xvEPA
                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                                                  (Do Not
                                                  Cite or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)

                                             600AP92001b
                                       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.

-------

-------
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DO NOT QUOTE OR CITE                                                   August 1992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

-------
                             DRAFT-DO NOT QUOTE OR CITE



                                       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.
                                                                                      08/14/92

-------
                        DRAFT-DO NOT QUOTE OR CITE









                                  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
                                                                       08/14/92

-------
                        DRAFT-DO NOT QUOTE OR CITE
                               LIST OF FIGURES
2-1  Mechanism of TCDD Action	   2-10
                                      iv                                08/14/92

-------
                             DRAFT-DO NOT QUOTE OR CITE









                                LIST OF ABBREVIATIONS









 ACTH             Adrenocorticotrophic hormone




 Ah                Aryl hydrocarbon




 AHH              Aryl hydrocarbon hydroxylasc




 ALT              L-alanine aminotransferase




 AST              L-asparate aminotransferase




 BDD              Brominated dibcnzo-p-dioxin




 BDF              Brominated dibenzofuran




 BCF              Bioconcentration factor




 BGG              Bovine gamma globulin




 bw                Body weight




 cAMP             Cyclic 3,5-adenosine monophosphate




 CDD              Chlorinated dibenzo-p-dioxin




 cDNA             Complementary DNA




 CDF              Chlorinated dibenzofuran




 CNS               Central nervous system




 CTL               Cytotoxic T lymphocyte




 DCDD             2,7-Dichlorodibenzo-p-dioxin




 DHT              5a-Dihydrotestosterone




DMBA             Dimethylbenzanthracene




DMSO             Dimethyl sulfoxide




DNA              Deoxyribonucleic acid
                                                                                  08/14/92

-------
                            DRAFT-DO NOT QUOTE OR CITE
                            LIST OF ABBREVIATIONS (cont.)
ORE




DTG




DTK
ECOD




EGF




EGFR




ER




EROD




EOF




FSH




GC-ECD




GC/MS




GGT




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-deethylase




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
                                                                 08/14/92

-------
                             DRAFT-DO NOT QUOTE OR CITE
                             LIST OF ABBREVIATIONS (cont.)
HpCDD




HpCDF




HPLC




HRGC/HRMS




HxCDD




HxCDF
Heptachlorinated dibenzo-p-dioxin




Heptachlorinated dibenzofuran




High performance liquid chromatography




High resolution gas chromatography/high resolution mass spectrometry




Hexachlorinated dibenzo-p-dioxin




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-elTect level




Lowest-observed-effect level




6-Methyl- 1 ,3,8-trichlorodibenzofuran




Mixed function oxidase




Messenger RNA




Af-methyl-7V-nitrosoguanidine




Nicotinamide adenine dinucleotide phosphate




Nicotinamide adenine dinucleotide phosphate (reduced form)




Natural killer
                                             vn
                                                                 08/14/92

-------
                             DRAFT-DO NOT QUOTE OR CITE
                             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 dibenzo-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
                                            vni
                                                                 08/14/92

-------
                            DRAFT-DO NOT QUOTE OR CITE
                            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 lipoprotein




Volume per volume




Weight by weight
                                            IX
                                                                08/14/92

-------
                            DRAFT-DO NOT QUOTE OR CITE
                            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
                                                                                    08/14/92

-------
                         DRAFT—DO NOT QUOTE OR CITE






                            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




                                      2-1                              08/14/92

-------
                          DRAFT—DO  NOT QUOTE OR CITE






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-




                                     2-2                              08/14/92

-------
                          DRAFT—DO NOT QUOTE OR CITE

 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., -105
 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

                                     2-3                              08/14/92

-------
                         DRAFT—DO NOT QUOTE OR CITE






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.




                                     2-4                             08/14/92

-------
                          DRAFT—DO  NOT  QUOTE  OR  CITE






     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




                                      2-5                              08/14/92

-------
                          DRAFT—DO NOT QUOTE  OR CITE






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




                                     2-6                              08/14/92

-------
                          DRAFT—DO NOT QUOTE OR CITE






 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 DMA-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.
                                      2-7
                                                                      08/14/92

-------
                         DRAFT—DO NOT QUOTE OR CITE






     Hankinaon 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,




                                     2-8                              08/14/92

-------
                          DRAFT—DO  NOT QUOTE OR CITE






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 required 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).








                                     2-9                             08/14/92

-------
               DRAFT—DO NOT QUOTE OR CITE
•  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)
                           2-10                             08/14/92

-------
                         DRAFT—DO NOT QUOTE OR CITE


     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 51
the enhancer.  Studies with an [ 125I]-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
                                      2-11                              08/14/92

-------
                          DRAFT—DO NOT QUOTE OR CITE






 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




                                     2-12                             08/14/92

-------
                          DRAFT—DO NOT QUOTE OR  CITE






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 interleukin-l|J, 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




                                      2-13                             08/14/92

-------
                           DRAFT—DO NOT QUOTE OR CITE






 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









                                     2-14                              08/14/92

-------
                          DRAFT—DO  NOT  QUOTE OR  CITE






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








                                     2-15                             08/14/92

-------
                          DRAFT—DO NOT QUOTE OR CITE






 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









                                     2-16                              08/14/92

-------
                          DRAFT—DO NOT QUOTE  OR CITE






be the establishment of  experimental  systems  in which such complex biological



phenomena are amenable to study at the molecular level.



2.8.   REFERENCES



Bailar, J.C.,  III.   (1991)   How  dangerous is  dioxin?  N. Eng.  J.  Med. 324:




260-262.








Bjeldanes,  L.F.,  J.Y.  Kim, K.R.  Grose, J.C.  Bartholomew  and C.A. Bradfield.



1991.   Aromatic  hydrocarbon  responsiveness-receptor  agonists  generated from



indole-3-carbinol in vitro  and in vivo: Comparisons with  2,3,7,8-tetrachloro-



dibenzo-p-dioxin.  Proc.  Natl. Acad.  Sci., USA.  88: 9643-9547.








Bradfield,  C.A.,  E. Glover  and A. Poland.   1991.  Purification and N-terminal



amino acid sequence of the Ah  receptor from the C57BL/6J mouse. Mol. Pharmacol.




39:  13-19.








Burbach, K.M., A. Poland and C.A.  Bradfield.   1992.   Cloning of the Ah-receptor



cDNA reveals  a  novel ligand activated transcription  factor.   Proc. Natl. Acad.




Sci. USA.   (In  press)








Carrier,  F.,  R.A. Owens,  D.W. Nebert  and  A. Puga.   1992.  Dioxin-dependent



activation  of  murine  Cypla-1  gene  transcription  requires protein  kinase



C-dependent phosphorylation.   Mol.  Cell. Biol.   12:  1856-1863.








Choi,  E.J., D.G. Toscano,  J.A. Ryan, N.  Riedel and W.A.  Toscano, Jr.  1991.



Dioxin induces transforming growth factor-alpha in human keratinocytes.  J. Biol.




Chem.   266: 9591-9597.








Clark,  A.J.   1933.   The Mode of Action of Drugs on  Cells.  E. Arnold  and Co.,




London.
                                      2-17                              08/14/92

-------
                           DRAFT—DO NOT QUOTE OR CITE

 Cohen, G.M., W.M. Bracken, R.P. Iyer, D.L. Berry, J.K. Selkirk and T.J. Slaga.
 1979.    Anticarcinogenic  effects   of  2,3,7,8-tetrachlorodibenzo-p-dioxin  on
 benzo(a)pyrene  and  7,12-dimethylbenz(a)anthracene  tumor  initiation  and  its
 relationship to DNA binding.  Cancer Res.  39: 4027-4033.

 Cook, J.C. and  W.F.  Greenlee.   1989.  Characterization  of  a  specific binding
 protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin in human thymic epithelial cells.
 Mol.  Pharmacol.  35:  713-719.


 Couture,  L.A.,  B.D.  Abbott and L.S. Birnbaum.  1990.   A critical review of the
 developmental toxicity and teratogenicity of 2,3, 7,8-tetrachlorodibenzo-p-dioxin:
 recent advances toward understanding the  mechanism.   Teratology.   42: 619-627.

 Cuthill,  s.,  A. Wilhelmsson and L.  Poellinger.   1991.   Role of the  ligand  in
 intracellular receptor function: Receptor affinity determines activation in vitro
 of  the latent  dioxin  receptor to  a  DNA-binding  form.    Mol.  Cell Biol.   11:
 401-411.


 Czuczwa,  J.M.,  B.D.  McVeety and R.A. Kites.   1984.   Polychlorinated dibenzo-
 p-dioxins  and dibenzofurans  in sediments  from Siskicoit  Lake,  Isle  Royale.
 Science.   226:  568-569.

 Denison,  M.S.  and E.F.  Yao.    1991.   Characterization of  the  interaction of
 transformed  rat  hepatic  cytosolic  Ah  receptor with  a   dioxin  responsive
 transcriptional enhancer.  Arch. Biochem. Biophys.  284:  158-166.

Denison, M.S., L.M. Vella and A.B. Okey.  1987.  Structure and function of the
Ah receptor:   sulfhydryl groups required for binding of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin  to  cytosolic  receptor  from rodent  livers.   Arch.  Biochem.
Biophys.  252: 388-395.
                                     2-18                             08/14/92

-------
                          DRAFT—DO NOT  QUOTE  OR CITE






Denison, M.S., J.M.  Fisher and J.P. Whitlock, Jr.   1989.   Protein-DNA inter-



actions at recognition sites for the dioxin-Ah receptor complex.   J. Biol. Chem.




264: 16478-16482.








DiGiovanni, J., D.L.  Berry,  G.L.  Gleason,  G.S.  Kishore and T.J. Slaga.  1980.



Time-dependent  inhibition  by  2,3,7,8-tetrachloro-dibenzo-p-dioxin  of  skin



tumorigenesis with polycyclic hydrocarbons.  Cancer Res.  40: 1580-1587.








Dunn, T.J., R. Lindahl and  H.C. Pitot.  1988.  Differential gene expression in



response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).   J.  Biol.  Chem.  263:




10878-10886.








Durrin, L.K. and J.P.  Whitlock, Jr.  1989.   2,3,7,8-Tetrachlorodibenzo-p-dioxin:



Ah  receptor-mediated change  in  cytochrome Pj-450  chromatin  structure occurs




independent of transcription.  Mol. Cell Biol.   9:  5733-5737.








Elferink, C.J. and J.P. Whitlock, Jr.  1990.  2,3,7,8-Tetrachlorodibenzo-p-dioxin



inducible, Ah receptor-mediated bending of enhancer DNA.  J. Biol. Chem.  265:




5718-5721.








Elferink,  C.J.,   T.A.  Gasiewicz  and  J.P.  Whitlock,  Jr.   1990.   Protein-DNA



interactions  at  a dioxin-responsive enhancer: Evidence that the transformed Ah



receptor is heteromeric.   J.  Biol.  Chem.   265:  20708-20712.








Ema, M.,  K.  Sogawa,  N. Wanatabe  et al.  1992.   cDNA cloning and structure of



mouse putative Ah receptor.   Biochem. Biophys.  Res. Commun.  184: 246-253.








Favreau, L.V. and C.B.  Pickett.   1991.   Transcriptional regulation of  the rat




NAD(P)H: Quinone  reductase gene.   J.  Biol. Chem.   266: 4556-4561.
                                      2-19                              08/14/92

-------
                          DRAFT—DO  NOT QUOTE OR CITE






Fiering, S., J.P.  Northrup,  G.P.  Nolan,  P.S. Mattila, G.R.  Crabtree and L.A.



Herzenberg.   1990.   Single cell assay  of a  transcription factor  reveals a



threshold in transcription activated by signals emanating from the T-cell antigen



receptor.  Genes Dev.  4: 1823-1834.








Fisher, J.M., K.W. Jones and J.P.  Whitlock, Jr.  1989. Activation of transcrip-



tion  as a general  mechanism of  2,3,7,8-tetrachloro-dibenzo-p-dioxin action.



Molec. Carcinogen.  1: 216-221.








Fisher, J.M., L.  Wu, M.S.  Denison and J.P. Whitlock,  Jr.  1990. Organization and



function of a dioxin-responsive enhancer.  J. Biol.  Chem.   265: 9676-9681.








Fujisawa-Sehara,  A.,  K.  Sogawa,  M.  Yamane  and  Y.  Fujii-Kuriyama.   1987.



Characterization  of  xenobiotic responsive  elements  upstream  from  the  drug-



metabolizing cytochrome P450c gene: A similarity  to glucocorticoid regulatory



elements.  Nucleic Acids Res.  15: 4179-4191.








Gasiewicz, T.A.  and T.A.  Bauman.   1987.   Heterogeneity of  the rat hepatic Ah



receptor and evidence for transformation in  vitro and in  vivo.   J. Biol. Chem



262: 2116-2120.








Gasiewicz,  T.A.,  C.J.  Elferink  and  E.G.  Henry.    1991.  Characterization of



multiple forms of the Ah receptor: Recognition of  a dioxin-responsive enhancer



involves heteromer formation.  Biochemistry.   30:  2909-2916.








Gillner, M., J. Bergman, C.  Cambillan, B. Fernstrom  and J.A. Gustafsson.  1985.



Interactions of  indoles with specific binding sites  for  2,3,7,8-tetrachloro-



dibenzo-p-dioxin in rat liver.  Mol. Pharmacol.  28: 357-363.








Gillner, M., J. Bergman, C.  Cambillau  and J.A. Gustafsson.  1989.   Interactions



of rutaecarpine alkaloids with specific binding sites for 2,3,7,8-tetrachloro-



dibenzo-p-dioxin in rat liver.  Carcinogenesis.  10: 651-654.








                                     2-20                             08/14/92

-------
                         DRAFT—DO NOT QUOTE OR CITE






Hankinson, O.  1979.  Single-step selection of clones of a mouse hepatoma cell



line deficient in aryl hydrocarbon hydroxylase.  Proc. Natl. Acad. Sci.  USA.  76:




373-376.







Hankinson,  O.   1983.   Dominant  and recessive aryl  hydrocarbon hydroxylase-



deficient mutants  of the mouse hepatoma  line,  Hepa 1,  and  assignment of the



recessive mutants  to three  complementation groups.   Somat. Cell  Genet.   9:




497-514.







Hapgood,  J.,  S.  Cuthill, M. Denis,  L. Poellinger  and J.A. Gustafsson.  1989.



Specific protein-DNA interactions at a xenobiotic-responsive element:  Copurifica-



tion of dioxin receptor  and  DNA-binding activity.   Proc. Natl.  Acad.  Sci. USA.




86: 60-64.







Harris, M.,  J.  Piskorska-Pliszczynska,  T. Zacharewski, M. Romkes and  S. Safe.



1989.   Structure-dependent induction of aryl hydrocarbon hydroxylase  in human



breast  cancer cell lines and characterization of the  Ah receptor.   Cancer Res.




49: 4531-4545.







Harris, M.,  T. Zacharewski and S. Safe.  1990.   Effects of 2,3,7,8-tetrachloro-



dibenzo-p-dioxin and related compounds on  the occupied nuclear estrogen receptor




in MCF-7  human breast  cancer cells.   Cancer Res.   50:  3579-3584.








Henry,  E.G., G. Rucci  and  T.A.  Gasiewicz.  1989.   Characterization  of multiple



 forms  of  the Ah receptor: Comparison of species and tissues.   Biochemistry.  28:




 6430-6440.







 Hoffman,  E.G.,  H.  Reyes, F.-F.  Chu et al.  1991.   Cloning of a factor required



 for activity of  the Ah (dioxin)  receptor.  Science.  252:  954-958.








 Jaiswal,  A.K.,  O.W. McBride, M.  Adesnik and D.W.  Nebert.  1988.  Human dioxin-



 inducible cytosolic NAD(P)H:  menadione  oxidoreductase.   J.  Biol.  Chem.   263:




 13572-13578.



                                      2-21                             08/14/92

-------
                          DRAFT—DO NOT QUOTE OR CITE






 Jones, K.W. and J.P. Whitlock,  Jr.  1990.  Functional analysis of the transcrip-



 tional promoter  for the CYP1A1 gene.  Mol. Cell. Biol.  10:  5098-5105.








 Jones, P.B.C., L.K. Durrin, D.R.  Galeazzi and J.P. Whitlock, Jr.  1986.   Control




 of  cytochrome  Pj-450 gene  expression: Analysis of a dioxin-responsive enhancer




 system.  Proc. Natl. Acad.  Sci.  USA.  83: 2802-2806.








 Kadonaga, J.T. and R. Tjian.  1986.   Affinity purification of  sequence-specific



 DNA-binding proteins.  Proc. Natl. Acad. Sci. USA.  83: 5889-5893.








 Karenlampi,  S.O.,  C. Legraverend, J.  Gudas,  N. Carramanzo  and O. Hankinson.



 1988.  A third genetic  locus affecting the Ah (dioxin)  receptor.  J.  Biol. Chem.



 263: 10111-10117.








 Kester,  J.E.  and  T.A.  Gasiewicz.    1987.   Characterization of the  in vitro



 stability of the rat hepatic  receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin



 (TCDD) .  Arch. Biochem. Biophys.  252: 606-625.








Landers, J.P. and N.J. Bunce.  1991.   The Ah receptor and the mechanism of dioxin



toxicity.  Biochem. J.  276: 273-287.








Landers, J.P.,  J. Piskorska-Pliszczynska, Jr., T. Zacharewski, N.J.  Bunce and S.



Safe.  1989.   Photoaffinity labeling  of the nuclear Ah receptor  from mouse Hepa



Iclc7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin.  J. Biol. Chem.  264:




18463-18471.








Limbird, L.E.   1986.  Cell Surface Receptors:  A  short course  on Theory and



Methods.  Martinus Niihoff, Boston.








Lorenzen, A.  and  A.B. Okey.  1991. Detection and characterization of Ah receptor



in tissue and cells  from human tonsils.  Toxicol. Appl.  Pharmacol. 107: 203-214.
                                     2-22                             08/14/92

-------
                          DRAFT—DO  NOT  QUOTE  OR CITE






Lucier, G.W.,  A.  Tritscher, T. Goldsworthy et  al.   1991.   Ovarian hormones



enhance TCDD-mediated increases  in cell  proliferation and preneoplastic foci in



a two stage model for rat hepatocarcinogenesis.  Cancer Res.  51: 1391-.








Lui, F.H.,  G.  Clark, L.S.  Birnbaum,  G.W.  Lucier  and  J.A. Goldstein.   1991.



Influence of the Ah locus on the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin



on the hepatic  epidermal growth factor receptor.  Molec.  Pharmacol. 39: 307-313.








Miller, A.G. and  J.P.  Whitlock,  Jr.  1981.   Novel variants in benzo(a)pyrene




metabolism.  J. Biol. Chem.  256:  2433-2437.








Miller, A.G., D.I. Israel and J.P. Whitlock, Jr.  1983.  Biochemical and genetic



analysis  of  variant mouse hepatoma cells  defective  in the  induction  of



benzo(a)pyrene-metabolizing  enzyme  activity.  J. Biol.  Chem.  258: 3523-3527.








Mocarelli, P., L.L. Needham, A.  Marocchi et al.   1991.  Serum concentrations of



2,3,7,8-tetrachlorodibenzo-p-dioxin and test results from  selected residents of




Seveso, Italy.  J. Toxicol.  Environ.  Health.  32:  357-366.








Nebert, D.W.   1989.  The Ah locus:  Genetic  differences  in toxicity, cancer,



mutation, and  birth defects.  Grit. Rev.  Toxicol.   20: 153-174.








Nebert, D.W.  and W.W.  Weber.  1990.   Pharmacogenetics,  in Principles of Drug



Action, W.B. Pratt and P. Taylor, Ed.  Churchill Livingstone, New York.








Nemoto, T., G.G.  Mason,  A.  Wilhelmsson et al.   1990.  Activation  of  the dioxin



and glucocorticoid receptors to a DNA binding state under  cell-free conditions.




J. Biol. Chem.  265: 2269-2277.








Neuhold,  L.A., F.J.  Gonzalez,  A.K.  Jaiswal and D.W. Nebert.   1986.  Dioxin-



inducible enhancer  region upstream  from the mouse Pl-450 gene  and interaction




with a heterologous SV40 promoter.  DNA.   5:  403-411.








                                      2-23                            08/14/92

-------
                          DRAFT—DO NOT QUOTE OR CITE






Neuhold,  L.A.,  Y. Shirayoshi,  K.  Ozato, J.E.  Jones and D.W.  Nebert.    1989.



Regulation of mouse  CYP1A1  gene expression  by dioxin:  Requirement of two cis-



acting elements during induction.  Mol. Cell. Biol.  9: 2378-2386.








Okino, S.T., U.R. Pendurthi and R.H. Tukey.  1992.  Phorbol esters inhibit the



dioxin receptor-mediated  transcriptional activation of the  mouse Cypla-1 and



Cypla-2  genes  by 2,3,7,8-tetrachlorodibenzo-p-dioxin.   J.  Biol.  Chem.   267:



6991-6998.








Parascandola, J.  1980.  Origin of the receptor theory.  Trends in  Pharmacol.



Sci.  1:  189-192.








Perdew, G.H.  1988.  Association of the Ah receptor with the 90-kDa heat  shock



protein.  J. Biol. Chem.  263: 13802-13805.








Perdew, G.H.  and C.E. Hollenbeck.  1990.  Analysis  of photoaffinity-labeled aryl



hydrocarbon  receptor heterogeneity  by  two-dimensional gel  electrophoresis.



Biochemistry.  29: 6210-6214.








Poland,  A.  and E. Glover.   1990.   Characterization and  strain distribution




pattern of the  murine Ah receptor specified by the Ahd and Ahb"3 alleles.  Molec.




Pharmacol.  38: 306-312.








Poland, A. and  J.C.  Knutson.    1982.   2,3,7,8-tetrachlorodibenzo-p-dioxin and



related aromatic hydrocarbons: Examination of the mechanism of toxicity.  Ann.



Rev. Pharmacol. Toxicol.  22:  517-554.








Poland, A., W.F.  Greenlee and A. Kende.  1979.   Studies on the mecahnism of




action of the chlorinated dibenzo-p-dioxins and related compounds.  Ann. NY Acad.



Sci.  320: 214-230.
                                     2-24                             08/14/92

-------
                          DRAFT—DO NOT  QUOTE  OR CITE






Poland, A., D. Palen and E. Glover.  1982.  Tumor promotion by TCDD in skin of



HRS/J hairless mice.  Nature.  300: 271-273.








Poland, A., E. Glover, F.H. Ebetino and A.S. Kende.  1986.  Photoaffinity labeling



of the Ah receptor.  J. Biol. Chem.  261: 6352-6365.








Poland, A., E. Glover and C.A. Bradfield.  1991.  Characterization of polyclonal



antibodies to the Ah receptor prepared by immunization with a synthetic peptide



hapten.  Molec. Pharmacol.  39: 20-26.








Pongratz, I., P.E.  Stromstedt, G.G.F. Mason and L. Poellinger.  1991.  Inhibition



of  the specific DNA  binding activity  of  the dioxin  receptor  by phosphatase



treatment.  J. Biol. Chem.  266:  16813-16817.








Quattrochi,  L.C.  and  R.H.  Tukey.   1989.   The human cytochrome  CyplA2 gene



contains regulatory elements responsive to 3-methylcholanthrene. Mol. Pharmacol.




36: 66-71.








Rannung, A.,  U.  Rannung,  H.S.  Rosenkratz et al.  1987.  Certain photooxidized



derivatives of tryptophan bind with very high affinity  to  the Ah receptor and are



likely to  be  endogenous  signal substances.  J.  Biol.  Chem.  262: 15422-15427.








Roberts, E.A., K.C. Johnson, P.A.  Harper and A.B. Okey.  1990. Characterization



of the Ah receptor mediating aryl  hydrocarbon hydroxylase induction in the human



liver  cell line HepG2.   Arch. Biochem.  Biophys.  276:  442-450.








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-hexachlorodibenzofuran treatment of pregnant




mice.  Toxicol. Appl.  Pharmacol.  98, 454-464.
                                      2-25                              08/14/92

-------
                           DRAFT—DO NOT QUOTE OR CITE

 Saatcioglu, F., D.J. Perry, D.S.  Pasco  and J.B. Fagan.  1990a.  Aryl hydrocarbon
 (Ah) receptor DNA-binding activity.  Sequence specificity and Zn2 + requirement.
 J. Biol. Chetn.  265: 9251-9258.

 Saatcioglu, F., D.J. Perry, D.S.  Pasco and  J.B.  Fagan.   1990b.   Multiple DNA-
 binding factors interact with  overlapping specificities at the aryl hydrocarbon
 response  element  of  the  cytochrome  P450IA1  gene.   Mol.  Cell.  Biol.   10:
 6408-6416.

 Safe, S.H.   1986.  Comparative toxicology and mechanism of action of polychlori-
 nated dibenzo-p-dioxins and dibenzofurans.   Ann.  Rev. Pharmacol.  Toxicol.  26:
 371-399.

 Shen, E.S.  and J.P. Whitlock, Jr.   .1989. The potential role of DNA methylation
 in the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.   J. Biol.  Chem.   264:
 17754-17758.

 Shen, E.S.  and J.P. Whitlock, Jr.   1992.  Protein-DNA interactions at  a dioxin-
 responsive  enhancer: Mutational analysis of the DNA binding site for the liganded
 Ah receptor.  J. Biol.  Chem.   267:  6815-6819.

 Silbergeld,  E.K. and T.A. Gasiewicz.  1989.   Dioxins  and the Ah receptor.  Am.
 J.  Ind. Med.  16:  455-474.

 Skene, S.A., I.e. Dewhurst and  M.  Greenberg.   1989.  Polychlorinated dibenzo-p-
 dioxins and polychlorinated dibenzofurans: The risks to human health.   A review.
 Hum.  Toxicol.  8:  173-203.

 Spink, D.C., D.C. Lincoln,  II,  H.W. Dickerman and J.F.  Gierthy.  1990.   2,3,7,8-
 Tetrachloro-dibenzo-p-dioxin causes  an  extensive  alteration  of  170-estradiol
metabolism  in  MCF-7 breast  tumor cells.   Proc.  Natl. Acad.  Sci. USA.   87:
 6917-6921.


                                     2-26                             08/14/92

-------
                         DRAFT—DO NOT QUOTE OR CITE






Stephenson, R.P.   1956.   A modification  of  the  receptor  theory.   Brit.  J.




Pharmacol.  11: 379-393.








Sunahara, G.I.,  G.W.  Lucier, Z. McCoy,  E.H.  Bresnick, E.R.  Sanchez and K.G.



Nelson.  1989.  Characterization  of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated



decreases  in  dexamethasone  binding  to  rat hepatic  cytosolic glucocorticoid



receptor.  Molec. Pharmacol.  36: 239-247.








Sutter, T.R.,  K.  Guzman, K.M. Dold and W.F.  Greenlee.  1991.  Targets for dioxin:



Genes for plasminogen activator  inhibitor-2 and interleukin-10. Science.  254:




415-418.








Telakowski-Hopkins,  C.A.,  R.G.  King and  C.B. Pickett.    1988.    Glutathione



S-transferase Ya subunit gene: Identification of regulatory elements required for



basal level and  inducible  expression.   Proc.  Natl. Acad.  Sci. USA.  85: 1000-




1004.







Umbreit,  T.H.  and M.A. Gallo.  1988.   Physiological  implications  of estrogen



receptor modulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.  Toxicol. Lett.  42:




5-14.








Waithe, W.I., M. Michand, P.A. Harper, A.B.  Okey and A. Anderson.  1991.   The Ah



receptor, cytochrome P450IA1 mRNA induction, and aryl hydrocarbon hydroxylase in



a human  lymphoblastoid cell  line.  Biochem. Pharmacol.  41: 85-92.








Watson, A.J. and O. Hankinson.   1988.  DNA transfection of a gene repressing aryl



hydrocarbon hydroxylase induction.   Carcinogenesis.  9: 1581-1586.








Wen,  L.P., N.  Koeiman and  J.P.  Whitlock, Jr.    1990.   Dioxin-inducible,  Ah



receptor-dependent transcription  in vitro.  Proc. Natl.  Acad. Sci. USA.  87:




8545-8549.
                                      2-27                              08/14/92

-------
                          DRAFT—DO NOT QUOTE OR CITE

Whitlock, J.P., Jr. 1990.  Genetic and molecular aspects of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin action.  Ann. Rev. Pharmacol. Toxicol.   30: 251-277.

Wilhelmsson, A., S. Cuthill,  M.  Denis,  A.C.  Wikstrom,  J.-A. Gustafsson and L.
Poellinger.  1990.   The specific DNA binding  activity of the dioxin receptor is
modulated by the 90 kd heat shock protein.  EMBO J.  9: 69-76.

Wu, L. and J.P. Whitlock, Jr.  1992.  Mechanism of dioxin action: Ah receptor-
mediated increase in promoter accessibility in vivo.  Proc.  Natl. Acad. Sci. USA.
89: 4811-4815.
                                    2-28                              08/14/92

-------

-------

-------

-------
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268

Official Business
Penalty for Private Use
$300
Please make all necessary changes on the below label,
detach or copy, and return to the address In the upper
left-hand comer.

If you do not wish to receive these reports CHECK HERE D;
detach, or copy this cover, and return to the address In the
upper left-hand comer.
      BULK RATE
POSTAGE & FEES PAID
          EPA
   PERMIT No. G-35
EPA/600/AP-92/001b

-------