PCB and TCDD derived embryonic cardiac defects result from a novel
AhR pathway
Corinna Singleman a,b
, Nathalia G. Holtzman a,b,
a Department of Biology, Queens College, City University of New York, 65-30 Kissena Blvd, Queens NY 11367-1597, USA b The Graduate Center, City University of New York, 365 Fifth Avenue, New York, NY 10016, USA
ARTICLE INFO
Keywords:
Zebrafish
Embryo
TCDD
PCB 126
Heart development
Aryl hydrocarbon receptor (AhR)
Src
VE-cadherin/Cadherin 5
SU6656
Atlantic sturgeon
ABSTRACT
Polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are environmental contami￾nants known to impact cardiac development, a key step in the embryonic development of most animals. To date,
little is understood of the molecular mechanism driving the observed cardiac defects in exposed fishes. The
literature shows PCB & TCDD derived cardiac defects are concurrent with, but not caused by, expression of
cyp1A, due to activation of the aryl hydrocarbon receptor (AhR) gene activation pathway. However, in this
study, detailed visualization of fish hearts exposed to PCBs and TCDD show that, in addition to a failure of
cardiac looping in early heart development, the inner endocardial lining of the heart fails to maintain proper cell
adhesion and tissue integrity. The resulting gap between the endocardium and myocardium in both zebrafish and
Atlantic sturgeon suggested functional faults in endothelial adherens junction formation. Thus, we explored the
molecular mechanism triggering cardiac defects using immunohistochemistry to identify the location and
phosphorylation state of key regulatory and adhesion molecules. We hypothesized that PCB and TCDD activates
AhR, phosphorylating Src, which then phosphorylates the endothelial adherens junction protein, VEcadherin.
When phosphorylated, VEcadherin dimers, found in the endocardium and vasculature, separate, reducing tissue
integrity. In zebrafish, treatment with PCB and TCDD contaminants leads to higher phosphorylation of VEcad￾herin in cardiac tissue suggesting that these cells have reduced connectivity. Small molecule inhibition of Src
phosphorylation prevents contaminant stimulated phosphorylation of VEcadherin and rescues both cardiac
function and gross morphology. Atlantic sturgeon hearts show parallels to contaminant exposed zebrafish cardiac
phenotype at the tissue level. These data suggest that the mechanism for PCB and TCDD action in the heart is, in
part, distinct from the canonical mechanism described in the literature and that cardiac defects are impacted by
this nongenomic mechanism.
1. Introduction
Co-planar (dioxin-like) polychlorinated biphenyls (PCBs) and
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are man-made chemicals
contaminating many environments worldwide. Although these con￾taminants are chemically inert, they have significant biological action.
Both PCBs and TCDD bioaccumulate in fatty tissue, are detrimental to
faunal survival and reproduction, cause cancers in the liver and thyroid,
and are linked to developmental problems in the brain and heart
(Birnbaum, 1995; Mayes et al., 1998; Ulbrich and Stahlmann, 2004;
Thackaberry et al., 2005; Nesan and Kurrasch, 2020). Often, early
disruptions in embryonic or larval development cause cessation of
growth and maturation, ultimately leading to death. However, embryos
that survive likely retain effects of exposure through maturation
impacting growth, reproduction, and long-term survival (Gamperl and
Farrell, 2004; Berninger and Tillitt, 2018; Singleman et al., 2021). This is
because small changes in organ development can produce functional,
but compromised organs. Fish living and breeding in contaminated
waters suffer the consequences of chronic exposure both through initial
direct contact with the contaminants and bioaccumulation of contami￾nants, resulting in multi-generational impacts on populations (Bush
et al., 1989; Monosson et al., 2003; Hicken et al., 2011; Incardona et al.,
Abbreviations: PCB, Polychlorinated biphenyls; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, Aryl hydrocarbon Receptor; VEcad, Vascular Endothelial cad￾herin (VEcadherin/Cad5); Src, Sarcoma protein tyrosine kinase; dpf, days post fertilization; hpf, hours post fertilization.
* Corresponding author at: Department of Biology Queens College, City University of New York 65-30 Kissena Blvd Queens, NY, 11367-1597 USA.
E-mail address: [email protected] (N.G. Holtzman).
Contents lists available at ScienceDirect
Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aqtox
Received 10 October 2020; Received in revised form 30 January 2021; Accepted 20 February 2021
Aquatic Toxicology 233 (2021) 105794
2014).
PCBs and TCDD are common contaminants in waterways and thus
have a great impact on fish viability. PCB 126 and TCDD exposure
impact heart maturation, thus reducing fitness and likelihood of fish
survival and reproduction rates (Gamperl and Farrell, 2004; Li et al.,
2004; Bartman and Hove, 2005; Singleman et al., 2021). The importance
of heart development to fish survival reaffirms the importance of
studying embryonic heart development to identify potential long-term
consequences of aquatic toxicant exposure.
While many studies use model fish species including zebrafish, Danio
rerio, comparisons of models to wild species impacted by contamination
is needed to realize the breadth of contamination impacts and the
environmental relevance of results. The use of the well-studied zebrafish
enables the utilization of many genetic and molecular tools to study PCB
and TCDD impacts on development (Andersson et al., 2001; Antkiewicz,
2005; Segner, 2009; Daouk et al., 2011; Singleman et al., 2021).
Meanwhile, Atlantic sturgeon, Acipenser oxyrhynchus oxyrhynchus, is a
federally designated endangered species in the U.S., native to PCB and
TCDD contaminated waters such as the Hudson and Delaware rivers.
Chambers et al. (2012) established that Atlantic sturgeon sustain
developmental consequences from PCB 126 and TCDD exposure, shown
by morphological defects and reduced survival to hatching. Compari￾sons of the impact of these contaminants on heart development between
evolutionarily divergent zebrafish and Atlantic sturgeon will provide
valuable information for understanding local contamination impacts in
wild species. Further, use and modification of zebrafish-based tech￾niques in Atlantic sturgeon enable broader use of these tools in other fish
species impacted by PCBs and TCDD.
Co-planar PCBs, including PCB 126, and TCDD have similar chemical
structures and act on a set of molecular pathways, causing comparable
effects in a wide array of exposed organisms. The most well-established
mechanism for PCB 126 and TCDD action begins with the binding of the
contaminant to the cytoplasmic aryl hydrocarbon receptor (AhR).
Activation of the AhR complex, likely AhR2 in fishes (Souder and Gor￾elick, 2019), results in the initiation of the expression of genes involved
in metabolic processes broadly in the liver and heart, specifically in
endothelial cells (Andreasen et al., 2002; Teraoka et al., 2010; Fu et al.,
2019). When activated, the AhR complex enters the nucleus by binding
the aryl hydrocarbon receptor nuclear translocator (ARNT), and pro￾motes expression of many genes, including cyp1A. cyp1A expression is
increased in the heart, liver and intestine of PCB 126 treated shortnose
and Atlantic sturgeon embryos demonstrating that these organs are
directly impacted by contaminant exposure (Roy et al., 2011). This is
defined as the canonical genomic AhR pathway. In zebrafish, cyp1a is
also upregulated in PCB 126 and TCDD exposed embryos (Carney et al.,
2004; Handley-Goldstone, 2005). Further, co-planar PCB exposure im￾pacts overall fish morphology and PCB 126 treated fish have hearts that
are smaller and misshapen (Grimes et al., 2008; Li et al., 2014).
The heart is the first functioning organ in a developing embryo, and
in zebrafish begins contracting by 24 h post fertilization (hpf). The first
functional stage of heart development is when the heart is a linear tube
which undergoes morphological changes looping the heart tube into an
“S” shape from which chambers balloon to form the ventricle and
atrium, resulting in rounded, laterally-oriented chambers (Glickman and
Yelon, 2002). Proper heart formation requires molecular instruction and
environmental inputs, including directional blood flow which induces
shear stress. Factors that impact the integrity of the heart shift the blood
flow and alter shear stress, resulting in defects in heart formation. PCB
126 and TCDD treated hearts in both Atlantic sturgeon and zebrafish do
not undergo proper cardiac looping or cardiac ballooning, key points of
development for proper function of the heart (reviewed in Kopf and
Walker, 2009).
There is clearly a critical role for activation of the AhR pathway in
PCB and TCDD mediated cardiac defects (Fu et al., 2019), it is likely not
exclusively via the canonical genomic pathway. While cyp1A is
expressed in cells with an activated AhR, there is little evidence that the
cyp1A gene plays a significant role in cardiac or endothelial defects.
Cardiac defects persist in PCB exposed embryos when ARNT and cyp1A
expression are knocked down, though removal of AhR function results in
rescue of the cardiac phenotypes (Andreasen et al., 2002; Antkiewicz,
2005; Scott et al., 2011; Van Tiem and Di Giulio, 2011). Thus, an
alternate, cyp1A independent, non-genomic, AhR pathway that works in
the cardiac cells is likely causing observed cardiac defects (Scott et al.,
2011; Larigot et al., 2018; Brown et al., 2016). Indeed, there have been
many studies that have explored the non-genomic pathways of AhR
activation in the development of other organs (Randi et al., 2008;
Mathew et al., 2009; Matsumura, 2009; Oesch-bartlomowicz and Oesch,
2009; Vezina et al., 2009). AhR activation is known to cause the
up-regulation and phosphorylation of various proteins, including the
tyrosine kinase Src (Larigot et al., 2018; Randi et al., 2008; Dong et al.,
2011). Src can phosphorylate the adhesion molecule Vascular Endo￾thelial cadherin (VEcadherin/Cad5), a membrane bound protein found
in cardiovascular endothelial cells (Vestweber, 2007) responsible for
maintaining vascular integrity. VEcadherin (VEcad) phosphorylation
reduces the ability of endothelial cells to remain bound resulting in
separation of neighboring endothelial cells lining the inside of the heart,
the endocardium, and vasculature (Gavard and Gutkind, 2006; Dejana
et al., 2008). VEcad morpholino knockdown in zebrafish presents
analogous phenotypes to those seen in PCB 126 and TCDD exposed
zebrafish. These phenotypes include pericardial edema, linear hearts
and separation of the endocardium and myocardium (Mitchell et al.,
2010). We propose that PCB 126 and TCDD exposure impacts VEcad
binding, decreasing the ability of vascular cells to bind appropriately
leading to loss of vascular integrity, fluid accumulation between the
endocardium and myocardium and thus separation of these two key
cardiac layers. Maintaining proper connection between the endocar￾dium and myocardium is essential for proper cardiac formation and
development. As blood flows through the heart and is pumped through
the body, the forces of blood flow apply shear stress to the endocardium
signaling shape changes in myocardial cells directing changes in overall
cardiac morphology (Brutsaert, 2003). Separation of these cardiac layers
prevents correct signaling and thus obstructs myocardial response and
normal growth of the heart. Based on the published data and our ob￾servations, we hypothesize that phosphorylation of Src by PCB 126 and
TCDD activated AhR leads to separation of endothelial cells by phos￾phorylation of VEcad.
2. Methods
2.1. Zebrafish contaminant exposure
Zebrafish were maintained as described in Westerfield (2000) using
an IACUC approved protocol. Wildtype AB adults (for immunohisto￾chemistry) or Tg(myl7:DsRed); Tg(flk:GFP) transgenic fish were mated,
the resulting eggs were collected, and screened for infertile eggs, then
incubated at 28.5◦C until the onset of gastrulation (Westerfield, 2000).
PCB 126 (99 % purity) and TCDD (AccuStandard, New Haven, CT)
were dissolved in acetone (Fisher Scientific, Waltham, MA), then ali￾quots were removed to make stock solutions. Treatment solutions were
made fresh for each experiment by adding acetone to the appropriate
volume of PCB 126 or TCDD stock solution in stender dishes for final
concentrations of 1, 10, and 100 μg/L (PCB 126) and 0.05, 0.5 and 5 μg/
L (TCDD) with 0.75 % acetone in 10 mL 1X Embryo Media (EM) as
vehicle controls. Blank controls and vehicle controls (0.75 % acetone)
were made concurrently for each treatment replicate. Embryos were
incubated at 28.5◦C beginning at approximately 4− 5 hours post fertil￾ization (hpf) for 24 h in the treatment solutions. Live embryos were then
transferred to fresh EM for continued incubation until 3 days post
fertilization (dpf).
C. Singleman and N.G. Holtzman
Aquatic Toxicology 233 (2021) 105794
2.2. Inhibitor exposure
Src phosphorylates VEcad at tyrosine residues (Dejana et al., 2008;
Yoshioka et al., 2011), so embryos were treated with a tyrosine kinase
specific inhibitor, 10 μM SU6656 (95 % purity, Cayman Chemicals, MI),
for 24 h following treatment of 100 μg/L PCB 126 or 5 μg/L TCDD,
beginning at 31 hpf. The inhibitor solution was made in the same way as
contaminant solutions by adding the appropriate volume of SU6656
stock solution in DMSO (Sigma-Aldrich, MO) for a final concentration of
10 μM and 0.75 % DMSO in a stender dish with 10 mL EM.
2.3. Atlantic Sturgeon contaminant exposure
Atlantic sturgeon embryos were acquired from hatchery fertiliza￾tions using wild broodstock from the Saint John River, New Brunswick
in late May 2015 and June 2016. Embryos were transported to NOAA
facilities in Sandy Hook, NJ where they were maintained in recirculating
1 part per thousand (ppt) salt water overnight, then sorted into treat￾ment dishes. Treatments included 1 μg/L PCB 126, 0.5 and 1.0 μg/L
TCDD in an acetone carrier (AccuStandard, CT). Following 24 h
contaminant treatment, embryos were grown in 1 ppt salt water in glass
dishes at 22◦C.
2.4. Immunohistochemistry
Zebrafish embryos were collected, anesthetized in MS-222 and
incubated in 0.5 M KCl for 5− 10 min to stop their hearts in diastole.
Wildtype embryos were fixed in 1% formalin for immunohistochemical
staining (Singleman and Holtzman, 2012). Atlantic sturgeon embryos
were fixed in 1% formalin.
Wholemount immunofluorescence experiments were performed in
both zebrafish and Atlantic sturgeon using MF20 (DSHB, IA), to visu￾alize cardiac musculature, and CYP1A primary antibodies (Cayman
Chemical, MI) (a marker for AhR activation), with AlexaFluor anti￾IgG2b-546 and AlexaFluor anti-IgG3− 488, respectively, as secondary
antibodies as previously described (Singleman and Holtzman, 2012).
Zebrafish were also stained with an additional primary antibody,
anti-phospho VEcadherin (pVEcad) (Sigma Aldrich, MO) with Alexa￾Fluor goat anti-rabbit-405 secondary antibody. Zebrafish embryos that
were fixed in 1% formalin for 1 h, and had tails removed before being
blocked in a 10 % sheep serum, 0.2 % saponin in 2 mg/mL BSA/PBS
solution for 1.5 h at room temperature. Embryos were then incubated
overnight at 4 ◦C in primary antibody at 1:10 MF20, 1:400 CYP1A, 1:50
pVEcad dilutions. Secondary antibodies (1:500 dilution, Invitrogen, CA)
were diluted in 0.2 % saponin in 2 mg/mL BSA/PBS and embryos were
incubated overnight at 4 ◦C. Upon completion, stained embryos were
stored in PBS at 4 ◦C for up to one week until photography of samples
was completed.
After fixation in 1% formalin, Atlantic sturgeon larvae were per￾meabilized with proteinase K dilution (10 μg/mL). The tail and peri￾cardium of Atlantic sturgeon larvae were removed during fixation to
enhance penetration. During incubation in blocking solution, primary
antibodies (MF20 and CYP1A), and associated secondary antibodies,
larvae were each kept overnight at room temperature on a shaker, much
longer than for zebrafish to ensure proper penetration of the reagents
because of the significantly larger size of the embryos.
2.5. Photography and measurements
Transgenic embryos were imaged using a stereoscope microscope
(Zeiss SteREO Discovery V12) and camera (Zeiss AxioCamMRc) with its
associated programming (Zeiss AxioVision) or on Leica SP5 Confocal
with its associated programming (LAS AF Version 2.0.2).
Antibody stained zebrafish embryos were mounted in agar on glass
bottom dishes following removal of the pericardial tissue, while Atlantic
sturgeon hearts were dissected and cross-sectioned before mounting on
glass bottom dishes with agar. All hearts were imaged using a Leica SP5
Confocal. All samples for each replicate were photographed on the same
day to avoid changes in fluorescence over time and all photos were taken
using the same settings across samples within each replicate. 3D
maximal projections and individual slice snapshots of confocal image
stacks were generated using confocal programming. Images shown are
representative of at least 10 samples.
Photographs of zebrafish hearts were analyzed using ImageJ (NIH) to
assess differences in brightness of pVEcad expression within replicate
groups. Images of MF20 staining were outlined manually using the
polygon selection tool. Outline was copied and transferred to pVEcad
images and mean value of pixel brightness was measured within the
outline and recorded. Data were analyzed using ANOVA followed by the
Tukey post-hoc tests.
3. Theory
This study expands on previous investigations by looking at specific
consequences of PCB 126 and TCDD activation of the hypothesized
nongenomic AhR pathway in zebrafish and Atlantic sturgeon hearts. To
establish that VEcad is in fact phosphorylated in the same tissues
impacted by contaminant exposure, tissues were stained with CYP1A
antibody as a marker for AhR activation and phosphorylated VEcad
specific antibody. Results confirm endothelial cell exposure to the con￾taminants results in phosphorylation of VEcad. Contaminant-exposed
fish treated with SU6656, a Src inhibitor, have a partially rescued
phenotype and which establishes a connection between AhR activation
and resulting VEcad phosphorylation (due to Src phosphorylation). We
have tested the hypothesis that developmental consequences caused by
PCB 126 and TCDD observed in zebrafish are mimicked in sturgeon. This
was done with the use of immunohistochemical staining using MF20 and
CYP1A, which showed similar phenotypes in tissue development to
exposed zebrafish. Current evidence suggests a common non-canonical
pathway for cardiac phenotypic response to contaminant exposure in
both species.
4. Results
Treatment with PCB 126 or TCDD resulted in separation of the
endocardial and myocardial layers of the heart, as seen in transgenic
zebrafish embryos (Fig. 1, arrowheads). With increasing concentration
from 1–100 μg/L PCB 126 and 0.05–5 μg/L TCDD, hearts in zebrafish
exposed to higher concentrations of either contaminant retain the pre￾mature linear heart tube phenotype, as they do not loop properly and
their chambers cannot balloon appropriately. This concentration
dependent mechanism suggests a direct connection between the amount
of contaminant exposure and response of the heart tissue (Fig. 1).
We proposed that exposure to PCB 126 and TCDD result in inap￾propriate phosphorylation of VEcadherin (pVEcad). To test whether
VEcad is in fact phosphorylated in response to PCB 126 and TCDD
exposure, hearts of treated zebrafish embryos were immunohis￾tochemically stained with an antibody specific to pVEcad to visualize
changes in VEcad phosphorylation (Fig. 2). There were increases in
expression levels of pVEcad in fish hearts treated with both contami￾nants when compared to the unexposed control embryos (Fig. 2B,C).
Interestingly, there was an unexpected increase in expression of MF20 in
the myocardium of PCB 126 and TCDD exposed hearts. The expression
of MF20 may reflect a direct impact of the contaminant on the
myocardium or may be in response to failed interactions between the
endocardium and myocardium. For this study, MF20 was used as a
morphological marker and thus this increase in expression does not
modify the primary analysis of the endocardium.
To determine if the pVEcad cells are responsive to contaminant
exposure by the activation of AhR we assessed these embryos for
expression of CYP1A, a classic marker (reviewed in Whitlock, 1999).
The observed co-localization of CYP1A with pVEcad in endothelial cells
C. Singleman and N.G. Holtzman
Aquatic Toxicology 233 (2021) 105794
after PCB 126 or TCDD exposure implies a direct link between PCB 126
or TCDD exposure and VEcad phosphorylation (Fig. 2). It is worth noting
that while CYP1A levels are highly elevated in the endocardium, CYP1A
is also upregulated in the myocardium in response to contaminant
exposure.
To further evaluate the link between ligand activated AhR and
pVEcad, we assessed the activation of the classic AhR target Src. Src is
known to be phosphorylated by activated AhR (Dong et al., 2011). To
test the hypothesized connection between activated AhR and phos￾phorylated VEcad occurring via Src, we treated contaminant-exposed
embryos with a Src inhibitor. SU6656 is a small molecule inhibitor for
the tyrosine kinase specific function of Src (Blake et al., 2000). If VEcad
is phosphorylated by the Src kinase, we predict that VEcad in cardiac
endothelial cells would not become phosphorylated in the presence of a
contaminant (PCB 126 or TCDD) and Src inhibitor SU6656, resulting in a
rescue of the observed cardiac defect in contaminant exposed fish and
reduced pVEcad. As predicted, treatment of PCB 126 and TCDD exposed
embryos with 10 μM SU6656 drastically reduced gross morphological
phenotypes including cardiac edema, short body length, and reduced
eye and head size (Fig. 3). When treated at 31 hpf, there is a 75 % rescue
in PCB 126 (100 μg/L) treated embryos (Fig. 3C) and 30 % rescue in
TCDD (5 μg/L) treated embryos (Fig. 3F).
Overall development of the fish was more normal, embryos grew to a
similar length as control siblings, craniofacial defects and pericardial
edemas were greatly reduced in inhibitor treated embryos. The heart
contractions were more regular and pumped blood more effectively in
SU6656 treated embryos than in exposed embryos not treated with Src
inhibitor. Immunohistochemical staining with MF20 (a cardiac muscle
marker), CYP1A (to demonstrate AhR activation) and pVEcad show
reduced separation of endocardial and myocardial layers in PCB 126
exposed embryos (Fig. 4C) and reduced pVEcad expression in both PCB
126 and TCDD (Fig. 4C & 4E) compared with expression in PCB 126 and
TCDD exposed embryos not treated with SU6656 (Fig. 4B & 4D). This
suggests Src inhibition results in improved maintenance of endothelial
integrity and less fluid leakage between the cardiac layers. In addition,
treated hearts maintain expression of CYP1A, suggesting that AhR is still
Fig. 1. Contaminant exposure results in
endocardial- myocardial separation in
zebrafish. Hearts of 3 dpf Tg(myl7:DsRed);Tg
(flk:GFP) zebrafish in which myocardial cells
express red fluorescence (shown as magenta)
and endocardial cells express green fluorescent
protein. (A) control fish with endocardium and
myocardium tightly associated. (A’) schematic
of heart in A, ventral view and anterior to the
top in all images. White arrowheads show sep￾aration between the endocardial and myocar￾dial cell layers in PCB 126 (B, C, D) and TCDD
(E, F, G) treated fish. Contaminant dosing
indicated in each panel. Ventricle = v, atrium =
Fig. 2. Increased phosphorylation of pVE￾cad in zebrafish hearts with PCB 126 and
TCDD exposed hearts. Immunohistochemical
staining using MF20 (myocardium, magenta),
CYP1A (green) and pVEcad (blue) primary an￾tibodies. (A) Untreated control 3 dpf zebrafish
hearts have little to no expression of CYP1A and
pVEcad in either the endocardium or myocar￾dium and the endocardium and myocardium
are tightly associated. Exposure with (B) 100
μg/L PCB 126 and (C) 5 μg/L TCDD results in
hearts that have increased expression of CYP1A
in both the endocardium and myocardium as
well as increased expression of pVEcad. The
final column shows a midsection optical section
showing a clear gap between endocardium and
myocardium in contaminant treated 3 day em￾bryos. Ventricle = v, atrium = a.
C. Singleman and N.G. Holtzman
Aquatic Toxicology 233 (2021) 105794
being activated in those tissues, while VEcad is less often phosphory￾lated in inhibitor treated embryos (Fig. 4).
We have been able to establish a link between the cardiac phenotype
induced by AhR activation upon exposure to PCB 126 and TCDD and
phosphorylation of VEcad. While there are few studies comparing wild
species to lab models, establishing the validity of zebrafish as a model
for wild fishes is crucial to increase the value of such studies. This study
used established and modified techniques for zebrafish for use in
Atlantic sturgeon larvae to characterize the cardiac phenotype in stur￾geon upon PCB 126 and TDCC exposure. Atlantic sturgeon were first
Fig. 3. SU6656 inhibition of Src phosphorylation reduces gross morphological impacts from PCB 126 and TCDD. 3 dpf control embryos with normal gross
morphology without (A) or with (D) the addition of SU6656. (B) PCB 126 exposed embryos have severe cardiac edema (arrow head), short body length, and reduced
eye and head size (*). (C) TCDD exposed embryos also have severe cardiac edema (arrow head), short body length, and reduced eye and head size (*). (E) PCB 126
and (F) TCDD exposed embryos treated with SU6656 show less severe gross morphological impacts in defects in the eye, head, and body length and have reduced
pericardial edema (arrow head).
Fig. 4. SU6656, Src inhibitor, reduces pVE￾cad expression and separation of tissue
layers while CYP1A levels are not affected.
Immunohistochemical staining using MF20
(magenta), CYP1A (green) and pVEcad (blue)
primary antibodies. (A) Untreated control
hearts have very little expression of CYP1A and
pVEcad while the endocardium and myocar￾dium are tightly associated. (B) 100 μg/L PCB
126 and (D) 5 μg/L TCDD exposed hearts have
increased expression of all cardiac markers and
a space between the endocardium and
myocardium. (C, E) The addition of SU6656, a
Src inhibitor rescues much of the observed
phenotype, while increased expression of
CYP1A is observed, but pVEcad expression level
is close to control levels. Midsections show little
separation between endocardium and myocar￾dium. Ventricle = v, atrium = a.
C. Singleman and N.G. Holtzman
Aquatic Toxicology 233 (2021) 105794
stained for just MF20 to visualize the myocardium and assess the cardiac
morphology upon contaminant exposure (Fig. 5 D–F). Analysis of car￾diac morphology demonstrates that, like zebrafish (Fig. 5 A–C), hearts of
Atlantic sturgeon larvae are more linear than their untreated siblings.
The second key phenotype in zebrafish hearts is the gap that forms be￾tween the endocardium and the myocardium, indicative of a loss of
endothelial integrity. To assess Atlantic sturgeon for the presence of this
gap, sturgeon larvae were stained for CYP1A (Fig. 5 J–L). Like their
zebrafish counterparts (Fig. 5 G–I), CYP1A is expressed at higher levels
in the endocardium and a gap was observed in treated larvae between
the endocardium and the myocardium.
In zebrafish, there is minimal expression of CYP1A in the endocar￾dium of untreated embryos (Fig. 5A), while embryonic hearts exposed to
10 μg/L PCB 126 or 5 μg/L TCDD have increased CYP1A expression in
the endocardium as well as low levels of expression in the myocardium
(Fig. 5 H and I). Atlantic sturgeon, treated with lower doses of 1 μg/L
PCB 126 and 1 μg/L TCDD, have expression levels of CYP1A in the heart
that are nearly equivalent between untreated and treated larvae (Fig. 5 K
and L). The concentrations of toxin exposure are much lower in the
Atlantic sturgeon than zebrafish (1 μg/L versus 100 & 5 μg/L), which
might explain the similarities to controls in expression levels of CYP1A
as well as the subtler separation of layers seen in Atlantic sturgeon as
compared to zebrafish. Though there are differences in expression be￾tween zebrafish and Atlantic sturgeon, they both show separation be￾tween the endocardial and myocardial layers.
5. Discussion
At the cellular level, TCDD and PCB 126 enter the cell and bind to the
cytoplasmic AhR, eventually initiating expression of cyp1A. Although
cyp1a is expressed in cardiac cells with contaminant activated AhR,
evidence indicates that CYP1A is not responsible for cardiac or endo￾thelial abnormalities, signifying an alternate AhR pathway as the cause
of these defects (Fig. 6) (Andreasen et al., 2002; Antkiewicz, 2005; Van
Tiem and Di Giulio, 2011). TCDD is also known to act on the epicardium
to impact heart development, however, the epicardium is not fully
formed at the early developmental stages of this study and thus the
observed cardiac defects are not likely impacted by epicardium at these
early time points. Activated AhR is known to activate other cytoplasmic
proteins, including phosphorylating the tyrosine kinase, Src (Enan and
Fig. 5. Parallel heart defects in contaminant
treated zebrafish and Atlantic sturgeon. (A￾F) MF20 antibody staining is shown in magenta
and marks the myocardium allowing visualiza￾tion of the shape of the heart tube in zebrafish
(A-C) and Atlantic sturgeon (D-F). Cardiac
looping and chamber ballooning is observed in
control fish (A and D). Exposure to PCB 126 (B
and E) and TCDD (C and F) results in more
linear heart tubes. In addition, exposure to PCB
126 or TCDD causes a gap to form between the
endocardium and myocardium in both zebrafish
(G-I) and Atlantic sturgeon (J-L) with CYP1A
expressed in the endocardium (green) and
myocardium (magenta) labeled with MF20. (G)
Control zebrafish embryo and (J) Atlantic stur￾geon control larvae have endocardium and
myocardium closely connected with low levels
of CYP1A. (H and K) PCB 126 exposed and (I
and L) TCDD exposed hearts have clear sepa￾ration between layers and increased levels of
CYP1A in both the endocardium and myocar￾dium. Ventricle = v, atrium = a.
C. Singleman and N.G. Holtzman
Aquatic Toxicology 233 (2021) 105794
Matsumura, 1996; Backlund and Ingelman-Sundberg, 2005; Tomkie￾wicz et al., 2012). Activated, phosphorylated Src in the cytosol has many
functions, including phosphorylating VEcad (Jopling and Hertog, 2007;
Tomkiewicz et al., 2012; Klomp et al., 2019). VEcad has been shown to
function as an adhesion protein between neighboring cells, thus tightly
binding these epithelial cells with each other. Phosphorylation of VEcad
results in the dissociation of intercellular dimers, allowing the cells to
separate (Vestweber, 2007; Dejana et al., 2008; Orsenigo et al., 2012)
(Fig. 6). The resulting increased permeability of the endothelium and
associated vascular leakage likely drives separation of the endocardium
and myocardium (inner and muscle layers) of the heart leading to fluid
accumulation between the layers (Orsenigo et al., 2012; Benn et al.,
2015). TCDD and PCB 126 exposure leads to a decrease of vascular cells
binding to each other, resulting in many of the observed heart de￾formities after exposure.
Zebrafish hearts exposed to PCB 126 and TCDD have improperly
developed linear hearts with a gap between the endocardial lining and
the myocardium (Fig. 1). Our data suggest these contaminants cause a
cardiac defect by activation of AhR ultimately leading to VEcad phos￾phorylation through a non-canonical pathway involving Src. Specif￾ically, we show that VEcad is phosphorylated in tissues with activated
AhR (as visualized by CYP1A expression) while the canonical pathway
remains active in these cells, as elevated levels of CYP1A are also
observed (Fig. 2). To verify Src’s involvement in this pathway we treated
contaminant-exposed zebrafish embryos with Src inhibitor, SU6656, to
rescue the heart defects from PCB 126 and TCDD exposure (Fig. 3,4).
This non-canonical pathway does not diminish the potential for PCB 126
and TCDD to interact with the canonical AHR pathway, which may still
play a role in myocardial defects. This study demonstrates a role for non￾canonical involvement of pVECad in endocardium of exposed embryos.
The results in zebrafish were compared to data collected in Atlantic
sturgeon exposed to PCB 126 and TCDD at similar concentrations. When
exposed to these contaminants, both species have hearts that do not loop
properly with endocardial-myocardial separation (Fig. 5). These organ
and tissue level defects are similar between the fishes, suggesting a
related mechanism is involved in causing these cardiac phenotypes. This
further implies that there might be a common mechanism driving car￾diac defects seen in many fish species exposed to AhR activating
contaminants. We hypothesized a novel nongenomic mechanism in
which activated AhR drives cardiac defects (Fig. 6), in which activated
AhR phosphorylates Src, in turn phosphorylating VEcad, resulting in
cardiac defects. This mechanism could explain the cardiac defects
observed in fishes after PCB 126 and TCDD exposure.
We found increased VEcad phosphorylation in PCB 126 and TCDD
exposed zebrafish hearts, which was rescued by Src inhibition with
SU6656 treatments (Fig. 3,4). Unfortunately, we were not able to
replicate the pVEcad results in the Atlantic sturgeon as the pVEcad
antibody does not appear to work efficiently in this species when sam￾ples are stored in fixative for extended periods of time. However, while
the Src inhibitor does have a function in rescuing gross morphological
defects, cardiac function and reduced phosphorylation of VEcad, this
rescue was incomplete. Src and VEcad are likely part of a complex
interaction of cellular responses to AhR mediated contaminant
responses.
In this paper we provide evidence for a non-canonical pathway by
which TCDD and PCB activation of AhR drive cardiac defects (Carney
et al., 2004; Antkiewicz, 2005; Scott et al., 2011). The alternate pathway
presented here is simple and there is likely a more complex interaction
between AhR, Src and VEcad yet to be determined. Studies exploring the
consequences of activated AhR in vascular adhesion shows potential for
interactions between VEcad and β-catenin (Barbieri et al., 2008; Chang
et al., 2012). While there is still more to be done, this alternate mech￾anism provides the framework for future studies exploring the cellular
mechanisms for cardiac defects in fishes exposed to AhR activating
contaminants.
Contributions
C.S. and N.G.H. contributed to the design and implementation of the
research, to the analysis of the results and to the writing of the manu￾script. All experiments were conducted by C.S.
CRediT authorship contribution statement
Corinna Singleman: Conceptualization, Methodology, Validation,
Formal analysis, Investigation, Writing – original draft, Visualization.
Fig. 6. Model of non-genetic mechanism. (A)
In the absence of PCB 126 or TCDD, there is
little activation of the AhR pathway. (B) Upon
exposure to the contaminants, activated AhR
translocates to the nucleus, binds to ARNT and
activates cyp1a expression. (C) In addition to
the genomic activation of cyp1A, some acti￾vated AhR phosphorylates Src in the cytoplasm.
Activated Src phosphorylates VEcadherin
resulting in separation of the VEcadherin dimer
which is required for endothelial integrity. (D)
Application of the Src phosphorylation inhibi￾tor SU6656 inhibits this nongenomic pathway,
preventing the loss of endothelial integrity
while still activating the genomic pathway of
cyp1A expression.
C. Singleman and N.G. Holtzman
Aquatic Toxicology 233 (2021) 105794
Nathalia G. Holtzman: Conceptualization, Methodology, Validation,
Resources, Writing – review & editing, Supervision, Visualization.
Declaration of Competing Interest
The authors reported no declarations of interest.
Acknowledgements
Thank you to our collaborators from NOAA for supplying treated
Atlantic sturgeon larvae: Dr. Chris Chambers, Ehren Habeck, and Kristin
Habeck. All toxins for Atlantic sturgeon experiments were provided by
Dr. Isaac Wirgin (NYU Langone Medical Center). Further thanks go to
Dr. Isaac Wirgin for his generosity in editing the final drafts of this
manuscript.
Funding
This research project was supported by the Hudson River Founda￾tion’s Mark B. Bain Graduate Fellowship Program. Additional funds
provided by the CUNY Graduate Center’s Doctoral Student Research
Grant (to C.S.), PSC-CUNY PSCREG-40-1135 and PSCREG-39-61492 and
Queens College Research Enhancement funds (to N.G.H). Some of the
experiments were done on the equipment from the Core Facilities for
Imaging, Cellular and Molecular Biology at Queens College, Queens, NY
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