United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
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Research and Development
EPA/600/S1-88/006 Jan. 1989
&EPA Project Summary
Characterization of the Ah
Receptor
Stephen H. Safe
The rat liver cytosolic receptor
protein containing the Ah-receptor
protein was purified and studied
using a photochemical assembly of
2,3,7,8-TCOD. The receptor protein
was purified using various
chromatographic procedures. The
unbound receptor protein rapidly lost
its capacity to bind 2,3,7,8-TCDD.
However, the 2,3,7,8-TCDD bound Ah
receptor did not readily dissociate,
probably reflecting the high potency
and persistence of the toxicity of
2,3,7,8-TCDD.
Results are based on a new one-
step methodology which allows
activation parameters to be calcu-
lated directly from raw experimental
measurements which allows the
uncertainty in the activation enthalpy,
expressed as a 95% confidence
interval, to be obtained unam-
biguously.
The enthalpies of activation for
both the formation and the
interaction of the receptor-ligand
complex are the same within the
statistical uncertainty. This led to a
kinetic model in which the receptor
was activated to an intermediate
followed by competitive degradation
of the unoccupied receptor and
formation of the receptor-ligand
complex, both of these latter steps
being fast compared with the first.
The conclusion is that ligand binding
and receptor degradation both
involve the protein in a conform-
ational reorganization.
This Project Summary was devel-
oped by EPA's Health Effects
Research Laboratory, Research
Triangle Park, NC, to announce key
findings of the research project that
is fully documented In a separate
report of the same title (see Project
Report ordering information at back).
Introduction
Complex halogenated compounds
such as dibenzo-p-dioxins (PCDDs),
dibenzofurans (PCDFs), chlorinated
biphenyls (PCBs) and brominated
biphenyls (PBBs) are industrial
compounds or by-products with a
number of common biologic and toxic
effects. The activities of these toxic
halogenated aryl hydrocarbons (HAH) are
structure dependent. It has been
proposed that the effects of the toxic
HAH are dependent on the initial
interaction of these compounds with a
cytosolic receptor protein (the Ah
receptor) in the target tissues.
Procedure
Evidence for receptor-mediated
mechanism:
1. Saturable Binding Criteria.
The synthesis of radiolabeled 2,3,7,8-
TCDD with a high specific activity
triggered several important mechanistic
studies; it was apparent that in the
soluble fraction of hepatic and extra-
hepatic tissues from several species
there was a protein which exhibited
saturable binding with the radioligand.
Moreover, several reports have shown
that 3-methylchloanthrene, benzo[a]
pyrene and dibenz[a,h]anthracene also
exhibit saturable binding with this
cytosolic receptor protein.
2. Tissue or Cellular Specificity.
Endogenous receptor ligands such as
steroid hormones and neurotrans-
mitters interact with receptors which are
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located within specific tissues or cells.
Tissue specificity has also been
demonstrated with 2,3,7,8-TCDD
receptor in rats and mice; C57B1/6J
mice and Sprague-Dawley rats which
are highly responsive to 2,3,7,8-TCDD
exhibit tissue-dependent concentrations
of the receptor which vary from 0-54
fmol/mg cytosolic protein. In contrast,
non-detectable levels of the receptor
protein are observed in cytosol from
DBA/2J which are relatively nonre-
sponsive to the effects of 2,3,7,8-TCDD
and related toxic HAHs.
3. High Affinity Ligand-Receptor
Binding.
2,3,7,8-TCDD, 3-MC and several
other toxic HAHs bind with high affinity to
the cytosolic receptor protein with Kd
values in the range of 0.1 to 10 nM which
approximate Kd values for steroids
binding to their cytosolic receptor
proteins.
4. Correlation Between Structure-
Dependent Binding and Their Biologic
and Toxic Responses.
Several studies with polychlorinated
dibenzodioxins and polychlorinated
dibenzofurans congeners clearly
demonstrate the effects of structure on
their binding affinities, AHH induction
potencies and toxicities. The most active
compounds contain 4 lateral (2,3,7 and 8)
Cl substituents and the removal of these
groups or the addition of 2 or more
non-lateral Cl substituents gives
cogeners with overall diminished
activities.
Results and Discussion
1. The interaction of several photolabile
chemicals with the receptor protein will
be demonstrated using a photochemical
assembly which has been set up for this
study. The first phase utilizes
hydrocarbons which exhibit highbinding
affinities for the Ah receptor and which
are available as radiolabled [3-H]
compounds with a high specific activity.
These compounds include 2,3,7,8-
TCDD, 3-methylcholanthrene and
benzo[a]pyrene. A second series of
photolabile compounds, including azido
derivatives of radiolabeled 2,3,7,8-
TCDD and benzo[a]pyrene used in these
photoaffinity studies. The competitive
binding of several ligands to the
covalently modified receptors were
investigated to probe the possible
differences in ligand binding site(s) on
the receptor protein(s) and the 3-H-
photoaffinity labeled protein adducts
isolated and used as markers for the
purification studies.
2. The receptor protein was purified
using a series of chromatographic
procedures including ion exchange
column chromatography, hydroxyapatite
column chromatography affinity chrom-
atography and gel permeation high
pressure liquid chromatography. The key
step in this approach is the preparation
of several affinity column supports which
have been functionalized with synthetic
substituted chlorinated dibenzo-p-
dioxins. These functionalized column
supports are utilized to preferentially
adsorb the receptor protein from the
cytosol and therefore facilitate
purification. Since it had been reported
that ligand binding activity of the Ah
receptor is labile, the purification scheme
used the covalently modified radio-
labeled ligand-receptor complex as a
marker protein.
3. The third objective focused on the
preparation of monochlonal antibodies to
the purified Ah receptor. This approach
was to facilitate the detection and
quantitation of the receptor in animal and
human tissue which would serve as a
probe for the determination of individual
susceptibilities to the toxic HAHs.
Progress
1. Ligand Binding Studies
The initial on understanding receptor-
ligand interactions resulted in the
development of two possible kinetic
models. The receptor-ligand inter-
actions resulted in the development of
two possible kinetic models.
R + L
kf
•RL
(1)
kr
scheme 1.
-> Inactivationn (2)
As shown in scheme 1, the unbound
receptor rapidly loses its capacity to bind
TCDD. Therefore, the concentrations of
R, L and RL at saturation will not
represent equilibrium concentrations and
can lead to inaccurate estimation of Kass
and the initial concentration (Ro) of
receptor binding sites. The values for Kf
and Kd were determined and the value
for Kr and Ro were estimated b
matching the experimental results wit
the computer simulated curve. Kr was to<
small to measure experimentally. Sine
Kr was a maximum estimate for thi
dissociation constant, the Kass values an
minimum estimates. The Kass values (:
x 10.10 to 3 x 10.11 M-1) were at leas
two orders of magnitude greater thai
those corresponding to the publishei
values of Kd. Consistent with this highe
estimate for Kass is the argument that i
Kf and Kd had the values determine!
experimentally in this study and if Kr hai
a magnitude consistent with the literatun
values of Kd, the complex should readil
dissociate with time. This effect was nc
observed experimentally.
The enthalpies of activation for bot
the formation and inactivation of thi
receptor-ligand complex are the sarm
both graphically and computationaly
within the statistical uncertainty. This lei
to the consideration of a second kinetii
(Scheme 2) in which the receptor i
activated to an intermediate, I, followei
by competitive degradation of thi
unoccupied receptor and formation of thi
RL complex with both of these latte
steps being fast compared with the first.
scheme 2
-> I
degradation
RL
[L]
Scheme 2 provides a ready explanation
of why the enthalpies of activation fo
complex formation and recepto
degradation should be the same, sine
both depend on the temperatun
coefficient of Ko only. The entropies c
activation are different due to thi
multiplying term [Ro] in the case c
complex formation. The conclusion i
that ligand binding and recepto
degradation both involve the protein in
conformational manner. The recepto
degradation occurs competitively will
binding over the whole range o
temperatures from 4 to 37°C; there is a
yet no direct evidence for comple
dissociation, and as a result, the bindini
of the ligand stabilizes the receptor to a
extent much greater than is found will
steroid hormones.
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Photoaffinity Labeling Studies
Attempts to photoaffinity label (PAL)
.he Ah receptor(s) have been
complicated by the low concentration of
this protein in most tissues (i.e., hepatic
< 100 fmol/mg) which is a problem not
encountered with many other receptors.
The photolysis of 2,3,7,8-TCDD in H20
was carried out and the results indicate
that 2,3,7,8-TCDD is rapidly photolyzed
to unknown product(s) when irradiated
with ultraviolet (UV-A, 250-400 nm)
light. The observed loss of 70% of the
starting material within the first 15
minutes of photolysis indicates that this
ligand is significantly photolabile within
the time frame of stability of.the liganded
Ah receptor in the cytosolic preparations.
Precipitation of the photolyzed cytosolic
protein with acetone (which solvates and
removes excess unbound ligand) pro-
vides direct evidence for photocovalent
attachment of the radioligand. In the
absence of photolysis (time 0), acetone
treatment removes all radioactivity from
the precipitated protein pellet whereas,
with increasing photolysis time, an
increase in unextractable radioactivity
was observed in the protein pellet.
With evidence that the radioligand
[3H]-2,3,7,8-TCDD was covalently
adducted to cytosolic protein, the
proteins were separated with sodium
dodecyclsulphate polyacrylamide gel
electrophoresis (SDS-PAGE). The gel
was impregnated with a fluorographic
enhancer, dried and loaded on ultra-
sensitive x-ray film and stored at
-70°C for exposure. A detectable
pattern was observed only after film
exposure of the gel for a minimum of 24
weeks; however, very little significant
information could be obtained. This
problem was circumvented by utilizing a
method involving slicing the acrylamide
gel lanes into 2-3mm slices and
determining the radioactivity content of
each slice. This was accomplished using
an oxidizer. The radioactivity contained in
the gel slices was determined by liquid
scintillation counting of the recovered
tritiated water (>95% recovery).
The typical gel profile had specific
labeling of a 95, 90 and 71 kDa protein
subunit, which was in good agreement
with some published results.
Purification of the Ah Receptor
Attempts to purify this receptor in its
unbound form have been carried out with
a variety of biochemical techniques
including column chromatography and
sucrose density centrifugation. It
appeared that biochemical manipulation
of the unliganded receptor resulted in a
rapid loss of specific ligand binding.
However, significant stabilization of the
receptor occurs when ligand, i.e.,
2,3,7,8-TCDD, was bound. When 3H-
TCDD liganded receptor from Long-
Evans rat hepatic cytosol was separated
on an equilibrated Sephacryl-300
column, there were two specifically
bound radioactive peaks and this pro-
cedure resulted in up to a 10-fold
purification of the receptor protein. A
comparable 10-fold purification was
obtained by centrifugation on a 5-25%
sucrose density gradient. Current studies
have been initiated to utilize a series of
column chromatographic and velocity
sedimentation procedures to further the
Ah receptor.
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Stephen H. Safe is with Texas A&M University, College Station, TX 77843.
K. Diane Courtney is the EPA Project Officer (see below).
The complete report entitled, "Characterization of the Ah Receptor," (Order No.
PB 89-118 657/AS; Cost: $13.95, cost subject to change) will be available only
from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S1-88/006
000032? PS
U S EHVIR PROTECTION AGENCY
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CHICAGO IL 60604
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