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Dioxin II

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Small liver size is due to deficient formation of hepatic vasculature AhRshunt.pdf ... mice show defective development of the vasculature. An endogenous ligand ? ... – PowerPoint PPT presentation

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Title: Dioxin II


1
Dioxin- II
  • Chemicals, Risk Cancer 7
  • David R. Bell

2
Dioxin human toxicity
Suspected poisoning dose of 1-2 mg
2004
1998
Viktor Yuschenko Ukrainian president
26 ng/g blood fat
Normal population 20 pg/ g blood fat
144 ng/g blood fat
EHP109 865, CMAJ 172873
3
TCDD and cancer
  • Dioxin reassessment
  • Is TCDD a human carcinogen ?
  • AhR Knockout mice- variation and use
  • PAHs vs TCDD- why is there such a difference ?
    AhR ancillary proteins

4
Assessment of dioxin risks-USA
  • Effects of TCDD
  • And related congeners (furans, PeCDD, Hex, Hept,
    etc)
  • Human risk assessment
  • Mandate
  • Based on the best scientific evidence available
  • NOT safety factors

5
Dioxin assessment
  • http//www.epa.gov/ncea/dioxin.htm
  • Previous dioxin assessment was rejected on
    grounds of poor science
  • US regulatory policy frequently leads world
    opinion
  • Reassessment by 2001

6
Dioxin exposure
  • Total TCDD Equivalent Dose (TEQ)
  • Includes TCDD and other congeners
  • TCDD 10
  • 1980s- 10 pg/kg/day
  • 2000- 1-2 pg/kg/day
  • US regulatory limit 10 fg/kg/day
  • WHO/Europe 1-4 pg/kg/day

7
Dioxin exposure 2
  • 1.4 ng/kg/day is the lowest dose producing
    tumours in rats
  • But half-life of dioxin 100x longer in human
  • 20 days in rats
  • 2100 days in human
  • TCDD accumulates to higher levels in human
  • Thyroid tumours not a human problem

8
Is TCDD a human carcinogen ?
  • Data for TCDD, vs other congeners, is not clear
  • Animal data unequivocal
  • What about human
  • Requirement for highly exposed cohort of workers
  • Known exposure
  • Sufficient time to see a tumour response

9
TCDD as a human carcinogen
  • See
  • Final.pdf p.43-50
  • Chapter1-6.pdf p.31-38
  • Evidence of human carcinogenicity highly
    controversial
  • Distinction between assuming it will be
    carcinogenic, and whether it actually is

10
Human studies
  • Confounding exposure to other carcinogens
  • Smoking, industrial exposure
  • Poor measurement of dose
  • Measurement of serum/ adipose TCDD after the
    event
  • What is important ?Peak dose ? Length of
    exposure ?

11
Dioxin and human cancer
12
Human Results
  • Increase in all tumour sites at high doses
  • 1.4 fold
  • Biologically non-plausible
  • All known human carcinogens show site-specificity
  • High doses are 100-1000 x normal background
    levels of exposure

13
What does the AhR do ?
  • Is there an endogenous ligand ?
  • What does the endogenous ligand do ?
  • AhR clears nasty foreign compounds
  • Look for potential endogenous ligands ICZ.pdf
  • Study AhR -/- mice

14
What does the AhR do ?
  • AhR -/- mice phenotypes Science 268722-726
    FujiiKO.pdf chrisKO.pdf
  • Small liver (all)
  • Immune system impairment and lethality Gonzalez
    only. Infection ?
  • Resistance to TCDD toxicity TCDDtoxinko.pdf
  • Small liver size is due to deficient formation of
    hepatic vasculature AhRshunt.pdf
  • Hepatic portal-venous shunting
  • Defective blood vessel development

15
AhR is important
  • Dioxin toxicology requires AhR
  • AhR null mice are resistant to dioxin toxicity
  • Liver
  • Thymus
  • Teratogenesis
  • Nulls, congenics, etc

PNAS 10116677, JBC 27817767
16
AhR and vascular biology
  • AhR null mice show defective development of the
    vasculature

17
An endogenous ligand ?
  • AhR hypomorphic mutants
  • Low levels of the AhR
  • Defective liver vascularisation in controls
  • TCDD rescues !

18
PAHs vs TCDD
  • Why is TCDD so much more potent than PAHs, eg
    3-MC ?
  • In rat liver, TCDD is 30,000 x more potent than
    3MC
  • In non-responsive mouse, no dose of 3MC induces,
    whereas TCDD induces
  • J. Biol. Chem. 269 12118-12128

19
Binding etc
  • Affinity of ligands for AhReceptor
  • 0.5nM for TCDD
  • 1.3 for 3MC
  • 4-fold difference in competitive binding assay
  • 2 hours after dosing, induction of CYP1A1 RNA
    shows a 10-fold difference in potency

20
TCDD and 3MC
  • 14hrs after dosing, TCDD is 1000x more potent
    than 3MC at inducing AHH (CYP1A1)
  • AHH induction rapidly falls off after 3MC
  • 3MC metabolism is rapidly induced in cells- as
    early as 4 hours
  • Little apparent difference in AhR activation, or
    DNA binding, between 3MC TCDD

21
Problems
  • Why does 3MC fail to induce in the non-responsive
    mouse ?
  • It cannot be due to hyperinduction of P450
  • Does activation of the Ah Receptor act like an
    on switch ?
  • Rapid switch off of AHH activity in 3MC treated
    cells

22
AhR is a signalling molecule
  • Signalling molecules should be short-lived
  • Cytosolic (unbound) AhR should be long-lived
  • Mol. Pharm. 49 391-398 (1996)
  • TCDD induces rapid degradation of the AhR (gt90
    after 4 hours)
  • The partner protein, arnt, is not degraded by TCDD

23
AhR turnover
  • TCDD-induced AhR degradation requires functional
    AhR and arnt proteins
  • Involves nuclear translocation of AhR, followed
    by export of AhR
  • Degradation in the proteasome
  • AhR has a short half-life when activated by TCDD
  • AhRdegrad.pdf

24
AhR production is regulated
  • AhR is produced in a ligand-binding conformation
    in reticulocyte lysate
  • Inactive in bacteria, or plant cell lysate
  • AIP.pdf, ara9.pdf, ara9a.pdf, ara9b.pdf
  • Indentification of a protein which interacts with
    AhR in yeast
  • Dramatic potentiation of b-NF-induced AhR
    signalling by ara9/AIP in yeast

25
Role of ara9/AIP and AhR ?
  • Ara9 forms a complex with AhR and hsp90
  • Ara9 stabilises the functional AhRs
    ligand-binding activity
  • Ara9 also places the AhR within the cell
  • Potential for explaining how AhR translocates to
    the nucleus after TCDD treatment
  • Production and intracellular localisation of AhR
    is regulated
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