[Dioxin-l] Reply to Jon

[Oram, Louise]

Jon
Thanks so much to Louise for guiding us in this endeavor, even though she
and Dr. David Bell (who were apparently colleagues in the UK) did so quite
inadvertently. This short paper makes clear the molecular nature of the Ah
Receptor site.

>This is a useful link. But as you will probably find from reading it, Mark
tends to generalise and over-simplify, for the general reader. His papers
are a little more hardcore, some of the chemistry goes straight over my
head. And as for you finding this page inadvertently. You could have asked
if I had a web page, or any useful links. (my web page isn't really that
useful, as I never finished it) I've never hidden who I was and who I work
for.  And I wasn't that hard to find, was I?

Notice the names in the reference list of Denison's (below) mechanism of
action; Okay, Hankinson, Brad field, Safe. And make the connection to the
references I recommended you read. 


    Note that as I had originally hypothesized, a single molecule of TCDD
can bind to the Ah receptor and the bound complex is then transmitted to
nuclear DNA where it begins to disrupt the normal functioning of the cell.
My original sources of the hypothesis were popularized versions of the
analysis below.


>Jon, to be correct.  Your theory was peppered with such poor understanding
of basic cell biology that it was, when taken as a whole, nonsense. To say
you have found validation for some small part (?) is a bizarre assumption.
And I corrected you because you were way off base. The issues I have been
addressing with you are;  not whether dioxin binds, if it didn't bind we
wouldn't be exchanging any emails, or if in fact that it has a downstream
effect. The issues were; how much ligand needs to bind to how many receptors
to see a response? Is there a linear dose response. For which the data is
poor, at best. And finally, is there therefore a threshold dose? These are
more relevant to risk assessment which seems to be the underlying theme of
this list (i.e how much is too much?) Now you have your thinking hat on
don't just jump on the nearest available explanation. Ask yourself the basic
questions and go and look for the data. But you seem to be on the right
track, FINALLY!!


Louise



MOLECULAR MECHANISM OF DIOXIN ACTION

Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated
dibenzo-p-dioxins, biphenyls, dibenzofurans, and related compounds represent
a diverse group of persistent, widespread environmental contaminants.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin), the most
biologically-active and toxic member of this class of compounds produces a
wide variety of species- and tissue-specific effects (1,2) including: tumor
promotion, immuno- hepato- and dermal toxicity, lethality, birth defects,
endocrine disruption and induction of numerous enzymes, most notably that of
microsomal cytochrome P4501A1 and its associated monooxygenase activity,
aryl hydrocarbon hydroxylase (AHH) (1,2). The P4501A1 isozyme contributes to
the metabolic activation and detoxification of polycyclic aromatic
hydrocarbons, many of which are carcinogens (3). 

The induction of hepatic AHH activity is perhaps the best studied of the
biochemical effects resulting from exposure to TCDD and related HAHs (4,5).
Early experiments examining the induction of AHH activity by a series of
halogenated dibenzo-p-dioxin and dibenzofuran congeners resulted in the
identification of a specific receptor which bound these compounds saturably
and with high affinity. Qualitative structure-activity relationship studies
revealed that the ability of a compound to bind to this receptor was well
correlated not only with its ability to induce AHH activity (1,2,6) but also
its ability to induce toxic effects, such as thymic involution, wasting and
epidermal keratinization (1,2,6,7), suggesting that the receptor also
mediates the toxicity of these compounds. This TCDD receptor has been
identified and characterized in a wide variety of species and tissues (8,9)
and has been designated as the aromatic hydrocarbon receptor (AhR). 

Induction of hepatic P450IA1-dependent AHH activity has been utilized as a
model system to examine the molecular mechanism of action of HAHs. The
current model for the AhR-dependent P4501A1 induction mechanism (above) is
in several ways similar to that described for some steroid hormone receptors
and steroid-responsive genes (10,11). The unliganded AhR complex exists in
the cytosol complexed with at least three additional proteins (12).
Following high affinity ligand (TCDD) binding, the TCDD:AhR complex
undergoes a poorly defined process of transformation, during which hsp90 (a
heat shock protein of 90 kDa) and other proteins appear to dissociate from
the TCDD:AhR complex, the AhR complex acquires the ability to bind to DNA
with high affinity and transformed TCDD:AhR complexes subsequently
accumulate within the nucleus (12-16). High affinity DNA binding of the AhR
complex appears to require its association with at least one additional
protein, the Ah receptor nuclear translocator (ARNT) protein (17-20). The
binding of these transformed heteromeric TCDD:AhR complexes to specific DNA
sequences (Dioxin Responsive Elements (DREs)) adjacent to the cytochrome
P4501A1 (CYP1A1) gene leads to DNA bending, chromatin disruption, increased
promoter accessibility and increased rates of transcription initiation of
the CYP1A1 gene with the subsequent accumulation of cytochrome
P450IA1-specific mRNA (4,21-27). The presence of the AhR complex in a wide
variety of species and tissues and with its ability to act as a
ligand-dependent transactivator of gene expression suggests that many of the
toxic and biological effects of HAHs result from differential alteration of
gene expression in susceptible cells. 


 REFERENCES 
1. Poland,A. and Knutson,J.C. (1982) 2,3,7,8-tetrachlorodibenzo-p-dioxin and
related halogenated aromatic hydrocarbons: examination of the mechanism of
toxicity, Ann. Rev. Pharmacol. Toxicol. 22, 517-554. 

2. Safe,S.H. (1986) Comparative toxicology and mechanism of action of
polychlorinated dibenzo-p-dioxins and dibenzofurans, Ann. Rev. Pharmacol.
Toxicol. 26, 371-399. 

3. Gelboin,H.V. (1980) Benzo[a]pyrene metabolism, activation and
carcinogenesis: role and regulation of mixed-function oxidases and related
enzymes, Physiol. Rev. 60, 1107-1166. 

4. Whitlock,J.P. (1986) The regulation of cytochrome P-450 gene expression,
Ann. Rev. Pharmacol. Toxicol. 26, 333-369. 

5. Whitlock,J.P.,Jr. (1990) Genetic and Molecular Aspects of
2,3,7,8-tetrachlorodibenzo-p-dioxin action, Ann. Rev. Pharmacol. Toxicol.
30, 251-277. 

6. Goldstein,J.A. and Safe,S. (1989) Mechanism of action and
structure-activity relationships for the chlorinated dibenzo-p-dioxins and
related compounds, in: Halogenated Biphenyls, Terphenyls, Naphthalenes,
Dibenzodioxins and Related Products (Kimbrough,R.D. and Jensen,J., eds), pp.
239-293, Elsevier Science Pub, Amsterdam. 

7. Knutson,J.C. and Poland,A. (1980) Keratinization of mouse teratoma cell
line XB produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin: an in vitro model
of toxicity, Cell 22, 27-36. 

8. Denison,M.S. and Wilkinson,C.F. (1985) Identification of the Ah receptor
in selected mammalian species and induction of aryl hydrocarbon hydroxylase,
Eur. J. Biochem. 147, 439-435. 

9. Denison,M.S., Wilkinson,C.F. and Okey,A.B. (1986) Ah receptor for
2,3,7,8-tetrachlorodibenzo-p-dioxin: comparative studies in mammalian and
nonmammalian species, Chemosphere 15, 1665-1672. 

10. Yamamoto,K.R. (1985) Steroid receptor regulated transcription of
specific genes and gene networks, Ann. Rev. Genet. 19, 209-252. 

11. Carson-Jurica,M.A., Schrader,W.T. and O'Malley,B.M. (1990) Steroid
receptor family: structure and functions, Endo. Rev. 11, 201-214. 

12. Perdew,G.H. (1992) Chemical cross-linking of the cytosolic and nuclear
forms of the Ah receptor in hepatoma line 1c1c7, Biochem. Biophys. Res.
Comm. 182, 55-62. 

13. Perdew,G.H. and Poland,A. (1988) Purification of the Ah receptor from
C57BL/6J mouse liver, J. Biol. Chem., 263, 9848-9852. 

14. Henry,E.C., Rucci,G. and Gasiewicz,T.A. (1989) Characterization of
multiple forms of the Ah receptor: comparison of species and tissues,
Biochem. 28, 6430-6440. 

15. Whitlock,J.P., Jr. and Galeazzi,D.R. (1984)
2,3,7,8-Tetrachlorodibenzo-p-dioxin receptors in mouse hepatoma cells:
nuclear location and strength of nuclear binding, J. Biol. Chem. 259,
980-985. 

16. Henry,E.C. and Gasiewicz,T.A. (1993) Transformation of the aryl
hydrocarbon receptor to a DNA-binding form is accompanied by release of the
90 kDa heat-shock protein and increased affinity for
2,3,7,8-tetrachlorodibenzo-p-dioxin, Biochem. J. 294, 95-101. 

17. Dolwick,K.M., Schmidt,J.V., Carver,L.A., Swanson,H.I. and Bradfield,C.A.
(1993) Cloning and expression of human Ah receptor cDNA, Molec. Pharm. 44,
911-917. 

18. Whitelaw,M., Pongratz,I., Wilhelmsson,A., Gustafsson,J-A. and
Poellinger,L. (1993) Ligand-dependent recruitment of the arnt coregulator
determines DNA recognition by the dioxin receptor, Molec. Cell. Biol. 13,
2504-2514. 

19. Probst,M.R., Reisz-Porszasz,S., Agbunag,R.V., Ong,M.S. and Hankinson,O.,
(1993) Role of the aryl hydrocarbon receptor nuclear translocator protein in
aryl hydrocarbon (dioxin) receptor action, Molec. Pharmacol. 44, 511-518. 

20. Hoffman,E.C., Reyes,H., Chu,F-F., Sander,F., Conley,L.H., Brooks,B.A.
and Hankinson,O. (1991) Cloning of a factor required for activity of the Ah
(dioxin) receptor, Science 252, 954-958. 

21. Denison,M.S., Fisher,J.M. and Whitlock,J.P.,Jr. (1988) Inducible,
receptor-dependent protein-DNA interactions at a dioxin-responsive
transcriptional enhancer, Proc. Natl. Acad. Sci. 85, 2528-2532. 

22. Elferink,C.J. and Whitlock,J.P.,Jr. (1990)
2,3,7,8-Tetrachlorodibenzo-p-dioxin-inducible, Ah receptor-mediated bending
of enhancer DNA, J. Biol. Chem. 265, 5718-5721. 

23. Morgan,J. E. and Whitlock,J.P.,Jr. (1992) Transcription-dependent and
transcription-independent nucleosome disruption induced by dioxin, Proc.
Natl. Acad. Sci. USA 89,11622-11626. 

24. Durrin,L.K. and Whitlock,J.P.,Jr. (1989)
2,3,7,8-Tetrachlorodibenzo-p-dioxin-inducible aryl hydrocarbon
receptor-mediated change in CYP1A1 chromatin structure occurs independent of
transcription, Mol.Cell. Biol. 9:5733-5737. 

25. Wu,L. and Whitlock,J.P.Jr., (1992) Mechanism of dioxin action: Ah
receptor-mediated increase in Promoter accessibility in vivo, Proc. Natl.
Acad. Sci. 89, 4811-4815. 

26. Denison,M.S., Fisher,J.M. and Whitlock,J.P.,Jr. (1988) The DNA
recognition site for the dioxin-Ah receptor complex: nucleotide sequence and
functional analysis, J. Biol. Chem. 263, 17221-17224. 

27. Denison,M.S., Fisher,J.M. and Whitlock,J.P.,Jr. (1989) Protein-DNA
interactions at recognition sites for the dioxin-Ah receptor complex, J.
Biol. Chem. 264, 16478-16482. 



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