Toxicology 313 (2013) 103–112

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Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Northern contaminant mixtures induced morphological and functional changes

in human coronary artery endothelial cells under culture conditions typifying
high fat/sugar diet and ethanol exposure
Maria Florian a,b , Jin Yan a , Saad Ulhaq a,b , Melanie Coughlan a , Mahemuti Laziyan a,b ,
William Willmore b , Xiaolei Jin a,∗
a
Toxicology Research Division, Food Directorate, HPFB, Health Canada, Ottawa, Ontario, Canada K1A 0K9
b
Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6

a r t i c l e i n f o a b s t r a c t

Article history: It has been reported that Northern populations are exposed to mixtures of various environmental con-
Received 19 April 2012 taminants unique to the Arctic (Northern contaminant mixtures – NCM) at a large range of concentrations,
Received in revised form depending on their geological location, age, lifestyle and dietary habits. To determine if these contam-
22 November 2012
inants may contribute to a cardiovascular health risk, especially when combined with a high fat and
Accepted 27 January 2013
sugar diet and ethanol exposure, we treated human coronary artery endothelial cells (HCAEC) with two
Available online 4 February 2013
mixtures of 4 organic (NCM1) or 22 organic and inorganic (NCM2) chemicals detected in Northerners’
blood during 2004–2005 in the presence or absence of low-density lipoprotein (1.5 mg/ml), very-low-
Keywords:
Northern contaminant mixtures
density lipoprotein (1.0 mg/ml) and glucose (10 mmol/L) (LVG), and in the absence or presence of 0.1%
Human coronary artery endothelial cells ethanol. After 24 h of exposure, cell morphology and markers of cytotoxicity and endothelial function
Cardiovascular disease were examined. NCM1 treatment did not affect cell viability, but increased cell size, disrupted cell mem-
High fat and sugar diet brane integrity, and decreased cell density, uptake of small peptides, release of endothelin-1 (ET-1) and
Ethanol exposure plasminogen activator inhibitor (PAI), while causing no changes in endothelial nitric oxide synthase
(eNOS) protein expression and nitric oxide (NO) release. In contrast, NCM2 decreased cell viability, total
protein yield, uptake of small peptides, eNOS protein expression, and NO release and caused membrane
damage, but caused no changes in the secretion of ET-1, prostacyclin and PAI. The presence of LVG and/or
alcohol did or did not influence the effects of NCM1 or NCM2 depending on the endpoint and the mixture
examined. These results suggested that the effects of one or one group of contaminants may be altered
by the presence of other contaminants, and that with or without the interaction of high fat and sugar
diet and/or ethanol exposure, NCMs at the concentrations used caused endothelial dysfunction in vitro.
It remains to be investigated if these effects of NCMs also occur in vivo.
Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Although a declining trend was observed for certain persis-

There has been an emergence of major circulatory illnesses
as a leading cause of death in some Canadian arctic populations.
Rapid changes in lifestyle and the departure from traditional diets
have been considered as important contributing factors. However,
increasing evidence suggests that chronic exposure to contami-
nants may also play a role in the pathogenesis of obesity, diabetes,
and cardiovascular disease in the Canadian arctic populations.
∗ Corresponding author at: Toxicology Research Division, Bureau of Chemical
Safety, Food Directorate, HPFB, Health Canada, PL 2202C, 251 Sir Frederick Bant-
ing Driveway, Ottawa, Ontario, Canada K1A 0K9. Tel.: +1 613 954 2045;
fax: +1 613 941 6959.
E-mail address: Dawn Jin@hc-sc.gc.ca (X. Jin).
tent organic pollutants (POPs) in the Arctic biota, levels of human
exposure remain of concern (Dewailly et al., 2006). Total mercury
concentrations in the Arctic environment and biota have remained
constant during the last decade. An Inuit Health Survey conducted
in Nunavik in 2004 and 2005 detected numerous contaminants in
the blood samples of Inuit between the age of 17 and 74 years. The
impact of these contaminants upon Inuit health remains unclear
(Dewailly et al., 2006). Polybrominated diphenyl ethers (PBDEs)
and perfluorooctanesulfonate (PFOS) are emerging contaminants
of potential concerns due to their rapidly increasing levels found in
the Northern wildlife, and their presence in Northern populations.
Their potential impact on human cardiovascular and metabolic
health warrants immediate investigation.
Increasing evidence suggests that exposure to POPs may con-
tribute to the development of cardiovascular diseases (Ha et al.,

0300-483X/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tox.2013.01.018
104 M. Florian et al. / Toxicology 313 (2013) 103–112
2007). A significant direct relation between serum PCB levels and
triglyceride levels was found among workers exposed to PCBs.
In an earlier study conducted in Triana, Alabama, 458 individ-
uals over 12 years of age, who had relatively elevated blood PCB
concentrations, primarily through eating contaminated fish, were
examined for serum levels of lipids and PCB (Kreiss et al., 1981).
It was found that serum PCB levels ranged from 3.2 to 157.9 ppb,
and were positively correlated with serum cholesterol levels and
blood pressure. In a more recent study, the incidence of coronary
heart disease and myocardial infarction was examined in a subpo-
pulation of New York State residents living along the Hudson River
within 200 miles of a National Priority Site highly contaminated
with PCBs (Sergeev and Carpenter, 2005). Despite a higher socio-
economic status and healthier life style, this population had over
30% higher rate of coronary heart disease and myocardial infarc-
tion than other populations. Similarly, the relationships among
the concentrations of total serum PCBs, mirex, dichloro-diphenyl-
dichloro-ethylene (DDE), and hexachlorobenzene (HCB), and total
serum lipids were investigated in a Native American population
(Goncharov et al., 2008). It was found that high serum PCB levels
were associated with elevated serum lipids levels and cardiovascu-
lar disease. The National Health and Nutrition Examination Survey
(NHANES) 1999–2002 conducted in U.S. revealed a positive associ-
ation between serum levels of PCBs and organochlorine pesticides
and prevalence of self-reported cardiovascular diseases among
female adults over 40 years of age, as well as a positive correlation
between polychlorinated dibenzo-p-dioxins (PCDDs) and preva-
lence of cardiovascular disease in both males and females (Ha et al.,
2007).
Emerging evidence from epidemiological studies in Finland and
the Brazilian Amazon suggests that MeHg exposure may increase
the risk of cardiovascular disease (Fillion et al., 2006; Stern, 2005),
although the mechanisms involved remain unclear. In a birth cohort
study conducted in Faroe Island, prenatal exposure to increasing
mercury concentrations was found to be associated with increased
diastolic and systolic blood pressure, and decreased heart rate vari-
ability in children at 7 years of age, and the effect of mercury was
stronger in children with lower birth weight (Sorensen et al., 1999).
A recent study in Nunavik found that blood mercury was signifi-
cantly and positively correlated with systolic and diastolic blood
pressure after adjusting for confounding factors (Dewailly et al.,
2009; Valera et al., 2008). The potential link between contami-
nants and cardiovascular disorders has been proposed in a number
of epidemiological studies (Baker et al., 1980; Fillion et al., 2006;
Goncharov et al., 2008; Ha et al., 2007; Kreiss et al., 1981; Sergeev
and Carpenter, 2005; Sorensen et al., 1999; Valera et al., 2008).
The Canadian arctic populations are exposed to a mixture of a
wide range of environmental contaminants. The exposure levels
of these populations vary tremendously. Although the geometric
mean of blood contaminant levels is relatively low (Dewailly et al.,
2006), a small percentage of Arctic populations can accumulate
NCM to a very high level due to their location, age, lifestyle and
dietary habits. The health effects of an individual contaminant may
be altered by the presence of other contaminants, as well as dietary
factors and lifestyles. It remains unclear if and how contaminant
mixtures such as those detected in the blood of Northerners may
affect metabolic and cardiovascular health, and if and how the high
fat/sugar diet of Western culture, as well as alcohol consumption,
modulate the effects of these contaminants.
In this study, we examined the effects of NCMs on endothe-
lial cell structure and function, which are known key factors in the
development of cardiovascular and metabolic diseases, in cultured
human coronary artery endothelial cells (HCAEC) under conditions
typifying a high fat plus high sugar diet or a standard nutritional
diet, and in the presence or absence of ethanol at a concentration
that is known to be associated with signs of intoxication in humans.
2. Materials and methods
2.1. Cell line and chemicals
HCAEC (Cat. # C-12221), Endothelial Cell Basal Medium MV 2 phenol red-free
(ECBM-MV2-PRF) (Cat. # C-22226) and SupplementPack for the Endothelial Cell
Growth Medium MV 2 (Cat. # C-39221) were obtained from PromoCell GmbH (Hei-
delberg, Germany). Trypsinizing reagent TrypLETM Express (Cat. # 12604013) and
calcium- and magnesium free Hank’s Balanced Salt Solution (Cat. # 14175095) were
purchased from Gibco, Invitrogen (Grand Island, NY, USA). Human plasma very low
density lipoprotein (VLDL) (Cat. # 12-16-221204) and low density lipoprotein (LDL)
(Cat. # 12-16-120412), both of which were stabilized in 150 mM NaCl + 0.01% EDTA,
were obtained from Athens Research & Technology (Athens, GA, USA). HPLC grade
absolute ethanol (200 proof) was purchased from Commercial Alcohols Inc. (Cat. #
P016EAAN, The Industrial & Beverage Alcohol Division of GreenField Ethanol Inc.,
Brampton, Ontario).
2.2. Northern contaminant mixtures
The contaminant mixtures were composed of 22 chemicals (except PFOS) that
were the major pollutants found in the Inuit blood during the 2004 and 2005
Inuit Health Survey (Dewailly et al., 2006), which included heavy metals, PCBs,
organochlorines, and PBDEs (Table 1). Although PFOS was not measured in the
2004–2005 Inuit Health Survey, it was detected in human plasma in both South-
ern and Northern Canadian populations (Dallaire et al., 2009; Kubwabo et al., 2004).
In preliminary experiments, five different mixtures were prepared and screened
for cytotoxicity. Two of the most potent mixtures were studied further for their
effects on endothelial function and interactions with fat/sugar and alcohol factors.
The two mixtures were designated NCM1 (a mixture of toxaphene, PBDE 47, 2,3,4,6-
tetrabromophenol, and PFOS), and NCM2 (a mixture of all the chemicals listed in
Table 1). To prepare NCM1, all the components were dissolved in dimethyl sulfoxide
(DMSO). NCM2 was a conceptual mixture, which consisted of two physical mix-
tures and one single chemical solution, which were (a) mixture of CdCl2 and PbCl2
dissolved in Milli Q water, (b) mixture of all organic chemicals listed in Table 1,
dissolved in DMSO, and (c) methylmercury chloride dissolved in 5 mM Na2 CO3 in
water. A 100-fold concentrated dosing solution was prepared for each of the dif-
ferent mixtures and the single chemical. Dosing volumes were calculated based on
medium volume per well. The chemical concentrations are listed in Table 1.
2.3. Culture and dosing of HCAEC
HCAEC (passages 2–5) were cultured in complete ECBM-MV2-PRF medium con-
taining 10% fetal bovine serum to a 70% confluence. Cells were trypsinized and
reseeded at 40,000 cells/ml in 6, 12, or 96 well plates or 6 cm dish. After overnight
culturing at 37 ◦ C and 5% CO2 , cells were treated with vehicle solvents, NCM1, or
NCM2 for 24 h in the presence or absence of LDL (1.5 mg/ml) plus VLDL (1.0 mg/ml)
and glucose (10 mmol/L) (LVG), reflecting human blood levels associated with
increased health risk and resulted from high fat and sugar diet, and in the presence
or absence of ethanol (EOH) (0.1%, v/v) reflecting a human blood ethanol concen-
tration associated with known signs of intoxication. The vehicle control group for
NCM1 was treated with DMSO. The vehicle control group for NCM2 was treated with
1% 5 mM Na2 CO3 , DMSO, and Milli Q water. For cells given LVG, a master mixture of
LDL plus VLDL and glucose was added to the culture. For cells given no LVG, the same
amount of 150 mM NaCl + 0.01% EDTA + Milli Q water, which was used to dissolve
LDL, VLDL, and glucose, was added to the culture. For cells not given EOH, the same
amount of Milli Q water was added to the culture. At least three replicates were
included in each experiment, and the same experiment was repeated at least three
times using different passages of cells.
2.4. Cell morphology
After 24 h of exposure to vehicle solvents or NCMs, cell morphology was exam-
ined in culture plates using an inverted Zeiss microscope (Axiovert 40 CFL, Gottingen,
Germany) at 10–20× power. Images were captured using a SPOT RT3 2.0 MP Slider
digital camera and processed with SPOT Basic Software (Diagnostic Instruments Inc.,
Sterling Heights, MI, USA).
2.5. Cell viability and protein mass
HCAEC were exposed to vehicle solvents or NCMs in complete ECBM-MV2-PRF
for 24 h. Effects of NCMs on cell viability were measured using three enzyme-based
assays. The protease activity assay was performed using a MultiTox Assay kit from
Promega (Cat. # G9201, Promega Corporation, Madison, WI, USA) following the man-
ufacture’s instruction. This assay measures intracellular protease activity of live cells
and the activity of extracellular protease released from dead cells. To determine if the
effects of NCMs on live cell protease activity were partly due to changes in the uptake
of small peptides which were used as substrates of protease in the MultiTox assay,
a time course kinetics of live cell protease activity was followed for the first 50 min
of exposure to vehicle solvents or NCMs in the absence of LVG and EOH. The adeny-
late kinase (AK) activity assay was conducted using a ToxiLight® Non-Destructive
 M. Florian et al. / Toxicology 313 (2013) 103–112 105

Table 1
Chemical composition and concentrations of NCMs.

Mixture name Mixture components Chemical composition Levels found in human Concentrations used in
blood (ng/L) this study (␮g/L)

NCM 2 Vehicle solvents 5 mM Na2 CO3 (vehicle for metal mixture) in H2 O 1%

DMSO (vehicle for organic mixtures) 1%
Metals Cadmium chloride 3035 303.5
Methylmercury chloride 10,997 1099.7
Lead chloride 39,368 3936.8
PCBs PCB 99 170 17.0
PCB 138 534 53.4
PCB 146 180 18.0
PCB 153 1333 133.3
PCB 163 221 22.1
PCB 170 216 21.6
PCB 180 813 81.3
PCB 187 287 28.7
PCB 194 182 18.2
PCB 201 167 16.7
PCB 203 105 10.5
Organochlorine Oxychlordane 431 43.1
p,p -DDE 3232 323.2
Trans-nonachlor 725 72.5
Pentachlorophenol 914 91.4
NCM1 Toxaphene (Parlar # 50) 142 14.2
PBDE IUPAC # 47 72 7.2
2,3,4,6-Tetrabromophenol 36 3.6
PFOS 29,000 2900

Total concentrations 101,929 10,192.9

Cytotoxicity BioAssay kit from Lonza (Cat. # LT07-217, Rockland, ME, USA) and fol-
lowing the protocol provided with the kit. This assay measures the activity of AK
released from membrane-injured cells. The lactate dehydrogenase (LDH) activity
assay was performed using a LDH Assay kit from Roche (Cat. # 11644-793-001,
Roche Applied Science, Laval, Quebec, Canada) and according to the manufacture’s
instruction. This assay measures the activity of LDH released from severely damaged
or dead cells.
2.6. Release of endothelial function markers
Release of endothelin-1 (ET-1) (Cat. # 900-020A, Assay Design Stressgen, Ann
Arbor, MI), 6-keto prostaglandin-F1␣ (6KPF or PGI2 ) (Cat. # 515211, Cayman Chem-
ical Company, Ann Arbor) and plasminogen activator inhibitor-1 (PAI-1) (Cat. #
DSE100, R&D Systems, Minneapolis, MN, USA) was measured in culture super-
natants collected after 24 h of exposure of HCAEC to vehicle solvents or NCMs in
complete ECBM-MV2-PRF medium and in the presence or absence of EOH and LVG
using ELISA kits according to the manufacturer’s instruction.
2.7. Endothelial nitric oxide synthase (eNOS)
HCAEC were exposed to vehicle solvents or NCMs in complete ECBM-MV2-PRF
medium with or without LVG in 6 cm culture dish for 24 h. A volume of 25 ␮l ice-cold
cell lysis buffer provided with the eNOS kit (Cat. # DEN00, R&D Systems, Minneapo-
lis, MN, USA) was added to each dish. Cells were scraped and centrifuged at 300 × g
at 4 ◦ C for 10 min. Supernatants were transferred into clean Eppendorf vials and
protein concentrations were determined using DC protein assay (Cat. # 500-0111,
Bio-Rad Laboratories Ltd., Mississauga, Ontario). eNOS was measured in cell lysates
using an ELISA kit from R&D Systems (Cat. # DEN00, R&D Systems, Minneapolis, MN,
USA) and according to manufacturer’s instruction.
3. Results
3.1. Cell morphology
HCAEC grown in medium containing LVG were generally thinner
and elongated as compared to those grown in medium without
LVG, and slightly larger in medium with than without EOH (Fig. 1).
In the absence of LVG with or without EOH, NCM1 increased cell
size and decreased cell density (Fig. 1). Some of these cells also lost
cell–cell connection. In the presence of LVG, the effects of NCM1
were less pronounced than those in the absence of LVG, leaving
some cell sizes unaffected, especially in the presence of EOH. In
addition, NCM1 also caused deformation of cell plasma membrane.
The effects of NCM2 on cell morphology were diverse, with
some cells severely disrupted or completely lifted off the plate
(not shown in the picture due to distance from focus), while oth-
ers remained normal in appearance (Fig. 1). This effect of NCM2
seems to be more pronounced in the absence of LVG with or with-
out EOH. In the presence of both LVG and EOH, NCM2 dramatically
increased cell size and decreased cell density. In the presence of
LVG, but absence of EOH, however, NCM2 caused vacuolization of
cells without markedly decreasing cell density and increasing cell
size.

2.8. Nitric oxide (NO) production 3.2. Effects of NCMs on cell viability and membrane integrity

HCAEC were exposed to vehicle solvents or NCMs in complete ECBM-MV2-PRF
medium in the presence or absence of LVG and absence of EOH for 24 h. Culture
supernatants were collected after centrifugation at 10,000 × g and 4 ◦ C for 10 min.
NO levels in supernatants were measured using a Nitric Oxide Fluorometric Assay Kit
from Biovision (Cat. # K252-200, Mountainview, CA, USA) following manufacturer’s
instruction.
2.9. Statistics
Three- or two-way ANOVA was performed using SigmaPlot 11 (Systat Software,
Inc., San Jose, CA, USA) to determine main effects of and interactions between fac-
tors: LVG, EOH and NCMs. All pairwise comparisons for all treatment groups were
conducted using Tukey’s test. Data failed normality and/or equal variance tests were
transformed or ranked before analysis.
The live cell protease activities in the vehicle control groups
were significantly higher in the cells grown in the absence (i.e.
−) than presence (i.e. +) of LVG (Fig. 2A and B). Regardless of
LVG and EOH, NCM1 dramatically (up to 70%) decreased live cell
protease activity (Fig. 2A). In contrast, only under the −LVG+EOH
condition, the NCM2 significantly decreased live cell protease activ-
ity, although a trend of decrease was seen for all LVG and EOH
conditions (Fig. 2B). It is interesting that under all LVG and EOH con-
ditions, a trend of increase in dead cell protease activity was found
in NCM1- or NCM2-treated cells (Fig. 3A and B). However, only
under the (+EOH+LVG) condition, the increase by NCM1 reached
statistical significance (Fig. 3A), and under both −EOH+LVG and
106 M. Florian et al. / Toxicology 313 (2013) 103–112

Fig. 1. HCAEC cell morphology observed after 24 h of exposure to vehicle solvents (VS), NCM1 or NCM2 in the absence (−) or presence (+) of EOH and LVG. Images were
captured under light microscope with a 10× objective and a digital camera and processed using ImagePro software.

+EOH+LVG conditions (Fig. 3B), the increase by NCM2 reached sta-
tistical significance.
To confirm that the decreased live cell activity by NCM1 and
NCM2 were not due to direct inhibition of protease activity by
these mixtures, a screening test was conducted and found that nei-
ther NCM1 nor NCM2 inhibited proteinase activity directly (data
not shown). However, a 50 min substrate uptake kinetic assay
showed that NCM1 inhibited substrate uptake after 5 min of incu-
bation (Fig. 4A), and NCM2 after 20 min (Fig. 4B). Substrate uptake
assays with individual chemical component of NCM1 revealed that
2,3,4,6-tetrabromophenol (TBP) was mostly inhibitory (Fig. 4C),
PFOS somewhat inhibitory (Fig. 4D), and PBDE 47 and toxaphene,
not inhibitory (data not shown).
To further confirm the cytotoxicity of the two chemical mix-
tures, LDH release was also measured. NCM2, but not NCM1,
significantly increased the release of LDH after 24 h of exposure,
especially under −EOH−LVG and −EOH+LVG conditions (Fig. 5),
indicating a severe membrane damage or cell death. These results
confirmed that after 24 h of exposure, NCM2 significantly decreased
cell viability, while NCM1 did not. Although both NCM1 and NCM2
decreased protease substrate uptake, the effects of NCM1 is much
more pronounced than those of NCM2. The presence of LVG did not
seem to influence the effects of NCM2 on cell viability.
To determine the effects of NCMs on cellular membrane
integrity, we examined the effects of NCMs on AK (≤31 kDa) release,
which reflects certain degree of membrane damage, assuming that
NCMs do not affect AK activity directly. After 24 h of exposure,
NCM1 caused significant increase in the release of AK from the
dosed cells, but only under +EOH+LVG and −EOH−LVG conditions
(Fig. 6A). In contrast, NCM2 significantly increased the release of
AK in all LVG and EOH conditions except −EOH−LVG condition
(Fig. 6B). These results imply that the presence of LVG and EOH
had differential influence on the effects of NCM1 and NCM2 on
membrane integrity.
3.3. Effects of NCMs on release of endothelial function markers
After 24 h of exposure, a significantly lower amount of ET-1 was
found in the supernatants of NCM1-dosed cells regardless of LVG
and EOH conditions (Fig. 7). No significant differences in super-
natant ET-1 concentrations were found between vehicle control
and NCM2 treatment groups at any LVG and EOH conditions used
(data not shown).
More 6-keto-PGF1␣ was released to the supernatants under the
+LVG than −LVG condition after 24 h of exposure (Fig. 8). Under the
+EOH+LVG condition, NCM1 decreased 6-keto-PGF1␣ release after
24 h of exposure (Fig. 8). This was due to the fact that in the presence
of LVG, EOH significantly increased release of 6-keto-PGF1␣, while
NCM1 abolished the effects of EOH. No significant differences in
supernatant 6-keto-PGF1␣ concentrations were detected between
vehicle control and NCM2 treatment groups regardless of EOH and
LVG conditions (data not shown).
 M. Florian et al. / Toxicology 313 (2013) 103–112 107

6000
A VS NCM1
Regardless of LVG and EOH conditions, NCM1 significantly
decreased PAI release after 24 h of exposure (Fig. 9). In addition,
Live Cell Protease Activity

5000 ** supernatant PAI concentrations were higher for the cells cultured
(fluorescent intensity)

4000 A
in the presence than absence of LVG with or without EOH or NCM1
B
treatment. No significant differences in supernatant PAI concen-
3000
C D trations were detected between vehicle control and NCM2-treated
cells after 24 h of exposure regardless of LVG and EOH conditions
2000 (data not shown).
aaa bbb ccc ddd
1000

3.4. Effects of NCMs on eNOS protein expression and NO release

0
-EOH +EOH -EOH +EOH
-LVG +LVG
LVG significantly decreased eNOS protein expression in HCAEC
after 24 h of treatment (data not shown). NCM1 caused no changes
5000
B VS NCM2 in either eNOS protein expression or NO release (Fig. 10) in
Live Cell protease Activity

4000 *** HCAEC with or without LVG. In contrast, NCM2 caused a signifi-
(fluorescent intensity)

cant decrease in eNOS protein expression under the −EOH−LVG

A
3000
condition which was associated with a significant decrease in NO
release (Fig. 10).
a

2000

4. Discussion
1000

In this study, cytotoxicity of NCMs was assessed by measur-

0
-EOH +EOH -EOH +EOH ing live cell or intracellular and dead cell or extracellular activities
-LVG +LVG of enzymes. Assuming that the NCMs do not directly affect these
enzyme activities, these measurements reflect cell viability, pro-
Fig. 2. Live cell protease activity of HCAEC measured after 24 h of exposure to vehi-
cle solvents (VS), NCM1 (A) or NCM2 (B) in the absence (−) or presence (+) of EOH
liferation and membrane integrity. Our microscopic observation
and LVG. Vertical bars stand for the mean values of 4–6 experiments. Error bars are revealed that regardless of LVG and EOH, NCM1 at the dose used
the standard errors of the means. “A”, “B”, “C”, and “D” are significantly different caused cell swelling after 24 h of exposure, which paralleled the
from “aaa”, “bbb”, “ccc”, and “ddd”, respectively, at p < 0.001. ** indicates a signifi- dramatic decrease in live cell protease activity but only minimal
cant difference between the two treatment factors indicated under the two lines at
amount of increase in dead cell protease activity. This suggests
p < 0.01. “A” is significantly different from “a” at p < 0.05.
that the decreased live cell protease does not completely reflect
cell death or severe membrane damage, although may partly reflect
decreased cell proliferation as revealed under the microscope. Time
A
18000
VS NCM1 course kinetics assay revealed that NCM1 dramatically decreased
15000 a the live cell protease substrate uptake during the first 50 min
Dead Cell Protease Activity

of incubation. The same effects were also found for PFOS and
(fluorescent intensity)

12000
A
2,3,4,6-tetrabromophenol alone, but not PBDE 47 and toxaphene,
all of which are the chemical components of NCM1. Therefore,
9000
the effects of NCM1 could be attributed to both PFOS and 2,3,4,6-
tetrabromophenol.
6000
PFOS at micro molar concentrations has been shown to
3000 increase the permeability of cell membranes to hydropho-
bic ligands (Hu et al., 2003; O’Brien and Wallace, 2004;
0 Starkov and Wallace, 2002). In addition, it was demonstrated
-EOH +EOH -EOH +EOH
-LVG +LVG that incorporation of PFOS into the cell membrane decreased
transmembrane potential gradient resulting in hyperpolarizing
20000
B VS NCM2 shifts of both the activation and inactivation of ion channels
*** for Ca, Na, and K (Harada et al., 2006). PFOS induced actin fila-
Dead Cell Protease Activity

16000
ment remodeling and endothelial permeability changes in human
(fluorescent intensity)

b a microvascular endothelial cells as a result of increasing production

12000
B of reactive oxygen species (ROS) (Qian et al., 2010), and increased
A
the brain endothelial cells permeability through the activation of
8000 phosphatidylinositol-3-kinase/Akt signaling (Wang et al., 2011).
Proton-coupled peptide transporter 1 (PepT1 or SLC15A1) and pep-
4000 tide transporter 2 (PepT2 or SLC15A2) are known to be expressed in
HCAEC and to efficiently transport 400 dipeptides and 8000 tripep-
0 tides and many other structurally similar molecules (Daniel, 2004;
-EOH +EOH -EOH +EOH
-LVG +LVG
Gilbert et al., 2008; Groneberg et al., 2001). It is very likely that
the peptide substrate, GF-AFC, for the intracellular protease is also
Fig. 3. Dead cell protease activity of HCAEC measured after 24 h of exposure to transported into cells by these transporters. Membrane potential
vehicle solvents (VS), NCM1 or NCM2 in the absence (−) or presence (+) of EOH and has been found to be essential to the direction and rate of pep-
LVG. Vertical bars stand for the mean values of 4–6 experiments. Error bars are the
tide transport by PepT1 and/or PepT2 (Chen et al., 2010; Daniel
standard errors of the means. “A” and “B” are significantly different from “a” and
“b”, respectively, at p < 0.001. *** indicates a significant difference between the two et al., 2006). It is reasonable to speculate that the decreased live
treatment groups indicated under the two lines at p < 0.001. cell protease activity in PFOS-treated HCAEC observed in this study
108 M. Florian et al. / Toxicology 313 (2013) 103–112

20000
A 20000
B
VS VS
16000 16000

Protease Subatrate Uptake

Protease Substrate Uptake
NCM1 NCM2
(fluorescent intensity)

(fluorescent intensity)
12000 12000

8000 8000

4000 4000

0 0
0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000
Time of Incubation (s) Time of Incubation (s)

20000
C 20000
D
VS VS
16000 16000
Protease Substrate Uptake

Protease Substrate Uptake

PFOS TBP
(fluorescent intensity)

(fluorescent intensity)
12000 12000

8000 8000

4000 4000

0 0
0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000
Time of Incubation (s) Time of Incubation (s)

Fig. 4. Uptake of peptide substrate (glycyl-phenylanalyl-aminofluorocoumarine) by HCAEC during the first 50 min of exposure to vehicle solvents (VS), NCM1 or NCM2 in
the absence (−) of EOH and LVG, and measured using intracellular protease activity assay. Data are expressed as means of 4 independent experiments. The error bars are the
standard errors of the means.

could be partly due to the decreased transport of peptide substrate
resulting from altered cell membrane potential by PFOS.
It was striking that 2,3,4,6,-tetrabromophenol at the dose used
markedly decreased live cell protease activity during the first
few minutes of exposure to an even greater degree than PFOS.
The effects of 2,3,4,6-tetrabromophenol on cellular membrane
potential or peptide transport have not been reported, although
2,4-dibromophenol and 2,4,6-tribromophenol have been shown
0.5
VS
0.4
(Absorbance at 490 nM)
to disturb calcium channel current (Hassenklover and Bickmeyer,
2006). It was well recognized that chlorinated phenols exert
toxicity by disrupting energy conversion processes in cellular
and subcellular membranes (Weinbach and Garbus, 1965). Pen-
tachlorophenol at even 0.1 ␮M concentration has been shown to
decrease mitochondrial membrane potential of rat spermatozoa
after 10 min of exposure, which was associated with decreased
motility (Gravance et al., 2003). Pentachlorophenol was also found
to induce membrane depolarization, increase Ca2+ uptake (Dong
et al., 2009; Nwoga et al., 1996) and inhibit gap junctional inter-
cellular communication (Sai et al., 1998). Knowing the similarity
NCM in chemical properties between pentachlorophenol and tetrabro-
mophenol, it is not irrational to speculate that the decrease in live
b

a
cell protease activity in HCAEC caused by 2,3,4,6-tetrabromophenol
LDH in Supernatants

was at least partly attributed to the alterations in membrane prop-

0.3
erty induced by this chemical. Data presented here suggest that
the membrane permeability and/or integrity may be one impor-
0.2 B
A tant target of PFOS and 2,3,4,6-tetrabromophenol, and therefore,
also the target of NCM1.
0.1 The aforementioned results imply that chemicals present in the
circulation at concentrations affecting cell membrane properties
0.0
NCM1 NCM2
in vitro may also disrupt uptake of amino acids, small peptides
NCM1 NCM2
-EOH-LVG -EOH+LVG and other molecules transported through the same mechanism to
peripheral tissues, possibly leading to malnutrition status of and/or
Fig. 5. LDH activity detected in the supernatants of HCAEC after 24 h of exposure insufficient drug delivery to the tissues.
to vehicle solvents (VS), NCM1 or NCM2 in the absence of EOH and the absence (−)
In contrast to dead cell protease assay and LDH assay, which
or presence (+) of LVG. Data are expressed as means of 4 independent experiments.
The error bars are the standard errors of the means. “A” and “B” are significantly showed minimal effect of NCM1, the AK release assays revealed a
different from “a” and “b”, respectively, at p < 0.05. significant increase in membrane damage by this mixture after 24 h
 M. Florian et al. / Toxicology 313 (2013) 103–112 109

500 800
A VS NCM1 VS NCM1

6-keto-PGF1a in Supernatants (pg/ml)

***
400 *
Adenylate Kinase Release
(Relative Luminescence)

600
b
aa **
A
300

400
a
200
B
A
200
100

0 0
-EOH +EOH -EOH +EOH +EOH
-EOH -EOH +EOH
-LVG +LVG
-LVG +LVG

1000
B VS NCM2 Fig. 8. Levels of 6-keto-PGF1␣ detected in the supernatants of HCAEC after 24 h of
exposure to vehicle solvents (VS) or NCM1 in the absence (−) or presence (+) of
Adenylate Kinase Release

EOH and LVG. Data are expressed as means of 5–6 independent experiments. The
(Relative Luminescence)

800
a error bars are the standard errors of the means. “A” is significantly different from
ccc “a” at p < 0.05. “**” and “***” indicate significant differences between the two groups
600
b located under the lines at p < 0.01 and 0.001, respectively.

400 A 50
VS NCM1
C B

200 **

PAI in Supernatants (ng/ml)

40

B C D
0
-EOH +EOH -EOH +EOH 30 A
d
-LVG +LVG cc
b
Fig. 6. Adenylate kinase activity detected in the supernatants of HCAEC after 24 h 20 aa
of exposure to vehicle solvents (VS), NCM1 (A), or NCM2 (B) in the absence (−)
or presence (+) of EOH and LVG. Data are expressed as means of 3–7 independent
experiments. The error bars are the standard errors of the means. “A” and “B” are 10
significantly different from “a” and “b”, respectively, at p < 0.05. “A” is significantly
different from “aa” at p < 0.01. “C” is significantly different from “ccc” at p < 0.001.
0
-EOH +EOH -EOH +EOH
-LVG +LVG
of exposure in the presence or the absence of both LVG and EOH.
This could be partly attributed to the differences in size and half-life Fig. 9. Levels of PAI-1 detected in the supernatants of HCAEC after 24 h of exposure
of the AK, protease and LDH, reflecting different degrees and time to vehicle solvents (VS) or NCM1 in the absence (−) or presence (+) of EOH and LVG.
Data are expressed as means of 5–6 independent experiments. The error bars are
course of membrane damage and/or cell death induced by NCM1
the standard errors of the means. “A” and “C” are significantly different from “aa”
under different LVG and EOH conditions. and “cc”, respectively, at p < 0.01. “B” and “D” are significantly different from “b” and
In this study, we observed a trend of decrease in live cell protease “dd”, respectively, at p < 0.05. “**” indicates a significant difference between the two
activity in NCM2-treated cells under all EOH and LVG conditions, groups located under the lines at p < 0.01.
but to a much smaller degree as compared with NCM1-treated
cells. LDH and protease release assays suggested that NCM2 was NCM1 and NCM2 on cytotoxicity endpoints suggested that the
more toxic than NCM1 to HCAEC cell integrity under the condi- action of one contaminant may be altered by the presence of
tions used. NCM2 is composed of the same chemical components other contaminants. In the case of NCM2, the effects of PFOS and
of NCM1 and many other organic and inorganic chemicals that 2,3,4,6-tetrabromophenol on the uptake of small peptide or live cell
have been detected in human blood. The differential effects of
20 20
VS
1000
VS NCM1 A NCM
eNOS in HCAEC (ng/mg protein)

15 15
ts (µM)
ET-1 in Supernatant (pg/ml)

800
10 a 10
NO in Supernatants (

C
600 5 5
A D 3 3
B
400
2 B 2
ccc dd
aaa bbb
bb
200 1 1

0 0 0
-EOH +EOH -EOH +EOH NCM1 NCM2 NCM1 NCM2
-LVG +LVG -EOH-LVG

Fig. 7. Levels of ET-1 detected in the supernatants of HCAEC after 24 h of exposure
to vehicle solvents (VS), or NCM1 in the absence (−) or presence (+) of EOH and LVG.
Data are expressed as means of 5–6 independent experiments. The error bars are
the standard errors of the means. “A” is significantly different from “aa” at p < 0.01.
“B” and “C” are significantly different from “bbb” and “ccc”, respectively, at p < 0.001.
“D” is significantly different from “dd” at p < 0.01.
Fig. 10. eNOS protein expression (on the left) and NO release (on the right) in HCAEC
after 24 h of exposure to vehicle solvents (VS), NCM1, or NCM2 in the absence
(−) of EOH and LVG measured using ELISA and a fluorescent assay, respectively.
Data are expressed as means of 4 independent experiments. The error bars are the
standard errors of the means. “A” is significantly different from “a” at p < 0.05. “B” is
significantly different from “bb” at p < 0.01.
110 M. Florian et al. / Toxicology 313 (2013) 103–112
protease activity seemed to be weakened by other contaminants
present in the mixture. The effects of NCM1 on membrane leakage
as measured by release of AK were affected to a minimal extent by
the presence of other chemicals. On the other hand, the effects of
NCM2 on LDH release seemed to be purely attributed to its chemical
components other than those of NCM1. These results suggest that
multiple endpoints should be used when assessing cytotoxicity of
chemical mixtures, especially in the presence of other factors such
as LVG, which may alter the effects of chemicals on one toxicological
endpoint, but not the others.
ET-1 is the most potent vasoconstrictor currently identi-
fied. However, it is not solely a vasoconstrictor, as it also
stimulates angiogenesis, induces astrocyte proliferation, constricts
bronchi, and stimulates production of inflammatory mediators in
macrophages (Tykocki et al., 2010). ET-1 is a peptide hormone with
diverse biological actions in neuronal development, pulmonary
physiology, autoimmune response, and cancer biology (Stow et al.,
2011). The biological action of ET-1 is mediated through activa-
tion of two receptors, ETA and ETB , which often act oppositely in
the vasculature (Kohan et al., 2011). Activation of ETA located in
the vascular smooth muscle cells, will lead to vasoconstriction, and
promote cell proliferation and fibrosis. Activation of ETB located
in the endothelial cells will promote NO and prostacyclin produc-
tion leading to vasodilation (Kohan et al., 2011; Rodriguez-Pascual
et al., 2011). It is believed that both ET-1 production and secre-
tion are largely regulated at the gene transcription level (Kohan
et al., 2011; Thorin and Webb, 2010). In this study, we observed
a significant decrease in ET-1 concentrations in the supernatants
of HCAEC treated with NCM1 with or without LVG or EOH, which
paralleled a decreased cell density, and increased cell membrane
leakage, but did not affect protein yield. Although the mechanisms
of ET-1 synthesis and secretion are not fully elucidated, it is gener-
ally believed that a prepropeptide of 212 amino acids is synthesized
in the endothelial cells, which is then cleaved by an endopepti-
dase to generate Big ET-1 with 38 amino acids (Kohan et al., 2011).
The Big ET-1 is packed in Weibel–Palade bodies along with ET
converting enzymes (ECE), and secreted upon demand. ECE has
been found in intracellular vesicles and extracellular surface of the
plasma membrane, where it converts Big ET-1 to ET-1. It is unclear
if NCM1 inhibited ECE activity leading to decreased conversion of
Big ET-1 to ET-1, down-regulated ET-1 protein expression leading
to decreased ET-1 release, or altered membrane property resulted
in blockage of ET-1 release. Constitutive basal levels of ET-1 are
known to play an important role in regulating blood vessel tone and
blood flow (Thorin and Webb, 2010). The decreased ET-1 secretion
from HCAEC by NCM1 will disrupt the normal regulation of blood
vessel tone and blood flow if occurs in vivo. In contrast to NCM1,
NCM2 showed no significant effects on secretion and/or expression
of ET-1, implying that other contaminants counteracted against the
effects of the contaminant present in NCM1. Actually, cadmium,
which is a component of NCM2, has been found to increase the
storage of ET-1 in the Weibel–Palade bodies of aortic endothelium,
which was associated with elevated serum ET-1 levels (Doi et al.,
1996).
Prostanoids are a family of bioactive lipid mediators that are
formed by cyclooxygenase (COX) from arachidonic acid contained
in the cell membrane (Kawabe et al., 2010). Prostacyclin (mea-
sured as 6-keto-PGF1␣) is thought to be one of the most important
prostanoids in regulating the homeostasis of the cardiovascular
system. In addition to being a vasodilator, prostacyclin is also an
inhibitor of platelet aggregation, leukocyte adhesion, and smooth
muscle cell proliferation. Prostacyclin is a major anti-atherogenic
prostanoid produced by prostaglandin-endoperoxide synthase 2
(COX-2), counteracting the atherogenic effects of thromboxane
produced by COX-1. These actions of prostacyclin are mediated
through specific cell surface receptor(s). It is interesting that the
cells cultured in the presence of LVG released more prostacyclin
than in the absence of LVG with or without NCMs, which is con-
tradictory to the findings of Smith et al. (2002) who reported that
addition of free cholesterol or LDL to cultured human umbilical
vein endothelial cells (HUVEC) decreased production of prostacy-
clin. However, it has also been shown that 25-hydroxycholesterol
increased COX-2 protein expression and activity in HUVEC in a
time- and concentration-dependent manner (Plasen et al., 2010).
It is possible that the commercial LDL/VLDL used in this study con-
tained 25 hydroxycholesterol, or that LDL/VLDL cholesterols were
transformed to 25-hydroxycholesterol in culture medium contain-
ing hydroxylase, leading to increased prostacyclin release from
HCAEC.
Another interesting observation is that addition of EOH to
HCAEC in the presence of LVG further increased prostacyclin
release, which is consistent with the reported increasing effects of
EOH on prostacyclin production both in vivo and in vitro (Landolfi
and Steiner, 1984). However, this effect of EOH in HCAEC was
abolished by NCM1 exposure. This could be a result of increased
metabolism of EOH by alcohol dehydrogenase or cytochrome P450
2E1 (CYP2E1), which might be induced by NCM1, although this
speculation needs to be confirmed by further experiments.
PAI-1 is a serine protease inhibitor and functions to stabilize
clots via inhibition of tissue plasminogen activator with subsequent
inhibition of fibrinolysis (Gramling and Church, 2010). PAI-1 is
expressed in almost all cell types, but most prominently in endothe-
lial cells, adipocytes, and hepatocytes (Nagamine, 2008). Exposure
to IL-1, TNF-alpha, reactive oxygen species (ROS), sheer stress, and
hypoxia have been shown to increase PAI-1 synthesis in endothelial
cells (Gramling and Church, 2010). PAI-1 plays an important role
in angiogenesis and thrombosis, and has a well-documented asso-
ciation with the development of cardiovascular disease and type
II diabetes. It was unexpected that NCM1 significantly decreased
the concentration of PAI-1 in the supernatants of HCAEC after 24 h
of exposure under all LVG and EOH conditions. This is in contrast
to the increased membrane damage induced by NCM1. In contrast
to NCM1, NCM2 treatment had no effects on PAI concentrations
in the supernatants regardless of LVG and EOH conditions, further
suggesting that the effects of one or one group of contaminants
may be altered by the presence of other contaminants. PAI-1 plays
multifunctional role in various processes associated with vascular
remodeling, which could be either antiapoptotic or proapoptotic,
and either promoting or repressing vascular proliferative response,
depending on the vascular environment/conditions and its interac-
tion with other proteases in the vasculature (Diebold et al., 2008).
Therefore, the consequences of decreased PAI-1 release by NCM1,
if happens in vivo, can be complicated and difficult to anticipate.
eNOS is an endothelial heme protein with an oxygenase
domain and (6R-)5,6,7,8-tetrahydrobiopterin (BH4) as a cofactor
(Forstermann, 2010). eNOS catalyzes the production of NO from
oxygen, l-arginine and NADPH. NO is a key regulator of endothelial
function, as it relaxes blood vessels, prevents platelet aggregation
and adhesion, limits oxidation of LDL, decreases the expression of
inflammatory genes, and inhibits smooth muscle cell proliferation.
In this study, we observed no significant effects from NCM1 either
on eNOS protein expression or on NO release under the conditions
used. However, NCM2 suppressed eNOS protein expression and
decreased NO release. This effect of NCM2 was more prominent
in the absence than the presence of LVG. We suspected that this
effect of NCM2 was due to the heavy metals present in NCM2. In
fact, our unpublished data demonstrated that methylmercury at
the same dose as that used in the NCM2 significantly decreased
eNOS protein expression in HCAEC after 24 h of exposure, which is
consistent with other published studies (Kishimoto et al., 1995;
Ohno et al., 1995). These findings point to NCM2 at high expo-
sure levels as a potential risk factor for hypertension and other
 M. Florian et al. / Toxicology 313 (2013) 103–112 111

Table 2
Comparison of NCM1 and NCM2 for their effects on cytotoxicity and endothelial function markers in HCAEC under different EOH and LVG conditions.

−LVG +LVG
Category End points
−EOH +EOH −EOH +EOH

NCM1 NCM2 NCM1 NCM2 NCM1 NCM2 NCM1 NCM2

Cytotoxicity Morphologya MS FS MS FS MS E FS MS
Live cell proteaseb +++ – +++ + +++ – +++ –
Dead cell protease – – – – – + + +
Substrate uptakec GE SE NT NT NT NT NT NT
LDH – + NT NT – + NT NT
Protein yield – + NT NT – – NT NT
AK + – – +++ – + ++ +

Function ET-1 +++ – +++ – +++ – ++ –

6-Keto PGF1␣ – – – – – – + –
PAI-1 ++ – + – ++ – + –
eNOS – + NT NT – – NT NT
NO – + NT NT – + NT NT

The bold text shows differences between the effects of NCM1 and those of NCM2.
a
“MS” represents many swelling cells. “FS” represents few swelling cells. “E” represents elongated cells.
b
“+” indicates significant effect at p < 0.05. “+++” indicates significant effects at p < 0.001. “–” represents no significant effects.
c
“++” indicates significant effects at p < 0.01. “GE” represents greater effects. “SE” represents smaller effects. “NT” means not tested.

cardiovascular disorders, although it remains to be confirmed if
this also occurs in vivo.
In summary, our results as summarized in Table 2 demonstrated
that both NCM1 and NCM2 at the concentrations used may cause
structural and functional changes in HCAEC with or without inter-
action of LDL/VLDL plus glucose and/or alcohol, although some of
these changes were only present under a specific LDL/LVG plus
glucose and/or alcohol condition. The changes induced by NCM1
seemed to be more related to membrane integrity and the processes
operated through membrane, resulting in significant changes in
release of endothelial functional markers, but little influence on cell
viability, at the conditions used. In contrast, the changes induced
by NCM2 were more severe leading to compromised intracellular
protein expression and cell death, and consequently decreased pro-
duction of endothelial functional markers. These results supported
the notion that different chemical mixtures may cause endothelial
dysfunction through different mechanisms, and that the endothe-
lial toxicity of one or one group of contaminants may be altered by
the presence of other contaminants. An in vivo exposure study is
currently being conducted in our laboratory to confirm these in vitro
findings using a rodent model.
Conflict of interest statement
We state that there is no conflict of interest related to the work
described in this manuscript.
Acknowledgements
This study was supported by the grant awarded to Dr. Jin and
team members by the Northern Contaminants Program, Aboriginal
Affairs and Northern Development Canada. We thank the con-
tributions of Toxicological Research Division, Bureau of Chemical
Safety, Food Directorate, Health Canada and Institute of Bio-
chemistry, Carleton University. Dr. Willmore was supported by
a Discovery Grant from the Natural Sciences and Engineering
Research Council (NSERC) of Canada. Dr. Florian was holding a
NSERC-Visiting Fellowship with Health Canada.
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