ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 72 (2009) 1985–1993

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Proteomic studies in zebrafish liver cells exposed to the brominated flame

retardants HBCD and TBBPA
Peter Kling , Lars Förlin
Department of Zoology/Zoophysiology, University of Gothenburg, Box 463, SE-405 30 Gothenburg, Sweden

a r t i c l e in fo abstract

Article history: Proteomic effect screening in zebrafish liver cells was performed to generate hypotheses regarding
Received 6 October 2008 single and mixed exposure to the BFRs HBCD and TBBPA. Responses at sublethal exposure were analysed
Received in revised form by two-dimensional gel electrophoresis followed by MALDI-TOF and FT-ICR protein identification.
16 April 2009
Mixing of HBCD and TBBPA at sublethal doses of individual substances seemed to increase toxicity.
Accepted 18 April 2009
Available online 23 May 2009
Proteomic analyses revealed distinct exposure-specific and overlapping responses suggesting novel
mechanisms with regard to HBCD and TBBPA exposure. While distinct HBCD responses were related to
Keywords: decreased protein metabolism, TBBPA revealed effects related to protein folding and NADPH production.
BFR Overlapping responses suggest increased gluconeogenesis (GAPDH and aldolase) while distinct mixture
Proteomics
effects suggest a pronounced NADPH production and changes in proteins related to cell cycle control
Zebrafish
(prohibitin and crk-like oncogene). We conclude that mixtures containing HBCD and TBBPA may result
MALDI-TOF
FT-ICR in unexpected effects highlighting proteomics as a sensitive tool for detecting and hypothesis
HBCD generation of mixture effects.
TBBPA & 2009 Elsevier Inc. All rights reserved.
Mixture toxicity

1. Introduction Recent technology including transcriptomics, metabonomics and

Brominated flame retardants (BFRs) are widely used in textiles,
electronic equipment and insulating materials to reduce the risk
of fire. Hexabromocyclododecane (HBCD) and tetrabromobisphe-
nol-A (TBBPA) belong to the most widely used BFRs and their
environmental levels are increasing (BFR, 2004). Both substances
are known to associate to estuarine sediments and may therefore
pose a health risk to fish (de Wit, 2002). HBCD in particular have
the potential to bioaccumulate in aquatic species such as fish
(Asplund et al., 1999; Eljarrat et al., 2005; Morris et al., 2004;
Sellström et al., 1998). Both HBCD and TBBPA exposure can result
in adverse effects on thyroid metabolism and reproduction
(Hamers et al., 2006; Kuiper et al., 2007b) and can induce
oxidative stress (Ronisz et al., 2004; Shi et al., 2005). Moreover,
TBBPA has been demonstrated to reduce egg production and
juvenile survival in zebrafish (Kuiper et al., 2007a). In addition,
TBBPA and HBCD have been observed to exhibit thyroxin (T4)
competition in fish in vitro systems (Hamers et al., 2006; Meerts
et al., 2000). To date the knowledge of the underlying mechanisms
of HBCD and TBBPA-induced toxicity is scarce. As two of the
major BFRs in the environment there is a need to identify
their molecular mechanisms and their potential to interact with
biological systems.
proteomics has the potential to provide insight into the underly-
ing mechanisms of chemically induced toxicity, and to identify
molecular response patterns. Such patterns may possibly reflect
sensitive and specific biomarkers. The large amount of data that can
be collected using these techniques allow for multi-endpoint effect
screening, and the potential for identifying unexpected adverse
effects. In the present study we have utilised cytotoxicity tests and
comparative proteomics in order to generate hypotheses regarding
mechanisms underlying HBCD- and TBBPA-induced toxicity. Liver
has been identified as a major target organ for both substances in
mammals (Gebbink et al., 2008; Tada et al., 2006). In addition HBCD
has been shown to accumulate in fish liver (Janak et al., 2005),
whereas TBBPA has been indicated to elicit potentially toxic hepatic
effects in zebrafish (De Wit et al., 2008). Therefore a zebrafish liver
(ZFL) cell test system was selected. The study was also conducted in
order to test the potential of a proteomic strategy as a sensitive tool
to screen for single substance and mixture effects. In this proteomic
strategy we used both MALDI-TOF and FT-ICR mass spectrometry
to increase the confidence of protein identifications.
2. Materials and methods
2.1. Chemicals

 Corresponding author. Fax: +46 31 41 67 29. Amfolyte 3–10, Coomassies Blue, bromophenol blue, chaps, Criterion precast
E-mail address: peter.kling@zool.gu.se (P. Kling). gel 8–16%, dithiothreitol (DTT), glycine, iodoacetamide, prestained SDS–PAGE

0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2009.04.018
 ARTICLE IN PRESS
1986 P. Kling, L. Förlin / Ecotoxicology and Environmental Safety 72 (2009) 1985–1993
standard broad range, RC-DC protein assay, ready strips IPG 11 cm NL 3–10 and
urea were from Bio-Rad, Hercules, CA, USA. Glycerol was supplied by BHD
Laboratory Supplies, England. CH3CN, thiourea and tris(hydroxymethyl)amino-
methane were products of MERCK, Darmnstadt, Germany. Endonuclease, PMSF and
sodium dodecyl sulfate (SDS) were from Sigma Chemicals Co., St Louis, MO, USA.
Zebrafish liver cells (ZFL) were purchased from European Cell Culture Collection
(ECCC). All chemicals for cell culture maintenance were purchased from GIBCO,
Invitrogen. HBCD were purchased from Sigma Chemicals Co., St Louis, MO, USA
and TBBPA from Promochem. The LDH cytotoxicity kit were purchased from Roche
Diagnostics.
2.2. Cell culture, exposure and cytotoxicity assays
ZFL cells were maintained in L-15 (50%); DMEM (35%); Ham-F-12 (15%)
supplemented with 0.15 g/L sodium bicarbonate; 15 mM Hepes; 0.01 mg/ml
insulin; 50 ng/L epidermal growth factor (EGF) and 5% heat inactivated (HI) foetal
bovine serum (FBS) under atmospheric conditions at 26 1C. Cytotoxicity tests using
the LDH leakage test were performed in 96-well plates using a reduced FBS
concentration (1%) according to the instructions given in the LDH cytotoxicity kit.
Stock solutions of HBCD and TBBPA were freshly prepared in DMSO and added to
the media prior to start of exposure. The substances were stored under appropriate
conditions to ensure quantitative and qualitative stability according to Sigma
chemicals (HBCD) and Promochems (TBBPA) storage conditions, and were
carefully weighed before dissolution in DMSO. All working solutions were freshly
prepared prior to cell exposure. Cells were exposed to single and mixed substances
of HBCD (0.05, 0.5, 5 and 187 mM) and TBBPA (0.05, 0.5, 5 and 220 mM). During
mixed exposure the cells were exposed to 1, 10, 50 and 100 mM of each substance.
Thus the total concentrations during mixed exposure were 2, 20, 100 and 200 mM,
respectively. Cells were exposed for 24 and 72 h. At the start of the experiment
30,000 cells were seeded in each well and allowed to settle overnight. Cells were
rinsed once with complete growth medium containing 1% FBS and thereafter the
exposure was started. 200 ml media was added to each well at a final concentration
of 0.2% DMSO. Control cells were maintained in 0.2% DMSO alone. Cytotoxicity was
monitored and determined spectrophotometrically at A492 nm–A620 nm.
2.3. Cell culture, exposure and protein extraction for proteomic analysis
ZFL cells were grown in 5% FBS and 2  106 cells were seeded in 5 cm plates.
ZFL cells were exposed to HBCD (5 mM), TBBPA (5 mM) and mixed exposure
(5+5 mM) for 72 h. Prior to harvesting cells were rinsed once in conditioned PBS
(at 26 1C) containing 10 mM Tris pH 8.5; 250 mM sucrose. Cells were quickly
frozen with liquid nitrogen and 150 ml of sample buffer (40 mM Tris, 7 M urea, 2 M
thiourea, 4% CHAPS, 0.2% (v/v) Bio-lytes, 100 mM DTT and 0.002% (v/v) BPB,
endonuclease and 1 mM PMSF) was added and cells were harvested. To allow
complete cellular lysis, samples were incubated on ice for 10 min prior to
ultracentrifugation at 100,000  g at 18 1C for 1 h. Supernatants were stored at
80 1C until proteomic analysis was performed.
2.4. Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis was run on protein extracts quantified
using the RC-DC kit (BIO-RAD). The first and second dimension was run according
to Albertsson et al. (2007) applying 100 mg of protein (dissolved in 200 ml of sample
buffer) onto immobilized pH strips (11 cm, pH 3–10 NL). For the second dimension
strips were applied onto precast criterion 8–16% resolving gels. Electrophoresis
was carried out in a criterion dodeca cell (Bio-Rad) using a Power Supply model
200/2.0. Following the second dimension, the gels were stained with BIO-SAFE
Coomassie according to the manufacturers instructions (BIO-RAD).
2.5. Scanning and computer-assisted gel analysis
Gel scanning and analysis were performed according to Albertsson et al.
(2007). Digitalised images of 16 bit greyscale and 400 dpi were created as TIFF files
and analysed using the PDQuest version 7.3.0 two-dimensional gel software. The
protein intensity of each spot was normalised to the total intensity of each gel
image. Protein expression in control (843783 spots), HBCD (906761 spots),
TBBPA (882753 spots) and mixture (8917137 spots)-treated cells was evaluated
in one single match-set to determine significant changes (po0.05) in protein
expression. The number of spots detected between control and treated cells was
not statistically different. Each experimental group consisted of 6 replicates.
2.6. MALDI-TOF mass spectrometry
Spots differentially expressed as compared to control samples, were manually
excised using a scalpel. Each spot was identified twice. Samples were analysed
using a MALDI-LR mass spectrometer (Micromass, Manchester, UK) in reflectron
mode. 0.7 ml of tryptic digest was mixed with 0.5 ml matrix solution (12 mg/ml
a-cyano-4-OH-cinnamic acid in acetonitrile/water 1:1, 0.1% TFA) directly on the
MALDI probe and allowed to dry at ambient conditions. ACTH (18–39) MH+
2465.199 was used as external, and tryptic autodigest MH+ 2211.105 as internal
lock mass. Monoisotopic mass values were used for peptide mass mapping using
MASCOT available at www.matrixscience.com, against the nonredundant database
at NCBI. All identifications had a score above 58, well above the limit for the 95%
level of confidence. The theoretical molecular weight (MW) and isoelectric point
(pI) of identified proteins (as determined by the Swiss-Prot database) were
compared to gel migration of respective proteins to confirm and validate MW
and pI.
2.7. Fourier transform ion cyclotron resonance (FT-ICR)
The tryptic peptides were separated using liquid chromatography (LC). For the
LC an Agilent 1100 binary pump was used, together with a reversed phase column,
200  0.050 mm, packed in-house with 3 mm particles Reprosil-Pur C18-AQ
(Dr. Maisch, Ammerbuch, Germany). The flow through the column was reduced
by a split to approximately 100 nl/min. A 40 min gradient 10–50% CH3CN in 0.2%
COOH was used for separation of the peptides. The nanoflow LC-MS/MS were
performed on a 7-T (Linear Trap Quadrupole-Fourier Transform) LTQ-FT mass
spectrometer (Thermo Electron) equipped with a nanospray source modified in-
house. The spectrometer was operated in data-dependent mode, automatically
switching to MS/MS mode. MS spectra were acquired in the FT-ICR, while MS/MS
spectra were acquired in the LTQ-trap. For each scan of FT-ICR, the three most
intense, doubly or triply charged, ions were sequentially fragmented in the linear
trap by collision induced dissociation. All the tandem mass spectra were searched
by MASCOT (Matrix Science, London) against the nonredundant database at NCBI.
The search parameters were set to MS accuracy 5 ppm, MS/MS accuracy 0.5 Da, one
missed cleavage by trypsin allowed, fixed propionamide modification of cysteine
and variable modification of oxidized methionine.
2.8. Statistical analyses
Cytotoxicity data were expressed as the means7SE and evaluated using the
Student T-test assuming two-tailed distribution and two-sample equal variance.
Protein expression changes between control and exposed cells were evaluated
using the nonparametric Mann–Whitney U-test. Statistical significance level was
set to po0.05. MALDI-TOF ms protein scores 458 were statistically significant.
For FT-ICR protein identifications, the minimum criteria were one tryptic peptide
matched at or above the 99.9% level of confidence and one additional peptide
match at the 95% level.
3. Results
3.1. HBCD and TBBPA-induced cytotoxicity
A time-dependent increase in cytotoxicity was observed
following both single and mixed exposure. No substantial toxicity
was observed for either HBCD or TBBPA in the lower mM range
(0.05–5 mM) following exposure for 24 or 72 h (Fig. 1A–D). There
was a substantial time-dependent (from 24 to 72 h) increase in
toxicity following equimolar mixing of HBCD and TBBPA even in
the low mM range (Fig. 1E). While 10 mM HBCD and TBBPA
exposure for 72 h resulted in 75% and 85% survival respectively
(Fig. 1A and B), equimolar mixing (5+5 mM) appeared to further
reduce cell survival to 50% (Fig. 1F).
3.2. Identification of exposure-specific and overlapping responses
Two-dimensional gel electrophoresis of extracted proteins
from ZFL cells resulted in a high separation and resolution with
respect to MW and pI (Fig. 2). There were 63 significant responses
(up/downs) as compared to control expression (Figs. 2–4).
Twenty-six of these proteins were identified using MALDI-TOF
and/or FT-ICR MS (Tables 1 and 2). In general we used both
MS strategies for protein identification. We consider these
identifications more confident than those identified only by
MALDI-TOF ms. A substantial amount of responses did not
retrieve significant hits following database search of MALDI-TOF
or FT-ICR MS data. The present study identified proteomic
responses that were HBCD, TBBPA and mixture specific. In
 ARTICLE IN PRESS
P. Kling, L. Förlin / Ecotoxicology and Environmental Safety 72 (2009) 1985–1993 1987

Fig. 1. Dose–response cell survival (%) of HBCD (A and B), TBBPA (C and D) and HBCD and TBBPA (E and F) exposed ZFL-cells at 24 and 72 h, respectively. The toxicity at 5 mM
of single/mixed exposure are shown (arrows) in B, D and F for comparison of toxicity following single substance and mixed exposure, respectively. Statistically different cell
survival (%) from cells exposed for 72 h is indicated (*) at the po0.05 level.
 ARTICLE IN PRESS
1988 P. Kling, L. Förlin / Ecotoxicology and Environmental Safety 72 (2009) 1985–1993
Fig. 2. Master image representing spots significantly regulated following exposure of ZFL cells to single substance or mixed exposure of HBCD and TBBPA for 72 h. Cells
were exposed to 5 mM of each substance. six replicates were used for each treatment.
addition, proteomic analysis also identified responses that were
overlapping between treatments, occurring in at least two of the
three exposure conditions. Proteomic analyses revealed that
HBCD, TBBPA and mixed exposure triggered 38, 13 and 33
responses respectively (Figs. 3 and 4).
3.3. Distinct protein responses elicited by HBCD
Twenty-two responses were unique following HBCD exposure
(6 ups/16 downs) and 7 identifications were retrieved using
MALDI-TOF and/or FT-ICR ms. A majority of regulated proteins
were downregulated including SSP 4804 (not identified) which
were 7-fold downregulated (Fig. 3). A majority of identified
proteins were related to protein metabolism (SSP 6414, 6611, 6808
and 7406) exhibiting a 1.5-fold downregulation. In addition,
responses related to cellular defence (SSP 4512) and cytoskeleton
dynamics (SSP 7202) were observed following sublethal HBCD
exposure (Table 1).
3.4. Distinct protein responses elicited by TBBPA
Seven proteins were shown to be uniquely regulated in
response to TBBPA exposure (3 ups/4 downs). SSP 7409 was
downregulated around 3-fold (Fig. 3), although we did not
manage to identify this protein. Both MALDI-TOF and FT-ICR
peptide mapping retrieved significant hits for Hsp 70 ( 1.5-fold,
SSP 2612) and transketolase (2.2-fold, SSP 5608) which may
indicate that TBBPA exposure results in an overall change in
protein folding and NADPH production (Table 1).
3.5. Distinct protein responses elicited by mixed HBCD and TBBPA
exposure
There were 17 proteins (6 ups/11 downs) uniquely regulated
following mixed exposure of HBCD and TBBPA (Fig. 4). Three of
the upregulated spots matched the NADPH generating enzymes
isocitrate dehydrogenase 2 (SSP 7404) and transaldolase 1 (SSP
2313 and SSP 4308). In addition two proteins related to cell cycle
control (SSP 1102 and 4305) were upregulated. Furthermore there
was a 3-fold downregulation of the Hsp70 kDa 9B protein (SSP
3613). Two proteins that could not be identified were observed to
be substantially upregulated including SSP 2313 (7-fold) and SSP
6705 (4-fold).
3.6. Overlapping protein responses
Seventeen proteins were observed to be regulated in response
to at least two of three exposure conditions (overlapping
responses). Four of these spots (SSP 3803, 6202, 7607 and 8209)
were regulated following both single and mixed exposure of HBCD
and TBBPA (Table 2), indicating common molecular targets. The
glycolytic enzymes including GAPDH (SSP 6202) and aldolase (SSP
8209) exhibited substantial inductions (7 and 32-fold respec-
tively) following mixed exposure. Moreover, 11 proteins were
regulated following HBCD and mixed exposure, while TBBPA did
not contribute to these responses (Fig. 3D). Five of these proteins
were identified (Table 2). Three of these proteins including
adenosylhomocysteinase (SSP 6403), GDP-mannose 4, 6-dehydra-
tase (SSP 7305) and transketolase (SSP 7802) were not further
regulated following mixed exposure, suggesting that HBCD alone
contributed to the observed responses. In addition, two proteins
were regulated by HBCD alone and mixed exposure including
transaldolase 1 (SSP 2310) exhibiting a 1.6-fold (HBCD exposure)
to 2.8-fold (mixed exposure) increase, while Hsp 70 (SSP 4607)
exhibited a 3.3-fold (HBCD exposure) to 2.1-fold (mixed
exposure) change in expression. There was only one protein (SSP
6503) observed to be regulated following TBBPA (7-fold increase)
and mixed exposure (8-fold increase), suggesting that TBBPA
mainly contributed to the observed upregulation (Fig. 3D).
3.7. Discrepancies between observed and theoretical MW and pI of
identified proteins
In general, a good agreement between predicted (from SWISS-
Prot database) and observed MW and pI was achieved (Tables 1
and 2). However, there are a few examples of proteins that have a
Table 1
Unique protein responses elicited by HBCD and TBBPA following single and mixed exposure of ZFL cells.

Exposure SSP no. Fold Protien identity Cellular function MALDI-TOF data FT-ICR data Obs./Theo. Obs./Theo. pId Accession

P. Kling, L. Förlin / Ecotoxicology and Environmental Safety 72 (2009) 1985–1993

regulation MW (kDa)c code
(7)
Masses Expect Mascot Masses
matched valuea score matchedb

HBCD 4508 1.3 Sb:cb825 protien Metobolism/reductase 13 4.8E 10 139 – 57.7/54.7 6.2/6.3 Q803D7

ARTICLE IN PRESS
4512 +1.2 Aldehyde dehydrogenase 2b Cellular defence 14 4.8E 11 149 – 52.7/56.4 6.3/6.3 Q1JPX8
6414 1.4 Aspartate aminotransferase Aminoacid metabolism 13 4.8E 9 129 – 47.2/46.0 7.0/6.5 Q7ZUW8
6611 1.5 Chaperonin cont. TCP1 subunit 6A Protien folding 17 9.5E 14 176 17 64.9/57.6 7.1/6.7 Q7ZYX4
6808 1.6 Eucaryotic translation elongation Translational elongation 14 1.9E 7 113 – 103.5/95.5 6.9/6.3 Q6P3J5
factor 2
7202 1.6 Arp 2/3 complex, subunit 2 Cytosceleton dynamics 6 6.7E 3 68 – 35.9/34.5 7.2/6.6 Q6P2T5
7406 1.8 Eucaryotic elongation factor 1 Translational elongation 11 2.4E 5 92 13 49.5/50.2 7.4/7.1 Q6NYN8

TBBPA 2612 1.5 Hsp 70 protien 8 Protein folding 15 3.8E 11 150 27 74.9/71.2 5.8/5.3 Q6NYR4
5608 +2.2 Transketolase NADPH generation 7 6.2E 2 58 17 63.4/67.8 6.4/6.8 Q7T2Q9

Mixed 301 2.1 Laminin receptor 1 Translation 9 2.6E 4 82 – 44.5/34.0 4.8/4.7 Q803F6
501 +1.2 Calumenin A Haemostasis 10 9.5E 7 106 – 51.9/37.1 4.5/4.4 Q61QP3
1102 +1.8 Prohibitin Cell cycle control 10 4.8E 9 129 7 32.7/29.7 5.3/5.3 Q7T1D8
2313 +7.2 Transaldolase 1 NADPH generation – – – 10 40.5/38 5.7/6.1 Q6NVC4
3613 3.0 Hsp 70 9B protein Protien folding 10 1.5E 4 84 31 73.3/74.0 6.1/6.7 Q5XJ12
4305 +1.8 V-crk homolog (crkl) Cell cycle control 6 3.7E 2 60 8 44.7/33.8 6.2/6.0 Q6PH06
4308 +2.8 Transaldolase 1 NADPH generation 12 3.8E 8 120 12 42.1/37.9 6.2/5.9 Q6P6Y0
4604 1.5 Seryl-tRNA synthetase Seryl-tRNA aminoacylation 12 3.0E 7 111 – 65.4/58.7 6.1/5.7 Q6DRC0
7404 +2.2 Isocitrate dehydrogenase 2 NADPH generation 7 4.6E 2 59 19 48.3/50.4 7.3/8.3 Q7ZUP6

a
Expectation value—odds of retrieving the same score from a random database without the target protein of interest. bFor FT-ICR protein identification at least two matched peptides were required at the 99.5 and 95% confidence
level respectively. Observed (Obs.) and theoretical (Theo.) molecular weight (MW)c and isoelectric point (pI)d was obtained from gel migration and from the Swiss-Prot. database respectively.

1989
 1990
Table 2
Overlapping (co-regulated) protein responses following single/mixed HBCD and TBBPA exposure of ZFL cells.

SSP Exposure and fold regulation Protien identity Cellular function MALDI-TOF data FT-ICR Obs./Theo. Obs./Theo. pId Accession
No. (7) data MW (kDa)c code

HBCD TBBPA Mixed Masses Expect Mascot Masses

matched valuea score matchedb

2310 +1.6 n.d. +2.8 Transaldolase 1 NADPH generation 11 6.0E 8 118 12 41.5/37.9 5.6/5.9 Q6P6Y0
4607 +3.3 n.d. 2.1 Hsp 70 protien 8 Protein folding 9 3.3E 3 71 16 75.7/71.2 6.2/5.3 Q6NYR4
6202 +2.5 +2.8 +7.4 GAPDH Glukoneogenesis 10 2.6E 5 92 6 37.9/36.1 6.6/6.5 Q6NYM9
6403 1.3 n.d. 1.3 Adenosylhomocysteinase Cysteine metabolism 8 1.7E 2 63 17 48.3/47.9 6.5/6.3 Q803T5
7305 1.7 n.d. 1.7 GDP-mannose 4,6-dehydratase Carbohydrate metabolism 7 2.8E 3 71 13 44.3/41.8 7.4/6.9 Q1LVF8
7607 +2.3 +1.8 +3.8 Methylcrotonoyl Co-A carboxylase 2 Ligase activity 9 3.2E 4 81 11 64.8/61.7 7.7/8.4 Q6NW15
7802 2.0 n.d. 2.1 Transketolase NADPH generation 8 2.1E 3 73 2 98.0/67.8 7.4/6.8 Q7T2Q9
8209 +6.1 +17 +32 Aldolase A Gluconeogenesis 5 9.1E 2 56 7 37.5/39.7 8.1/8.4 Q803Q7

P. Kling, L. Förlin / Ecotoxicology and Environmental Safety 72 (2009) 1985–1993

a
Expectation value—odds of retrieving the same score from a random database without the target protein of interest. bFor FT-ICR protein identification at least two matched peptides were required at the 99.5 and 95% confidence
level respectively. Observed (Obs.) and theoretical (Theo.) molecular weight (MW)c and isoelectric point (pI)d was obtained from gel migration and from the Swiss Prot.database respectively. Protein expression change not
detected (n.d.).

ARTICLE IN PRESS
This may be an explanation to the observed discrepancy between
tional processing of certain proteins is known to greatly affect pI.
et al., 2007; Zhan and Desiderio, 2003). In addition, posttransla-
but may also be due to SDS-resistant multimer formation (Coling
including posttranslational modifications such as glycosylation
reasons to the discrepancy between observed and theoretical MW
7802) than predicted (Tables 1 and 2). There may be several
significantly higher observed MW (SSP 301, 501, 4305, 6611 and

2313 (C), 6202, 6503 and 8209 (D) and is indicated by broken bars.
following exposure. A more than 5-fold change was observed for SSP 4804 (A),
ments (D). Standard spot numbers (SSP) indicate respective protein regulated
TBBPA-specific (B), mixture-specific (C) or overlapping between different treat-
were quantified and grouped according to responses that were HBCD-specific (A),
Fig. 3. Quantity of regulated responses following 72 h of exposure. The responses
 ARTICLE IN PRESS
P. Kling, L. Förlin / Ecotoxicology and Environmental Safety 72 (2009) 1985–1993
Fig. 4. Qualitative view of exposure-specific and exposure-overlapping responses
in ZFL cells presented in a Venn diagram. All significant responses were compared
with control expression of the corresponding protein.
observed and theoretical pI for some proteins including SSP 4607
(obs./theo. 6.2/5.3) and SSP 7404 (obs./theo. 7.3/8.3).
4. Discussion
To gain realistic insights into environmental impacts on
aquatic organisms, there is an urgent need to develop sensitive
and reliable methods to monitor potential mixture effects. Due to
increasing levels of BFRs in the environment, including HBCD and
TBBPA (BFR, 2004) these substances are of ecotoxicological
importance. The mechanisms of HBCD and TBBPA toxicity are to
date poorly understood. We have therefore conducted a compara-
tive two-dimensional gel approach in order to generate hypoth-
eses regarding single and mixture effects from HBCD and TBBPA at
sublethal concentrations in zebrafish liver cells.
Cytotoxicity tests were performed to determine sublethal
levels of HBCD and TBBPA in ZFL cells. TBBPA exhibited a more
pronounced cytotoxicty after 24 h of exposure, while HBCD
toxicity was increased at 72 h of exposure. This might reflect the
shorter half-life of TBBPA and its previously demonstrated acute
toxicity in fish (Birnbaum and Staskal, 2004). The observation that
HBCD toxicity was substantially increased (in the higher mM
range) following 72 h of exposure is in support with the view of a
slow metabolism of HBCD and potential to bioaccumulate (Morris
et al., 2004), but may also suggest a slower cellular uptake when
compared to TBBPA. While 5 or 10 mM (estimated from dose–
response curve) exposure of individual substances for 72 h did
not result in substantial toxicity, mixing at equimolar (5+5 mM)
concentrations seem to result in an increased toxicity. However,
further dose–response studies are required to make firm conclu-
sions regarding possible more than additive effects. While limited
in vitro data on individual substance toxicity are available, mixture
effects of HBCD and TBBPA are lacking. The in vitro toxicity of
HBCD and TBBPA in the ZFL cell line is comparable to other studies
using liver cells as test system (Nakagawa et al., 2007; Zhang et al.,
2008), but has been shown to differ between cell types with EC50
ranging from 50 to 200 mM TBBPA exposure (Strack et al., 2007).
The present study identified proteomic responses that were
HBCD, TBBPA and mixture specific. In addition, proteomic analysis
1991
also identified responses that were overlapping between different
treatments, suggesting common modes of action. In total we
observed 63 significant responses in our test set. This is the first
study describing proteomic responses from single and mixed
HBCD and TBBPA exposure. In our attempt to identify these
responses we used both MALDI-TOF and FT-ICR MS to confirm and
increase the confidence of the protein identifications. To our
knowledge five proteomic studies on BFRs have been performed.
Hepatic effects in zebrafish exposed to TBBPA or a selected BFR
mixture suggest increased cellular stress and effects on proteins
related to energy metabolism (De Wit et al., 2008; Kling et al.,
2008). Proteomic studies in mussels and crabs exposed to PBDE 47
suggest that the responses might be used as toxicological
signatures and involve responses related to xenobiotic stress,
amino acid metabolism, oxidative stress and effects on the cyto-
skeleton (Apraiz et al., 2006; Gomiero et al., 2006). In addition
proteomic responses in mice exposed to PBDE 99 revealed
responses that were related to neurotoxicity (Alm et al., 2006).
Sublethal exposure of HBCD resulted in 22 unique protein
responses. Although only a fraction of the regulated proteins was
identified it appears that the HBCD-distinct responses are
relatively mild including downregulation of housekeeping protein
metabolism and a decrease in cytoskeleton dynamics. Sublethal
TBBPA exposure resulted in 7 unique protein responses indicating
responses related to protein folding (Hsp 70) and increased
NADPH generation (transketolase).
Proteomic analysis revealed 4 responses that were common to
all three exposures. Identification of two of these responses
suggests increased cellular energy conservation since a substan-
tial induction in gluconeogenic proteins (GAPDH and aldolase)
was observed. Both TBBPA and HBCD have been demonstrated to
induce oxidative stress (Canesi et al., 2005; Ronisz et al., 2004; Shi
et al., 2005). Oxidative stress has previously been demonstrated to
slow down glycolysis and increase gluconeogenesis in order to
conserve cellular glucose and glucose-6 phosphate to be shunted
for NADPH production (Lou, 2003). Hence, the substantial
induction of gluconeogenic proteins in response to single and
mixed exposure of HBCD and TBBPA may indicate cellular adverse
effects including oxidative stress. These observations are in
support of proteomic studies in zebrafish liver suggesting
increased production of gluconeogenic proteins following TBBPA
exposure (De Wit et al., 2008). Moreover, a high expression of
GAPDH and aldolase have been linked to adverse effects including
hepatocellular carcinoma and apoptosis (Castaldo et al., 2000;
Hara et al., 2006; Hara and Snyder, 2006; Sirover, 1997). In this
study 11 protein responses were co-regulated after HBCD and
mixed exposure, while only one protein was uniquely regulated by
TBBPA and mixed exposure. Hence a substantial portion of
overlapping responses was mediated by HBCD, which might
indicate that HBCD mainly contributes to increased cytotoxicity
observed at 72 h of mixed exposure.
Although single substance exposure of HBCD and TBBPA in ZFL
cells resulted to some extent in increased production of proteins
related to NADPH production (transaldolase and transketolase,
respectively), mixed exposure resulted in a more pronounced
upregulation of proteins related to NADPH production (two
transaldolase isoforms and isocitrate dehydrogenase 2). Thus
mixing may specifically enhance cellular NADPH that may be used
for production of cellular defence systems involved in xenobiotic
metabolism and oxidative stress including cytochrome P450 and
reduced glutathione, GSH (Mayes, 1993). Transaldolase is involved
in the pentose phosphate shunt, generating NADPH important
for GSH production and hence important for resistance against
oxidative stress (Mayes, 1993). A high expression of transaldolase
has been suggested to increase redox-dependent apoptotic
sensitivity (Banki et al., 1996). The pronounced increase in
 ARTICLE IN PRESS
1992 P. Kling, L. Förlin / Ecotoxicology and Environmental Safety 72 (2009) 1985–1993
different isoforms of transaldolase following mixing may contri-
bute to the increased cytotoxicity observed following mixed
exposure of HBCD and TBBPA. Furthermore mixture-specific effects
related to changes in cell cycle control (upregulation of prohibitin
and v-crk oncogene homologue) were observed. The tumor
suppressor prohibitin is overexpressed in gastric cancer tissues
as shown by two-dimensional gel electrophoresis (Kang
et al., 2008; Wang et al., 2004) and has been shown to protect
against oxidative stress (Theiss et al., 2007). The observed mixture-
specific increase in prohibitin may therefore suggest an increased
need for cellular protection against oxidative stress. Furthermore, a
mixture-specific upregulation of v-crk oncogene homologue (crkl)
was observed. Crkl has been shown to induce cell transformation,
cell migration and has also been suggested as a metastasis
associated biomarker (Liu et al., 2008; Nievers et al., 1997; Tsuda
et al., 2002). Crkl transformation is known to result in overactivity
of the ras-MAPK signalling pathway resulting in overproliferation
(Ishimaru et al., 1999). TBBPA-mediated activation of MAPK
signalling has earlier been suggested in mussel hemocytes (Canesi
et al., 2005) and in zebrafish liver (De Wit et al., 2008). Although
there were no TBBPA-specific effects related to MAPK activation,
there was an induction of crkl following mixed HBCD and TBBPA
exposure, possibly suggesting interference with the MAPK signal-
ling pathway at a different molecular target.
5. Conclusions
The proteomic approach used in this study has generated new
hypotheses regarding both single and mixture related effects
following HBCD and TBBPA exposure. While single substance
specific responses and responses that were overlapping suggest
increased energy conservation including gluconeogenesis, the
observed mixture-specific effects suggest a pronounced increase
in proteins related to NADPH production and cell cycle control.
Hence the proteomic approach used in this study clearly points
out some differences with regard to exposure that may suggest
early biomarkers for single as well as mixed HBCD and TBBPA
exposure. Exposure was performed at sublethal levels and the
effects observed may represent early toxicological markers that in
the future may be developed into robust biomarkers. In addition,
the present study suggests that environmental mixtures contain-
ing HBCD and TBBPA may result in unexpected biological effects.
In the future the proteomic approach will hopefully be applicable
for detecting mixture effects from other pollutants and therefore
used as a routine tool in ecotoxicity testing.
Acknowledgments
The authors would like to thank FORMAS and Mistra-NewS for
financial support. We would also like to thank Elisabet Carlsohn
and Sjoerd van der Post at Bio-X-Med proteomic facilities at the
University of Gothenburg for help with the MALDI-TOF and FT-ICR
MS.
Funding sources: This study was financially supported by the
research foundation MISTRA-NewS and the research council
FORMAS.
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