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