Characterization of toxicity pathways of TCDD,
PCB 77, and BaP in white sturgeon using whole
transcriptome and proteome analysis
By: Jon Doering
Dioxin-like Compounds
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Include Dioxins, Furans, and PCBs
Found in complex mixtures in the environment
Different DLCs have vastly different potencies
Fish being among most sensitive organisms
Embryos
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Craniofacial
Cardiovascular
Skeletal
Edema
Mortality
Juveniles and Adults
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Hepatotoxicity
Fin necrosis
Wasting syndrome
Immune suppression
Reproductive failure
Aryl Hydrocarbon Receptor
TEF Approach
Additive toxicity of dioxin-like compounds due to
their common and specific mechanism of toxic
action.
Effects and severity of
effects on embryos of
equipotent mixtures of
DLCs are indistinguishable.
TEF Approach
But do equipotent mixtures induce the same
responses with regard to more subtle effects or
effects on juvenile or adult fishes?
Omics
• Recently developed and emerging technologies
have significantly improved our ability to
identify mechanisms of toxicity.
• “Omics”
• Global analysis
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
1) Are global
responses among
chemicals similar?
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
1) Are global
responses among
chemicals similar?
2) Are responses of
transcriptome and
proteome similar?
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
1) Are global
responses among
chemicals similar?
2) Are responses of
transcriptome and
proteome similar?
3) Are similar
pathways perturbed?
Test Species
Juvenile White Sturgeon (Acipenser transmontanus)
• Prototypical DLC
• Among most potent
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
• Prototypical DLC
• Among most potent
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
• 500-fold less potent
than TCDD
3,3’,4,4’-tetrachlorobiphenyl (PCB 77)
• Prototypical DLC
• Among most potent
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
• 500-fold less potent
than TCDD
3,3’,4,4’-tetrachlorobiphenyl (PCB 77)
Benzo[a]pyrene (BaP)
• Polycyclic aromatic
hydrocarbon (PAH)
• Among strongest
AhR-agonists of
PAHs
• AhR and non-AhR
effects
Methods
Raise animals
Methods
Raise animals
I.P. exposure
(Equipotent doses)
Methods
Raise animals
I.P. exposure
Excise livers
Methods
Raise animals
I.P. exposure
Transcriptomics & Proteomics
Excise livers
CYP1A
Equal activation of Ah Receptor
ANOVA followed by Tukey Post-hoc (P < 0.05)
Transcriptomics
(global analysis of gene expression)
Illumina MiSeq
Sequencer
Transcriptomics
(global analysis of gene expression)
1) Isolate RNAs
Illumina MiSeq
Sequencer
Transcriptomics
(global analysis of gene expression)
1) Isolate RNAs
2) Prepare “library”
Illumina MiSeq
Sequencer
Transcriptomics
(global analysis of gene expression)
1) Isolate RNAs
2) Prepare “library”
3) Sequence
Illumina MiSeq
Sequencer
Transcriptomics
(global analysis of gene expression)
1)
2)
3)
4)
Illumina MiSeq
Sequencer
Isolate RNAs
Prepare “library”
Sequence
Mapped “reads”
to a reference
transcriptome
Transcriptomics
(global analysis of gene expression)
1)
2)
3)
4)
Isolate RNAs
Prepare “library”
Sequence
Mapped “reads”
to a reference
Illumina MiSeq
transcriptome
Sequencer
5) Downstream
Analysis
Proteomics
(global analysis of protein expression)
Orbitrap Liquid Chromatography –
Mass Spectrometry (Orbitrap LC-MS)
Proteomics
(global analysis of protein expression)
Orbitrap LC-MS
1) Isolate Proteins
2) Sequence
3) Mapped
“reads” to a
reference
proteome
4) Downstream
Analysis
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
1) Are global
responses among
chemicals similar?
2) Are responses of
transcriptome and
proteome similar?
3) Are similar
pathways perturbed?
Transcriptome
Up-regulated
14% of altered
transcripts shared by all
3 chemicals
37% of altered
transcripts shared by 2
or more chemicals
Transcriptome
9% of altered transcripts
shared by all 3
chemicals
38% of altered
transcripts shared by 2
or more chemicals
Down-regulated
Transcriptome
Up-regulated
Down-regulated
Transcriptome
Up-regulated
Transcriptome
Up-regulated
Greater fold-change
for PCB 77
Transcriptome
Up-regulated
Greater fold-change
for BaP
Transcriptome
Up-regulated
Equal fold-change
for all 3 chemicals
Transcriptome
Up-regulated
Down-regulated
Proteome
Up-regulated
36% of altered proteins
shared by all 3
chemicals
76% of altered proteins
shared by 2 or more
chemicals
Proteome
26% of altered proteins
shared by all 3
chemicals
76% of altered proteins
shared by 2 or more
chemicals
Down-regulated
Proteome
Up-regulated
Down-regulated
Proteome
Up-regulated
Down-regulated
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
1) Are global
responses among
chemicals similar?
Significant similarity in
response and
magnitude of response
among 3 chemicals. The
proteomes being more
similar than the
transcriptomes.
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
1) Are global
responses among
chemicals similar?
2) Are responses of
transcriptome and
proteome similar?
3) Are similar
pathways perturbed?
Association between transcriptome and
proteome (68.22% similarity)
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
2) Are responses of
transcriptome and
proteome similar?
Genes altered at
transcriptome level
and proteome level
were similar for all 3
chemicals.
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
1) Are global
responses among
chemicals similar?
2) Are responses of
transcriptome and
proteome similar?
3) Are similar
pathways perturbed?
Pathway Analysis
Transcriptome
PCB 77
Great similarity in perturbed
pathways, even where altered
transcripts differed.
TCDD
BaP
Proteome
PCB 77
Great similarity in perturbed
pathways, even where altered
proteins differed.
TCDD
BaP
Question
Investigate the whole
transcriptome and proteome
response following exposure
to equipotent doses of 3
model AhR agonists
3) Are similar
pathways perturbed?
Preliminary
investigation shows
that the same major
pathways were
perturbed between
chemicals, even
where exact genes
differed.
Conclusions
Great similarity in global transcriptomic and
proteomic response to equipotent concentrations of
3 model agonists of the Ah receptor.
Similar pathways were perturbed and by similar
magnitude.
Suggests similar adverse effects and severity of
effects at equipotent concentrations.
Acknowledgements
Co-authors:
Song Tang
Steve Wiseman
Hui Peng
Bryanna Eisner
Jianxian Sun
John Giesy
Markus Hecker
Questions ??