c/EPA
United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
P.O. Box 93478
Las Vegas NV 89193-3478
EPA/600/4-89/362
November 1989
Research and Development
Cell Receptor-Xenobiotic
Complexes as Exposure
Biomarkers
Literature Summary and
Recommendations
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CELL RECEPTOR-XENOBIOTIC COMPLEXES AS EXPOSURE BIOMARKERS
LITERATURE SUMMARY and RECOMMENDATIONS
by
J. A. Santolucito
Environmental Research Center
University of Nevada-Las Vegas
Las Vegas, Nevada 89154-4009
Cooperative Agreement No. 814342-01
Work Assignment Manager
Charles H. Nauman
Exposure Assessment Research Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193-3478
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193-3478
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NOTICE
This document is intended for internal Agency use only.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
u
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ABSTRACT
This report presents a brief review of the literature relevant to the interaction between
cell receptor macromolecules and chemicals in their immediate environment. An overview of
the physiological role of cell receptors in relation to endogenous chemical regulators such as
hormones is presented together with the essentials of receptor kinetics.
While a specific receptor type usually binds only one kind of endogenous chemical, any
number of man-made molecules having the required structural characteristics (therapeutic
drugs for example) may also be bound. Literature is cited demonstrating that certain toxic
environmental chemicals, in addition to drugs, can also bind to receptor active sites. Further,
their toxic action is dependent upon the binding.
The evidence at hand suggests that receptor-bound xenobiotic, rather than total tissue
concentration, may provide a more accurate measure of exposure to environmental chemicals.
However, since most tissues are not readily accessible for routine monitoring, a surrogate
biomarker of exposure may be developed using receptor-bound xenobiotics present in the
formed elements of the blood.
Recommendations are made for research needed to demonstrate the feasibility of blood
cell receptor biomarkers of exposure.
111
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CONTENTS
Page
Notice ii
Abstract iii
List of Figures and Tables v
Glossary vi
Introduction 1
Cell Receptors-Background 4
Xenobiotic Modulation of Receptor Numbers/Affinity 10
Xenobiotic-Immune System Interaction 23
Research Needs for Development of Receptor Biomarkers 24
Specific Recommendations 31
Literature Cited 35
IV
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LIST OF FIGURES
Number Page
1 6
2 9
LIST OF TABLES
I 11
H 25
III 26
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Santolucito, John A.
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Q245 10 Cell receptor-xenobiotic complexes as exposure biomarkers : *b
literature summary and recommendations / *c by J.A. Santolucito; United States
Environmental Protection Agency. Environmental Monitoring Systems Laboratory.
Q260 Las Vegas, NV : =t=b Environmental Monitoring Systems Laboratory. Office
of Research and Development. U.S. Environmental Protection Agency, *c 1989.
Q300 47 p. : *b ill. ; *c 28 cm.
0500 "EPA/600/4-89/362"
0 Biochemical markers *x Bibliography.
0 Cell receptors *x Bibliography.
0 Xenobiotics *x Bibliography.
Environmental Monitoring Systems Laboratory (Las Vegas, Nev.)
United States. *b Environmental Protection Agency. *b Office of
Research and Development.
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GLOSSARY
BIOMARKER
A change in composition or function of a biological system or sample which signals the
occurrence of an event of concern.
CYTOSOL
The non-membranous cytoplasm bounded by the cell membrane.
DEXTRAN
A polymer of glucose having both 1-4 and 1-6 linkages, with branches about every five
units, giving it considerable resistance to enzymic hydrolysis. Molecular weight ranges from
30,000-25,000.
DIOXIN
A family of planar poh/chlorinated organic compounds consisting of two benzene rings
connected by two oxygen bridges. The congeners differ in their structure and toxicity according
to the number and positioning of chlorine atoms on the molecule.
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ENDOCYTOSIS
A mechanism whereby cells can internalize molecules aggregated on its outer surface.
An infolding of the membrane results in vesicle formation with envelopment of the aggregated
material. The vesicle then separates from the boundary membrane, opens, and releases its
contents to the cytoplasm.
GLUCOCORTICOIDS
Steroid hormones, produced by the adrenal cortex, which regulate metabolic
interconversions between carbohydrates, fats and proteins and also exert anti-inflammatory
effects.
HALOGENATED
The chemical designation of a molecule containing one or more halogen (e.g., chlorine
bromine, etc.) atoms.
IMMUNOGLOBULIN
Any of five structurally and antigenically distinct antibodies produced by lymphoid
tissue and present in the serum. Specifically, IgA, IgD, IgE, IgG, and IgM
INVAGINATE
To fold inward, creating a pocket or vesicle.
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LIGAND
An organic molecule attached to a specific site on a macromolecule or membrane
surface.
MICROSOMAL MONOOXEGENASE
Any of a group of enzymes, located in microsomes, that catalyze the oxidation of
organic compounds.
MONOCLONAL ANTIBODES
Antibodies derived from one B-lymphocyte cell line.
MUSCARINIC RECEPTOR
Receptors for acetylcholine located on smooth and cardiac muscle cells in contrast to
those located on skeletal muscle cells, termed nicotinic receptors.
NEUROTRANSMITTER
Amine or peptide chemicals released by neurons which result in transmission of nerve
impulses across synaptic junctions.
OUABAIN
A gtycoside prepared from Strophanthus gratis. Acts similar to digitalis on the heart,
resulting in increased strength and regularity of beat.
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PHARMACOKINETICS
The mathematical description of the fate of drugs in the body including absorption,
distribution, metabolism, and excretion.
POLYCLONAL ANTIBODIES
A mixture of antibodies produced by more than one type of B-lymphocyte.
STEREOSPECIFIC
Three-dimensional organic structures whose molecules have an ordered (crystalline)
orientation rather than the random (amorphous) form.
XENOBIOTIC
Pertaining to organic substances foreign to the body.
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CELL RECEPTOR BIOMARKERS
INTRODUCTION
Under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), and other
environmental protection laws, the U.S. Environmental Protection Agency (EPA) is responsible
for pesticide regulation and to ensure that unreasonable risks to human health or the
environment do not result from exposure to pesticides. Quantitative health risk assessment
requires that both toxicity and exposure assessments be made (1). Thus, accurate exposure
assessments become critical to the evaluation of environmental and human health risks of
pesticides and to the subsequent development of control regulations that may be needed for
their protection.
Assessments may be made of external exposure, i.e., the sum of the xenobiotic material
presented to the absorptive surfaces of an organism, or of internal dose, i.e., the amount of
xenobiotic actually absorbed (2). Biological markers of internal dose may obviate the need for
estimating the amount of xenobiotic absorbed, which must be done when exposure assessments
are derived from environmental monitoring data. Indexes of internal dose range from
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measurements of the tissue/body fluid concentration of a xenobiotic and/or its metabolites, to
the measurement of a biochemical or physiological reaction at the cell, organ, or organism level.
At the molecular level, a chemical pollutant or its reactive metabolite binds to a
receptor protein which may in turn produce a disturbance in intermediary metabolism at the
cellular level. When the disturbance is of sufficient degree, a perceptible and measurable
biochemical, physiological, or behavioral dysfunction occurs. The degree of dysfunction, as
determined by controlled laboratory studies, provides a measure of the toxicity of the chemical.
The close association of xenobiotic-receptor concentration with the metabolic processes leading
to toxicity suggests that exposure assessments based on the amount of xenobiotic bound to
receptor protein may support more accurate risk assessments than those based on the total
amount of xenobiotic present in the organism or one of its tissue compartments.
In order to prevent unacceptable health risks, the measurement methods used must be
sensitive enough to detect concentrations of receptor-bound xenobiotic below the threshold of
physiological dysfunction. Immunoassays have the required sensitivity (3), and the antigenicity
of receptor protein to which the xenobiotic is bound will facilitate production of the antibodies
needed for the assay procedure. Biomarkers that measure exposure (i.e., receptor-bound
pesticide) to the most sensitive (target) tissues will have the greatest relevance to risk
assessment. However, sampling of that tissue often requires the use of invasive procedures and
are thus nnsuited for routine exposure monitoring of human populations. Accordingly, this
report will evaluate, through a review of the literature, the occurrence of and potential for using
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measures of xenobiotic compounds bound to receptor macromolecules in the formed elements
of the blood as surrogate biomarkers of human exposure. The ease of access of blood samples
compared to other tissues makes them a realistic choice. Receptors associated with cells or cell
fragments found in other body fluids will also be addressed briefly when appropriate.
With certain exceptions (e.g., steroid hormones), receptors for hormones, neurotrans-
mitters, and other chemical signals are believed to be located on the cell membrane surface (4).
Cell membrane receptors are an integral part of the plasma membrane structure and serve to
discriminate, with high specificity and efficacy, the ligand(s) present in the immediate
environment surrounding the cell (5). The high affinity of receptor macromolecules assures
binding of ligand at low concentrations.
The early conceptual framework for the study of receptor-ligand interaction evolved
from pharmacologic investigations of drug action during the second half of the 19th century.
Since most drugs bind to receptors designed for interaction with endogenous hormones and
neurotransmitters, these studies also demonstrated receptor mediation of physiologic hormonal
regulation. Xenobiotic-receptor interaction was also suggested in this period with the finding
that lead has a greater affinity for certain tissues, notably those of the central nervous system
(CNS), than for others (6). Experimental evidence for the binding of xenobiotics to cellular
receptors has developed relatively rapidly in the past decade. For example, the Ah receptor,
which is responsible for the induction of microsomal monooxygenases by polycyclic aromatic
hydrocarbons, has also been found to mediate the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin
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(TCDD) (7). Also, three major classes of chlorinated hydrocarbon insecticides have been
found to be potent, competitive, and stereospecific inhibitors of the 7-aminoburyric acid
(GABA) receptor in brain tissue (8).
The foregoing emphasizes the three cornerstones of the rationale for developing
membrane receptor biomarkers. First, the amount of xenobiotic bound to cell-receptor
macromolecules at a specific time may afford a physiologically meaningful biomarker of
exposure. This view is supported by the observation that the polybrominated biphenyl (PBB)
concentration of the white blood cell fraction correlates better with severity of lymphocytic
dysfunction than does the PBB concentration in the plasma fraction (9). Second, at very low
concentrations xenobiotic-receptor complexes are measurable using immunoassay techniques.
And third, xenobiotic-receptor binding also mediates a pollutant-induced adverse effect when
the internal dose becomes sufficiently high. Therefore, information gained from investigations
of xenobiotic binding to receptor macromolecules may advance the development of exposure-
as well as effects-biomarkers.
CELL RECEPTORS - BACKGROUND
Receptors have been identified and have been shown to mediate physiologic as well as
pathologic reactions for hormones, neurotransmitters, viruses, bacteria, immunoglobulins, and
chemoattractants (10,11,12,13). Receptor-ligand interactions are reversible reactions involving
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all the components of the membrane, viz., protein, carbohydrate, and lipid. Binding sites may
be competitively inhibited by a molecule that resembles the ligand. They may also be inhibited
non-competitively by molecules that react with sites other than the ligand binding site, causing a
structural change that interferes with the signal transduction function of the receptor. The
membrane-receptor macromolecule, composed of an assembly of proteins or glycoproteins, is
embedded in the lipid bilayer. Receptor active sites are small portions of the molecule
generally making up less than 10% of the total amino acid residues. They are often clefts in the
surface of the macromolecule, composed mostly of hydrophobic residues but also containing
polar groups that participate in binding (14). In glycoprotein receptors, the carbohydrate units
are usually clustered on the periphery of the molecule so that this hydrophilic area is exposed to
the external side of the cell membrane.
Some of the glycoproteins may extend their contact to the cytoplasmic side of the
membrane, providing a means of internalizing an external chemical stimulus such as a hormone.
The carbohydrate units of glycoproteins are covalentry bonded to the polypeptide backbone.
Carbohydrate prosthetic groups can, of course, be part of the ligand molecule as well as of the
receptor macromolecule. In general, carbohydrate moieties of glycoproteins contribute to the
stabilization of protein conformation and the regulation of metabolic half-life in the circulation
of the intact animal, uptake by cells, message transmission, etc. (5). Some of the receptor
macromolecule features described are represented in Fig. 1.
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Outer
Protein
Layer
Receptor Macromolecule
Lipid Bilayer
Inner
Protein
Layer
Channel
Receptor
Active Sites
o>
c
(0
-Q
E
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In most cells the number of receptors is under some regulation at the level of gene
expression. Also, many receptor macromolecules contain one or more binding sites for specific
regulatory molecules in addition to the ligand-specific binding site. Interaction between the
subunits are of two types: either an alteration in the activity of the regulatory-molecule
binding-site when ligand binds to the active site, or an alteration in the activity of the
ligand-binding-site when the regulator site is occupied. The number of cell surface binding sites
is also regulated by cycles of endocytosis, reutilization, and degradation (14).
From this brief overview, it can be seen that the magnitude of an effect resulting from a
given concentration of hormone, drug, or xenobiotic may vary according to the available
number of cell membrane receptors in existence at the time. Data from a number of systems
suggest that all the necessary information for activation of the effector is contained in the
receptor, i.e., some receptors phosphorylate, others aggregate, while still others exist in
multiple-binding states. Receptor macromolecules can consist of subunits and can undergo
reversible interactions in the lateral plane of the plasma membrane. Experimental techniques
allow measuring the physical characteristics of the receptor while still a component of the
membrane or whole cell as well as measuring its characteristics in purified form following
membrane solubilization with suitable detergents (12).
A receptor is defined or identified, in the first place, in terms of its pharmacokinetics.
Substances that effectively mimic or block the effects of the principal ligand should compete for
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binding sites at low concentration. Others should compete only at very high concentrations, if
at all.
To distinguish receptor-binding (specific) from "other" binding (non-specific) there must
be a finite number of binding sites of rather high affinity, i.e., having dissociation constants in
the nanomolar range or lower. This can be demonstrated by adding more and more
radiolabeled compound and noting that binding increases until saturation occurs, i.e., all sites
are occupied. In a binding assay, the membrane component (receptor) is selectively exposed to
a radioactively labeled ligand to determine the rate at which the ligand attaches-kj
(association), and is removed--!^ (dissociation), i.e.,
kj = the rate constant for [L] + [R] => [L.R], and
kj = the rate constant for [L.R] => (L] + [R].
The apparent equilibrium dissociation constant of the receptor, Kd, can then be calculated (=
ICj/lCj) and is usually on the order of 10~9 - 10~12 M. The Kd represents the concentration of
ligand that results in one-half the total number of receptor sites being occupied at equilibrium.
An increase in Kd correlates with a decrease in affinity. In addition, the maximum number of
binding sites can be estimated by measuring the quantity of ligand necessary to saturate all the
available binding sites (B^).
This approach is complicated by the fact that radioligands will bind to a variety of
membrane constituents, only one of which represents the receptor of interest. However, this
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non-specific binding can be corrected by measuring the total radioligand bound following its
addition alone, then determining its binding in the presence of excess (usually 100 times the K.
concentration) unlabeled ligand (blank). The difference (total - blank) represents
receptor-specific binding (14). Binding properties of receptors are quantitated by Scatchard
plots in which the ratio of bound-ligand to free-ligand is plotted on the ordinate against bound
ligand on the abscissa (15). When a single ligand is interacting with a single population of
receptors possessing a single affinity for the ligand,
-l/Kd = the slope of the straight line obtained,
Bfflax/Kd = the y-intercept, and
Bmax = the x-intercept (Fig. 2).
•o
a
Slope = -1/K.
[Bound]
B
Fig. 2. Illustration of a Scatchard plot showing derivation of Kd and B
max
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However, as pointed out by Rosenthal (16), Bmax must be interpreted as representing
the total number of binding sites rather than the number of receptor macromolecules, since
each macromolecule may have more than one binding site. Until the receptor is purified and
its structure known, Bmax is determined in units of fmol/mg membrane protein.
A number of mammalian cell membrane receptor systems which bind endogenous
regulatory chemicals have been characterized functionally as well as structurally to varying
degrees (Table I). Purification of some receptors, e.g., insulin receptors, may be hampered by a
low occurrence or physical similarity to surrounding membrane protein, or both. On the other
hand, the muscarinic cholinergic receptor that binds acetylcholine is relatively more abundant
and easily separated and has been well characterized.
XENOBIOTIC MODULATION OF RECEPTOR NUMBERS/AFFINITY
Membrane Receptors
Ah Receptor The induction receptor for the potycyclic aromatic hydrocarbon-inducible
microsomal monooxygenases, cytochrome(s) Pa-450, (sometimes designated the TCDD" or
"dioxin" receptor) was identified by Poland et al.(\T). Later studies revealed that this protein
regulates the expression of a gene battery which includes the structural genes for cytochrome(s)
Pj-450 and in certain tissues, in concert with at least one other regulatory locus, alters patterns
of cell proliferation and differentiation. For a review of these developments, see Poland and
Knutson (18).
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TABLE I. Verified hormone/transmitter receptor systems
Designation
^-adrenergic
dp a2-adrenergic
Acetyl Choline
Insulin
Glucocorticoid
Estrogen
Thyroxine
Growth Hormone
Vasopressin/
Oxytocin
Substance P
Endorphins
Histamine
GABA
Dopamine
Ligand(s)1
E,NE
E,NE
Ach
Insulin
Cortisol
Estradiol
T3
Somatotrophin
Vasopressin/
Oxytocin
Substance P
^-Endorphins
Histamine
TT-Aminobutyric acid
Dopamine
CeU
Location2
PM
PM
PM
PM
C;N
C;N
PM,C,M,N
PM
PM
PM
PM
PM
PM
PM
Occurrence3
wbc;s;sk;c;hp;ns
hp;s;sk;c;wbc;ns
wbc;ns;s;sk;c
wbc;wd
wbc;wd
ut, wd
wbc;wd
wbc;wd
wbc;ns;sm
wbc
wbc
wbc
ns
ns
Ref.
(11,13)
(11,13)
(10,13)
(11)
(13)
(20)
(10,13)
(12,13)
(13,21)
(13)
(13)
(13)
(12)
(13)
1 E = epinephrine; NE^norepinephrine; Ach=Acetylcholine; T3=triidothyronine.
2 PM=plasma membrane; N»nucleus; C=cytosol
3 s=smooth muade; sk=skeletal muscle; c=cardiac muscle; hp=liver; ut = uterus; pi=platelet;
rbc=erythrocyte; wbc=leukocyte; ns=nervous system; pnc=pancreas; wd=widely distributed.
11
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The administration of 3-methylcholanthrene (MC) and structurally related polycyclic
aromatic hydrocarbons (PAH) results in the induction of cytochrome P1-450 and associated
monooxygenase activities, including aryl hydrocarbon hydroxylase (AHH) in some but not all
strains of mice (19). PAH responsiveness in some strains of mice is inherited as a simple
autosomal dominant trait. The gene locus controlling this trait was designated the Ah locus.
Available data indicate that the receptor protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), MC, and other inducers is the product of the Ah locus (17). Studies to determine the
subcellular distribution of the Ah receptor in mouse hepatic tissue suggest a cytosolic as well as
a nuclear compartment. Comparisons have been made between species of the relative
concentrations of Ah receptor in hepatic and extrahepatic tissues. Interspecies differences exist
with respect to the tissue distribution of the Ah receptor as well as the relative concentrations
of this protein in those tissues possessing the protein (19).
The direct demonstration of the occurrence of a receptor protein for TCDD in human
lymphocytes (22) and human squamous cell carcinoma lines (23) has been reported. Binding
studies using [3H]TCDD demonstrated the presence of the Ah receptor in the cytosolic fraction
from cultures of human squamous cell carcinoma of epidermal origin (17). The relative
amount of receptor measured in different cell lines correlated with the maximally induced
activity of the monooxygenase 7-ethoxycoumarin O-deethylase (ECOD)(23). Studies using cell
cultures of cortical thymocytes (T-lymphocyte precursors) and medullary thymic epithelial cells
treated with TCDD have suggested impaired differentiation of intrathymic precursor cells for
12
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the observed TCDD-induced thymic atrophy seen in the intact animal (24). The potential for a
direct effect of TCDD on cultured human lymphocytes has not been as extensively explored.
An excellent correlation was demonstrated between the binding affinities of 23
halogenated dibenzo-p-dioxin and dibenzofuran congeners in vitro, and a measure of their
potencies in vivo (25). The charcoal-dextran binding assays were performed using rat hepatic
cytosol receptor protein. At a given concentration, the amount of receptor-chemical complex
formed is proportional to the affinity constant of the ligand. Potencies (ED^) to induce AAH
activity were determined in the intact rat.
Thus, the amount of receptor-chemical complex may provide a more physiologically
meaningful measure of exposure to TCDD and related compounds than would total tissue
concentration. Further, the ease of acquiring a blood sample for measuring complexed
lymphocyte Ah receptor would facilitate routine monitoring of human exposures to these
chemicals.
Gamma aminobutvric acid Receptor Neurotransmitters (neurohormones) are chemical
signals used by nerve cells to communicate with each other and with muscle or gland cells. They
are amino acids or small potypeptides released by one cell and received by another via
stereospecific receptor sites. Binding of the neurotransmitter to its receptor causes a change in
receptor conformation and initiates a change in membrane potential
13
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Neurons in the brain utilize a number of neurotransmitters in addition to the familiar
acetylcholine and norepinephrine found in peripheral nerves. One of these, ir-aminobutyric
acid (GABA), and its receptor complex have received considerable attention in recent years.
The receptor model which has emerged is that of a protein having at least three interacting
components: one that binds GABA, another that binds benzodiazepine (BZ) tranquilizers, and
another which binds the convulsants picrotoxinin (PTX) and bicyclophosphates (26). These sites
interact allosterically and affect each other's binding through conformational changes (27, 28).
Activation of most GABA receptors (GABAR) in mammals leads to an influx of Cl" causing
membrane hyperpolarization (29).
The ligand of choice for studying agonists and antagonists of the PTX binding site
within the GABAR complex is t-Butylbicyclophosphorothionate (TBPS) (30). It was selected
because of its specificity (no more than 33% non-specific binding), high affinity (Kd = Ca 17 x
10"9 M), and because t-butylbicyclophosphite is easily labeled with ^S in a single step to form
[^SjTBPS. The relatively high specific activity (about 30 Ci/mmole at the start), and the
relatively short half-life of ^S (about 90 days), permit a fresh batch of pSITBPS to be used for
about 6 months. Its binding and displacement properties remained constant over this period.
Most insecticides are neurotoxins that inhibit acerylcholinesterase at synapses or disturb
sodium channels on nerve or muscle cell membrane. Some, however, exert their primary toxic
action on the GABAR found in the vertebrate and invertebrate central nervous systems as well
as in invertebrate neuromuscuiar junctions. Included are certain cyclodiene (e.g., dieldrin) and
14
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hexachlorocyclohexane (e.g., lindane) insecticides (29), and three of the components of
toxaphene, which probably account for most of the toxicity of the commercial product which has
at least 188 components (31). These insecticides inhibit GABA-induced chloride permeability
and may account for their stimulant and convulsant effects. On the other hand, competitive
binding studies with some of the potent cr-cyano-3-phenoxybenzyl pyrethroid insecticides
suggest that they bind to a site closely associated with, but possibly distinct from, that of TBPS
and PTX (32).
The density of specific [^SJTBPS binding sites varies in different regions of the brain,
being highest in cerebral cortex (Ca. 65 pmoles/g) and lowest in the pons-medulla (Ca. 14
pmoles/g) (30). Relatively few studies report a systematic search for the presence of GABAR
in non-nerve tissues. Squires (30) found specific TBPS binding in rat liver and lung tissue to be
about 7% and 2%, respectively, of that seen in cerebral cortex, while no specific binding was
demonstrated in kidney tissue. [3H]-muscimol, a potent GABA agonist used to label GABAR
sites in vertebrate brain, was found to bind specifically to sites in a crude membrane fraction
prepared from bovine cerebral blood vessels (33). GABA receptors have been reported in
guinea pig ileum and vas deferens (34).
The BZ receptor in brain tissue occurs in a macromolecular complex that includes a
binding site for the inhibitory neurotransmitter GABA. While most BZ binding sites are
associated with GABA receptors, a few studies have demonstrated the existence of one without
the other in some rat brain preparations (35). The GABA-BZ receptor complex also includes
15
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recognition sites for barbiturates and possibly ethanol. In initial studies of 3H-diazepam
binding, peripheral tissues were examined as controls (36). Surprisingly, high densities of
diazepam binding sites were observed in a variety of peripheral tissues with similar nanomolar
affinity for diazepam as the central receptors. High densities of these receptors have been
found in rat adrenal cortex (but not medulla), salivary glands, testes, and ovaries. Pancreas and
pituitary tissue have relatively low densities of receptors. The physiological significance of
these peripheral BZ receptors is unclear.
Nevertheless, the association of BZ and GABAR sites in central nervous system tissues
and the occurrence of diazepam receptor sites in peripheral tissues give reason to investigate
the possible occurrence of receptors for pesticides (e.g., dieldrin, lindane, toxaphene, and
cypermethrin) in blood cell membranes.
Acetylcholine Receptor The acetylcholine receptor (AchR) is a well-characterized
component of synaptic and neuromuscular transduction systems. This receptor translates the
binding of the neurotransmitter acetylcholine (Ach) into a rapid increase, then decrease, in the
permeability of the membrane to the passage of cations (Na*, K*). AchR sites are sub-units of
a macromolecular receptor complex containing other agonist and/or antagonist binding sites.
The enzyme acetylcholinesterase (AchE) and the AchR either occupy neighboring sites on the
same macromolecule or sites on separate molecules that are coupled in such a way as to permit
coordination of function (37). AchE is an extrinsic protein (i.e., it is readily dissociated from
16
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membrane preparations of vertebrate muscle endplate), while AchR is an intrinsic protein
embedded within the membrane structure (38).
The presence of AchE on erythrocyte membranes is well documented, and the
inhibition of this enzyme's activity is used to assess exposure to organophosphate insecticides
(39). The AchR has been reported in rat lymphocyte membranes (13). Whether the close
association of AchR and AchE seen in neural and muscular tissue also holds for RBC and/or
WBC membranes has not been reported.
There is evidence for interaction of organophosphate insecticides with RBC membrane
protein other than AchE. Erythrocytes from humans exposed to organophosphate insecticides
were found to have altered membrane stability (40). Human subjects with serum or blood
AchE inhibition between 40-60% were considered mildly intoxicated, and those with more than
60% inhibition as severely intoxicated. Erythrocyte membranes from mildly intoxicated
subjects were more resistant, while those from severely intoxicated subjects were less resistant
to hypotonic hemotysis. Also, a significant increase in membrane phosphatidylcholine was seen
in the severely intoxicated. This suggests a binding of organophosphate to RBC membrane
macromolecules. Whether this binding involves the AchR receptor and/or can be utilized as a
biomarker of exposure is unknown.
Dnpaming Receptor The dopamine-sensitive site in the brain and pituitary to which
neuroleptics bind with high affinity has been studied intensively in the search for new
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therapeutic agents. Its functional molecular size is known, though it has not been characterized
structurally. There appear to be multiple receptors for dopamine, some linked to the adenylate
cyclase and others not (13).
The ligand spiroperidol binds specifically to dopamine receptors and is commonly used
to study dopamine receptor agonists and antagonists. The specific binding of this ligand in
membrane preparations from brain cortical tissue was found to be inhibited in the presence of
lead and mercury (41). Tri-n-butyl lead was a more potent inhibitor than lead acetate, and
mercurous chloride was more potent than methylmercuric chloride. Although the specificity of
the heavy metal binding to receptor protein was not reported, clearly the affinity of the receptor
site for dopamine was altered. The blocking action was not indiscriminate, since the heavy
metals were relatively ineffective in blocking giycine, GABA, or diazepam receptors. However,
they did inhibit the binding of 3H-quinuclidinyl benzilate (^-QNB), a specific ligand for the
muscarinic AchR.
Adrenergic Receptor Adrenergic receptors (adrenoceptors) have high specificity for
the catecholamine neurotransmitters epinephrine and norepinephrine. While they are
abundant in nerve and smooth muscle tissues, they can be found in most human tissues
including erythrocytes and lymphocytes (42). ^-receptor concentration increases from late fetal
life to adulthood; in most tissues and decreases with age thereafter. As rat reticulocytes mature
to erythrocytes, the 0-adrenergic receptors decrease by ca. 66%.
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Four main receptor sub-types are recognized. The ^- and 02-adrenergic receptors
stimulate the membrane-bound enzyme, adenylyl cyclase, thus raising intracellular cyclic AMP
levels. The or2-adrenergic receptors inhibit this enzyme, whereas the or.-adrenergic receptor
leads to the hydrolysis of polyphosphoinositides which, in turn, generates two secondary
messengers, inositoltriphosphate and diacylglycerol (43).
Methyl mercury exposure to mice in utero produced a dose-dependent increase in the
adrenergic receptor binding in liver and kidney tissue which persisted throughout the first 5
weeks of postnatal life. The animals' physiological reactivity to adrenergic stimulation was
altered in a manner consistent with the increased receptor binding capacity (44). The authors
suggest that methyl-mercury exposure in utero alters adrenergic responses through targeted
effects on post-synaptic receptor populations in specific tissues. Modulation of ^-adrenergic
receptor activity by cardiac glycosides has also been reported. A single neonatal treatment with
either ouabain or digoxin resulted in increased responsiveness to adrenergic excitation of
two-month-old adult rats (45).
Additional work would be required to determine first, whether the receptor population
increase would persist with continued exposure, and second, whether the phenomenon has
potential as a biomarker of in utero and/or postnatal exposure. However, the results reported
(44, 45) verify the link between receptor population and potential for response and lends
further support for evaluating receptor-bound xenobiotic concentration as the physiologically
meaningful measure of exposure.
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Cytosolic/Nuciear Receptors
Receptors for some steroid hormones have been identified in target tissue cytosolic-
and/or nuclear-fractions as well as in cell membrane-fractions. Receptors which are
membrane-bound interact primarily with neighboring macromolecules, while the soluble and
more mobile cytosolic receptors can interact with a greater variety of systems within the
cytoplasm, including nuclear DNA. A review of the experimental data suggests the
membrane-mediated effect of the hormone is probably different from the cytosolic/nuclear
receptor-mediated effect (46). For example, the cytosolic/nuclear receptor for a steroid
hormone may mediate a protein anabolic response, while the membrane-bound version of the
same receptor may mediate an increase in intracellular cyclic AMP.
Receptors for most non-steroidal hormones/neurotransmitters are found only in the
plasma membrane. One exception is the receptor for thyroxine, (T3), which occurs as both
membrane-bound and cytosolic (10), a finding which is consistent with the many intracellular
effects induced by T3.
Estrogen Receptor The receptor macromolecule for estrogen is found in a number of
tissues including pituitary glands, gonads, smooth muscle of the reproductive tract, brain, etc.,
reflecting the rather widespread actions of estrogenic hormones.
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An anti-estrogenic effect on rat pituitary cell cultures was reported for the pesticide
chlordecone (Kepone) (47). Pretreatment of pituitary gonadotrophs with estradiol-170 (E,)
alone enhanced their response to gonadotrophin-releasing hormone (GnRH) in tissue culture.
This response could be inhibited by pretreatment with E2 plus Kepone. The approximate
two-fold increase in secretion of follicle stimulating hormone (FSH) and luteinizing hormone
(LH) caused by E2 pretreatment was depressed by Kepone in a dose-dependent manner. A
related pesticide, Mirex, did not suppress the GnRH response at any concentration. It was
noted that Kepone had little or no effect on basal FSH and LH secretion.
An estrogenic action has been reported for several pesticides which appear to bind to
estrogen receptor macromolecules. Among these are o,p'-DDT and/or o,p'-DDE, Kepone, and
the mono- and bis-phenol metabolites of Methoxychlor (48). The apparent disagreement
concerning the effect of Kepone (47 vs. 48) may result from its ability to competitively inhibit
E2 in the in vitro test system, while expressing an inherent weak estrogenic activity in an in vivo
test system lacking other sources of estrogen.
A rather extensive literature confirms the estrogenic activity exhibited by a number of
pesticidal compounds or their metabolites, as evidenced by their ability to stimulate
reproductive tract tissues in vitro and in vivo. On the other hand, demonstration of estrogenic
effects on the CNS and/or behavior have been less consistent. For example, administration of
these compounds to neonatal animals fails to influence sex differentiation. However, a recent
report clearly shows a CNS/behavtoral effect of methoxy-chlor when administered to
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ovariectomized female rats and hamsters (49). These animals exhibited increased acyclic
running wheel activity as well as persistent vaginal estrus, and displayed reproductive behaviors
following progesterone administration.
While recent studies have demonstrated that TCDD significantly reduces uterine and
liver estrogen receptor levels in rats (50), earlier studies had shown that several other inducers
of the mixed function oxidase system decreased estrogen binding in rat liver preparations (51).
Whether these compounds compete for the estrogen receptor or in some way alter its affinity
was not determined.
Glucocorticoid Receptor Glucocorticoid receptors (GRc) are found in most tissues, an
observation consistent with the regulatory action of the glucocorticoid hormones on fat,
carbohydrate, and protein metabolism. GRc are also present in lymphocytes (Table I).
The maximal binding capacity (BMX) of skeletal muscle cytosolic GRc was reduced in
rats treated with TCDD (52). A similar reduction of hepatic cytosolic GRc binding capacity
was effected in pregnant mice treated with either 2,3,4,7,8-pentachlorodibenzofuran or
1,2,3,4,7,8-hexachlorodibenzofuran (53). Interestingly, these polychlorinated dibenzofurans
also decreased the binding capacity of hepatic plasma membrane epidermal growth factor
(EGF) receptors. Since both the GRc and EGF receptor systems have roles in controlling
normal growth and differentiation, they are receiving considerable attention with respect to
their possible involvement in xenobiotic-induced teratogenesis.
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XENOBIOTIC-IMMUNE SYSTEM INTERACTION
Depressed antibody (B-lymphocyte) and/or cell-mediated (T-lymphocyte) immunity,
indicative of impaired immunocompetence, occur after exposure to small but not essentially
toxic amounts of certain environmental pollutant chemicals (9). Included are representatives
from various chemical types. For example:
Halogenated aromatic compounds-2.3.7.8-tetrachlorodibenzo-p-dioxin. polybromo-biphenyls,
polychloro-biphenyls, and hexachlorobenzene,
Heavy metals-cadmium, cobalt, chromium, nickel, and lead,
Organometallics-methylmercuric chloride, and
Pesticides-Carbaryl Carbofuran, DDT, Dieldrin, and Methyl Parathion.
The examples of immune system suppression presented in Table n were excerpted from
a recent review of the subject (9) and are representative of the kinds of responses which have
been observed. Additionally, the route and timing of the xenobiotic exposure appear to
influence the immune response. For example, a single dose of Cd augmented the antibody
response to an antigen if injected intraperitonealy and diminished the response when
administered orally. Also, injection of Cd 14 days before the administration of antigen
increased antibody synthesis, while injection 7 days before decreased it.
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Despite the considerable attention given to immunosuppressive effects of
environmental chemicals in recent years, relatively little is known concerning their mechanisms
of action. While a discussion of the relevant research is beyond the scope of the present report,
it is noteworthy that immunotoxicity by TCDD and PCBs is associated with stereospecific
binding to the Ah receptor present in lymphoid tissue and cells (54).
RESEARCH NEEDS FOR DEVELOPMENT OF RECEPTOR BIOMARKERS.
A number of receptor systems that have been identified in various tissue types also are
found in the formed elements of the blood. Much of the investigative effort concerning
hormone receptors in blood cells originally focused on leukocytes and concerned interactions
between the neuroendocrine and immune systems. Examples of these interactions include
stress-induced immunosuppression mediated by epinephrine and glucocorticoids, and
modulation of the neuroendocrine system by neuroactive peptides that are released by
leukocytes responding to foreign stimuli (13).
In recent years, studies have extended to other formed elements in the blood. A
serious, though not exhaustive, search of the literature revealed a respectable number of
receptor systems that have been identified in blood cells (Table HI). Clearly, the information
base exists to warrant a systematic search for blood cell receptor systems that bind pesticidal
and other toxic chemicals that have high Agency priority. Complexes thus identified can then
be further evaluated for their potential use as biomarkers of exposure.
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TABLE n. Xenobiotic Alteration Of Immunocompetence
Chemical1 Response
Pb Incr. susceptibility of mice to Salmonella typhimurium.
Pb Incr. susceptibility of mice to Encephalomyocarditis virus.
Pb Incr. susceptibility of mice to Langat virus.
Cd Incr. susceptibility of rats to inoculation with bacterial endotoxin.
Cd Enhanced mortality of mice inoculated with Encephalomyocarditis virus.
Cd Enhanced mortality of mice naturally infected with Hexamitis muris.
Hg., Enhanced mortality of mice inoculated with Encephalomyocarditis virus. .
Pb.Cd.Hgj . Lowered antibody response in mice to infectious agents.
Pb, Cd Reduced I G antibody synthesis in rodents.
Hj^ Suppressed both primary I M and secondary I G responses.
Heavy metals Evidence of direct effect on B-lymphocytes to suppress humoral response.
PCB, HCB Lowered rodent antibody synthesis to sheep RBC (antigen).
HCB Suppressed cell-mediated and enhanced humoral immunity in rodents.
PBB Decreased population of T-lymphocytes.
PBB Better correlation of Lymphocyte dysfunction with PBB concentration in WBC
fraction than in plasma fraction.
Dioxin Progeny of mice exposed perinataily showed thymic atrophy.
1- Pb = lead ;Cd = cadmium; Hgj . = inorganic/organic mercury; HCB = hexachlorobenzene;
PCB=polychlorinated bipheiiyls; PBB=poh/brominated biphenyls.
An early step in the evaluation of a candidate complex requires in vitro as well as in vivo
studies which will: (1) compare the affinity constants, for a given xenobiotic, between receptors
recovered from blood cells and those recovered from target tissue cells; (2) verify that the
amount of receptor-bound xenobiotic in a blood sample is potentially detectable; and (3)
determine the pharmacokinetics of the complex under different exposure scenarios in vivo, and
the stability of the complex in vitro following withdrawal of the blood sample.
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TABLE m
Receptor Systems Found In the Formed Elements of Blood
As Well As in Other Tissues
Receptor Location1 Ref.
ACTH (m)Leukocyte (13)
Acetylcholine (m)Leukocyte (13)
a-Adrenergic (m)Leukocyte, Platelet (13,11)
^-Adrenergic (m)Leukocyte, platelet (13,55)
Ah (c)Leukocyte (22)
Endorphin (m)Leukocyte (13)
Enkephalin (m)Leukocyte (13)
Dopamine (m)Leukocyte, Pituitary Cells (56,57)
Glucocorticoid (m)Leukocyte (13)
Growth Hormone (m)Leukocyte (13)
Histamine (m)Leukocyte (13)
Insulin (m)Leukocyte, erythrocyte (13,58)
Interferon (m)Leukocyte (12)
Parathyroid (c)Leukocyte (13)
Prolactin (m)Leukocyte (13)
Prostaglandin E1&2 (m)Leukocyte, Erythrocyte (59, 60)
Serotonin (m)Leukocyte, Platelets (13,61)
Somatomedin (m)Leukocyte, Erythrocyte (12)
Somatostatin (m)Leukocyte (13)
Substance? (m)Leukocyte (13)
Thyroxine (c)Leukocyte (13)
Vasopressin (m)Leukocyte (13)
1. (c) - receptor occurs in cytosolic and/or nuclear fraction
(m) = receptor occurs in membrane fraction.
Regarding the comparison of blood-borne and target tissue receptors, the literature
suggests that the same receptor system obtained from different tissues possess similar in vitro
binding properties. Total binding capacity, reflective of receptor numbers rather than receptor
binding kinetics, are the most frequently reported differences. Examples include: the AcR
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(62); estrogen receptor (63); the Ah receptor (64); benzodiazepine receptors (36); adrenergic
receptors (42); and insulin receptors (65). Insulin receptors from different human tissues
appear to possess structural as well as functional similarity (66).
It is worth noting that interspecies similarities in functional and/or structural
characteristics of GABA (35, 67), Ah (19), GnRH (68), and corticosteroid receptor systems
(69) have also been reported.
Potential for detectability of the complex cannot be addressed directly because data on
numbers of xenobiotic binding sites per blood cell are lacking. However, a calculation based on
reported numbers of insulin receptors per erythrocyte suggests the feasibility of an
immunoassay on a one milliliter blood sample. The abundance of insulin receptors per human
erythrocyte has been estimated from 6.3-9.9 per RBC (70), up to >400 binding sites per
erythrocyte (71). For purposes of illustration, taking the lower mean value of 8 sites per
erythrocyte and 4xl06 RBC/mm3 of blood, a one ml sample of blood contains 4xl09
erythrocytes and 3.2xl010 receptor sites. Dividing by 6 x ICr^approx. Avogadro's number) gives
about 5xlO'14moles of binding potential, assuming one molecule of ligand per receptor site.
Using the higher value for RBC insulin receptors, provides about 2xlO"12 moles of ligand
binding potential per ml of blood Enzyme-linked immunosorbent assays using monoclonal
antibodies are capable of detecting femtomole (10*15 moles) quantities of benzo[a]pyrene-DNA
adduct (72). The numbers of hormone receptor sites reported for leukocytes are generally
greater than those for erythrocyte insulin receptors, as can be seen from the following
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examples: 2,000-7,000 0-adrenergic sites/cell, 200or-adrenergic sites/cell, 300 T3 sites/cell, 4,000
growth hormone sites/cell, 7,000 substance P sites/cell, and 3,000 ACTH sites/cell (13), and
240 prostaglandin E2 sites/cell (60). A similar calculation using 3,000 receptors per leukocyte
and 7,000 WBC per mm3 would provide for 3xlO"12 moles of ligand binding. Thus, the potential
for immunoassays of receptor-bound xenobiotic in the blood does appear to exist. Further,
should a receptor type occur on both leukocytes and erythrocytes and have similar binding
kinetics, an immunoassay screen could be performed on the total cell population of the sample.
Finally, the kinetics of ligand-receptor interaction which includes the association (k^)
and dissociation (k,) rate constants, and the apparent equilibrium dissociation constant, Kd,
(kj/lCj) of the receptor can be readily determined (5). For exposure biomarker use, the Kd of
the complex must be indicative of specific binding. Also important is the rate of dissociation of
the Ligand-receptor complex. The temperature of separation of bound from free ligand
determines the loss of complex in the interval from removing the complex from its equilibrium
melieu to its separation from the mixture (73). Thus, complexes that dissociate rapidly require
separation techniques that minimize dissociation (assumes that separation began while binding
reaction was at steady state. Since free ligand is infinitely diluted in the procedure prior to
separation for all practical purposes, reassociation does not occur.
The loss of binding due to dissociation has been shown to increase approximately
linearly through the first half-life (t1/2) of dissociation of ligand-receptor complexes that follow
first-order kinetics (73). When percent binding loss is plotted on the y-axis against separation
28
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time (in dissociation half-life units) on the x-axis, it is seen that a separation procedure
completed in less than approximately 0.15 t: ,2 avoids losing more than 10% of bound ligand.
Since
Kd * [L][R]/[LR]
an association rate constant, (1^), of 106 M'1 sec'1 (a value frequently obtained for
neurotransmitter receptors) can be used to show that a ligand-receptor complex having a K . =
10"8 M permits a separation time of about 10 sec., i.e.,
10-8 M = 0.693/t1/2/106 M'1 sec'1
and
t1/2 = 69.3 sec. and 0.15t1/2 = 10 sec. (66).
Continuing the calculations, Yamamura (73) shows: for Kd = 10"7M, separation must
be complete within 0.10 sec; for Kd - 10"9, 1.7 rain; for Kd • 10'10, 17 min; and for Kd = HT11,
2.9 hr. For the lower affinity constants, and depending on the separation time, it may be
necessary to accommodate and correct for higher percentage losses.
Assessment of an exposure rate from the concentration of receptor-bound xenobiotic in
the blood requires an understanding of the pharmacokinetics of receptor macromolecules in
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vivo. This information is available for many receptor-bound therapeutic drugs,
neurotransmitters, and hormones, but not for receptor-bound environmental chemicals.
Important phannacokinetic parameters of receptor-bound chemical following acute
exposure, assuming first order kinetics, include the rise-time and biological half-life of the
receptor-xenobiotic complex in vivo for each exposure route of concern. These values must be
estimated either from laboratory animal experiments, and then extrapolated to humans, or
determined from successive measurements made on humans following a single accidental
exposure.
Interpretation of receptor-bound xenobiotic levels resulting from chronic exposure
requires, in addition, information on the total number of receptors present. The phenomenon
of internalization of extracellular material has been described in which occupied receptor
macromolecules migrate laterally, aggregate, invaginate, and enter the cytoplasm within
vesicles. Endocytosis of receptors may be followed by return of the receptors to the surface
unchanged or by receptor degradation and replacement (74). It is hypothesized that receptor
numbers could remain unchanged, be decreased if replacement lagged internalization, or be
increased if replacement was overcompensated. Thus, the total number of receptors in exposed
individuals, compared to those found in non-exposed individuals, may help to establish a
chronic exposure scenario.
-------
Finally, the existence of body fluids, other than blood, containing cells and/or cell
fragments should be acknowledged. The presence of renal tubular epithelial cells in urine and
spermatozoa in semen may provide a source of receptor macromolecules either not found in
blood, or found in greater numbers. Some of the evidence in support of exposure biomarker
development using these fluids was presented in an earlier report (75).
SPECIFIC RECOMMENDATIONS
A study to evaluate the potential use of pesticide-receptor complex levels in human
blood samples as biomarkers of exposure is timely for several reasons. Foremost is the
Agency's recognition of the need for rapid, cost-effective screening tests as expressed in the
research strategy for the 1990s (76). Further, the separate technologies needed to conduct a
feasibility study, viz. receptor macromolecule-complex recovery, determination of receptor
binding kinetics, and immunoassays have developed sufficiently to be well documented in the
literature. Relatively minor modifications would be required for their application to a specific
biomarker. Finally, immunoassays can be incorporated into biosensor devices which can
measure the amount of analyte in the blood in real time, i.e., at the time of sample acquisition.
Should feasibility be demonstrated, method standardization, protocol development, method
comparison studies, QA/QC procedures, etc., would follow.
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Ten specific tasks are recommended in order to evaluate receptor-xenobiotic complex
concentration in the blood as a biomarker of human exposure. The approximate order of
optimal undertaking is reflected in the following sequence:
1. Verify the occurrence of AhR and GABAR in experimental animal and human blood
cells using radiolabeled ligands with high specific binding, e.g., [3H]TCDD for AhR and
[35S]TBPS for GABAR.
2. For each receptor, determine the total binding capacity (Bmax), association and
dissociation rate constants (1^ and kj), apparent equilibrium dissociation constant (Kd),
and stability of the receptor-xenobiotic complex within the sample in vitro. A
considerable information base is already in existence for TCDD, making it the logical
choice for the AhR. Of the insecticidal compounds bound by the GABAR, endrin is
preferred because of its high affinity constant.
3. If blood cell GABA receptor concentrations are extremely low, the development of an
immunoassay for one of the known pesticide ligands may not be practical. Accordingly,
an alternative approach should be investigated concurrently with steps 2-5. Specifically,
brain GABAR should be used to bind the pesticide of interest in a plasma sample
followed by isolation of the complex and quantitation using an immunoassay procedure.
4. Obtain potyclonal antibodies for each receptor complex. Develop and evaluate an
immunoassay for each complex. The specificity and/or sensitivity may be improved
with the use of monoclonal antibodies if necessary.
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5. Using the immunoassay procedure developed, identify the useful dose-response range
by determining the concentration of complex formed at different xenobiotic
concentrations in vitro.
6. Plan and conduct a controlled study using experimental animals to verify the methods
and determine the relationship between exposure and receptor-bound xenobiotic
concentration.
7. Initiate the development of a biosensor system which will transduce a quantitative
chemical/physico-chemical reaction in the immunoassay system developed above into
an electrical or optico-electrical signal which can be quantitatively processed.
8. When feasible, identify a human population with known exposure to one or both of the
above chemicals and for which an epidemiological study is ongoing. Compare the
concentration of receptor-bound pesticide in the blood with other measures of exposure
such as total blood concentration, urinary metabolite concentration, etc.
9. A concurrent study should be carried on in conjunction with number one above. This
would involve selecting four or five additional high priority pesticides to determine
whether these chemicals show specific binding to blood cell membrane preparations in
vitro. Small groups of xenobiotics, examined periodically as time permits, provide a
reservoir of potential biomarkers to be developed as funds and need permit.
10. Three-dimensional molecular modeling software should be used to study the potential
for binding of pesticides to specific receptors prior to initiating laboratory animal
experiments. These programs permit quantitative information to be gained about
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goodness of fit between a particular pesticide structure and the receptor based on
comparisons with ligands with known structures and affinities.
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LITERATURE CITED
1. Lippmann, M., and G.D. Thurston. 1988. Exposure assessment: input into risk
assessment. Arch. Environ. Health 43(2): 113-123.
2. Committee on Biological Markers of the National Research Council. 1987. Biological
markers in environmental health research. Environ. Health Perspect. 74:3-9.
3. Van Emon, J., J. Seiber, and B. Hammock. 1987. Application of an enzyme-linked
immunosorbent assay (ELJSA) to determine paraquat residues in milk, beef, and
potatoes. Bull. Environ. Contam. Toxicol. 39:490-497.
4. Triggle, D.J. 1980. Receptor-hormone interrelationships. In Membrane Structure and
Function, Vol. Three, E. Edward Bittar (ed.), John Wiley & Sons, p. 6.
5. Sairam, M.R. 1985. Protein gtycosylation and receptor-ligand interaction. In: The
Receptors, Vol. n, P.M. Conn (ed.), Academic Press, Inc., pp. 307-340.
6. Limbird, Lee E. 1986. Cell Surface Receptors: A Short Course on Theory and
Methods. Martinus Nijhoff Publishing, Boston.
35
-------
7. Poland, A., and J.C. Knutson. 1982. 2,3,7,8-Tetrachlorodibenzo:p-dioxin and related
halogenated aromatic hydrocarbons: Examination of the mechanism of toxicity. Ann.
Rev. Pharmacol. Toxicol. 22:517-554.
8. Lowell, J.L., and John E. Casida. 1984. Interactions of lindane, toxaphene and
cyclodienes with brain-specific t-Butylbicyclo-phosphorothionate receptor. Life
Sciences 35:171-178.
9. Hathaway, D.E. 1984. Molecular Aspects of Toxicology. The Royal Society of
Chemistry, London, pp. 277-291.
10. Conn, P.M., ed. 1984. The Receptors, Vol. I, Academic Press, Inc.
11. Conn, P.M., ed. 1985. The Receptors, VoL n, Academic Press, Inc.
12. Conn, P.M., ed. 1986. The Receptors, Vol. m, Academic Press, Inc.
13. Conn, P.M., ed 1986. The Receptors, Vol. IV, Academic Press, Inc.
14. Wallach, Donald F.H. 1987. Fundamentals of Receptor Molecule Biology. Mercel
Decker, Inc.
36
-------
15. Scatchard, G. 1949. The attractions of Proteins for small molecules and ions. Ann.
N.Y. Acad. Sci. 51:600-672.
16. Rosenthal, H.E. 1967. A graphic method for the determination and presentation of
binding parameters in complex systems. Anal. Biochem. 20:525-532.
17. Poland, A., E. Glover, and A.S. Kende. 1976. Stereospecific, high-affinity binding of
2,3,7,8-Tetrachlorodibenzo-p-dioxin by hepatic cytosol: evidence that the binding
species is receptor for induction of aryl hydrocarbon hydroxylase. J. Biol. Chem.
251:4936-4946.
18. Poland, A. and J.C. Knutson. 1982. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related
halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Ann.
Rev. Pharmacol. Toxicol. 22:517-554.
19. Greenlee, W.F., and R.A. NeaL 1985. The Ah receptor: A biochemical and biologic
perspective. In: The Receptors, VoL n, P.M. Conn, ed. Academic Press, Inc., pp.
89-129.
20. Clark, C.R., ed. 1987. Steroid hormone receptors: their intraceilular localization, Ellis
Horwood Ltd., Chichester (England).
37
-------
21. Reid, Eric, G.M.W. Cook, and D.J. Morr6, eds. 1984. Investigation of
membrane-located receptors, Plenum Press, New York.
22. Carlstedt-Duke, J., R. Kurl, L. Poellinger, M. Gillner, L.-A. Hannson, R. Toftgard, B.
Hogberg, and JA. Gustafsson. 1982. The detection and function of the cytosolic
receptor for 2,3,7,8-Tetrachlorodibenzo-/7-dioxin (TCDD) and related carcinogens. In:
Chlorinated dioxins and related compounds: impact on the environment. Hutzinger,
R.W. Frei, E. Merian, and F. Pocchiari, eds. Pergamon, New York, pp. 355-356.
23. Hudson, L.G., R. Shaikh, WA. Toscano, Jr., and W.F. Greenlee. 1983. Induction of
7-ethoxycoumarin O-Deethylase activity in cultured human epithelial cells by
2,3,7,8-Tetrachlorodibenzo-/7-dioxin (TCDD): evidence for TCDD receptor. Biochem.
Biophys. Res. Commun. 115:611-617.
24. Clark, D.A., G. Sweeny, S. Safe, E. Hancock, D.G. Kilbourn, and J. Gauldie. 1983.
Cellular and genetic basis for suppression of cytotoric T-cell generation by haloaromatic
hydrocarbons. Imrnunopharmacology 6:143-153.
25. Poland, A., and E. Glover. 1976. Stereospecific, high-affinity binding of
2,3,7,8-Tetrachlorodibenzo-/7-dioxin by hepatic cytosol. J. Biol. Chem.
251(16):4936-4946.
38
-------
26. Tallman, J.F. and D.W. Gallager. 1985. The GABA-ergic System: a locus of
benzodiazepine action. Ann. Rev. Neurosci. 8:21-44.
27. Tallman, J.F., J.W. Thomas, and D.W. Gailager. 1978. GABAergic modulation of
benzodiazepine binding site sensitivity. Nature 274:383-385.
28. Sigel, E. and E.A. Bernard. 1984. A 7-Aminoburyric Acid/Benzodiazepine receptor
complex from bovine cerebral cortex. J. Biol Chem. 259:7219-7223.
29. Eldefrawi, M.E., I.M. Abalis, M.T. Filbin, and A.T. Eldefrawi. 1985. Glutamate and
GABA receptors of insect muscles: biochemical identification and interactions with
insecticides. In: Approaches To New Leads For Insecticides, von Keyserlingk et at.,
eds., Springer-Verlag, pp. 101-116.
30. Squires, R.F., I.E. Casida, M. Richardson, and E. Saederup. 1983.
[^Sjt-Butylbicyclophosphorotnionate binds with high affinity to brain-specific sites
coupled to 7-Aminobutyric acid-A and ion recognition sites. Mol. Pharmacol.
23:326-336.
31. Lawrence, LJ., and J.E. Casida. 1984. Interaction of lindane, toxaphene and
cyclodienes with brain-specific t-Butylbicyclo-phosphorothionate receptor. Life
Sciences 35:171-178.
39
-------
32. Lawrence, L.J. and I.E. Casida. 1983. Stereospecific action of pyrethroid insecticides
on the 7-Aminobutyric acid receptor-ionophore complex. Science 221:1399-1401.
33. Krause, D.N., E. Wong, P. Degener, and E. Roberts. 1980. GABA receptors in bovine
cerebral blood vessels: binding studies with [3H]-muscimol. Brain Res. 185:51-57.
34. Dickenson, H.W., R.D. Allan, J. Ong, and G.A.R. Johnston. 1988. GABAB receptor
antagonist and GABAA receptor agonist properties of a 5- aminovaleric acid derivative,
Z-5-amino-pent-2-enoic acid. Neurosci. Lett. 86(3):351-355.
35. Olsen, R.W. 1982. Drug interactions at the GABA receptor-ionophore complex. Ann.
Rev. Pharmacol. Toxicol. 22:245-277.
36. Verma, A., and S.H. Snyder. 1989. Peripheral type benzodiazepine receptors. Ann.
Rev. Pharmacol. Toxicol. 29:307-322.
37. Cuatrecasas, P. 1974. Membrane receptors. Ann. Rev. Biochem. 43:169-214.
38. Malhotra, S.K. 1980. Organization, composition, and biogenesis of animal cell
membranes. In: Membrane Structure and Function, E.E. Bittar, ed. John Wiley &
Sons, N.Y., pp. 1-72.
40
-------
39. Limperos, G. and K.E. Ranta. 1953. A rapid screening test for the determination of the
approximate cholinesterase activity of human blood. Science 117:453-455.
40. de Potas, G.M., and A.M.P. de'Angelo. 1987. Perturbation effects of organophosphate
insecticides on human erythrocytes. Bull. Environ. Contam. Toxicol. 39:802-806.
41. Bondy, S.C., and A.K. Agrawal. 1980. The inhibition of cerebral high- affinity receptor
sites by lead and mercury compounds. Arch. Toxicol. 46:3-4.
42. Stiles, G.L., M.G. Caron, and RJ. Lefkowitz. 1984. 0-Adrenergic receptors:
biochemical mechanisms of physiological regulation. Physiol. Rev. 64:661-743.
43. O'Dowd, B.F., RJ. Lefkowits, and M.G. Caron. 1989. Structure of the adrenergic and
related receptors. Ann. Rev. Neurosci. 12:67-83.
44. Slotkin, TA., L. Orband, T. Cowdery, RJ. Kavelock, and J. Bartolome. 1987. Prenatal
exposure to methyl mercury alters development of adrenergic receptor binding sites in
peripheral tissue. Toxicol. Lett. (AMST) 35(2/3) :285-296.
45. Csaba, G., A. Inczefi-Gonda, O. Dobozy, A. Varro, and G. Rabloczky. 1983. Impact of
neonatal treatment with cardiac grycosides (digoxin, ouabain) on receptor binding
41
-------
capacity, blood level and cardiac function in the adult rat: expression of the imprinting
theory. Gen. Pharmacol. 14(6): 709-712.
46. Haukkamaa, M. 1987. Membrane-associated steroid hormone receptors. In: Steroid
hormone receptors: their intracellular localization, ed. C. R. Clark. Ellis Harwood, Ltd.,
pp. 155-169.
47. Huang, E. Su-Rong, and F.R. Nelson. 1986. Anti-estrogenic action of chlordecone in
rat pituitary gonadotrophs In Vitro. Toxicol. Appl. Pharmacol. 82:62-69.
48. Murphy, S.D., L.G. Costa, and B.W. Schwab. 1982. Pesticide interactions and
development of tolerance. In: Effects of chronic exposure to pesticides on animal
systems, eds. I.E. Chambers and J.D. Yarbrough, Raven Press, New York.
49. Gray, Jr. L.E., J.S. Ostby, J.M. Ferrel, E.R. Sigmon, and J.M. Goldman. 1988.
Methoxychlor induces estrogen-like alterations of behavior and the reproductive tract in
the female rat and hamster: effects on sex behavior, running wheel activity, and uterine
morphology. ToxicoL Appl. Pharmacol. 96:525-540.
50. Romkes, M., J. Piskorska-Pliezczyneka, and S. Safe. 1987. Effects of
23,7,8-Tetrachlorodibenzo^»-dioxin on hepatic and uterine estrogen receptor levels in
rats. ToxicoL AppL Pharmacol. 87:306-314.
42
-------
51. Duvivier, J., J. Van Cantfort, and I.E. Gielsen. 1981. The influence of five
monooxygenase inducers on liver cytosol estradiol receptor levels in the ovariectomized
adult rat. Biochem. Biophys. Res. Commun. 99: 252-258.
52. Max, S.R., and E.K Silbergeld. 1987. Skeletal muscle glucocorticoid receptor and
glutamine synthetase activity in the wasting syndrome in rats treated with
2,3,7,8-Tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 87:523-527.
53. Ryan, R.P., G.I. Sunahara, G.W. Lucier, L.S. Birnbaum, and K.G. Nelson. 1989.
Decreased ligand binding to the hepatic glucocorticoid and epidermal growth factor
receptors after 2,3,4,7,8-Pentachlorodibenzofuran and 1,2,3,4,7,8-Hexachloro-
dibenzo-furan Treatment of Pregnant Mice. Toxicol. Appl. Pharmacol. 98:454-464.
54. Luster, M.I., et al. 1987. Molecular and cellular basis of chemically induced
immunotoxicity. Ann. Rev. Pharmacol. and Toxicol. 27:23-49.
55. Wang, XX. and O.E. Brodde. 1985. Identification of a homogeneous class of
/?2-Adrenoceptors in human platelets by iodine-125 Levo-Iodopindolol binding. J. Cyclic
Nucleotide Protein Phosphorylation Res. 10:439-450.
43
-------
56. Shaskan, E.G., M. Ballow, M. Lederman, S.L. Margolis, and R. Melchreit. 1984.
Spiroperidol binding sites on mouse lymphoid cells: effects of ascorbic acid and
psychotropic drugs. J. Neuroimmunol. 6:59-66.
57. Cronin, M.J., M.O. Thorner, P. Hellman, and A.D. Rogol. 1984. Bromocriptine inhibits
growth hormone release from rat pituitary cells in primary culture. Pro. Soc. Exp. Biol.
Med. 175:191-195.
58. Schernathaner, G., R. Prager, C. Punzengruber, and A. Luger. 1985. Severe
hyperprolactinemia is associated with decreased insulin binding In Vitro and insulin
resistance In Vivo. Diabetologia 28:1386-142.
59. Dutta-Roy, A.K. and A.K. Sinha. 1985. Binding of prostagiandin Et to human
erythrocyte membrane. Biochim. Biophys. Acta 812:671-678.
60. Eriksen, E.F., B. Richelsen, H. Beck-Nielsen, F. Melsen, H.K Nielsen, and L.
Mosekilde. 1985. Prostagiandin E2 receptors on human peripheral blood monocytes.
Scand. J. Immunol. 21:167-172.
61. Hoffman, D. and W. Weswmann. 1985. Characterization of 5-Hydroxytryptamine
binding sites in the plasma membrane of pig blood platelets. J. Neural Transm.
61:193-218.
44
-------
62. Schimerlik, M.I. 1989. Structure and regulation of muscarinic receptors. Ann. Rev.
Physiol. 51:217-227.
63. Notides, A.C. 1970. Binding affinity and specificity of the estrogen receptor of the rat
uterus and anterior pituitary. Endocrinology 87:987-992.
64. Mason, M.E. and A.B. Okey. 1982. Cytosolic and nuclear binding of
2,3,7,8-Tetrachlorodibenzo-p-dioxin to the Ah receptor in extra-hepatic tissues of rats
and mice. Eur. J. Biochem. 123:209-215.
65. Mcelduff, A. and CJ. Eastman. 1981. The erythrocyte insulin receptor. Aust. J. Exp.
Biol. Med. Sci. 59:439-448.
66. Roth, RA., DJ. Cassell, K.Y. Wong, BA. Maddux, and I.D. Goldfine. 1982.
Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit
insulin action. Proc. NatL Acad. ScL U.SA 79:7312-7316.
67. Sieghart, W., A. Eichenger, P. Riederer, and K. Jellinger. 1985. Comparison of
benzodiazepine receptor binding in membranes from human or rat brain.
Neuropharmacology 24:751-760.
45
-------
68. Iwashita, M., and K.J. Catt. 1985. Photoaffinity labeling of pituitary and gonadal
receptors for gonadotropin-releasing hormone. Endocrinology 117:738-746.
69. Sutanto, W., J.A.M. Van Eekelen, J.M.H.M. Revi, and E.R. Dekloet. 1988.
Species-specific topography of corticosteroid receptor types in rat and hamster brain.
Neuroendocrinol. 47:398-40.
70. Hepp, R., H.E. Meyer, F. Peters, W. Passlack, and H. Reinauer. 1984. The influence of
tracers on insulin binding to human erythrocytes. J. Clin. Chem. Clin. Biochem.
21:829-834.
71. Gambihr, K.K, JA. Archer, S.G. Nerurkar, LA. Cruz, and M. Sanders. 1981.
Erythrocyte insulin receptors in chronic renal failure. Nephron 28:4-10.
72. Perera, F., R. Santella, and M. Poirier. 1986. Biomonitoring of workers exposed to
carcinogens: immunoassays to Benzo[a]Pyrene-DNA adducts as a prototype. J.
Occupational Med. 28:1117-1123.
73. Yamamura, ELL, SJ. Enna, and M. Kuhar. 1978. Neurotransmitter receptor binding,
Isted Raven Press, NY.
46
-------
74. Raffa, R.B., and RJ. Tallarida. 1985. The concept of a changing receptor
concentration: implications for the theory of drug action. J. Theor. Biol. 115:625-632.
75. Santolucito, JA. 1988. Biomarkers of exposure-introduction, literature summary, and
recommendations, EPA--/600/X-88/383.
76. The Report of the Research Strategies Committee, Science Advisory Board, U.S.
Environmental Protection Agency. September, 1988. Future Risk: Research Strategies
for the 1990s. SAB-EC-88-040.
47
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