Yinfei Yang
Environmental Toxicology Laboratory
Toxicology Centre
University of Saskatchewan, Saskatoon, SK, CANADA
SETAC North America 30th Annual Meeting
2009 New Orleans, LA
Dr.
Dr.
Dr.
Dr.
John P. Giesy
Paul Jones
Steve Wiseman
Yi Wan
Dr. John Newsted
Dr. Denise Kay
Dr. Sean Kennedy
Jessica Hervé
Reza Farmahin
Dr. Steve Bursian
Dr. Matt Zwiernik
Andrew Cohen-Barnhouse
Dr. Tim Fredricks
Patrick Bradley
Tittabawassee River Risk Assessment
 2003: Michigan
Department of
Environmental Quality
concluded that PCDDs
and PCDFs exist at
levels such that they
may present a risk to
wildlife. 110,000
PARTS PER TRILLION
TEQ!!!
 A study throughout the
Tittabawassee river
floodplain was initiated
to investigate the impact
of dioxin and furan
exposure from the
environment.
Tittabawassee River Risk Assessment ctd…
2,3,7,8-tetrachlorodibenzo-p-dioxin
2,3,7,8-tetrachlorodibenzofuran (TCDF)
(TCDD)
2,3,4,7,8-pentachlorodibenzofuran (PeCDF)
 Routes of Exposure
 Aquatic
 Terrestrial
 Tropic Level
 Residency
 Migratory
 Resident
Avian Health Assessment
Individual Condition
Measurement
General Health
Nestling Weight
Nestling Growth
Clutch Size (egg/nest)
Reference vs. Tittabawassee
No difference
No difference
No difference
No difference
Productivity (nestlings
fledged/nest)
No difference
Population Condition
Measurement
Abundance
Productivity
Nestling Return Rates
Adult Return Rates
Reference vs. Tittabawassee
No difference
No difference
No difference
No difference
The Aryl Hydrocarbon Receptor (AhR)
Ligand Binding Domain (LBD)
AATI K S
AATVK A
TAI I K A
AhR Signaling Pathway
Type 1: Chicken-like,
Very Sensitive
Type 2: Pheasant-like,
Moderately Sensitive
Type 3: Japanese Quaillike, Insensitive
Kennedy et al. (Abstract 247)
Study Design
What are the effects of TCDD and PCDFs on
differentially sensitive avian species?
Impact of In Ovo Exposure
to TCDD, PeCDF and TCDF
Biomarker reponses in
cultured hepatocytes
Biomarker Responses
Tittabawassee River Risk Assessment
Mortality Data
Egg Injection
oHatch Success
oMortality
oDeformities
Injection of 0.1 µl/g egg into the air
cell of eggs with:
oTriolein
* Doses of each chemical,
oTCDD
selected to bound the predicted
oPeCDF
White-leghorn Chicken
LD50 of that species!
oTCDF
(Gallus gallus domesticus)
14-d post
hatch
Inject egg
Incubate
Common Pheasant
(Phasianus colchicus)
Japanese quail
(Coturnix japonica)
oCYP1A mRNA
•CYP1A4
•CYP1A5
oEROD activity
Experimental Goal
 The objectives of these studies were to investigate the
CYP1A response to DLCs, specifically TCDD, PeCDF and
TCDF, in differentially sensitive avian species within the
Order Galliformes.
 The null hypotheses for this study are:
 Within each species, there is no difference among the
potencies of TCDD, TCDF and PeCDF.
 Within each chemical, there is no difference among the
sensitivities of White-leghorn chicken, Common pheasant
and Japanese quail.
White-leghorn Chicken
TCDD
PeCDF
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
TCDF
*
*
*
*
*
*
Kruskal-Wallis one-way ANOVA.
Significant changes from control are denoted
by * (P ≤ 0.1, Mann Whitney U Test)
Common Pheasant
TCDD
PeCDF
*
*
*
*
*
*
*
*
TCDF
*
*
*
Kruskal-Wallis one-way ANOVA.
Significant changes from control are denoted
by * (P ≤ 0.1, Mann Whitney U Test)
Japanese Quail
TCDD
PeCDF
*
*
*
*
*
*
**
*
*
*
*
*
*
A
TCDF
*
*
Kruskal-Wallis one-way ANOVA.
Significant changes from control are denoted
by * (P ≤ 0.1, Mann Whitney U Test)
*
Fold Change in CYP1A4 mRNA Abundance
(Change from Control)
LOEC Determination
25
20
Estimated LOEC value of Japanese
quail CYP1A4 mRNA abundance
in PeCDF exposed birds.
15
10
5
0
0
-5
1
2
3
4
[PeCDF] ng/g Egg
TCDD
Whiteleghorn
Chicken
≤ 0.0494
PeCDF
TCDF
CYP1A4
Common Japanese
Pheasant Quail
0.466
> 2.86
≤ 0.0438
0.0294
3.08
≤ 0.0742
0.654
4.90
LOEC Determination cont…
CYP1A5
White-leghorn
Chicken
Common
Pheasant
Japanese Quail
TCDD
0.0621
> 6.68
> 2.86
PeCDF
0.117
> 6.76
> 11.16
TCDF
0.0980
> 14.2
> 8.56
EROD
White-leghorn
Chicken
Common
Pheasant
Japanese Quail
TCDD
≤ 0.0494
> 6.68
> 2.86
PeCDF
≤ 0.0438
5.26
> 11.16
TCDF
≤ 0.0742
0.425
> 8.56
Summary
Interspecies Comparisons
White-leghorn Chicken
Common Pheasant
Japanese Quail
CYP1A4
Undetermined
PeCDF (15) > TCDD (1) > TCDF (0.75)
PeCDF (?) > TCDF (?) ≥ TCDD (?)
CYP1A5
TCDD (1) > TCDF (0.67) > PeCDF (0.5)
Undetermined
Undetermined
EROD
Undetermined
TCDF (?) > PeCDF (?) ≥ TCDD (?)
Undetermined
Rank (ReP)
TCDD not always the most potent!
(? = undetermined)
Intercompound Comparisons
Rank
(ReS)
TCDD
PeCDF
TCDF
CYP1A4
C>P>Q
P≥C>Q
C>P>Q
CYP1A5
C>P>Q
C>P≥Q
C>P≥Q
EROD
C>P>Q
C>P>Q
C>P>Q
Chicken not always the most sensitive!
Interspecies Comparisons
White-leghorn
Chicken
in
vitro*
Common Pheasant
Japanese Quail
EC50
TCDD = PeCDF ≥ TCDF PeCDF > TCDD ≥ TCDFPeCDF > TCDD > TCDF
Ecthr
TCDD ≥ PeCDF = TCDF PeCDF > TCDD ≥ TCDFPeCDF > TCDD > TCDF
Lethality** TCDF > TCDD ≥ PeCDFPeCDF > TCDF > TCDD PeCDF > TCDF > TCDD
in ovo
CYP1A4
Undetermined
PeCDF > TCDD > TCDF PeCDF > TCDF > TCDD
CYP1A5
TCDD > TCDF > PeCDF
Undetermined
Undetermined
EROD
Undetermined
TCDF > PeCDF > TCDD
Undetermined
* Hervé, et al. ToxSci Advance Access, 2009. **Cohen-Barnhouse et al. MP177, 178.
Intercompound Comparisons
in
vitro *
TCDD
PeCDF
TCDF
EC50
C>P>Q
Q=C≥P
C>P>Q
Ecthr
C>P>Q
Q>C=P
C>P>Q
Lethality**
C>P>Q
P>C>Q
C>P>Q
CYP1A4
C>P>Q
P≥C>Q
C>P>Q
CYP1A5
C>P>Q
C>P≥Q
C>P≥Q
EROD
C>P>Q
C>P>Q
C>P>Q
in ovo
* Hervé, et al. ToxSci Advance Access, 2009. **Cohen-Barnhouse et al. MP177, 178.
Discussion and Conclusions
 TCDD is not always the most potent.
 Chicken is not always the most sensitive.
 Different biomarkers have different responses, which
depends on species and chemicals as well.
 Different endpoints may give different ranks of
sensitivities for species or relative potencies for
compounds.
 Selection of samples from survivors in the in ovo
study for mRNA and enzymatic analysis might
introduce bias that should be corrected before using
for further studies or risk assessment.
Yinfei Yang yinfei.yang@usask.ca
Environmental Toxicology Laboratory
University of Saskatchewan
Saskatoon, SK, CANADA
http://www.usask.ca/toxicology/jgiesy/index.php
Yang et al., submitted.,
Dr. John P. Giesy
john.giesy@usask.ca
Dr. Steve Wiseman
steve.wiseman@usask.ca