Why are dioxins so much different than the other contaminants to measure?
Israel has excellent equipment in the Veterinary Institute for measuring contaminants. Over a hundred possible residues are checked on a regular basis in all products from animals using GC/MS and tandem MS as well as a variety of other analytical techniques. However, the dioxin require a special device called high resolution GC/MS. To date the machine is in the million dollar range and it is difficult to justify its purchase just to measure dioxins. Even with the machine the procedure is expensive and time consuming.
Environmental contaminants in the US are measured using the SW846.
What is SW-846?
The EPA publication SW-846,
entitled Test Methods for Evaluating Solid Waste, Physical/Chemical
Methods, is the official compendium of analytical and sampling methods
that have been evaluated and approved for use in complying with the RCRA (Resource conservation and recovery act) regulations. SW-846 functions primarily as a guidance document setting forth
acceptable, although not required, methods for the regulated and regulatory
communities to use in responding to RCRA-related sampling and analysis
requirements.
SW-846 is a multi-volume document
that changes over time as new information and data are developed. It has been
issued by EPA since 1980 and is currently in its third edition. Advances in
analytical instrumentation and techniques are continually reviewed by EPA and
incorporated into periodic updates to SW-846 to support changes in the
regulatory program and to improve method performance and cost effectiveness. To
date, EPA has finalized Updates I, II, IIA, IIB, III, IIIA, IIIB, IVA and IVB
to the SW-846 manual, and the updated and fully integrated manual contains
approximately 3500 pages.
What is high resolution gas chromatography?
Although conventional gas
chromatography mass spectrometry (GC/MS) provides relatively high efficiency
separations, the analysis of some complex, natural-matrix samples may require
the use of even higher resolution approaches. Comprehensive two-dimensional gas
chromatography (GC x GC) is an emerging high-resolution technology which uses
coupled GC columns to achieve separations that are not possible by conventional
GC. Separations in the “first dimension” that are carried out on a long GC
column are directed via a modulator to a short GC column with different
selectivity (the “second dimension”), and are detected by time of flight mass
spectrometry (TOF-MS). Because the entire sample is represented in the
analysis, the technique is described as “comprehensive.” Efforts at NIST are
being directed toward the study of issues related to quantitation by GC x
GC/TOF-MS, and to the value assignment of complex matrix Standard Reference
Materials. A particular emphasis is being placed on metabolomics and
environmental applications.
AccuTOF™ GCv Time-of-Flight Mass Spectrometer
Dioxin testing from the SW-846
METHOD 8290
POLYCHLORINATED DIBENZODIOXINS (PCDDs) AND POLYCHLORINATED
DIBENZOFURANS (PCDFs)
BY HIGH-RESOLUTION GAS CHROMATOGRAPHY/HIGH-RESOLUTION
MASS SPECTROMETRY (HRGC/HRMS)
1.0 SCOPE AND APPLICATION
1.1 This method provides procedures for the detection and quantitative
measurement of polychlorinated dibenzo-p-dioxins (tetra- through
octachlorinated
homologues; PCDDs), and polychlorinated dibenzofurans (tetra-
through
octachlorinated homologues; PCDFs) in a variety of environmental
matrices and at
part-per-trillion (ppt) to part-per-quadrillion (ppq)
concentrations. The
following compounds can be determined by this method:
_______________________________________________________________________________
Compound Name CAS No
2,3,7,8 -Tetrachlorodibenzo-p-dioxin (TCDD) 1746-01-6
1,2,3,7,8-Pentachlorodibenzo-p-dioxin (PeCDD) 40321-76-4
1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin (HxCDD) 57653-85-7
1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin (HxCDD) 39227-28-6
1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin (HxCDD) 19408-74-3
1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin (HpCDD) 35822-39-4
1,2,3,4,6,7,8,9-Octachlorodibenzo-p-dioxin (OCDD)
3268-87-9
2,3,7,8-Tetrachlorodibenzofuran (TCDF) 51207-31-9
1,2,3,7,8-Pentachlorodibenzofuran (PeCDF) 57117-41-6
2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) 57117-31-4
1,2,3,6,7,8-Hexachlorodibenzofuran (HxCDF) 57117-44-9
1,2,3,7,8,9-Hexachlorodibenzofuran (HxCDF) 72918-21-9
1,2,3,4,7,8-Hexachlorodibenzofuran (HxCDF) 70648-26-9
2,3,4,6,7,8-Hexachlorodibenzofuran (HxCDF) 60851-34-5
1,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCDF) 67562-39-4
1,2,3,4,7,8,9-Heptachlorodibenzofuran (HpCDF) 55673-89-7
1,2,3,4,6,7,8,9-Octachlorodibenzofuran (OCDF) 39001-02-0
_____________________________________________________________________
CAS is the Chemical
Abstract Service Registry Number.
.
1.2 The analytical method calls for the use of high-resolution gas chromatography
and high-resolution mass spectrometry (HRGC/HRMS) on purified sample
extracts. Table 1 lists the various
sample types covered by this analytical protocol, the 2,3,7,8-TCDD-based method
calibration limits (MCLs), and other pertinent information. Samples containing concentrations of specific
congeneric analytes (PCDDs and PCDFs) considered within the scope of this
method that are greater than ten times the upper MCLs must be analyzed by a
protocol designed for such concentration levels, e.g., Method 8280. An optional method for reporting the
analytical results using a 2,3,7,8-TCDD toxicity equivalency factor (TEF) is
described.
September 1994
1.3 The
sensitivity of this method is dependent upon the level of interferences within
a given matrix. The calibration range of
the method for a 1 L water sample is 10 to 2000 ppq for TCDD/TCDF and
PeCDD/PeCDF, and 1.0 to 200 ppt for a 10 g soil, sediment, fly ash, or tissue
sample for the same analytes (Table 1). Analysis of a one-tenth
aliquot of the sample permits measurement of concentrations up to 10 times the
upper MCL. The actual limits of
detection and quantitation will differ from the lower MCL, depending on the
complexity of the matrix.
1.4 This method is designed for use by analysts who are experienced
with residue analysis and skilled in HRGC/HRMS.
1.5 Because of the extreme toxicity of many of these compounds, the analyst
must take the necessary precautions to prevent exposure to materials known or
believed to contain PCDDs or PCDFs. It
is the responsibility of the laboratory personnel to ensure that safe handling
procedures are employed.
2.0 SUMMARY OF METHOD
2.1 This procedure uses matrix specific extraction, analyte specific cleanup,
and HRGC/HRMS analysis techniques.
2.2 If interferences are encountered, the method provides selected cleanup
procedures to aid the analyst in their elimination. A simplified analysis flow chart is presented
at the end of this method.
2.3 A specified amount (see
Table 1) of soil, sediment, fly ash, water, sludge (including paper pulp), still bottom, fuel oil, chemical
reactor residue, fish tissue, or
human adipose tissue is spiked with a solution containing specified amounts of
each of the nine isotopically ( C ) labeled PCDDs/PCDFs listed in Column 1 of
Table 2. The sample is then extracted
according to a matrix specific extraction procedure. Aqueous samples that are judged to contain1 percent or more solids, and
solid samples that show an aqueous phase, are filtered, the solid phase
(including the filter) and the aqueous phase extracted separately, and the
extracts combined before extract cleanup.
The extraction procedures are:
a) Toluene: Soxhlet
extraction for soil, sediment, fly ash, and paper pulp samples; b) Methylene
chloride: liquid-liquid extraction for
water samples; c) Toluene: Dean-Stark
extraction for fuel oil, and aqueous sludge samples; d)
Toluene extraction for still bottom samples; e)
Hexane/methylene chloride: Soxhlet
extraction or methylene chloride:
Soxhlet extraction for fish tissue samples; and f) Methylene
chloride extraction for human adipose tissue samples.
Soxhlet extractor from wikipedia
2.6 Two µL of the concentrated extract are injected into an HRGC/HRMS system
capable of performing selected ion monitoring at resolving powers of at least
10,000 (10 percent valley definition .
2.7 The identification of
OCDD and nine of the fifteen 2,3,7,8-substituted congeners (Table 3), for which a C-labeled standard is available in the sample
fortification and recovery standard solutions (Table 2), is based on their
elution at their exact retention time (within 0.005 retention time units measured
in the routine calibration) and the simultaneous detection of the two most
abundant ions in the molecular ion region.
The remaining six 2,3,7,8-substituted congeners (i.e., 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-HxCDD;
1,2,3,6,7,8-HxCDF; 1,2,3,7,8,9-HxCDF; 2,3,4,6,7,8-HxCDF, and
1,2,3,4,7,8,9-HpCDF), for which no carbon-labeled internal standards are
available in the sample fortification solution, and all other PCDD/PCDF
congeners are identified when their relative retention times fall within their
respective PCDD/PCDF retention time windows, as established from the routine calibration data, and the
simultaneous detection of the two most abundant ions in the molecular ion
region. The identification of OCDF is
based on its retention time relative to
C -OCDD and the simultaneous detection of the two most abundant ions in the molecular ion region. Identification also is based on a comparison of the ratios of the
integrated ion abundance of the molecular ion species to their theoretical
abundance ratios.
2.8 Quantitation
of the individual congeners, total PCDDs and total PCDFs is achieved in
conjunction with the establishment of a multipoint (five points) calibration
curve for each homologue, during which each calibration solution is analyzed
once.
6.2.1 Sample collection personnel should, to the extent possible, homogenize samples in the field before filling the sample
containers.This should minimize or eliminate the necessity for sample
homogenization in the laboratory. The
analyst should make a judgment, based on the appearance of the sample,
regarding the necessity for additional mixing. If the sample is clearly not homogeneous, the entire contents
should be transferred to a glass or stainless steel pan for mixing with a
stainless steel spoon or spatula before removal of a sample portion for
analysis.
6.2.2 Grab and composite samples must be collected in glass containers. Conventional
sampling practices must be followed. The
bottle must not be prewashed with sample before collection. Sampling equipment must be free of potential
sources of contamination.
6.3 Grinding or Blending of Fish Samples - If not otherwise specified
by the U.S. EPA, the whole fish (frozen) should be blended or ground to provide
a homogeneous sample. The use of a
stainless steel meat grinder with a 3 to 5 hole size inner plate is
recommended. In some circumstances,
analysis of fillet or specific organs of fish may be requested by the U.S.
EPA. If so requested, the above whole fish requirement is superseded.
6.4 Storage and Holding Times - All samples, except fish and adipose tissue
samples, must be stored at 4 C in the dark, extracted within 30 days and be completely
analyzed within 45 days of extraction.
Fish and adipose tissue samples must be stored at -20 C in the dark,
extracted within 30 days and completely analyzed within 45 days of
collection. Whenever samples are
analyzed after the holding time expiration date, the results should be
considered to be minimum concentrations and should be identified as such.
NOTE: The holding times listed in Sec. 6.4 are
recommendations. PCDDs and PCDFs are
very stable in a variety of matrices, and holding times under the conditions
listed in Sec. 6.4 may be as high as a year for certain matrices. Sample extracts, however, should always be analyzed
within 45 days of extraction.
6.5 Phase
Separation - This is a guideline for phase separation for very wet (>25
percent water) soil, sediment and paper pulp samples. Place a 50 g
portion in a suitable centrifuge bottle and centrifuge for 30 minutes at
2,000 rpm. Remove the bottle and
mark the interface level on the bottle. Estimate the relative volume of each phase. With a disposable pipet, transfer the liquid
layer into a clean bottle. Mix the solid
with a stainless steel spatula and remove a portion to be weighed and analyzed
(percent dry weight determination, extraction).
Return the remaining solid portion to the original sample bottle (empty)
or to a clean sample bottle that is properly labeled, and store it as
appropriate. Analyze the solid phase by
using only the soil, sediment and
paper pulp method. Take note of, and
report, the estimated volume of liquid before disposing of the liquid as a
liquid waste.
6.6 Soil, Sediment, or Paper Sludge (Pulp) Percent Dry Weight Determination
- The percent dry weight of soil, sediment or paper pulp samples showing
detectable levels (see note below) of at least one 2,3,7,8-substituted PCDD/PCDF
congener is determined according to the following procedure. Weigh a 10 g portion of
the soil or sediment sample (+ 0.5 g) to three significant figures. Dry it to constant weight at 110 C in an
adequately ventilated oven. Allow the
sample to cool in a desiccator. Weigh
the dried solid to three significant figures.
Calculate and report the percent dry weight. Do not use this solid portion of the sample
for extraction, but instead dispose of it as hazardous waste . NOTE: Until detection limits have been established (Sec. 1.3), the
lower MCLs (Table 1) may be used to estimate the minimum detectable
levels.
7.4.3 Fly Ash NOTE: Because of the tendency of fly ash to
"fly", all handling
steps should be performed in a hood in order to minimize
contamination.
7.4.3.1 Weigh about 10 g fly ash to two decimal places and transfer to an
extraction jar. Add 150 mL of 1 M HCl to
the fly ash sample. Seal the jar with
the
Teflon lined screw cap and shake for 3 hours at room temperature.
7.4.3.2 Rinse a glass fiber filter with toluene, and filter the sample
through the filter paper, placed in a Buchner funnel, into a 1 L flask. Wash the fly ash cake with approximately500 mL
organic-free reagent water and dry the filter cake overnight at room
temperature in a desiccator.
7.4.3.3 Add 10 g anhydrous powdered sodium sulfate,
mix thoroughly, let sit in a closed container for one hour, mix again, let sit for another hour, and mix again.
7.4.3.4 Place
the sample and the filter paper into an extraction thimble, and extract in a
Soxhlet extraction apparatus charged with 200 mL toluene for 16 hours using a
five cycle/hour schedule.
7.4.3.5 Cool
and filter the toluene extract through a
glass fiber filter into a 500 mL round bottom flask. Rinse the filter with 10 mL toluene. Add the rinse to the extract and concentrate
the combined toluene solutions to near dryness on a rotary evaporator at 50 C.
7.4.4 Transfer the concentrate to a 125 mL separatory funnel using 15 mL
hexane. Rinse the flask with two 5 mL
portions of hexane and add the rinses to the funnel. Shake the combined solutions in the
separatory funnel for two minutes with 50 mL of 5 percent sodium chloride
solution, discard the
aqueous layer
7.4.5 Aqueous samples
7.4.5.1 Allow the sample to come to ambient temperature, then mark the water meniscus on the side of the 1 L sample bottle for
later determination of the exact sample volume.
7.4.5.2 When the sample is judged to contain 1 percent or more solids, the
sample must be filtered through a glass fiber filter that has been rinsed with
toluene. If the suspended solids content
is too great to filter through the 0.45 µm filter, centrifuge the sample, decant, and then filter the aqueous phase
7.4.5.3 Combine
the solids from the centrifuge bottles with the particulates on the filter and
with the filter itself and proceed with the Soxhlet extraction
7.4.6.4. Remove and invert
the Snyder column and rinse it down into the KD apparatus with two 1 mL
portions of hexane.
Snyder column from wikipedia
7.4.5.4 Pour the aqueous filtrate into a 2 L separatory funnel. Add 60 mL methylene chloride to the sample
bottle, seal and shake for 30 seconds to rinse the inner surface. Transfer the solvent to the separatory
funnel and extract the sample by shaking
the funnel for two minutes with periodic venting.
7.4.5.5 Allow the organic layer to separate from the water phase for a
minimum of 10 minutes. If the emulsion
interface between layers is more than one third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the
phase separation (e.g., glass stirring rod)
7.4.5.6 Collect the methylene chloride into a KD apparatus mounted with a
10 mL concentrator tube) by passing the sample extracts through a filter funnel
packed with a glass wool plug and5 g anhydrous sodium sulfate.
7.4.5.7 Repeat the extraction twice with fresh 60 mL portions of methylene
chloride. After the third extraction,
rinse the sodium sulfate with an additional 30 mL methylene chloride to ensure
quantitative transfer. Combine all
extracts and the rinse in the KD apparatus. NOTE: A continuous liquid-liquid extractor may be used in place of
a separatory funnel when experience with
a sample from a given source indicates that a serious emulsion problem
will result or an emulsion is encountered when using a separatory funnel. Add 60 mL methylene chloride to the sample
bottle, seal, and shake for 30 seconds to rinse the inner surface. Transfer the solvent to the extractor. Repeat the rinse of the sample bottle with an
additional 50 to 100 mL portion of methylene chloride and add the rinse to the
extractor. Add 200 to 500
mL methylene chloride to the distilling flask, add sufficient organic-free
reagent water to ensure proper operation, and extract for24 hours. Allow to cool, then detach the distilling flask. Dry and concentrate the extract.
7.4.5.8 Attach a Snyder column and concentrate the extract on a water bath
until the apparent volume of the liquid is 5 mL. Remove the KD apparatus and allow it to drain and cool for at least10 minutes.
7.4.5.9 Remove the Snyder column, add 50 mL hexane, add the concentrate
obtained from the Soxhlet extraction of the suspended solids if applicable,
re-attach the Snyder
column, and concentrate to approximately 5 mL.
7.4.5.10 Rinse the flask and the lower joint with two 5 mL portions of
hexane and combine the rinses with the extract to give a final volume of about
15 mL.
7.4.6 Soil/Sediment
7.4.6.1 Add 10 g anhydrous powdered sodium sulfate to the sample portion
(e.g., 10 g) and mix thoroughly with a stainless steel spatula. After breaking up any lumps, place the
soil/sodium sulfate mixture in the Soxhlet apparatus on top of a glass wool
plug.
7.4.6.2 Add
200 to 250 mL toluene to the Soxhlet apparatus and reflux for 16 hours. The solvent must cycle completely through the
system five times per hour.
7.4.6.3 Cool and filter the extract through a glass fiber filter into a 500
mL round bottom flask for evaporation of the toluene. Rinse the filter with 10 mL of toluene, and
concentrate the combined fractions to near dryness on a rotary evaporator at50 C. Remove the flask from the water bath and
allow to cool for5 minutes.
7.4.6.4 Transfer
the residue to a 125 mL separatory funnel, using 15 mL of hexane. Rinse the flask with two additional portions
of hexane, and add the rinses to the funnel.
7.5 Cleanup
7.5.1 Partition
7.5.1.1Partition the hexane extract against 40 mL of concentrated sulfuric
acid. Shake for two minutes. Remove and discard the sulfuric acid layer
(bottom). Repeat the acid washing until
no color is visible in the acid layer (perform a maximum of
four acid washings.
7.5.1.2 Partition
the extract against 40 mL of 5 percent (w/v) aqueous sodium chloride. Shake for two minutes. Remove and discard the aqueous layer (bottom).
7.5.1.3Partition the extract against 40 mL of 20 percent (w/v) aqueous potassium
hydroxide (KOH). Shake for two
minutes. Remove and discard the aqueous
layer (bottom). Repeat the base washing
until no color is visible in the bottom layer (perform a maximum of four base
washings). Strong base (KOH) is known to
degrade certain PCDDs/PCDFs, so contact time must be minimized.
7.5.1.4 Partition
the extract against 40 mL of 5 percent (w/v) aqueous sodium chloride. Shake for two minutes. Remove and discard the aqueous layer (bottom). Dry the extract by pouring it through a
filter funnel containing anhydrous sodium sulfate on a glass wool plug, and collect it in a 50 mL
round bottom flask. Rinse the
funnel with the sodium sulfate with two 15 mL portions of hexane, add the rinses
to the 50 mL flask, and concentrate the hexane solution to near dryness on a
rotary evaporator (35 C water bath), making sure all traces of toluene (when
applicable) are removed.
7.5.2 Silica/Alumina Column Cleanup
7.5.2.1 Pack a gravity column (glass, 30 cm x 10.5 mm) fitted with a Teflon
stopcock, with silica gel as follows:
Insert a glass wool plug into the bottom of the column. Place 1 g silica gel in the column and tap
the column gently to settle the silica gel.
Add 2 g sodium hydroxide-impregnated silica gel, 4 g sulfuric acid-impregnated
silica gel, and 2 g silica gel. Tap the
column gently after each addition. A
small positive pressure (5 psi) of clean nitrogen may be used if needed. Elute with 10 mL hexane and close the
stopcock just before exposure of the top layer of silica gel to air. Discard the eluate.
7.5.2.2 Pack a gravity column (glass, 300 mm x 10.5 mm) fitted with a
Teflon stopcock, with alumina as follows:
Insert a glass wool plug into the bottom of the column. Add a 4 g layer of sodium sulfate. Add a 4 g layer of Woelm® Super 1 neutral
alumina. Tap the top of
the column gently. Woelm® Super 1
neutral alumina need not be activated or cleaned before use, but it should be
stored in a sealed desiccator. Add a 4 g
layer of anhydrous sodium sulfate to cover the alumina. Elute with 10 mL hexane and close the stopcock
just before exposure of the sodium sulfate layer to air. Discard the eluate.
7.5.2.3 Dissolve the residue from Sec. 7.5.1.4 in 2 mL hexane and apply the
hexane solution to the top of the silica gel column. Rinse the flask with enough hexane (3-4 mL)
to complete the quantitative transfer of the sample to the surface of the
silica
gel.
7.5.2.4 Elute the silica gel column with 90 mL of hexane, concentrate the eluate on a rotary evaporator (35 C water bath) to approximately
1 mL, and apply the concentrate to the top of the alumina column (Sec.
7.5.2.2). Rinse the rotary evaporator
flask twice with 2 mL of hexane, and add the rinses to the top of the alumina
column.
7.5.2.5 Add 20 mL hexane to the alumina column and elute until the hexane
level is just below the top of the sodium sulfate.
7.5.2.6 Add 15 mL of 60 percent methylene chloride in hexane (v/v) to the
alumina column and collect the eluate in
a conical shaped (15 mL) concentration tube. With a carefully regulated stream of
nitrogen, concentrate the 60 percent methylene
chloride/hexane fraction to about 2 mL.
7.5.3 Carbon Column Cleanup
7.5.3.1 Prepare
an AX-21/Celite 545® column.
7.5.3.3 Rinse the AX-21/Celite 545® column with 5 mL of toluene, followed
by 2 mL of 75:20:5 (v/v) methylene chloride/methanol/toluene, 1 mL of 1:1 (v/v)
cyclohexane/methylene chloride, and 5 mL
hexane. The flow rate should be less
than 0.5 mL/min. Discard the
rinses. While the column is still wet
with hexane, add the sample concentrate (Sec. 7.5.2.6) to the top of the column. Rinse the concentrator tube (which contained the sample concentrate)
twice with 1 mL hexane, and add the rinses to the top of the column.
7.5.3.4 Elute the column sequentially with two 2 mL portions of hexane, 2
mL cyclohexane/methylene chloride (50:50 v/v), and 2 mL methylene
chloride/methanol/toluene (75:20:5, v/v).
7.5.3.5 Turn
the column upside down and elute the PCDD/PCDF fraction with 20 mL
toluene.
7.5.3.6 Concentrate the toluene fraction to about 1 mL on a rotary
evaporator by using a water bath at 50 C.
Carefully transfer the concentrate into a 1 mL minivial and, again at
elevated temperature (50 C), reduce the volume to about 100 µL using a stream of
nitrogen and a sand bath.
Well that sounds like a lot of fun. I have never turned a Snyder column upside down. I think that most people use a Liquid/Liquid extractor today.
Note that there are twenty compounds being tested. The compounds found are then assigned a TEQ (toxic equivalent) based on testing the compounds in various toxicity tests. This introduces two sources of variance. (1) it is really hard to separate twenty compounds which have similar resolution. Just because you identify the peak as authentic, it doesn't mean the whole peak is the compound you think you are measuring. (2) the equivalence factor used based on a toxicology test is subject by definition is an arbitrary number. I really could not find how the TEQ were calculated. I guess that is why there are four different systems. However, everyone seems to agree binding with the Aryl receptor is the mechanism of action and the greater the binding the higher the TEF . So one analyses the 18 isomers, gives each a TEF based on the literature and adds them up to get a TEQ. It is simpler just to measure the substrate for Aryl Receptor binding and state the actual TEQ.
What is the Aryl hydrocarbon receptor? (based on wikipedia)
The Aryl hydrocarbon receptor (AhR or AHR) is a member of the family of basic helix-loop-helix transcription factors. The physiological ligands of this receptor are unknown, but it binds several exogenous ligands such as natural plant flavonoids, polyphenolics and indoles, as well as synthetic polycyclic aromatic hydrocarbons and dioxin-like compounds.
This means that it is a magic mystery ligand receptor. Nobody has a clear idea what the ligand was doing till dioxin came along. It is unlikely man evolved the receptor to respond to dioxin release from the family firepit.
I would like to make a few remarks about the flavanoid binding. The flavanoids appear to be antagonist to dioxin binding the AHR receptor. (This abbreviation is a problem for me because it looks too much like the abbreviation for Androgen receptor (AR) or lutienizing hormone receptor (LHR).
The flavonoid have been shown to have activity of the most interest as an antagonist is luteolin which a phytoestrogen produced by the alfalfa plant. The luteolin acts to activate the nod D gene in the rhizobium which induces them to nodulate the alfalfa root. However, I don't think one gets much luteolin when eating alfafa as quantitatively the principal estrogenic compound is coumestrol, which has not been test for the AhR.
On the other hand two isoflavones have been reported as AhR agonists, genistein and diadzein. Genistein is the phytoestrogen that activates the rhizobium for the soya plant and it is also a potent tyrosine kinase inhibitor. Both genistein and diadzein are found in mg quantities in soya products. No one seems to be touting them as dioxin like compounds like the flavanoids are being touted as health supplements based on the same data.
Now to make life even more complicated both PCBs and the dioxins both activate the AHR receptor which among other thing causes a reduction in estrogen receptor concentration. On top of this some PCBs can bind the estrogen receptor directly. I am mentioning all this because I gather it is a source of confusion for a lot of people.
Flavonoids
as aryl hydrocarbon receptor agonists/antagonists: effects of structure and
cell context. .
Suppression
mechanisms of flavonoids on aryl hydrocarbon receptor-mediated signal
transduction.
Arch Biochem Biophys. 2010 Sep 1;501(1):134-41. Epub 2010 May 5
Effect of highly bioaccumulated polychlorinated biphenyl congeners on estrogen and androgen receptor activity Eva Cecilie Bonefeld-Jørgensen, Helle Raun Andersen ,Thomas Høj Rasmussen,Anne Marie Vinggaard Toxicology 158 (2001) 141–153.Flavone Limitations to Root Nodulation and Symbiotic Nitrogen
Fixation in Alfalfa YORAM KAPULNIK, CECILLIA M. JOSEPH, DONALD A. PHILLIPS
Plant Physiol. (1987) 84, 1193-1196
Adaptive response
The adaptive response is manifested as the induction of xenobiotic metabolizing enzymes. Evidence of this response was first observed from the induction of cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyp1a1) resultant from TCDD exposure, which was determined to be directly related to activation of the Ahr signaling pathway. The search for other metabolizing genes induced by Ahr ligands, due to the presence of DREs (Dioxin responsive elements), has led to the identification of an “Ahr gene battery” of Phase I and Phase II metabolizing enzymes consisting of CYP1A1, CYP1A2, CYP1B1, NQO1, ALDH3A1, UGT1A2 and GSTA1. Presumably, vertebrates have this function to be able to detect a wide range of chemicals, indicated by the wide range of substrates Ahr is able to bind and facilitate their biotransformation and elimination. The AhR may also signal the presence of toxic chemicals in food and cause aversion of such foods.[
AhR activation seems to be also important for immunological responses and inhibiting inlammation.
Toxic response
Extensions of the adaptive response are the toxic responses elicited by Ahr activation. Toxicity results from two different ways of Ahr signaling. The first is a side effect of the adaptive response in which the induction of metabolizing enzymes results in the production of toxic metabolites. For example, the polycyclic aromatic hydrocarbon benzo(a)pyrene (BaP), a ligand for Ahr, induces its own metabolism and bioactivation to a toxic metabolite via the induction of CYP1A1 and CYP1B1 in several tissues. The second approach to toxicity is the result of aberrant changes in global gene transcription beyond those observed in the “Ahr gene battery.” These global changes in gene expression lead to adverse changes in cellular processes and function.
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