1. Effects of Environmentally
Relevant Transplacental
Arsenic Exposure on Mouse
Hepatic Protein Expression
Jay Knowlton
University of Maine
Honors Thesis Defense
16 April 2015 1
3. Arsenic Exposure
• WHO and US DEP set 10ppb standard in 2001, down from
50ppb standard used for decades prior
• Common exposure from:
• Ground Water- Unregulated well water
• Sodium arsenite exposure in drinking water for this study
• Food
• Pesticides
• Mining, metal working
• Industrial air pollution
• Some medicines
• Traditional Chinese Medicine
• New Leukemia treatments 3
4. Arsenic and human health
• Linked to several
diseases and disorders
• Bladder, lung, prostate,
skin and liver cancers
• Type II diabetes
• Black foot disease
• Mechanism of action
under investigation
• Altered gene expression
• Protein activation
• AKT/pAKT 4
7. Mouse arsenic exposure
Start F0♀
treatment
Start
mating
Remove
♂ F0
F1 pups
born
F1 tissue
harvest (21-
25 days old)
F2 birth
F2 tissue
harvest
(21-25
days old)
F1 mating
0, 10, 50 or 500 ppb sodium
arsenite (NaAsO2) in drinking water Mouse timeline
Arsenic exposure
day
0 4 11 32 53-58
7
Samples come from
this tissue harvest
Modified from Carlson et al., 2013
8. Patrick Carlson’s Work
• Identified several dozen proteomic expression differences in
zebrafish in response to arsenic
• Further investigation showed low-dose transplacental arsenic
exposure to alter mRNA expression of Wee1, Brca2, Cdkn1a,
Pparγ, and several others
• Each linked to AKT activity, proposed a pathway of molecular interaction
8
9. Suggested mechanism – arsenic alters
signaling pathways
Demonstrates not only the cell growth/progression regulatory properties of
AKT, but the interactions in metabolic pathways as well
Carlson et al., 2013
9
12. Methods
• Identify Samples
• Same used in Carlson mouse studies
• Prepare homogenates
• Phosphosafe buffer with protease inhibitors
• BCA Assay (quantify total protein content)
• Load equal protein samples
• Separate via SDS-Page
• Transfer to PVDF Membrane
• Block non-specific binding sites, incubate with target antibody,
wash, incubate with HRP-conjugated secondary antibody, wash
• Incubate in Chemiluminescent Substrate
• Image Blots (FUJIFilm LAS4000)
• Densitometry Analysis
• Gel Analyzer software
• Graphpad Prism software
BCA
Assay
SDS-Page Electrophoresis
Ponceau Stain
12
13. Hypothesis
• Transplacental exposure to sodium arsenite induces an
increase in pAKT/AKT expression in mouse hepatic
tissue that is dose-dependent
• Formed from literature review and Patrick Carlson’s proposed
mechanisms of molecular interaction
13
14. Western Blot
Results
Representative western blots of F1 transplacentally-exposed mouse liver
samples, with pAKT-stimulated Jurkat cell controls, showing lane structure and
antibody targets.
14
15. Sex-dependent analysis
Relative difference in pAKT/AKT expression
between male and female F1 samples; no
significant sex-dependent effect of
transplacental sodium arsenite exposure shown
in paired t-test. (±SEM, N= 12 for each sex)
Student's t-Test
Exposure P-Value
0ppb 0.2942
10ppb 0.7266
50ppb 0.5025
500ppb 0.7702
15
16. Dose-dependent analysis
No significant effect of dose in relative pAKT/AKT expression with
transplacental sodium arsenite exposure. (±SEM, N=5 for each exposure; sexes
combined)
16
17. Conclusion
• No significant effect of transplacental sodium arsenite
exposure on pAKT/AKT protein expression shown
• No sex- or dose- dependent effects observed
• Hypothesis not supported
17
18. Discussion
• Western Blotting for
analytics
• “Semi-quantitative”
• Large room for error
• AKT phospho-state
• Often reversed
within minutes
• Hepatic
heterogeneity
• Varying expression
profiles between
liver lobes
Varying pAKT levels after
stimulation of different cell types
18
19. Future
Directions
• Further analysis of samples using a variety of techniques (ie.
ELISA, IHC, qPCR, etc.)
• Expand analysis to include samples from F2 to determine
generational effects
• Downstream analysis of metabolic pathways involved in
glucose level regulation
19
20. Proposed Downstream
Analysis
• GSK3, FOXO1, PFK
• All downstream AKT substrates involved in glucose metabolism
• Hepatic relevance positions Van Beneden lab for study
20
22. Acknowledgements
• This Research was partially funded by Maine INBRE and the
University of Maine Center for Undergraduate Research
(CUGR) to JSK, and by the Maine Agricultural and Forest
Experiment Station (MAFES) to RJVB.
• Thanks to the members of the Van Beneden lab for
assistance with laboratory techniques
• Thanks to Adria Elskus, Angela Myracle, and Julie Gosse for
assistance with statistical analysis
• Thanks to Jared Arsenault and the Maine Journal staff for
producing a video synopsis of the project
• http://mainejournal.umaine.edu/transplacental-arsenic-
exposure-effects-on-mouse-hepatic/ 22
Thank everyone for being here
Let ‘em know questions are welcome
Leukemia stuff: arsenic trioxide used as a therapeutic in conjunction with other drugs
Black foot disease- artheriosclerosis endemic to Taiwan, has been mostly remediated since the implementation of tap water in rural villages within the past few decades
Altered gene expression is the primary focus of most of Pat’s work for his Transplacental paper
Note the change from hazard identification to hazard characterization
As(III) – reduced, trivalent
As(V) – oxidized, pentivalent
Reduction and Methylation involved in detoxifying process (methylated trivalent arsenicals found in urine of exposed individuals)
Timeline of experimental method
Note: after F1 mice were done nursing, there was no further arsenic exposure
I have focused my reading on the cell-cycle regulation of Akt
Final document will detail upstream mechanisms involved in arsenic exposure
I need to explore the metabolic pathways in greater detail
Wee1- Cell cycle regulator, Ser/thr kinase, Checkpoints: G2/M, cell size, DNA damage
CDK1- Cyclin-dependent kinase 1 (p34)- conserved ser/thr kinase, cell cycle regulator, acts on many substrates
CCNB1- Cyclin B1- G2/Mitotic specific, forms maturation-promoting factor (MPF) with Cdk1, “all-or-none switch” to commit to mitosis- kinase activity, important to degrade nuclear envelope
Brca2- Breast cancer 2- early onset, repairs double strand DNA breaks,
PPARg- Peroxisome proliferator-activated receptor gamma (glitazone receptor)- nuclear regulatory protein involved in lipid uptake and adipogenesis (regulated in part by FOXO1)
RXR- Retinoid x receptor- binds to activated PPARg to regulate transcription of various genes
Crot- Peroxisomal Carnitine O-octanoyltransferase- catalyzes transfer of fatty acyl groups between Coenzyme A and carnitine (between intermembranous space to matrix)
Fabp3- Heart-type fatty acid binding protein- transfer of fatty acids from membrane into mitochondria, growth inhibitor
Hmgcs1- HMG-CoA synthase- catalyzes an Actyl-CoA-related condensation reaction involved in cholesterol synthesis and ketogenesis (fatty acid breakdown), a reaction associated with over-activation in type-1 diabetes patients (clinical significance)
Mitochondrial Importance: Beta-oxidation- breakdown of fatty acids to generate Acetyl-CoA, NADH, and FADH2 for citric acid cycle and electron transport chain
The crux of complex pathway “cross-talk”
Growth factor binds to RTK (receptor tyrosine kinase), recruits PI3K (phosphoinositide-3 kinase), PIP2 phsphorylated to PIP3, recruitment of PDK1 (pyruvate dehydrogenase kinase), phospohorylates Akt, leads to downstream kinase activity:
AKT Pleckstrin Homology domain binds to phosphoinositides
Thr308 phosphorylated by PDK1, Ser473 phosphorylated automatically or by Insulin-like-kinase, or mTOC2 (not well understood)
Activated via phosphorylation by mTORC2 to some extent, but more well documented is PDK1
PI3K has 2 subunits, p85 (binds RTK) and p110 (catalytic). P110 activation can also be stimulated in a GPCR-induced RAS-GTP binding, thus initiating the downstream signaling
PTEN (Phosphatase and tensin homolog) tumor suppression regulation
BAD (Bcl2 associated death promoter): recruits Bcl2, inhibits cytochrome c release through mitochondrial membrane, inhibiting apoptosis via cytoplasmic caspase cascade
mTOR (mammalian target rapamyacin, ser/thr kinase): regulates cell growth, motility, survival, protein synthesis, and transcription. Integrated via IGFs (insulin-like growth factors)
MDM2: negative regulator of p53 tumor suppressor
JNK (c-Jun N-terminal Kinases): mitogen-activated kinases activated in response to stress stimuli (extensive literature available on the extremely broad and complex MAPK pathways involved in proliferation, gene expression, differentiation, mitosis, and apoptosis)
P27 (Cdkn1b) G1/S regulation
GH-IGF receptors
Note PTEN as an example of a negative regulator of AKT signaling
Note the diversity of downstream pathways, connection between cancer-linked pathways and metabolic pathways
Hepatic Role: detoxification and metabolism (glucose)
Potential link: Warburg effect: cancer cells showing increased levels of glucose metabolism relative to normal cells
BCA: bicinchoninic acid assay
apparent trend, no statistical significance
Note this is from a cell line, which is far more controlled than in vivo.
F2: Explore whether any expression changes are direct insult or epigenetic modification? Literature suggests epigenetic connections
GSK3: Glycogen synthase kinase
FOXO1 (forkhead box protein O1): transcriptional regulator of glucose metabolism: lowers hepatic glucose production and interferes with the differentiation of cells involved in metabolic control
PFK (phosphofructokinase): stimulates glycolysis, further raising glucose levels in hepatic tissue
Thank you all for taking the time to work with me
I look forward to working with you all throughout the remainder of the academic year
I want to make myself as available as possible
Please get in touch anytime with questions or concerns!
