
ER       
,AR,
GR,
MR,

&
PR       
TR       
,ERR       
,
VDR,

RAR       
,
PPAR       
Note:
Greek
letters
indicate
separate
(multiple)
forms
of
the
receptor
exist
action
ligand
specificity,
cell
distribution
may
vary
Vertebrates
only:
Steroid
Family
(??)
"Others"
also
restricted
to
a
few
phyla
Echinoderms
Hemichordates
Urochordates
Cephalochordates
Vertebrates
Implications
1.

Is
there
a
scientific
basis
or
rationale
to
extrapolate
an
EAT
based
screen
to
other
phyla?
At
this
time,

probably
not!
2.

This
means
we
have
no
screens
for
the
other
phyla.
They
are
unprotected!
We
have
serious
scientific
gaps
that
need
to
be
filled!!!
Metazoans
&

Nuclear
Receptors
Gastrotrichs
Nematodes
Priapulids
Kinorhynchs
Onychophorans
Tardigrades
Arthropods
Bryozoans
Entoprocts
Platyhelminthes
Pogonophorans
Brachiopods
Phoronids
Nemerteans
Annelids
Echiurans
Molluscs
Sipunculans
Gnathostomulids
Rotifers
??

??

??

RXR       
    
    
    
COUP       
   
   
   
EcdR,

KNIRPS       
   
   
   
For
invertebrates,
outside
Arthropods
far
less
known
about
NucRec's
and
their
transcriptional
roles
–
some
are
identified
only
as
genes
Echinoderms
Hemichordates
Urochordates
Cephalochordates
Vertebrates
Briefly,
the
P450s
Far
more
ancient
Far
more
diverse
But
a
parallel
way
to
analyze
the
problem
Lipid
synthetic
tree
of
ligands
DNA
element
Several
core
structures
intron­
exon
break
in
DNA
region
organization
abcd
abcd
abcd
dcba
nucleotide(
s)
spacing
Suggested
analysis
What
are
the
nuclear
receptors
that
are
potential
ED
targets?
What
are
the
ligands
(`

mimic
targets')?
What
are
the
synthetic
steps
(P450scc,
aromatase,

5­

alpha
reductase,
…)?
What
then
is
the
appropriate
phyla
for
which
a
screen
can
and
cannot
be
used?
(And
have
a
rationale
basis
to
validate)
N­

terminus
C­

terminus
DBD
–
DNA
Binding
Domain
Zinc
finger
interaction
Often
repeated
Several
configurations
distinguish
receptor
subclasses
LBD
–
Ligand
Binding
Domain
Large,

3
sided
cavity
Envelopes
ligand
Largely
hydrophobic
amino
acids
lining
the
cavity
Nuclear
Hormone
Receptor
Structure
Nuclear
Hormone
Receptor
Structure
N­

terminus
C­

terminus
AF­
1
Activation
Factor
1
(site
for
activation
by
several
kinase
or
phosphorylation
pathways)

AF­
2
Activation
Factor
2
(interaction
with
other
protein
families
–
one
represses
and
one
activates)
Essential
for
transcription
D
N
A
 
R
e
s
p
o
n
s
e
 
E
l
e
e
n
t
Nuclear
Hormone
Receptor
Ligand
Triggering
Action
#1
NH 
receptor
Li
and 

Bound
Heat 

Shock 
Prote
n
(HSP) 

chaperone
complex
CoRepressor
Dissociation
Receptor
Dimer
Formation
This `
free'
dimer 

ppe
rs
susceptible 
to
proteolysis
(ubiquitin)

Binding
DNA
Element
Sites
L
NH 
receptor
Li
and 

Bound
L
NH 
receptor
Li
and 

Bound
L
N
H
 

r
e
c
e
p
t
o
r
L
i
a
n
d
 
B
o
u
n
d
L
N
H
 

r
e
c
e
p
t
o
r
L
i
a
n
d
 
B
o
u
n
d
L
Transcribing
Genes
Into
mRNA
RNA 

polymerase 
II
or 

pol 
II 

complex
actually 

transcribes,
binds 
DNA 
at 

TATA 
box
DNA 
site 
TATA 

Box                                               
Gene 
Sta
t (

AUG 

codon)                   
1.

Pol
II
Binds
TATA
Box
2.

Pol
II
oves
Down
DNA
3.

Pol
II
Initiates
mRNA
transcription
Pol 
II
transcribin
Base 
0 
fo
 gene
Base "
X" # 
bases 
upst
eam 
fo
 gene
Nuclear
Hormone
ReceptorLigand
Complex
Action
#2
D
A 

Response 

Element                                            

TATA 

Box         
RNA 

polymerase 
II
or 

pol 
II 

complex
actually 

transcribes,
startin
 at 

TATA 
box
DNA 
site 
Response
element
for
receptor
is
upstream
of
TATA
box
RNA
pol
II
normally
cannot
bind
to
TATA
box
alone,
transcription
blocked
Upstre
m 
of 
t
rget
gene 
by 
X 
b
ses
Nuclear
Hormone
ReceptorLigand
Complex
Action
#2
D
A 

Response 

Element                                            

TATA 

Box         
RNA 

polymerase 
II
or 

pol 
II 

complex
must 
bind 
TATA 
box
DNA 
site 
to 

transcribe
tar
et 

ene
Receptor­
ligand
(

holo
­receptor)
does
not
interact
directly
with
the
Pol
II
complex
NH 
receptor
Li
and 

Bound
L
NH 
receptor
Li
and 

Bound
L
Upstre
m 
of 
t
rget
gene 
by 
X 
b
ses
Upstre
m 
of 

the 

TATA 
box 
Y 
b
ses
or 
X 

 Y 
of 
t
rget 
gene
Nuclear
Hormone
ReceptorLigand
Complex
Action
2
D
A 

Response 

Element                  

TATA 

Box         
RNA 

polymerase 
II
or 

pol 
II 

complex
actually 

transcribes,
startin
 at 

TATA 
box
DNA 
site 
NH 
receptor
Li
and 

Bound
L
NH 
receptor
Li
and 

Bound
L
Recruitment
of
Coactivators
is
Necessary
They
are
an
Obligate
`Bridge'

to
Pol
II
complex*
* 

Receptor­
li
and 
can 
bind 
DNA 
and 
NOT 

recruit 
pol
II = 
Anta
onist
Chemical
Numbers
vs
Chemical
`Doses'
Working
Examples
Perfume
Raw
Materials
Salvito
et
al.

Env.

Toxicol.
Chem.
21(
6):

1301­
1308
Prediction
of
Environmental
Concentrations
(PECs)
for
>

2,

100
chemicals
Prediction
of
PNEC
–
PEC/
PNEC
ratio
­

prioritization
Simonich
et
al.

Env.
Sci.

Tech.
36(
13):

2839­
2847.
Measurement
and
Validation
of
PECs
Thyroid
Toxicological
History
Adverse
Effects
and
Mechanisms
Thyroid
–
goiter/

developmental
toxicity
Mechanisms
elucidated
1940­
1990:
­

Iodide
deficiency
or
uptake
blockade
to
thyroid
­

Inhibition
of
thyroid
peroxidase
­

Blockage
of
thyroid
T3/
T4
release
­

Increased
T3/
T4
metabolism
­

Inhibition
of
5'­

deiodinase
­

Life
stage
sensitivity
vs
consequences
Clear
endpoints:
thyroid
histopathology,
circulating
T3/
T4
and
TSH
