Effects of Perfluorinated Phosphate Derivatives on Gene Expression in the H4IIE Rat Hepatoma Cell Line
Paul D. Jones1,2, Jonathan Naile2,*, Steve Wiseman2, and John P. Giesy2,3,4,5
1 School for Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada. 2 Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada3
Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada,
4 Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA, 5 Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China.
* Corresponding Author’s Phone Number: 306‐966‐5062, Email: paul.jones@usask.ca
ABSTRACT
Perfluorinated chemicals (PFCs) have gained much recent notoriety due to widespread occurrence in the environment and wildlife and their toxicological effects. Recognition of the potential adverse effects of PFCs has lead to development and use of a variety of alternative compounds, some
still containing perfluorinated moieties. Notable among these alternative chemicals are the perfluoroalkyl‐phosphates (PAPs). These compounds are either the phosphonic acid analogs of carboxylic acids or are mono‐, di‐ or tri‐phosphate esters, commonly of fluorotelomer alcohols. During
environmental and metabolic breakdown PAPs can be expected to yield a variety of perfluorinated moieties including the parent phosphonic acids and compounds such as FTOHs and potentially perflurocoaboxylates after further degradation of those FTOHs. The objectives of the current study
were to determine if PAPs caused the same changes in gene expression as PFCs and whether the gene expression effects of some PAPs were the same as the effects caused by the constituent PFC moieties. Rat hepatoma cells were treated with different PAPs and PFCs at concentrations of 1.0‐
100 uM for 24 and 72 hrs. RNA was isolated, purified and gene expression was quantitatively measured using Real‐Time PCR. Processes investigated included fatty acid synthesis, cellular communication, and thyroid development. Perfluorophosphonic caused differential gene expression
profiles relative to analogous perfluorocarboxylates. For example PFDPA at 10 uM caused 69‐fold induction of Apo A4 while the analogous PFDA caused only 6.4 fold induction of the same gene. Effects of phosphate ester compounds were different from comparable perfluoroalcohols which
would be expected to be released by metabolic breakdown. We therefore conclude that PAPs may act through mechanisms of action different from other PFCs and so greater research and understanding of the effects of this emerging chemical group is required.
INTRODUCTION
RESULTS
Perfluorinated compounds (PFCs) have been produced and used in large quantities since the
1950s. While some of the predominant compounds are no longer produced the search for
alternative perfluorinated chemicals continues.
 Perfluoroalkyl phosphates (PFAPs), analogues of the sulphonic and carboxylic acid PFCs have
recently been identified in the environment (D’eon et al 2009 Environ. Toxicol. Chem.
28:2101‐2107).
 Currently two classes of PFPAs are of
interest; Perfluoroalkyl phosphate esters
(PEs) and the phosphonic acids (PAs).
 PEs
have the potential to release
perfluoro moieties when hydrolysed.
These moieties may be metabolized to
carboxylates in organisms (Fig. 1).
 Phosphonic acids may possess unique
toxic potential due to the presence of the
C‐P bond.
 In this study PEs and PAs were compared
to other PFCS (Table 1) with respect to
their ability to modulate the expression of
several genes.
 Genes were selected based on the known
effects of other PFCs and on their ability
to act as markers for critical biochemical
functions (Table 2).

Abbreviation
PFBS
PFOS
PFBA
PFOA
PFOPA
PFDPA
PFHxPA
POFPP
PHFBP
PTEHP
Table 1 Chemicals Tested for Effects on Gene Expression
Type
Name
Sulphonic Acid
Pefluorobutyl sulphonic acid
Sulphonic Acid
Pefluorooctyl sulphonic acid
Carboxylic Acid
Perfluorobutyl carboxylic Acid
Carboxylic Acid
Perfluorooctyl carboxylic Acid
Phosphonic Acid
Perfluorooctyl phosphonic acid
Phosphonic Acid
Perfluorodecyl phosphonic acid
Phosphonic Acid
Perfluorohexyl phosphonic acid
Phosphate Ester
Tris (octafluoropentyl) phosphate
Phosphate Ester
Tris (heptafluorobutyl) phosphate
Phosphate Ester
Tris (trifluoroethyl) phosphate
Chain Length
C4
C8
C4
C8
C8
C10
C6
C5
C4
C2
Table 2 Genes Investigated (responses normalized to Glyceraldehyde‐3‐phosphate dehydrogenase)
Gene
Function
Na+/K+ ATPASE
Maintenance of cellular osmotic balance
PAX 8 (Paired box gene 8)
Related to thyroid follicular cell development
HEX (Homeobox)
Related to thyroid cell differentiation
Apo‐A4 (Apolipoprotein A‐IV)
Related to fatty acid metabolism
RESULTS
Fig. 3. Chemical Ordination Based on Gene Expression
Fig. 2. Chemical Induced Changes in Gene Expression
PFDPA
PFDPA
PFDPA
Tri C5 ester
40
Apo‐A4
40
30
20
PFDPA
PFDPA
Tri C5 ester
PTEHPTri C5 ester
PFHxPA
PFOPA
30
Fold Induction
50
Fold Induction
Na/K ATPase
APOA4
60
20
PTEHP
PFOS
Tri C4 ester
PFBA
10.0
1.0
0.1
1
0.0
C
0
0
0
entr
onc
n
atio
Carboxylates
)
(uM
PT
PH EH
PO FB P
FP P
P
PF FO P
H PA
PF xP
D A
PF PA
O
PF
A
BA
PF
O
S
PF
BS
PT
PH EH
PO FB P
FP P
P
PF FO P
Hx PA
PF P
D A
PF PA
O
PF
A
BA
PF
O
S
PF
BS
Ch
em
ic
al
Chemical Class
Phosphonic acids
Phosphate esters
Ch
em
ic
al
10.0
1.0
0.1
0.0
1
0
0
ntr
nce
Co
0
Tri C4 ester
Chemical Class
PTEHP
Tri C4 ester
Tri C4 ester
Carboxylates
PFBA PFOA
PFBA
PFBA
PFBS
PFOA
PFOA
PFOS PFBA
PFBA
PFBA
PFBA
PFBAPFBS
PFBS
Phosphonic acids
PFOA
PFOS
Tri C4 ester
PFOA
PFBA
PFOA
Phosphate esters
PFOS
PFOS
PFOA
PFOA
PFOA
PFOA
PFOS
PFOS
PFBS
PFBS
PFBS
PFBS
PAX8
Sulphonic acids
PFBS
PFBS
ATPASE
HEX
M)
n (u
atio
Fig. 4. Chemical Ordination Based on Thyroid Related Genes
PFOA
70
Fold Induction
30
20
10
PFOA PFOA
PAX
60
Tri C5 ester
PTEHP
PFOA
50
PFHxPA
PTEHP
PFHxPA
PTEHP
ester PFDPA
TriTri
C5C5
ester
PFOPA
PTEHP PFOPA
PFDPA
PFHxPA
10
40
HEX
HEX
Fold Induction
PTEHP
PFOS
PFBA
Tri C4 ester
PFOS
Sulphonic acids
40
Tri C5 ester
Tri C4 ester
PFBS
PFBS
00
100.
PTEHP Tri C5 ester
PFOPA
PFHxPA
PFHxPA
PFOPA
PFHxPA
PFOPA
Tri PTEHP
C5 ester
PTEHP
Tri C5 ester
Tri C5 ester
PFBA PFOA
PFOA
PFBS
0
PFDPA
PFDPA
Tri C5 ester
Tri C5 ester
Tri C4 ester
PFOS
.0
100
PTEHP
PTEHP
Tri C4 ester
Tri C4 ester
Tri C4 ester
PFOS
PFOS
10
10
PFHxPA
PFHxPA
PTEHP
Tri C5 ester
Tri C5 ester
PFOPA
PFDPA
PFDPA
PTEHP
PFOPA PFHxPA
PFHxPA
PFHxPA
PFOPA
PTEHP
30
20
0
.0
100
PT
PH EH
PO FB P
FP P
P
PF FO P
H PA
PF xP
D A
PF PA
O
PF
A
BA
PF
O
S
PF
BS
PT
PH EH
PO FB P
FP P
P
PF FO P
H PA
PF xP
D A
PF PA
O
PF
A
BA
PF
O
S
PF
BS
Ch
em
ic
al
10.0
0
Ch
em
ic
al
0
1.0
M)
n (u
0
atio
ntr
nce
Co
0.1
0.0
1
Carboxylates
Phosphonic acids
PFDPA
ester
Tri Tri
C4 C4
ester
PFOS
PFOS
PFOS
10
Chemical Class
Tri C5 ester
Phosphate esters
PFOS
Tri C4 ester
10.0
0
.0
100
0
0
1.0
M)
n (u
0
atio
ntr
nce
Co
0.1
0.0
1
1
PFBS
PFBS
PFBS
Sulphonic acids
PFBA PFBA
PFBA
PFBSPFBA
0.1
1.0
10.0
PAX8
METHODS
CONCLUSIONS
Rat hepatoma cells (H4IIE cell line) were cultured under standard conditions, 37ᴼC, 5% CO2.
 24 hrs after plating exposure chemicals were added, cell were incubated for 72 hrs.
 After 72 hrs mRNA was extracted from cultured cells and stored in ‘RNAlater’ until analysis.
 RT‐PCR was performed using standard methods, all expression values were normalized to
the expression of glyceraddehyde‐3‐phosphate dehydrogenase as a house keeping gene.
• PFAPs of both classes induced greater changes in Apo‐A4 expression than any of the carboxylic and sulphonic acids tested (Fig. 2). Increased expression of Apo‐A4 has been linked with decreases in feeding responses, and is believed to be a vital signal for lipid uptake, transportation and metabolism.
• Of the phosphonic acids tested the C10 (PFDPA) was generally the most potent for alterations in gene expression analogous of the greater potency of the C8‐10 PFCs.
• POFPP (C5 ester) induced increased the expression of Na+/K+ ATPase even at the lowest dose tested and was clearly the most potent chemical for this endpoint (Fig. 2). This transporter is involved in osmotic homeostasis this response may be indicative of alterations in membrane permeability.
• The expression profile of the phosphate esters overlaps or ‘approaches’ that of the carboxylates suggesting that the metabolic generation of carboxylates may be occurring.
• This study clearly demonstrates that PFAPs elicit a range of effects on gene expression that are distinct from those elicited by other PFCs (Fig. 3). Therefore, further studies on the potential modes of action of these emerging contaminants is warranted.
• Alteration of PAX 8 and HEX expression by PFAPs, particularly the phosphonic acids (PAs) may be indicative of potential impacts on thyroid function and homeostasis (Fig. 4).


Cell culture and exposure
(24 and 72 hrs)

RNA Extraction

cDNA Synthesis

Q‐PCR
 Normalize to G3P‐DH
Incubate at 37°C for 72 hrs
RNA Extraction and Q‐PCR
Acknowledgements
This research was supported by a Discovery Grant from the National Science and Engineering Research Council (NSERC) (Project # 326415-07)
and a grant from Western Economic Diversification Canada (Projects 6578, 6807). We acknowledge an instrumentation grant from the Canada
Foundation for Infrastructure. JPG was supported by the Canada Research Chair program and an at large Chair at the Department of Biology
and Chemistry and the Research Centre for Coastal Pollution and Conservation, City University of Hong Kong.