Toxicology Letters 224 (2014) 141–146
Contents lists available at ScienceDirect
Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Effects of novel brominated flame retardants on steroidogenesis in
primary porcine testicular cells
Rishikesh Mankidy a,∗ , Bibhuti Ranjan b , Ali Honaramooz b , John P. Giesy a,b,c,d
a
Toxicology center, University of Saskatchewan, Saskatoon, SK, Canada
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
d
Department of Biology and Chemistry and State Key Laboratory for Marine Pollution research, City University of Hong Kong, Kowloon, Hong Kong, China
b
c
h i g h l i g h t s
•
•
•
•
Primary cells were used to investigate the effects of NBFRs on steroidogenesis.
TBB exposure upregulated the pathway for aldosterone and cortisol biosysthesis.
TBCO and TBPH exposure produced greater amounts of sex hormones.
The study revealed subtle differences in the molecular targets of TBCO and TBPH.
a r t i c l e
i n f o
Article history:
Received 5 September 2013
Received in revised form 15 October 2013
Accepted 16 October 2013
Available online 25 October 2013
Keywords:
BFR
NBFR
TBB
TBCO
TBPH
In vitro steroidogenesis
a b s t r a c t
Brominated flame retardants are chemicals with fire quenching properties which are extensively used in
manufacturing. Historically, less regulated use of legacy brominated flame retardants (BFRs) for a number
of years has resulted in ubiquitous contamination of the environment. As a result, some of the more persistent BFRs have been phased out and are being replaced by a next generation of brominated compounds
for which there is little toxicological data. The study investigated effects of 2-ethylhexyl tetrabromobenzoate (TBB), 1,2,5,6-tetrabromocyclooctane (TBCO), and bis-(2-ethylhexyl) tetrabromophthalate (TBPH)
on steroidogenesis in a porcine primary testicular cell model. TBB did not affect sex-steroid production
in this cell model; rather the data suggest a flux towards synthesis of aldosterone and cortisol via upregulation of CYP21A2. At the greatest concentrations of TBCO and TBPH tested greater production of sex
hormones testosterone (T) and estradiol (E2) was observed. Effects were mediated by regulation of multiple molecular targets in the steroidogenesis pathway; CYP11A in the case of TBPH and CYP17A1 in the
case of TBCO. This investigation is the first of its kind to use a testicular mixed population cell model to
investigate mechanism(s) of action of three chemically diverse compounds currently used in commercial
fire retardants.
© 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Brominated flame retardants (BFRs) are chemicals added to
commercial and end-user consumer products to improve their
fire resistance. They are characterized by the presence of multiple bromine atoms covalently linked to carbon. Presence of the
electronegative bromine atoms aid in resisting fire by quenching
free radicals generated during combustion of organic chemicals,
thereby mitigating propagation. BFRs are extensively used in fire
retardant mixtures put into plastics, textiles, furniture, electrical
components as well as in various industrial applications. Since
∗ Corresponding author. Tel.: +306 966 8733/306 966 4680; fax: +306 966 4796.
E-mail address: mankidy@gmail.com (R. Mankidy).
0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxlet.2013.10.018
these additives are not covalently linked to the matrix, they have
the propensity to leach out of products. Legacy BFRs such as
polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) have been produced in large quantities for four
decades, and as a result are ubiquitous in the environment (de
Wit, 2002; Harrad et al., 2009; Law et al., 2006; Sellström et al.,
1998) biota (de Wit et al., 2006; Lam et al., 2009; Law et al., 2006;
McDonald, 2002; Thomsen et al., 2003; Vorkamp et al., 2012; Weiss
et al., 2004). The penta-, octa- and deca-versions of PBDEs, which
were primarily used in early commercial fire-retardant mixtures
have the tendency to bioaccumulate and biomagnify (Birnbaum
and Staskal, 2004; Burreau et al., 2004; Law et al., 2006). In addition
to being detected in urban industrial areas, these chemicals are of
concern as they have been detected in polar regions, which suggest
that they have a potential for long-range atmospheric transport
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R. Mankidy et al. / Toxicology Letters 224 (2014) 141–146
(de Wit et al., 2006). In the 2009 Stockholm Convention pentaand octa-BDEs were listed as persistent, organic pollutants and
have since been phased-out of production. Deca-BDE is slated to
be phased out by the end of 2013.
As there are still statutory requirements for fire resistance in
products such as textiles and electronics, and brominated organic
compounds are the most efficient and cost-effective ways to
impart such flame retardation properties, historically used BFRs
are being replaced with brominated compounds which maintain
the fire retarding properties of the technical mixtures (Covaci et
al., 2011). 1,2,5,6-tetrabromocyclooctane (TBCO) is a component
of Saytex BC-48 produced by Albermarle Corporation (Gauthier
et al., 2009). 2-Ethylhexyl tetrabromobenzoate (TBB) and bis(2-ethylhexyl) tetrabromophthalate (TBPH) are components of
Firemaster® (Stapleton et al., 2008) and BZ-54 is synthesized
by Chemtura corporation (Davis and Stapleton, 2009). This next
generation of fire retarding chemicals is often synthesized by
brominating backbones of chemicals used in other industrial processes. TBB is a brominated version of 2-ethylhexyl benzoate, a
non-phthalate plasticizer used in paint mixtures and cosmetic
products and TBPH is the brominated analog of diethylhexylphthalate, a commercially used plasticizer. While it is tempting to suggest
that the brominated analogs mimic their non-brominated versions
with respect to their physicochemical profiles, such extrapolations are not warranted without empirical evidence. Bromination
alters physical and chemical properties of parent compounds. These
new compounds require independent studies investigating their
presence, distribution, degradation, absorption, accumulation, toxicities, and critical mechanism(s) of action.
There is currently, little, publically available information on
these newer chemicals. TBPH was detected in fatty tissue of porpoises and dolphins in South China Sea (Lam et al., 2009) and
demonstrated a tendency to bio accumulate in fathead minnows
(Bearr et al., 2010). TBB and TBPH have been shown to cause damage to DNA in hepatic tissue of fish (Bearr et al., 2010) and have
been detected in air samples from the North American Great Lakes
region at 10-fold greater concentrations in urban areas compared
to remote areas (Ma et al., 2012). In humans, dust was the major
route of exposure to BFRs (Lorber, 2008; Wilford et al., 2005) and
though studies have reported the presence of TBB, TBCO and TBPH
in house dust (Dodson et al., 2012; Stapleton et al., 2008), the
bioavailability of these chemicals and implications for health of
humans due to these exposures are unclear. Attempts at identifying the metabolic products of TBB and TBPH have been made
using an in vitro model (Roberts et al., 2012), although toxicities of
the metabolites are not known. A correlation between concentrations of TBPH in house dust and concentrations of triiodothyronine
(T3) in blood plasma of humans has been reported (Johnson et al.,
2013). Recent investigations using the components of FM 550 have
demonstrated endocrine disruptive effects caused by TBB and TBPH
in rat tissue (Patisaul et al., 2013), whereas in vitro studies with
TBB, TBCO, and TBPH have demonstrated ER- and AR-mediated
antagonistic effects and elevated steroidogenesis of sex hormones
(Saunders et al., 2013). Based on these preliminary reports, we
investigated the effects of TBB, TBCO, and TBPH on steroidogenesis
in a primary neo-natal porcine testicular cell model and assessed
the molecular effects of the NBFRs on key targets in the steroid
biosynthesis pathway.
2. Materials and methods
2.1. Isolation and propagation of porcine testicular cells
Testes were collected from neonatal piglets (1to2 weeks of age) and placed in
Dulbecco Phosphate buffer solution (Lonza Walkersville Inc., Walkersville, MD) containing 10 ␮g/ml penicillin and 100 U/ml streptomycin. Isolation, purification, and
culturing were done as previously described (Bernier et al., 1983; Lejeune et al.,
1998). Briefly, the testes were decapsulated, minced, and digested with 0.5 mg/ml
of collagenase solution. Tissue was collected following 90 min digestion and filtered through 0.4 ␮m nylon filter. The cell suspension was centrifuged, and the
cell pellet was washed in DMEM medium. Resuspended cells were layered on a
percoll® gradient (21, 26, 34, and 60%) and centrifuged at 800 × g for 20 min. The
heterogeneous population of cells containing Leydig, Sertoli, and interstitial cells
was isolated from the 34% layer of the percoll gradient and resuspended in DMEM
-F12 medium (1:1) containing antibiotics, 10% Nuserum, and ITS premix. Viable cells
were enumerated by use of the trypan blue exclusion method. Cytochemical staining for 3␤-HSD activity was carried out by exposing the cells to a solution containing
0.2 mg/ml nitrotetrazolium blue 0.12 mg/ml Dehydroepiandrosterone and 1 mg/ml
of beta NAD in PBS (0.05 M, pH 7.4) (Sigma Aldrich, St Louis, MO). 60–70% of the
isolated cells were confirmed to be Leydig cells as they stained positive for 3␤-HSD
enzyme activity (Mendelson et al., 1975), S1. 3 × 105 cells were seeded in each well
of a 24-well plate and incubated for 72 h at 34 ◦ C and 5% CO2 . Following 72 h of
incubation, the medium was replaced with one containing the test chemicals TBB
(Wellington Laboratories, ON, Canada), TBPH (Waterstone Technology, IN, United
States), and TBCO (Specs, Delft, Netherlands) or DMSO solvent control. The system
was cultured under the control of porcine LH (Lutropin-V, Bioniche, ON, Canada) at
a concentration of 1 ng/ml (Lejeune et al., 1998).
2.2. Viability of cells
Metabolically active cells in the heterogeneous population were determined by
use of WST-1 reagent (Roche Applied Science, Indianapolis, IN) according to manufacturer’s protocol. Briefly, 3 × 104 cells were exposed to the test chemicals for 48 h.
The percent viable cells following exposure to chemicals was estimated by comparing absorbance at 440 (A440) of cells exposed to NBFRs with that of the solvent
control.
2.3. Quantification of hormones
Conditioned medium was collected after exposure of the heterogeneous cells to
the test chemicals TBB, TBCO, and TBPH for 48 h. Hormones (T and E2) were extracted
by organic solvent liquid–liquid extraction as described (Chang et al., 2010). In brief,
300 ␮l of media was extracted using 2.5 ml of hexane and Ethylacetate (1:1). The
organic fraction was collected following centrifugation at 400 g for 5 min. Following a second extraction, organic fractions were pooled and dried under a stream
of nitrogen. Dried organic content was reconstituted with 150 ␮l of appropriate
buffer (supplied with the ELISA kit). T and E2 quantification was done by the use
of an ELISA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s
recommendations.
2.4. Expression of genes
Primary cells exposed to test chemicals were harvested simultaneously with
conditioned media. RNA was isolated by use of Trizol (Life Technologies Corporation, Carlsbad, CA) and quantified by use of a NanoDrop ND-1000 Spectrophotometer
(Nanodrop Technologies, Welmington, DE, USA). cDNA was synthesized from 2.5 ␮g
of each RNA sample by the use of a QuantiTect Reverse Transcription Kit (Qiagen, Mississauga, ON) according to the manufacturer’s recommendations. Relative
quantification of transcript abundance was carried out using SYBR green by the
Ct method. Expression of target genes was reported relative to the expression of
housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT). The
thermal cycle profile was: denaturization for 15 s at 95 ◦ C followed by annealing
and extension for 1 min at 60 ◦ C for a total of 40 PCR cycles. In each case, product
homogeneity was confirmed by a melt curve analysis. The sequence of primers used
for the amplification of target genes has been published previously (Lervik et al.,
2011).
2.5. Statistical analyses
Statistical analyses were done by the use of IBM SPSS Statistics 20. For each
of the data sets, Shapiro–Wilk’s test was used to test for normality of data (significance > 0.05), and Levene’s test was applied to ensure homogeneity of variances
(significance > 0.05). Differences between treatment conditions and the solvent control were evaluated by a one-way ANOVA followed by a Dunnet’s post hoc test.
P-values less than 0.05 were considered significant.
3. Results
3.1. Determination of toxicity of NBFRs
Exposure of neo-natal porcine testicular cells to the three NBFRs
revealed the highest testable concentration that did not cause cellular toxicity (Fig. 1). TBB was not cytotoxic to primary cells at
concentrations from 5 × 10−1 to 5 × 10−5 mg TBB/L. TBPH caused
15% lesser viability relative to unexposed cells (P = 0.0029) at only
R. Mankidy et al. / Toxicology Letters 224 (2014) 141–146
143
Viable cells (percent control)
120
100
*
80
60
*
40
20
0
Solvent
0.00005
Control
0.005
0.5
TBB (mg/L)
0.03
3.0
TBCO (mg/L)
300
0.15
15.0
1500
TBPH (mg/L)
Fig. 1. Cytotoxicity of novel brominated fire retardants. Primary porcine testicular cells were exposed to NBFRs (TBB, TBCO, and TBPH) or to the solvent control for 48 h. Unit
of concentration is mg/L. Metabolically active cells were enumerated using WST-1 reagent. Results represent an average of 4 independent experiments. Asterisk indicates
significant cytotoxicity (P < 0.005) compared to the control. Error bars represent the standard errors of the mean (SEM).
the greatest concentration of 1.5 × 103 mg/L Similarly, Exposure to
3 × 102 mg TBCO/L resulted in 60% fewer viable cells than control
( P = 2.8 × 10−6 ). The number of viable cells exposed to lesser concentrations of TBPH and TBCO were not significantly different from
those exposed to solvent controls.
(P) served as controls; exposure to 4.11 mg F/L resulted in 2.2-fold
greater T production, exposure to 1.13 mg P/L resulted in 70% less
T in the medium (Fig. 3).
3.3. Effects of NBFRs on estradiol biosynthesis
3.2. Effects of NBFRs on testosterone biosynthesis
Concentration of T in the conditioned media of cells exposed
to 0.005 and 0.5 mg TBB/L was not significantly different from
the solvent control (Fig. 2). In the case of TBCO exposure, the
concentration of T in the conditioned media was elevated in a
concentration-dependent manner; exposure to 0.03 mg and 3.0 mg
TBCO/L resulted in 1.6-fold (P = 0.015) and 2.1-fold (P = 0.008)
greater production of testosterone. Incubation of the cells with
TBPH revealed a reciprocal relationship between the exposure concentration and T production by the cells. Cells exposed to the lesser
concentration of 0.15 mg TBPH/L resulted in a 2-fold increase in T
concentration over control (P = 0.016), however, exposure to 100fold greater concentration of TBPH resulted in 25% less production
of T (P = 0.03), and yet maintained a concentration 1.5-fold greater
that of the solvent controls (P = 0.006). Forskolin (F) and prochloraz
Cells exposed to 0.005 and 0.5 mg TBB/L demonstrated ∼2fold greater production of E2 in the conditioned media compared
to DMSO controls, but reached statistical significance only at the
lesser concentration of 0.005 mg/L (P = 0.042). Cells exposed to
0.03 mg TBCO/L produced 5.9-fold greater concentration of E2 in
the conditioned media (P = 0.006). Exposure to 100-fold greater
concentration of TBCO however, did not generate greater amounts
of E2 in the system. Exposure of cells to 0.15 mg TBPH/L resulted in
4.6-fold greater production of E2 in the conditioned medium compared to the solvent controls (P = 0.007). Despite a 100 fold greater
exposure concentration of 15 mg TBPH/L, E2 production was only
slightly augmented to a concentration 5.3-fold greater than the
control (P = 0.007). (F and P served as controls; exposure to 4.11 mg
F/L resulted in 5-fold greater E2 production, exposure to 1.13 mg
P/L resulted in 64% less E2 in the medium.
Fig. 2. Concentration of T in the conditioned media of primary testicular cells. Values represent the T concentration as a percentage of the solvent control. Cells were exposed
to NBFRs (TBB, TBCO, and TBPH) or to the solvent control for 48 h. The unit of concentration is mg/L. Values are an average of 3 independent replicates; error bars indicate the
standard errors of the mean. Asterisk indicates values significantly different than the solvent control. 4.11 mg F/L and 1.13 mg P/L served as positive and negative controls
for steroidogenesis respectively.
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R. Mankidy et al. / Toxicology Letters 224 (2014) 141–146
Fig. 3. Concentration of E2 in the conditioned media of primary testicular cells. Values represent the E2 concentration as a percentage of solvent controls. Cells were exposed
to NBFRs (TBB, TBCO, and TBPH) or to the solvent control for 48 h. The unit of concentration is mg/L. Values are an average of 3 independent replicates; error bars indicate
the standard errors of the mean. “*” indicate values significantly different than solvent controls (P < 0.05); “**” indicate values significantly different from controls (P < 4.11
mg F/L and 1.13 mg P/L served as positive and negative controls for steroidogenesis respectively.
3.4. Gene expression profile
3.7. Effects of TBPH on genes of steroidogenesis
Expression of key enzymes of steroidogenesis illustrates differing mechanisms of action of the NBFRs (Fig. 4).
Exposure of primary cells to TBPH did not affect expression of
StAR, CYP17A1, CYP21A2, 3-ˇHSD, or 17ˇ-HSD at either of the concentrations tested (Fig. 4C). Expression of CYP11A1 was 3.5-fold
(P = 0.01) and 6.6-fold (P = 0.03) greater in cells exposed to 0.15 and
15.0 mg TBPH/L, respectively, relative to that of solvent controls.
No change in expression of CYP19A1 was observed in cells exposed
to 0.15 mg TBPH/L, though expression of mRNA for CYP19A1was
3.3-fold greater (P = 0.048) when the cells were exposed to 15 mg
TBPH/L.
3.5. Effects of TBB on genes of steroidogenesis
Exposure of primary testicular cells to 0.005 and 0.5 mg TBB/L
did not alter expression of StAR, CYP11A1, 3ˇ-HSD, 17ˇ-HSD, or
CYP19A1 (Fig. 4A). Expression of CYP17A1 exposed to 0.5 mg TBB/L
(P = 0.03) was less than 16% of that of control cells. This effect
was more pronounced at a lesser exposure of TBB; <5% control
expression was observed a 0.005 mg/L (P = 0.02). Abundance of
Cyp21A2, the enzyme responsible for directing substrate away
from sex hormone synthesis, and towards the synthesis of cortisol and aldosterone appeared to be greater in cells exposed
to TBB. Transcription of CYP21A2 was 2-fold greater in cells
exposed to 0.005 mg TBB/L than that of controls, though statistical significance was achieved only at 0.5 mg TBB/L wherein the
observed transcript abundance was 2.5-fold greater than control
(P = 0.04).
3.6. Effects of TBCO on genes of steroidogenesis
Exposure of TBCO to the heterogeneous population of primary
testicular cells did not affect expression of StAR, CYP11A, 3ˇHSD, 17ˇ-HSD or CYP19A1 at either concentration tested (Fig. 4B).
Expression of CYP17A1 exhibited a TBCO concentration-dependent
increase in expression, though statistical significance was observed
only at the greater concentration. When exposed to 0.03 or 3.0 mg
TBCO/L expression of CYP17A1 was 1.8-fold (P = 0.22) and 3.5fold (P = 0.03) greater than that of controls. Expression of CYP21A
remained unaffected at the lesser concentration of TBCO tested,
but was significantly (P = 0.03) greater (4.2-fold) than that of cells
exposed to only the carrier solvent.
4. Discussion
This study is the first of its kind to investigate effects of three
novel brominated flame retardants on steroidogenesis. The heterogeneous primary cell culture system derived from pig testes
contained androgen synthesizing Leydig cells, supportive Sertoli
cells, and adrenal-like cells derived from the interstitium of testis
(Griswold, 1995; Val et al., 2006). It can thus be used to monitor
effects on biosynthesis of sex, glucocorticoid (cortisol), and mineralocorticoid (aldosterone) steroids simultaneously. This system
is easier to maintain and propagate than an ex-vivo organ culture
system, and is superior to traditional homogeneous cell culture systems as it allows cross-talk between the different component cell
types, reminiscent of an intact organ.
In the canonical steroid hormone synthesis pathway, cholesterol is the common precursor which can have multiple fates
dictated by the sequential action of enzymes (S2). Steroidogenic
acute regulatory protein (StAR) mediates transfer of cholesterol
to the inner membranes of mitochondria and is considered to be
the rate-limiting step in biosynthesis of steroids. Since none of
the NBFRs tested altered expression of StAR, it is unlikely that
the exposures resulted in greater influx of cholesterol into the
biosynthetic pathways; changes in concentrations of T and E2
observed in the conditioned media following exposures to the
R. Mankidy et al. / Toxicology Letters 224 (2014) 141–146
145
Fig. 4. Transcription profile of genes involved in steroidogenesis. Expression profile of genes involved in the process of steroidogenesis in neonatal porcine testicular cells.
Cells were exposed to NBFRs: TBB (panel A), TBCO (panel B), TBPH (panel C), or to appropriate solvent controls for 48 h. Values indicate expression of target genes relative to
controls and are an average of 3 independent replicates. Error bars indicate the SEM. Asterisk denotes values that are significantly different from solvent controls (P < 0.05).
test chemicals appear to be due to redirection/redistribution of
existing substrate. Exposure of primary cells to TBCO and TBPH
resulted in greater production of sex hormones suggestive of a
similar mechanism of action. However, careful scrutiny of the
underlying molecular events revealed critical differences between
the exposures. The result of both greater transcript abundance of
CYP11A1 caused by TBPH and selective up-regulated expression of
CYP17A1 caused by TBCO, was a greater flux of substrate through the
sex-steroid biosynthesis pathway. Another subtle observation was
evident in effects of TBPH on sex-steroid production. Expression
of CYP19A1 was significantly augmented over solvent control only
at the greater concentration (15 mg TBPH/L). Since Cyp19A1 catalyses conversion of T to E2, a significant reduction in concentration
of T was detected in the conditioned medium, but corresponding
increases in concentrations of E2 in the media were not observed.
This paradox could be explained based on the fact that E2 is in
equilibrium with its precursor estrone (E1) under the control of
17ˇ-HSD (Moeller and Adamski, 2009), and therefore any excess
amounts of E2 generated from T by the action of Cyp19A1 could be
re-distributed to E1 and thereby evade detection in the conditioned
medium.
Simultaneous measurements of steroids and gene expression
provide insight into the preferential effects of TBB on steroidogenesis. Exposure of porcine cells to TBB had no effect on T production
and elevated E2 concentration only at the lower concentration
tested. These data are in agreement with our previous report using
the H295R cell system (Saunders et al., 2013).Furthermore, abundances of CYP11A1, CYP17A1 or 3ˇ-HSD, key enzymes required for
biosynthesis of androstenedione (the common precursor of T and
E2) were not significantly altered by TBB. Instead, expression of
CYP21A2, the “gateway” enzyme responsible for directing the flux
of substrate (progesterone) towards glucocorticoid and mineralocorticoid endpoints (Miller, 1988), was greater following exposure
to TBB. This was reinforced by the observation that exposure to
the NBFR resulted in down-regulation of expression of CYP17A1.
This would have resulted in severe retardation in clearing of its
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R. Mankidy et al. / Toxicology Letters 224 (2014) 141–146
substrates, pregnenolone and progesterone. While it is conceivable
that up-regulation of CYP21A2 is a direct consequence of accumulation of progesterone, an alternate possibility could be that
both CYP21A2 and CYP17A1 are independent molecular targets of
TBB, and as a result of exposure to the NBFR, work in concert to
drive cholesterol biosynthesis away from sex steroid synthesis and
towards synthesis of aldosterone and cortisol. The data here however do provide unequivocal proof in favor of one theory over
another. It is important to note that we did not observe a dose
response for any of the end points measured (T, E2, or transcript
abundances) in the case of exposure to TBB. Absence of such a
response could be attributed to poor solubility of the compound
in an aqueous medium.
In conclusion, this study demonstrates the use of a simple, yet
elegant system in highlighting subtle difference between the mechanism(s) of action of NBFRs. All compounds in the study exhibited
different mechanism(s) of action in the primary cell model; TBB
exposure appeared to favor synthesis of cortisol and aldosterone,
while TBCO and TBPH exposures selectively produced more sex
hormones. It is important to note that while single compound
investigations as this one are instrumental in teasing apart the critical mechanism(s) of action, in reality, the receptor organism never
encounters exposure to a single chemical. In the case of exposure to
fire-mitigating technical mixtures, the components of the mixture
would simultaneously stress the steroidogenesis pathway, albeit,
at different molecular targets. It is therefore conceivable that the
cumulative stress on the receptor organism as a result of multiple
assaults would be compounded, and hence be significantly greater
than that predicted by the model.
Acknowledgements
The research was supported by a Discovery Grant from the Natural Science and Engineering Research Council of Canada (Project #
406497). The authors wish to acknowledge the support of an instrumentation grant from the Canada Foundation for Infrastructure.
Prof. Giesy was supported by the Canada Research Chair program,
an at large Chair Professorship at the Department of Biology and
Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, The Einstein Professor Program of the Chinese
Academy of Sciences.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.
2013.10.018.
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