Society of Environmental Toxicology and Chemistry
New Orleans, LA, USA
November 19-23, 2009
Authors
Jonathan Naile, Steve Wiseman,
Kali Bachtold, Paul Jones, and John Giesy
Toxicology Centre
University of Saskatchewan
Saskatoon, SK, Canada
Jonathan.naile@usask.ca
Website: http://www.usask.ca/toxicology
Outline

History and Background

Rationale for Study

Methods

Results

Conclusions
PFC History and Background





Large scale production and usage began in the 1950’s
Thought to be chemically stable and biologically inert in
the environment
Globally distributed in a wide variety of environmental
matrices
Most work has focused on only the two most widely
produced and detected chemicals (PFOS and PFOA)
The causative biochemical events leading to toxicity as
a result of PFC exposure are largely unknown
PFC History and Background

PFOS has been shown to cause reduced body weight,
increased liver weight, reduced cholesterol and steep dose
response curves for mortality in vertebrates including nonhuman primates

PFOS has been show to be an incomplete peroxisome
proliferator

PFOA has been shown to be a potent and complete
peroxisome proliferator, and causes liver and pancreatic
tumours

But most research so far has focused only on PFOS and
PFOA, with emphasis on distribution and transport
Rationale

Due to these toxicological concerns PFOS production
was phased out in 2000

Shorter chain-length replacement chemicals such as
PFBS and PFBA were chosen because of studies
showing them to have reduced half-lives and fewer
biological effects

There are few mechanistic and toxicological studies of
PFCs other than PFOS and PFOA but in general these
replacement chemicals have been shown to work
through similar mechanisms, but with reduced
toxicity
Rationale

PFCs have been shown to alter biochemical
process such as fatty acid metabolism,
cholesterol synthesis, peroxisome
proliferation, cellular communication and
thyroid hormone function

Specific genes were chosen based on
previous research showing alterations in
mRNA abundance as a result of PFOS
exposure
Objectives

To compare the potential effects of 10
widely distributed and frequently
identified PFCs on 7 important genes
related to processes potentially altered by
PFCs

Describe potential affects PFCs have on key
biochemical pathways and evaluate
relative potencies for altering these
important processes
Genes Related to the Thyroid

Paired box gene 8 (PAX8)
 Related to thyroid follicular cell development

Homeobox (HEX)
 Related to thyroid cell differentiation
Genes Related to Fatty Acid and
Cholesterol Synthesis

Apolipoprotein A-IV (ApoA4)
 Related to fatty acid synthesis

Squalene synthase (SqSyn)
 Important in the synthesis of sterols

Peroxisome 3-ketoacyl-CoA thiolase (Per Keto A)
 Involved in FA beta-oxidation

Mitochondria 3-ketoacyl-CoA thiolase (Mito Keto A)
 Involved in FA beta-oxidation

Mitochondria 3-ketoacyl-CoA thiolase (Mito Keto B)
 Involved in FA beta-oxidation
Chemicals Tested
Chemical Name
Acronym
Perfurobutane sulfonate
PFBS
Perfurohexane sulfonate
PFHS
Perflurooctane sulfonate
PFOS
Perflurobutyric acid
PFBA
Pefluropentanoic acid
PFPnA
Peflurohexanoic acid
PFHA
Perflurooctanoic acid
PFOA
Perfluroononanoic acid
PFNA
Perfluroundodecanoic acid
PFUnA
Perflurododecanoic acid
PFDoA
Methods
Seed plate at
300,000 cells/mL
Dose cells after 24 h
at 37oC
Incubate
at
Incubate at
oC for 48 h
37
37°C
for 72 hrs
medium

Cell culture and
exposure

RNA Extraction

cDNA Synthesis

Q-PCR
Cell
viability
cells
Hormone
andand
RNA
Extraction
Enzymatic
Analyses
Q-PCR
HEX
45.00
40.00
Fold Change
35.00
30.00
25.00
20.00
0.1 uM
15.00
1.0 uM
10 uM
10.00
100 uM
5.00
0.00
Chemicals
Sulphonates
35
30
Fold Change at 100 uM
25
MKA
APOA 4
PKA
MKB
SQSYN
HEX
PAX 8
20
15
10
5
0
-5
-10
PFBS
PFHS
Chemicals
PFOS
Carboxylates
35
30
Fold Change at 100 uM
25
20
MKA
APOA 4
PKA
MKB
SQSYN
HEX
PAX 8
15
10
5
0
-5
-10
Chemicals
Specific Conclusions

Both processes were affected by PFC exposure

All of the sulphonates and all but one of the
carboxylates caused up-regulation in the two
genes related to the thyroid

The response for the 5 genes related to fatty
acid and cholesterol processes were much
more variable, but significant changes were
seen for 35% of the chemicals
General Conclusions

mRNA expressions studies provide a useful tool
for identifying potentially effected pathways, and
allows for quick comparisons between chemicals

Not all PFCs cause the same affects on mRNA
expression and differences could not simply be
attributed to chain-length or head group

Further studies are needed to determine if these
differences are expressed at more toxicologically
relevant endpoints, such as at the protein level
Thank You!
40
Carboxylates
35
Fold Change at 100 uM
30
25
PFBA
20
PFPnA
PFHA
15
PFOA
PFNA
10
PFUdA
5
PFDoA
0
-5
-10
MKA
APOA 4
PKA
MKB
Genes
SQSYN
HEX
PAX 8
Sulphonates
35
Fold Change at 100 uM
30
25
20
15
PFBS
10
PFHS
PFOS
5
0
-5
-10
MKA
APOA 4
PKA
MKB
Genes
SQSYN
HEX
PAX 8