CPA/600/AP-92/OOlb
c/EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
Chapter 2.
Mechanisms of
Toxic Actions
EPA/600/AP-92/001b
August 1992
Workshop Review Draft
Review
Draft
(Do Not
Cite or
Quote)
Notice
This document is a preliminary draft. It has not been formally released by EPA and should not
at this stage be construed to represent Agency policy. It is being circulated for comment on
its technical accuracy and policy implications.
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DRAFT EPA/600/AP-92/001b
DO NOT QUOTE OR CITE August 1992
Workshop Review Draft
Chapter 2. Mechanisms of Toxic Actions
Health Assessment for
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
and Related Compounds
NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by the U.S.
Environmental Protection Agency and should not at this stage be construed to represent Agency
policy. It is being circulated for comment on its technical accuracy and policy implications.
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C.
Printed on Recycled Paper
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DISCLAIMER
This document is a draft for review purposes only and does not constitute Agency policy.
Mention of trade names or commercial products does not constitute endorsement or recommendation
for use.
Please note that this chapter is a preliminary draft and as such represents work
in progress. The chapter is intended to be the basis for review and discussion at
a peer-review workshop. It will be revised subsequent to the workshop as
suggestions and contributions from the scientific community are incorporated.
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CONTENTS
Figures iv
List of Abbreviations v
Authors and Contributors x
2. MECHANISM OF ACTION 2-1
2.1. INTRODUCTION 2-1
2.2. THE "RECEPTOR" CONCEPT 2-1
2.3. THE Ah (DIOXIN) RECEPTOR 2-3
2.4. BIOCHEMICAL PROPERTIES OF THE AH RECEPTOR 2-6
2.5. FUNCTION OF THE Ah RECEPTOR 2-8
2.6. IMPLICATIONS FOR RISK ASSESSMENT 2-14
2.7. FUTURE RESEARCH 2-16
2.8. REFERENCES 2-17
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LIST OF FIGURES
2-1 Mechanism of TCDD Action 2-10
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LIST OF ABBREVIATIONS
ACTH
Ah
AHH
ALT
AST
BDD
BDF
BCF
BGG
bw
cAMP
CDD
cDNA
CDF
CNS
CTL
DCDD
DHT
DMBA
DMSO
DNA
Adrenocorticotrophic hormone
Aryl hydrocarbon
Aryl hydrocarbon hydroxylase
L-alanine aminotransferase
L-asparate aminotransferase
Brominated dibcnzo-p-dioxin
Brominatcd dibenzofuran
Bioconcentration factor
Bovine gamma globulin
Body weight
Cyclic 3,5-adcnosine monophosphate
Chlorinated dibcnzo-p-dioxin
Complementary DNA
Chlorinated dibenzofuran
Central nervous system
Cytotoxic T lymphocyte
2,7-Dichlorodibenzo-p-dioxin
5a-Dihydrotestosterone
Dimethylbcnzanthraccne
Dimethyl sulfoxide
Deoxyribonucleic acid
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LIST OF ABBREVIATIONS (cont.)
DRE
DTG
DTH
ECOD
EGF
EGFR
ER
EROD
EOF
FSH
GC-ECD
GC/MS
GOT
GnRH
GST
HVH
HAH
HCDD
HDL
HxCB
Dioxin-responsive enhancers
Delayed type hypersensitivity
Delayed-type hypersensitivity
Dose effective for 50% of recipients
7-Ethoxycoumarin-O-deethylase
Epidermal growth factor
Epidermal growth factor receptor
Estrogen receptor
7-Ethoxyresurofm 0-dcethylase
Enzyme altered foci
Follicle-stimulating hormone
Gas chromatograph-electron capture detection
Gas chromatograph/mass spectrometer
Gamma glutamyl transpeptidase
Gonadotropin-releasing hormone
Glutathione-S-transferase
Graft versus host
Halogenated aromatic hydrocarbons
Hexachlorodibenzo-p-dioxin
High density lipoprotein
Hexachlorobiphenyl
VI
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LIST OF ABBREVIATIONS (cont.)
HpCDD Heptachlorinated dibenzo-p-dioxin
HpCDF Heptachlorinated dibenzofuran
HPLC High performance liquid chromatography
HRGC/HRMS High resolution gas chromatography/high resolution mass spectrometry
HxCDD Hexachlorinated dibenzo-p-dioxin
HxCDF Hexachlorinated dibenzofuran
I-TEF
LD50
LH
LDL
LPL
LOAEL
LOEL
MCDF
MFO
mRNA
MNNG
NADP
NADPH
NK
International TCDD-toxic-equivalency
Dose lethal to 50% of recipients (and all other subscripter dose levels)
Luteinizing hormone
Low density liproprotein
Lipoprotein lipase activity
Lowest-observable-adverse-effect level
Lowest-observed-effect level
6-Methyl-l ,3,8-trichlorodibenzofuran
Mixed function oxidase
Messenger RNA
W-methyl-yV-nitrosoguanidine
Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate (reduced form)
Natural killer
VII
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LIST OF ABBREVIATIONS (cont.)
NOAEL
NOEL
OCDD
OCDF
PAH
PB-Pk
PCB
OVX
PEL
PCQ
PeCDD
PeCDF
PEPCK
PGT
PHA
PWM
ppm
ppq
ppt
RNA
SAR
No-observable-adverse-effect level
No-observed-effect level
Octachlorodibenzo-p-dioxin
Octachlorodibenzofuran
Polyaromatic hydrocarbon
Physiologically based pharmacokinetic
Polychlorinated biphenyl
Ovariectomized
Peripheral blood lymphocytes
Quaterphenyl
Pentachlorinated dibcnzo-p-dioxin
Pentachlorinated dibenzo-p-dioxin
Phosphopenol pyruvate carboxykinase
Placental glutathione transferase
Phytohemagglutinin
Pokeweed mitogen
Parts per million
Parts per trillion
Ribonucleic acid
Structure-activity relationships
vin
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LIST OF ABBREVIATIONS (cont.)
SCOT
SGPT
SRBC
t*
TCAOB
TCB
TCDD
TEF
TGF
tPA
TNF
TNP-LPS
TSH
TTR
UDPGT
URO-D
VLDL
v/v
w/w
Serum glutamic oxaloacetic transaminase
Serum glutamic pyruvic transaminase
Sheep erythrocytes (red blood cells)
Half-time
Tetrachloroazoxybenzene
Tetrachlorobiphenyl
Tetrachlorodibenzo-p-dioxin
Toxic equivalency factors
Thyroid growth factor
Tissue plasminogen activator
Tumor necrosis factor
lipopolysaccharide
Thyroid stimulating hormone
Transthyretrin
UDP-glucuronosyltransferases
Uroporphyrinogen decarboxylase
Very low density lipoprotcin
Volume per volume
Weight by weight
IX
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AUTHORS AND CONTRIBUTORS
The Office of Health and Environmental Assessment (OHEA) within the Office of Research
and Development was responsible for the preparation of this chapter. The chapter was prepared
through Syracuse Research Corporation under EPA Contract No. 68-CO-0043, Task 20, with Carol
Haynes, Environmental Criteria and Assessment Office in Cincinnati, OH, serving as Project Officer.
During the preparation of this chapter, EPA staff scientists provided reviews of the drafts as
well as coordinating internal and external reviews.
AUTHORS
James Whitlock, Jr.
Department of Pharmacology
Stanford University School of Medicine
Stanford, CA
EPA CHAPTER MANAGER
William H. Farland
Office of Research and Development
Office of Health and Environmental Assessment
Washington, DC
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2. MECHANISM OF ACTION
2.1. INTRODUCTION
The environmental contaminant 2,3,7,8-TCDD has generated worldwide concern
because of its wide-spread distribution, its persistence, its accumulation within
the food chain and its toxic potency in experimental animals. Epidemiological
studies have not produced a well-defined estimate of the risk that the dioxin
poses to human health (Bailar, 1991). Knowledge of the mechanism of TCDD action
may facilitate the risk assessment process by imposing constraints upon the
assumptions used to estimate an acceptable exposure to the dioxin. Here, we
review our current knowledge of dioxin action, with emphasis on the contribution
of the Ah receptor to the mechanism. Other reviews provide additional background
on the subject (Couture et al., 1990; Landers and Bunce, 1991; Nebert, 1989;
Poland and Knutson, 1982; Safe, 1986; Silbergeld and Gasiewicz, 1989; Skene et
al., 1989; Whitlock, 1990).
2.2. THE "RECEPTOR" CONCEPT
The idea that a drug, hormone, neurotransmitter or other chemical produces
a physiologic response by interacting with a specific cellular target molecule
(i.e., a "receptor") evolved from several observations. First, many chemicals
elicit responses that are restricted to specific tissues. For example, ACTH
stimulates the secretion of cortisol by the adrenal cortex, but has no effect on
other tissues. This type of observation implied that the responsive tissue
(i.e., the adrenal cortex) contained a "receptive" component, whose presence was
required for the physiological effect (i.e., cortisol secretion). Second, many
chemicals are quite potent. For example, nanomolar concentrations of numerous
hormones and growth factors elicit biological effects. This type of observation
suggested that the target cell contained a site(s) to which the chemical could
bind with high affinity. Third, stereoisomers of some chemicals (e.g.,
catecholamines, opioids) differ by orders of magnitude in potency. This type of
observation indicated that the molecular shape of the chemical strongly
influenced its biological activity; this, in turn, implied that the binding site
on or in the target cell also had a specific geometric configuration. Together,
these three types of observation predicted that the biological responses to some
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chemicals involve stereospecific, high affinity binding of the chemical to
particular receptor sites, located on or in the target cell.
The availability of compounds of high specific radioactivity permitted the
quantitative analysis of their binding to tissue components in vitro. To qualify
as a potential "receptor," a binding site for a given chemical must satisfy
several criteria. (1) The binding should be saturable, (i.e., there should be
a limited number of binding sites per cell). (2) The binding should be
reversible. (3) The binding affinity measured in vitro should be consistent with
the potency of the chemical observed in vivo. (4) If the biological response
exhibits stereospecificity, so should the in vitro binding. (5) For a series of
structurally-related chemicals, the rank order for binding affinity should
correlate with the rank order for biological potency. (6) Tissues that respond
to the chemical should contain binding sites with the appropriate properties.
The binding of a ligand to its cognate receptor is assumed to obey the law
of mass action; therefore, a rectangular hyperbola represents the relationship
between the concentrations of ligand and ligand-receptor complex. Conceptually,
ligand-receptor binding is analogous to substrate-enzyme binding, and similar
analytical approaches are often applied to both types of interaction. For
example, the Michaelis-Menton model, developed to account for the kinetic
characteristics of some enzymatic reactions, may also account in some instances
for the kinetics of ligand-receptor binding. According to this model, at very
low concentrations of ligand, the formation of the ligand-receptor complex is
directly proportional to the ligand concentration; at very high concentrations
of ligand, the formation of the ligand-receptor complex is independent of the
ligand concentration. However, it is not clear that all receptors obey
Michaelis-Menton kinetics, just as some enzymes (such as those that exhibit
allosteric effects) do not.
Ligand binding constitutes only one aspect of the receptor concept. By
definition, a receptor transduces a chemical signal into a biological effect.
Thus, ligand binding must lead to a response, and the functional consequences of
ligand-receptor binding represent an essential component of the receptor concept.
Receptor theory attempts to quantitatively relate ligand binding to the
biological response. The classical "occupancy" model postulates (1) that ligand-
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receptor binding is a reversible, bimolecular reaction, (2) that the magnitude
of the biological response is directly proportional to the number of occupied
receptors, and (3) that the response is maximal when all receptors are occupied.
The expected rectangular hyperbolic "dose-response" relationships predicted by
the occupancy model have been observed for numerous drugs and other chemicals
(Clark, 1933; Limbird, 1986; Parascandola, 1980; Stephenson, 1956). One
empirical aspect of this type of relationship is that, when receptor occupancy
is sufficiently low, a small increase in ligand concentration may elicit no
detectable biological response. Thus, a drug or other chemical may produce no
observable effect, even though finite concentrations are attained at its
receptor.
2.3. THE Ah (DIOXIN) RECEPTOR
The remarkable potency of TCDD in eliciting its toxic effects suggested the
possible existence of a receptor for the dioxin. Poland and co-workers using
radiolabeled TCDD as a ligand, demonstrated that C57BL/6 mouse liver contained
a soluble, intracellular protein that bound the dioxin saturably (i.e., -10
binding sites per cell) and with high affinity (i.e., in the nanomolar range,
consistent with TCDD's biological potency (in vivo). Furthermore, competition
binding studies with congeners of TCDD revealed that ligands with the highest
binding affinity were planar and contained halogen atoms in at least three of the
four lateral positions; thus, ligand binding exhibited stereospecificity.
Together, these findings indicated that the intracellular, TCDD-binding protein
had the ligand-binding properties expected for a "dioxin receptor." In addition,
the binding of TCDD to the protein in vitro resembled a rectangular hyperbola
and, therefore, appeared to obey the law of mass action (Poland and Knutson,
1982).
Biochemical and genetic evidence implicates the TCDD-binding protein in the
biological response to dioxin. For example, studies of structure-activity
relationships among congeners of TCDD reveal a correlation between a ligand's
binding affinity and its potency in eliciting a biological response, such as
enzyme induction. Furthermore, inbred mouse strains in which TCDD binds with
lower affinity to the receptor exhibit decreased sensitivity to dioxin's
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biological effects. Thus, from both a ligand-binding standpoint and a functional
standpoint, the intracellular TCDD-binding protein satisfies the criteria
required for a receptor. The protein is designated as the "Ah receptor," because
it binds and mediates the response to other aryl hydrocarbons, in addition to
TCDD (Poland and Knutson, 1982).
The Ah receptor evolved prior to the introduction of halogenated aromatic
hydrocarbons into the environment (Czuczwa et al., 1984). Therefore, some other
compound(s) must represent the "natural" ligand(s) for the receptor. Other,
naturally-occurring high affinity ligands for the receptor are present in the
environment (Gillner et al., 1985, 1989; Rannung et al., 1987; Bjeldanes et al.,
1991). Presumably, the structure of TCDD is similar enough to such compounds
that it mimics their binding to the receptor.
Inbred mouse strains differ quantitatively in their sensitivities to
aromatic hydrocarbons, such as TCDD. This polymorphism is genetic in origin, and
in crossbreeding studies, the sensitive phenotype segregates as an autosomal
dominant trait. Numerous responses to aromatic hydrocarbons (e.g., enzyme
induction, thymic involution, cleft palate formation, hepatic porphyria) exhibit
a similar segregation pattern. For this reason, the genetic locus responsible
for the polymorphism is envisioned as regulatory in nature. It is designated as
the "Ah locus", and is thought to encode a regulatory proteins(s). Crossbreeding
studies also reveal that the segregation patterns for TCDD binding and aromatic
hydrocarbon responsiveness are identical. These observations imply that the Ah
locus encodes the Ah receptor (Nebert, 1989; Poland and Knutson, 1982).
Electrophoretic studies reveal the existence of several forms of the TCDD-
binding protein in inbred mouse stains. These observations imply the existence
of multiple alleles at the Ah locus in mice (Perdew and Hollenbeck, 1990; Poland
and Glover, 1990). The biochemical properties of the different forms of the Ah
receptor remain to be described. In particular, the extent to which the
different receptor forms affect the sensitivity of the host to TCDD is not known.
An analogous receptor polymorphism might exist for humans. If differences in the
properties of the Ah receptor do exist among humans, they might produce
interindividual differences in sensitivity to TCDD. However, this possibility
remains to be tested.
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Human cells contain an intracellular protein whose properties resemble those
of the Ah receptor in animals. Ligand-binding studies and hydrodynamic analyses
have identified an Ah receptor-like protein(s) in a variety of human tissues
(Cook and Greenlee, 1989; Harris et al., 1989; Lorenzen and Okey, 1991; Roberts
et al., 1990; Waithe et al., 1991). The human receptor has not been studied
extensively, and it is not yet clear if the properties of the human protein
differ substantially (in a functional sense) from those of the Ah receptor in
animals. However, by analogy with the existence of multiple receptor forms in
mice, we anticipate that the human population will also be polymorphic with
respect to Ah receptor structure and function. Therefore, we would expect humans
to differ from one another in their susceptibilities to TCDD.
Analyses of mouse hepatoma cells in culture reveal additional genetic and
biochemical aspects of the Ah receptor. It is possible to use physical
(fluorescence-activated cell sorting) and/or chemical (resistance to benzo(a)-
pyrene) techniques to select cells that are defective in the Ah receptor
(Hankinson, 1979; Miller and Whitlock, 1981). One class of cells exhibits a
defect in its ability to bind TCDD; however, the liganded receptors that do form
are able to accumulate normally within the cell nucleus. These cells respond
poorly to TCDD (as measured by aryl hydrocarbon hydroxylase induction). In
another class of variants, ligand binding is normal; however, the liganded
receptors are unable to bind to DNA and fail to accumulate in the nucleus. These
cells do not respond to TCDD. Genetic studies of these variant cells using cell
fusion reveal that the receptor-defective phenotype is recessive and that the
variants fall into different complementation groups. The latter observation
implies that more than one gene contributes to receptor function (Hankinson,
1983; Miller et al., 1983; Whitlock, 1990). It is possible, for example, that
the Ah receptor has both a ligand-binding component and a separate, DNA-binding
component. Furthermore, there could be multiple alleles for each component. In
principle, the fact that multiple genes influence receptor function increases the
potential for polymorphisms in receptor structure and function among both animals
and humans.
Hankinson and co-workers also have described mouse cells which exhibit
alterations in both ligand binding and nuclear accumulation; these variants
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comprise a third genetic complementation group (Karenlampi et al., 1988).
Furthermore, analyses of dominant nonresponsive variants suggests the existence
of a represser protein that may block Ah receptor function (Watson and Hankinson,
1988). Together, these observations imply that multiple genetic loci influence
Ah receptor function in mice. Presumably, the situation is similar for other
animals and humans; if so, we anticipate that humans will differ in their
responses to TCDD because of genetic differences in the Ah receptor.
2.4. BIOCHEMICAL PROPERTIES OF THE AH RECEPTOR
The Ah receptor has been difficult to purify in substantial quantity, and
insufficient knowledge of its structural and functional properties represents a
major impasse in our understanding of dioxin action (Bradfield et al., 1991).
The receptor is a soluble (as opposed to a membrane-bound) intracellular protein,
which, upon binding TCDD, acquires a high affinity for DNA. The ligand-binding
properties of the receptor have been studied in some detail, in order to define
the ligand characteristics important for binding and for eliciting a biological
response. The analysis of such structure-binding and structure-response
relationships is useful for evaluating the role of the Ah receptor in systems
where receptor-defective variants are not available. For example, studies with
congeners of TCDD reveal a good correlation between their binding affinities for
the Ah receptor and their potencies in inducing aryl hydrocarbon hydroxylase
activity (Poland et al., 1979). This finding suggests that the receptor
participates in the induction response. The results of analogous structure-
activity studies implicate the Ah receptor in a broad spectrum of biochemical,
morphological, immunologic, neoplastic, and reproductive effects produced by TCDD
(Poland and Knutson, 1982; Safe, 1986). For this reason, we envision that the
receptor participates, directly or indirectly, in every biological response that
the dioxin elicits.
The ligand-binding and hydrodynamic properties of the Ah receptor differ
relatively little across species and tissues; it is difficult to account for the
diversity of TCDD's biological effects by these criteria (Henry et al., 1989).
Relatively little is known about the human Ah receptor. In particular, the
variability among humans in the receptor's binding affinity for TCDD remains to
be determined; therefore, the extent to which this factor influences human
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sensitivity to TCDD is unknown. The hydrodynamic properties of the human
receptor are similar to those of the rodent protein. It is currently unknown
whether the rather small structural variations in the receptor observed among
species or tissues are associated with differences in receptor function.
Ligand binding increases the affinity of the Ah receptor for DNA in vitro
and is associated with the accumulation of the receptor in the cell nucleus in
vivo. Several investigators have used a gel retardation technique to show that
ligand binding converts the Ah receptor to a DNA-binding form, which is found
predominantly in nuclear extracts from TCDD-treated cells (Cuthill et al., 1991;
Denison et al., 1989; Denison and Yao, 1991; Hapgood et al., 1989; Nemoto et al.,
1990; Saatcioglu et al., 1990a,b). Thus, the binding of TCDD to the receptor
"transforms" the protein to a DNA-binding species. The process of transformation
is poorly understood. Biochemical studies indicate that transformation is
associated with changes in thermostability, surface charge, and sensitivity to
sulfhydryl reagents, implying that a conformational alteration (i.e., a change
in the shape of the receptor protein) occurs (Denison et al., 1987; Gasiewicz and
Bauman, 1987; Kester and Gasiewicz, 1987). Hydrodynamic studies suggest that
ligand binding leads to the dissociation from the receptor of another factor
(possibly, a heat-shock protein), a process which could expose the receptor's
DNA-binding domain (Perdew, 1988; Wilhelmsson et al., 1990). Other analyses
reveal that the transformed receptor undergoes an increase in its sedimentation
coefficient, implying that the ligand-binding component of the receptor
associates with a second protein(s) during the transformation process (Henry et
al., 1989). Thus, the transformation process appears to involve multiple events
and interactions between the Ah receptor and other proteins. These hydrodynamic
results are consistent with biochemical evidence that the DNA-binding form of the
receptor is composed of at least two different proteins. For example, the
results of both protein-DNA crosslinking experiments and protein-protein
crosslinking studies imply that the liganded Ah receptor binds to DNA as a
heteromer (Elferink et al., 1990; Gasiewicz et al., 1991). Both the biochemical
findings and the hydrodynamic results are consistent with the genetic evidence
that multiple genes and proteins contribute to receptor function.
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Hankinson and coworkers have used molecular genetic approaches to identify
a human cDNA that partially corrects the defect in variant mouse hepatoma cells
in which the liganded Ah receptor fails to bind DNA (Hoffman et al., 1991). The
cDNA encodes a protein (termed "Arnt" by Hankinson's group) whose function
remains to be determined. The protein apparently does not bind TCDD. It
contains a basic helix-loop-helix domain analogous to those present in other
proteins that bind DNA-binding regulatory proteins. Therefore, the Arnt protein
may be a DNA-binding component of the Ah receptor. It is also possible that the
Arnt protein is a translocase that transports the receptor from cytoplasm to
nucleus (Hoffman et al., 1991). Ema et al. (1992) and Bradfield et al. (1991)
have identified a mouse cDNA that appears to encode the ligand-binding component
of the Ah receptor (Burbach et al., 1992). Like the Arnt protein, the ligand-
binding component also contains a basic helix-loop-helix motif, which presumably
contributes both to DNA binding and to protein-protein interactions. It is
notable that neither component of the Ah receptor contains the zinc finger DNA-
binding motif that is characteristic of the steroid/thyroid/retinoid family of
receptors. Therefore, the Ah receptor presumably belongs to a different class
of regulatory factors proteins.
Electrophoretic studies reveal that multiple alleles exist for the ligand-
binding component of the Ah receptor (Poland and Glover, 1990). By analogy, it
is reasonable to expect that the DNA-binding component of the receptor will also
exhibit polymorphisms and exist in multiple forms. In principle, such a
situation raises the possibility that different functional forms of the receptor
can exist, created by the association of receptor subunits in different
combinations. Such combinatorial diversity could contribute to the variety of
biological responses produced by TCDD.
2.5. FUNCTION OF THE Ah RECEPTOR
TCDD induces microsomal AHH activity in many tissue types, as well as in
cells in culture. Hydroxylase activity is measured using a simple and sensitive
fluorescence technique. Therefore, the induction of AHH activity represents a
convenient TCDD-dependent response to study.
Much of our current understanding of the mechanism of dioxin action stems
from analyses of AHH induction in mouse hepatoma cells in culture (Whitlock,
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1990). Figure 2-1 outlines some of the molecular events that are thought or
known to contribute to the mechanism of TCDD action. Nuclear transcription
experiments reveal that TCDD induces hydroxylase activity by stimulating the
transcription of the corresponding CYP1A1 gene. The response to TCDD occurs
within a few minutes and is direct, in that it does not require ongoing protein
synthesis. Thus, the regulatory components reg_uired for the activation of CYP1A1
transcription are present constitutively within the cell. TCDD fails to activate
CYP1A1 transcription in Ah receptor-defective cells; therefore, the response is
receptor-dependent.
The observations that TCDD activates transcription and that the liganded Ah
receptor is a DNA-binding protein, led to the discovery of a regulatory DNA
domain, upstream of the CYP1A1 gene, which responds to TCDD in a receptor-
dependent manner. Recombinant DNA methods were used to construct chimeric genes,
in which potential regulatory DNA domains from the CYP1A1 gene were ligated to
a heterologous "reporter" gene. After transfection of the recombinants into
mouse hepatoma cells, TCDD was observed to activate the expression of the
reporter gene. This type of experiment revealed that the dioxin-responsive DNA
had the properties of a transcriptional enhancer (Jones et al., 1986; Neuhold et
al., 1986; Fujisawa-Sehara et al., 1987; Fisher et al., 1990). In addition, the
DNA upstream of the CYP1A1 contains a second control element (a transcriptional
promoter), which functions to ensure that transcription is initiated at the
correct site. Neither the enhancer nor the promoter functions in the absence of
the other, and the response of the CYP1A1 gene to TCDD requires that both control
elements function properly in combination (Jones and Whitlock, 1990) . Analyses
of stably-transfected mouse hepatoma cells reveals that the dioxin-responsive
enhancer can function in a chromosomal location distinct from that of the CYP1A1
gene (Fisher et al., 1989). These observations imply that analogous enhancer and
promoter elements, possibly in combination with additional regulatory components,
can mediate the transcriptional response of other genes to TCDD. This hypothesis
remains to be tested. However, the combinatorial model is attractive in that it
provides a plausible biological mechanism for generating the diversity of effects
that TCDD elicits (Whitlock, 1990).
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• Diffusion into cell
• Binding to ligand-binding component of Ah receptor
• Transformation of receptor to DNA-binding form
• Dissociation from heat-shock protein(s)?
• Phosphorylation?
• Association with DNA-binding component of receptor?
• Translocation from cytoplasm to nucleus?
• Binding of liganded receptor to enhancer DNA
• Enhancer activation
• DNA bending?
• Histone modification?
• Recruitment of additional proteins?
Nucleosome disruption
Increased accessibility of transcriptional promoter
Binding of transcription factors to promoter
Increased mRNA and protein synthesis
Primary biological response
Cascade of compensatory changes
Secondary biological effects
Figure 2-1
Mechanism of TCDD Action (see text for details)
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Transcriptional enhancement by TCDD requires a functional Ah receptor;
furthermore, the liganded receptor is a DNA-binding protein. Together, these
observations suggest that the induction of CYP1A1 gene transcription by TCDD
involves the binding of the liganded receptor to the dioxin-responsive enhancer.
Electrophoretic (gel retardation) studies, using enhancer DNA and nuclear extract
from uninduced and TCDD-induced cells, reveal the existence of an inducible,
receptor-dependent protein-DNA interaction in vitro, whose characteristics are
those expected for the binding of the liganded receptor to DNA (Denison et al.,
1989; Hapgood et al., 1989; Saatcioglu et al., 1990a,b). The liganded receptor
recognizes a specific nucleotide sequence
5'T-GCGTG 3'
, which is present in multiple copies within
3'A-CGCAC 5'
the enhancer. Studies with an [ I]-labeled dioxin indicate that the liganded
receptor binds in a 1:1 ratio to the recognition sequence (Denison et al., 1989).
Methylation protection and interference experiments in vitro reveal that the
liganded receptor lies within the major DNA groove and contacts the four guanines
of the recognition sequence (Shen and Whitlock, 1989; Neuhold et al., 1989;
Saatcioglu et al., 1990a,b). These properties of the liganded Ah receptor are
consistent with those described for other transcriptional regulatory proteins.
Experiments involving the use of phosphatases and/or protein kinase
inhibitors suggest that phosphorylation of the Ah receptor, possibly by protein
kinase C, plays a role in the biological response to TCDD (Pongrantz et al.,
1991; Carrier et al., 1992; Okino et al., 1992). The nature of the phosphoryla-
tion(s) and its precise functional role remain to be determined.
Use of a methylation protection technique to analyze receptor-enhancer
binding within the intact cell reveals evidence for TCDD-inducible, Ah receptor-
dependent protein-DNA interactions, which are similar to those observed in vitro
(Wu and Whitlock, 1992). These findings strongly imply that the studies of
receptor-enhancer interactions in vitro do, in fact, reflect biologically
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relevant effects of TCDD and provide accurate insights into the mechanism of
dioxin action in vivo.
The DNA recognition sequence for the liganded Ah receptor contains two CpG
dinucleotides. Studies in other systems reveal that cytosine methylation at CpG
is associated with decreased gene expression, often in tissue-specific fashion.
Cytosine methylation within the Ah receptor's recognition sequence diminishes
both the binding of the receptor to the enhancer (as measured by gel retardation)
and the function of the enhancer (as measured by transfection). These
observations suggest that DNA methylation may contribute to the observed
differences among tissues in their biological responses to TCDD (Shen and
Whitlock, 1989).
Gel retardation analyses reveal that the binding of the receptor to its
recognition sequence bends the DNA in vitro (Elferink and Whitlock, 1990). Thus,
the receptor-enhancer interaction has the potential to alter the configuration
of the DNA. Analyses of the CYP1A1 gene in intact nuclei reveal that the
enhancer/promoter regulatory region undergoes a dioxin-inducible, Ah receptor-
dependent increase in nuclease susceptibility within minutes exposure of the cell
to TCDD. This observation suggests that the receptor-enhancer interaction leads
to an alteration in chromosomal structure, such that the regulatory DNA region
becomes more accessible to the protein factors that are required for transcrip-
tion of the CYP1A1 gene (Durrin and Whitlock, 1989). Methylation protection
studies of the promoter region in vivo reveal that TCDD does, in fact, induce a
receptor-dependent increase in protein binding, which probably reflects the
increased accessibility of the promoter region in the intact cell (Wu and
Whitlock, 1992).
The evidence to date implies that the Ah receptor participates in every
biological response to TCDD (Poland and Knutson, 1982; Safe, 1986; Whitlock,
1990). This hypothesis predicts that TCDD will be found to activate the
transcription of other genes via a receptor- and enhancer-dependent mechanism
analogous to that described for the CYP1A1 gene. Preliminary data suggest that
this is the case. For example, TCDD induces the expression of a cytochrome
P4501A2 gene, a glutathione S-transferase Ya subunit gene, an aldehyde
dehydrogenase gene, and a quinone reductase gene; in some cases, induction is
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known to occur at the transcriptional level, to be Ah receptor-dependent, and to
involve a genomic regulatory element(s) analogous to that found upstream of the
CYP1A1 gene (Quattrochi and Tukey, 1989; Telakowski-Hopkins et al., 1988; Dunn
et al., 1988; Jaiswal et al., 1988; Favreau and Pickett, 1991). In addition,
recent observations suggest that, in human keratinocytes, TCDD activates the
transcription of plasminogen activator inhibitor-2 and inter leukin-1)}, as well
as other genes which remain to be identified (Sutter et al., 1991). The
mechanism by which dioxin activates the expression of these genes is currently
unknown. For dioxin-responsive genes other than CYP1A1, and especially for those
genes that respond in tissue-specific fashion, the presence of the
receptor/enhancer system may not be sufficient for dioxin action, and other,
tissue-specific regulatory components may play a dominant role in governing the
response to TCDD. Thus, future research may reveal the existence of additional
positive or negative gene regulatory components that can influence the response
of the cell to TCDD.
Compensatory changes, which occur in response to TCDD's primary effects, can
complicate the analysis of dioxin action in intact animals. For example, TCDD
can produce changes in the levels of steroid hormones, peptide growth factors
and/or their cognate cellular receptors (Choi et al., 1991; Harris et al., 1990;
Lui et al., 1991; Ryan et al., 1989; Sunahara et al., 1989; Umbreit and Gallo,
1988). In turn, such alterations have the potential to produce a series of
subsequent biological effects, which are not directly mediated by the Ah
receptor. Furthermore, the hormonal status of an animal appears to influence its
susceptibility to the hepatocarcinogenic effects of TCDD (Lucier et al., 1991).
Likewise, exposure to other chemicals can alter the teratogenic response to TCDD
(Couture et al., 1990). Therefore, in some cases, TCDD may act in combination
with other chemicals to produce its biological effects. Such phenomena increase
the difficulty of analyzing dioxin action in intact animals and increase the
complexity of risk assessment, given that humans are routinely exposed to a wide
variety of chemicals.
The fact that TCDD may induce a cascade of biochemical changes in the intact
animal raises the possibility that the dioxin might produce a response such as
cancer by mechanisms that differ among tissues. For example, in one case, TCDD
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might activate a gene(s) that is directly involved in tissue proliferation. In
a second case, TCDD-induced changes in hormone metabolism may lead to tissue
proliferation secondary to increased secretion of a trophic hormone. In a third
case, TCDD-induced changes in hormone receptors for growth factors or hormones
may alter the sensitivity of a tissue to proliferative stimuli. In a fourth
case, TCDD-induced toxicity may lead to tissue death, followed by regenerative
proliferation. At present, it is unknown whether any of these hypothetical
mechanisms actually occurs, whether they would exhibit similar sensitivities to
TCDD, or whether they would occur in all animal species exposed to the dioxin.
Under some circumstances, exposure to TCDD elicits beneficial effects. For
example, TCDD protects against the carcinogenic effects of polycyclic aromatic
hydrocarbons in mouse skin; this may reflect the induction of detoxifying enzymes
by the dioxin (Cohen et al., 1979; DiGiovanni et al., 1980). In other
situations, TCDD-induced changes in hormone metabolism may alter the growth of
hormone-dependent tumor cells, producing a. potential anti-carcinogenic effect
(Spink et al., 1990). These (and perhaps other) potentially beneficial effects
of TCDD complicate the risk assessment process for dioxin.
2.6. IMPLICATIONS FOR RISK ASSESSMENT
A substantial body of biochemical and genetic evidence indicates that the
Ah receptor mediates the biological effects of TCDD. This concept implies that
a response to dioxin requires the formation of ligand-receptor complexes. TCDD-
receptor binding appears to obey the law of mass action and, therefore, depends
upon (1) the concentration of ligand in the target cell; (2) the concentration
of receptor in the target cell; and (3) the binding affinity of the ligand for
the receptor. In principle, some TCDD-receptor complexes will form even at very
low levels of dioxin exposure. However, in practice, at some finite concentra-
tion of TCDD, the formation of TCDD-receptor complexes will be insufficient to
elicit detectable effects. Furthermore, biological events subsequent to TCDD-
receptor binding may not necessarily exhibit a linear response to dioxin. For
example, the ability of the liganded Ah receptor to activate transcription does
not correlate with its binding affinity for DNA in vitro (Shen and Whitlock,
1992). In addition, in some systems, the induction of gene transcription appears
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to require a threshold concentration of transcription factor(s) (Fiering et al.,
1990). An analogous situation may exist for the liganded Ah receptor.
The concentration of TCDD required to produce a biological effect may vary
among individuals. For example, inbred mice vary in their sensitivity to TCDD
according to the receptor's ligand-binding affinity (Poland and Knutson, 1982).
Analogous phenomena commonly occur in humans, who differ in their responses to
numerous xenobiotics (Nebert and Weber, 1990). Thus, a concentration of TCDD
that is sufficient to induce a response in one individual may be insufficient in
another. For example, studies of humans exposed to dioxin at Seveso fail to
reveal a direct relationship between blood TCDD levels and the development of
chloracne (Mocarelli et al., 1991).
The challenge for risk assessment is to use the known properties of the Ah
receptor, together with the known dose-response relationships and pharmacokinetic
data for TCDD, to estimate an intracellular concentration of TCDD below which no
detectable effects occur. This estimate will, in principle, make it possible to
set limits on acceptable human exposure.
Other issues also influence the risk assessment process. Given TCDD's
widespread distribution, its persistence, and its accumulation within the food
chain, it is likely that most individuals (at least in industrialized societies)
are exposed to the dioxin. Therefore, the population at potential risk is
extended and heterogeneous. This fact implies that individuals are likely to
differ in their susceptibility to dioxin, either because of genetic differences
or because of exposure to other chemicals.
Complex TCDD-induced effects (such as cancer) probably require multiple
steps and are likely to involve several genetic and/or environmental factors.
For example, a homozygous recessive mutation at the hr (hairless) locus appears
necessary for TCDD's action as a tumor promoter in mouse skin (Poland et al.,
1982). Furthermore, tumor induction requires exposure to a second chemical,
which acts as a tumor initiator. Presumably, a similar situation exists for
humans. If so, only certain individuals may be at risk from exposure to TCDD,
because of their particular genetic makeup and/or their exposure to other
chemicals. Continued analyses of the mechanism of dioxin action in the future
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may lead to methods for identifying individuals who are especially at risk from
exposure to TCDD.
2.7. FUTURE RESEARCH
The paucity of information about the structure and function of the Ah
receptor represents the major impasse to a better understanding of the mechanism
of dioxin action. The probable heteromeric composition of the receptor, implied
by both genetic and biochemical observations, imposes important constraints upon
approaches which are likely to be successful for the purification of the receptor
proteins themselves or the cloning of the corresponding genes. Recently-
developed biochemical methods, such as photoaffinity labeling (Poland et al.,
1986; Landers et al., 1989) or DNA affinity chromatography (Kadonaga and Tjian,
1986), as well as genetic techniques (Hoffman et al., 1991) constitute new
experimental approaches, which appear likely to generate novel insights into
receptor structure and function in the near future. Antibodies generated against
a receptor-related synthetic peptide also provide new information about the
biological properties of the Ah receptor (Poland et al., 1991). In vitro
transcription provides a new method for analyzing the function of the purified
Ah receptor (Wen et al., 1990). Together, these experimental approaches may help
to reveal the extent to which receptor heterogeneity contributes to inter-
individual differences in susceptibility to TCDD.
Studies of other tissues (e.g., skin, thymus) are likely to reveal
additional TCDD-responsive genes, which exhibit tissue-specific expression
(Sutter et al., 1991). Analyses of the mechanism of dioxin action in such
systems appear likely to reveal additional factors that influence the suscepti-
bility of a particular tissue to TCDD. In addition, studies of other TCDD-
inducible genes, such as glutathione S-transferase, quinone reductase, and
aldehyde dehydrogenase, may reveal whether differences in enhancer structure,
receptor-enhancer interactions, or promoter structure affect the responsiveness
of the target gene to TCDD (Whitlock, 1990).
Analyses of dioxin action may provide some insight into the mechanisms by
which TCDD and related compounds produce birth defects or cancer, effects which
are of particular public health concern. A major challenge for the future will
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be the establishment of experimental systems in which such complex biological
phenomena are amenable to study at the molecular level.
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