[Dioxin-l] molecular mechanism of dioxin action

[Tony Tweedale]

in case anyone was wondering, the below summary was written by the below.
---
>     Below is a short paper posted by dioxin researchers at UC Davis. I
>found a link on Louise Oram's university web page, which guided me to the
>article at http://dioxins-r-us.ucdavis.edu/Dioxin.HTML 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.
>
>Regards
>Jon Campbell
>
>MOLECULAR MECHANISM OF DIOXIN ACTION
>
>Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated
[SNIP]
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   Michael S. Denison, Professor of Environmental Toxicology
AREAS OF RESEARCH INTEREST
1. Molecular mechanisms of gene expression.
2. Molecular mechanisms of action of halogenated aromatic hydrocarbons.
3. Structure and function of receptors for hormones and xenobiotics.

	Dioxin Mechanism Information
Research in our lab is focused on two specific areas:

I. Examination of the specific interaction of the TCDD:AhR complex with DNA
and the relationship of this interaction to alteration of gene expression.
It is in this area that my laboratory has made the greatest progress. We
were the first laboratory to successfully develop an in vitro procedure for
transformation of the AhR complex into its high affinity DNA binding form
(faithfully mimicking that which occurs in vivo).  This procedure, now
extensively used by many investigators, has been utilized extensively by
our lab to characterize the biochemical properties of the transformed
TCDD:AhR complex and to examine its sequence-specific DNA binding.  Other
significant contributions from our laboratory in this area come from recent
results which have demonstrated the presence of multiple forms of the AhR
as well as the additional AhR subunits.  These results have some very
significant implications, especially relative to new approaches to studying
species- and tissue-specific differences in AhR action and
TCDD-responsiveness.

II. Development of novel and rapid bioassay systems for detection of HAHs
in environmental and biological samples.
The presence of HAHs in environmental samples as complex mixtures has made
it difficult to accurately predict their biological and toxic potency in
the animals at risk. We carried out to develop novel bioassay systems for
toxic HAHs.  Our most significant contribution has been the recent
development of novel recombinant species-specific cell bioassays for
detection and relative quantitation of bioactive HAHs in environmental and
biological extracts. We have utilized aspects from our work on the
molecular mechanism of HAH action to prepared a recombinant expression
plasmid which responds to HAHs with the induction of easily measurable
enzyme activity (alkaline phosphatase or firefly luciferase).  Our
resulting bioassay system is quite simple in that exposure of the
recombinant cells to any sample extract which contains bioactive HAHs will
increase the amount of reporter enzyme.  There is a significant amount of
interest in use of our cell lines for both commercial screening
applications and biochemical analysis as well as in the identification and
characterization of novel xenobiotic and endobiotic ligands. This
technology is being commercially exploited by a North Carolina-based
biotechnology company called Xenobiotic Detection Systems, Inc. (XDS).
(http://www.dioxins.com/)

Dioxin Analysis by         Xenobiotic Detection Systems, Inc.

   Dioxins are one of the most toxic man-made compounds known and like the
PCBs and dibenzofurans that they are related to, they are persistent
contaminants in the environment. Collectively, these groups of compounds
belong to a class of compounds called polychlorinated diaromatic
hydrocarbons (PCDH).  PCDH have been extensively studied and are known to
accumulate in animals causing toxic effects. Birth defects, immunotoxicity,
tumor production, changes in metabolism and even death have all been
observed as a result of exposure to PCDH.

The mechanism of action of these compounds has been extensively studied
over the past twenty years. These compounds bind to an intracellular
receptor called the aryl hydrocarbon receptor (Ah Receptor) and activate
the receptor. The PCDH-Ah Receptor complex then travels to the nucleus of
the cell and binds to specific sequences in the DNA called dioxin
responsive elements (DRE). The binding of the PCDH-Ah Receptor complex to
the DRE causes the expression of the associated genes to be altered. It is
this alteration in gene expression that causes the toxic effects that are
observed.

                            Figure describing the mechanism of action of PCDHs

XDS has used this information to produce a bioassay that detects the
presence of PCDH.  XDS has genetically engineered mammalian cell lines to
contain a firefly gene as a reporter for the presence of PCDH.  In a
nutshell, they have designed a cell that produces luciferase (the enzyme
that makes fireflies light up) when PCDH's interact with the cells'
aryl-hydrocarbon receptors (AhR) - this type of assay is also referred to
as a CALUX (Chemical-Activated LUciferase gene eXpression cell bioassay
system) assay.  Unlike normal cells that produce a wide variety of
responses when exposed to PCDH's, these cells light up.  By measuring the
amount of light generated XDS can tell how much PCDH is present in the test
sample.  These measurements are highly correlated to the
2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ's) that are typically
used to measure dioxin concentration.

                           Figure showing the difference in potency of
different PCDHs in                            activating the Dioxin
Responsive CALUX assay

 This assay is not as delicate as a traditional high resolution
gas-chromatography mass-spectroscopy (GC/MS) assay, but is every bit as
sensitive, able to detect femptograms of dioxin.  Unlike a GC/MS assay, the
fat in biological samples does not disturb the CALUX cells, so the
extraction does not have to be as 'clean' for this type of assay.  By
simplifying the cleanup step, XDS is able to offer both faster turnaround
and a reduced price.  This provides an additional benefit of requiring much
smaller samples than a GC/MS assay - only a couple grams are required.

 Rather than measuring the concentrations of specific congeners of dioxin,
the CALUX assay measures the TOTAL biological response to PCDH's.  This
makes the assay ideal for use both as a research tool and a screening
assay.  For research purposes, this assay is a very cost-effective way to
measure biological responses to specific TEQ's.  As a screening tool, this
assay allows quick and cheap determination of contamination, after which a
high-resolution GC/MS assay could be used to determine specifically which
congeners of dioxin were causing the contamination.

The assay can also be used to determine concentrations of specific classes
of compounds (for instance PCB's or dioxins/dibenzofurans).  By adjusting
the sample clean up methods we can isolate the specific classes of
compounds.  This requires an extra step and could add 2-5 days to the
turnaround time, depending on current demand for XDS's services.

 The assay has been used to analyze milk, sediment, water; ash, air and
extracts from commercial and consumer products.  A study by Aarts et al.,
showed a positive correlation (r=0.71) between the TEQ determined by the
CALUX assay and that determined by GC/MS. There was less than a factor of
two discrepancy between the results for the two analysis methods.

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