The role of DNA methylation in
AHR-mediated toxicity of PAHs in
chicken embryo
Jessica Head, Jonas Brandenburg
Department of Natural Resource Sciences
McGill University
Montréal, Québec, Canada
Predictive Adaptive Response
Experimental System
 Chicken embryo model
 Polycyclic aromatic hydrocarbons (PAHs)
 Aryl hydrocarbon receptor (AHR)
pathway
Benzo[k]fluoranthene (BkF)
Aryl hydrocarbon receptor (AHR)
 Well characterized
pathway
 Positive and
negative effects
 Isoforms CYP1A4/5
induced by AHR
ligands in birds
 CYP1A enzyme
metabolizes
xenobiotics
Experimental Design
 Control (not injected)
 Vehicle (inject corn oil)
 1 ug/kg BkF: High (typically <0.25 ug/kg)
 10 ug/kg BkF: Very high (possible)
 100 ug/kg BkF
 Not environmentally relevant
 Possible for total PAHs
 Induced significant mortality (35% at D19)
Experimental Design
 ED0: approx. 40,000 cells
 ED7: first liver functions
 ED10: hepatocytes mature
Endpoints:
• CYP1A4/5 mRNA
expression
• DNA methylation
 ED19: >90% injected PAHs metabolized (Näf et al. 1992)
 D2 post-hatch
ED0
ED7
ED10
ED19
D2
Hypothesis
Abundance
PAH exposure
[PAH] in embryo
AHR-mediated gene expression
Methylation of AHR responsive gene
Embryo
Chick
Adult
DEVELOPMENTAL STAGE
Hatching
CYP1A5 fold induction
Hatching
CYP1A4 fold induction
CYP1A4/5 Induction
Global DNA methylation
 Poster P25: “Use of
LUMA to measure DNA
methylation in
ecological organisms”
 No effect
60
DNA methylation (%)
 LUminometric
Methylation Assay
(LUMA)
40
20
N-I
Corn oil
10
100
Injected dose of B[k]F (μg/kg egg)
CYP1A4/5 promoter
 Shared promoter
 Target known and
predicted Xenobiotic
Response Elements (XREs)
 3 fragments screened via
Methyl Sensitive High
Resolution Melting Assay
(MS-HRM)
 Results confirmed with
bisulfite pyrosequencing
MS-HRM results
 Methy Sensitive High Resolution Melting Assay (MS-HRM)
CpG methylation (%)
 D2 hatchlings
Fragment 1
Fragment 2
Injected dose of B[k]F (ug/kg egg)
Fragment 3
Bisulfite pyrosequencing
CpG methylation (%)
 Fragment 1: no effect
across 10 CpG sites
CpG site within Fragment 1
(CYP1A4/5 promoter)
Bisulfite pyrosequencing
Fragment 2
 Fragment 2: Significant
hypermethylation at 100
ug/kg BkF
 Good correspondence
with MS-HRM data
CpG methylation (%)
 Fragment 1: no effect
across 10 CpG sites
CpG site within Fragment 2
(CYP1A4/5 promoter)
CpG methylation (%)
Developmental profile
*
ED10
ED19
D2
Developmental Stage
*
 Significant relationship
for CYP1A5 at D2
(basal expression)
 Not observed for
CYP1A4, or either
gene at ED19
CYP1A5 mRNA expression
Correlation
DNA methylation (%)
Conclusions
 Transient changes in gene expression
 10 - 25-fold induction of CYP1A4/5 at ED10
 Small, but persistent hypermethylation at CYP1A4/5
starting at ED19
 Environmental relevance?
 Biological relevance?
 Negative relationship between DNA methylation and
CYP1A5 mRNA expression at D2
Future directions
 “DNA methylation – a potential player in AHR adverse
outcome pathways”*
 Potential targets
 Phase I biotransformation: CYP1A
 Phase II biotransformation: NQO1 and GSTP1
 Positive regulator: AHR
 Negative regulator: AHRR
 Global: Reduced Representation Bisulfite Sequencing
(RRBS)
*Jonas Brandenburg, MSc thesis, 2016 (manuscript in preparation)
Future directions
 Re-exposure experiments
 Expose embryos during incubation (exposure 1)
 Re-expose…
 In vivo (chicks), or…
 In vitro (cultured liver tissue slices)
Exposure 1
Option 1
Re-expose
in vivo
Option 2
Re-expose
in vitro
ED19
D7
Acknowledgements
 Jonas Brandenburg (MSc)
 Jenny Eng
 McGill Ecotox Lab
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Emily Boulanger (MSc student)
Cynthia Franci (PhD student)
Mélanie Guigueno (post-doc)
Matt Alloy (post-doc)
Benjamin Barst (post-doc)
Ashley Hanas (MSc studen)
Lise Coquilleau (Honours student)
François Chamberland (Honours student)