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Citation: Rumph, J.T.; Stephens, V.R.;
Ameli, S.; Brown, L.K.; Rayford, K.J.;
Nde, P.N.; Osteen, K.G.; Bruner-Tran,
K.L. A Paternal Fish Oil Diet
Preconception Reduces Lung
Inflammation in a Toxicant-Driven
Murine Model of New
Bronchopulmonary Dysplasia. Mar.
Drugs 2023,21, 161. https://doi.org/
10.3390/md21030161
Academic Editor: Maria
Stefania Sinicropi
Received: 22 December 2022
Revised: 25 February 2023
Accepted: 25 February 2023
Published: 27 February 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
marine drugs
Article
A Paternal Fish Oil Diet Preconception Reduces Lung
Inflammation in a Toxicant-Driven Murine Model of New
Bronchopulmonary Dysplasia
Jelonia T. Rumph 1,2 , Victoria R. Stephens 1,3, Sharareh Ameli 1,3, LaKendria K. Brown 2, Kayla J. Rayford 2,
Pius N. Nde 2, Kevin G. Osteen 1,3,4 and Kaylon L. Bruner-Tran 1, *
1Women’s Reproductive Health Research Center, Department of Obstetrics and Gynecology,
Vanderbilt University School of Medicine, Nashville, TN 37232, USA
2Department of Microbiology, Immunology and Physiology, Meharry Medical College,
Nashville, TN 37208, USA
3Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine,
Nashville, TN 37232, USA
4VA Tennessee Valley Healthcare System, Nashville, TN 37232, USA
*Correspondence: kaylon.bruner-tran@vumc.org
Abstract:
New bronchopulmonary dysplasia (BPD) is a neonatal disease that is theorized to begin
in utero and manifests as reduced alveolarization due to inflammation of the lung. Risk factors for
new BPD in human infants include intrauterine growth restriction (IUGR), premature birth (PTB)
and formula feeding. Using a mouse model, our group recently reported that a paternal history of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure increased his offspring’s risk of IUGR, PTB,
and new BPD. Additionally, formula supplementation of these neonates worsened the severity of
pulmonary disease. In a separate study, we reported that a paternal preconception fish oil diet
prevented TCDD-driven IUGR and PTB. Not surprisingly, eliminating these two major risk factors
for new BPD also significantly reduced development of neonatal lung disease. However, this prior
study did not examine the potential mechanism for fish oil’s protective effect. Herein, we sought
to determine whether a paternal preconception fish oil diet attenuated toxicant-associated lung
inflammation, which is an important contributor to the pathogenesis of new BPD. Compared to
offspring of standard diet TCDD-exposed males, offspring of TCDD-exposed males provided a fish
oil diet prior to conception exhibited a significant reduction in pulmonary expression of multiple
pro-inflammatory mediators (Tlr4,Cxcr2,Il-1 alpha). Additionally, neonatal lungs of pups born to fish
oil treated fathers exhibited minimal hemorrhaging or edema. Currently, prevention of BPD is largely
focused on maternal strategies to improve health (e.g., smoking cessation) or reduce risk of PTB (e.g.,
progesterone supplementation). Our studies in mice support a role for also targeting paternal factors
to improve pregnancy outcomes and child health.
Keywords:
bronchopulmonary dysplasia; fish oil; toxicants; inflammation; neonatal; lung; dioxin;
formula; therapeutics
1. Introduction
New Bronchopulmonary Dysplasia (BPD) is a neonatal lung disease that is among the
leading causes of death in premature infants. Epidemiology and animal models suggest
that risk factors for this disease include intrauterine growth restriction (IUGR), premature
birth (PTB), and formula feeding [
1
,
2
]. Chorioamnionitis and maternal smoking have each
been linked to the development of new BPD in neonates [
3
–
5
] and supports the theory
that this disease begins during pregnancy. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is
a common environmental contaminant present in cigarette smoke as well as car exhaust
and forest fires. Our group previously reported that less than 50% of adult males with a
Mar. Drugs 2023,21, 161. https://doi.org/10.3390/md21030161 https://www.mdpi.com/journal/marinedrugs
Mar. Drugs 2023,21, 161 2 of 15
history of in utero exposure to TCDD (F1
TCDD
males) were fertile and that the placentae
arising in their unexposed partners exhibited heightened inflammation compared to control
pregnancies [
6
,
7
]. These pregnancies frequently resulted in spontaneous PTB as well as
pups born small for their gestational age [
7
,
8
]. Since IUGR and PTB are well-known risk
factors for new BPD, our group recently assessed the incidence of this disease in offspring
of TCDD-exposed males (F2
TCDD
mice). We demonstrated that new BPD was common in
F2
TCDD
pups, and that formula supplementation of these neonates heightened the severity
of disease [9,10].
Relevant to the current study, we also previously reported that providing F1
TCDD
males a fish oil supplemented diet prior to mating eliminated the risk of new BPD in
F2
TCDD
mice. These studies further demonstrated that a paternal fish oil diet preconception
improved postnatal growth, lung alveolarization, and the risk of interalveolar red blood
cell infiltration in F2 animals. Importantly, the presence of blood within the interalveolar
space is a sign of pulmonary hypertension in human infants and is often a consequence of
new BPD [9].
Human infants who develop new BPD are also susceptible to inflammation-driven
hemorrhaging and edema [
11
,
12
]. Although our previous studies did not specifically
explore lung inflammation in neonatal pups born to fish oil treated fathers, a number
of investigators have demonstrated that direct fish oil supplementation reduces lung
inflammation and edema in animal models [
13
,
14
]. Importantly, this treatment was not
associated with increased risk of hemorrhage in either human infants or mice [
14
,
15
].
Finally, inflammation-driven fibrosis has been documented in numerous tissues [
16
] as well
as in association with BPD [
17
]. Fish oil fatty acid supplementation was found to reduce
collagen deposition in an animal model [
18
] while human supplementation was shown
to protect against the development of interstitial lung disease [
19
]. There is also evidence
that long-term supplementation with fish oil fatty acids may benefit patients with cystic
fibrosis [
20
]. However, to our knowledge, the impact of the paternal preconception diet on
neonatal lung disease has not previously been investigated.
In the present study, we evaluated the impact of both paternal and neonatal diet on
lung inflammation and associated comorbidities in a TCDD-driven murine model of new
BPD. We found that offspring of control males exhibited normal morphology regardless
of the paternal diet; however, disease-associated changes were noted in the majority of
F2
TCDD
pups. Interestingly, neonatal formula supplementation had little impact on lung
health in the absence of paternal toxicant exposure. We further found that a paternal
fish oil diet preconception provided to F1
TCDD
males attenuated pulmonary expression
of pro-inflammatory cytokines in the F2 generation regardless of the neonatal diet. Reduced
inflammation was associated with a reduction in lung hemorrhaging, edema, and fibrosis.
Our findings demonstrate that intervening with a fish oil diet preconception reduces the
risk of toxicant-associated new BPD in the F2 generation by attenuating lung inflammation
and its sequelae.
2. Results
2.1. A Paternal Fish Oil Diet Reduced the Risk of Lung Hemorrhaging and Edema in F2
TCDD
Pups
Our first goal was to determine whether in utero TCDD exposure of fathers (F1
TCDD
males) was associated with lung hemorrhaging and edema in their offspring (F2
TCDD
pups) (Figure 1; The pup nomenclature used through the text is listed in Table 1). We also
examined the impact of both the paternal and neonatal diet on these parameters. To assess
these endpoints, all experimental groups were compared to offspring of unexposed controls
(F2None pups).
Mar. Drugs 2023,21, 161 3 of 15
Figure 1.
A paternal fish oil diet reduced toxicant-driven lung hemorrhaging. Representative images
of gross lung anatomy of neonatal lungs on postnatal day 11 taken at 15
×
magnification. Scale
bar = 11.5 mm.
Table 1. Description of pup nomenclature used throughout the manuscript.
F2 Generation
Nomenclature
Was the Pup’s Father
(F1 Generation)
Exposed to TCDD in
Utero?
Did the Pup’s Father
(F1 Generation)
Receive a Fish Oil
Preconception Diet?
Did the Pup (F2
Generation) Receive
Postnatal Formula
Supplementation?
F2NONE
(CT) No No No
F2NONE/Fish (CT) No Yes No
F2NONE/Form (CT) No No Yes
F2 NONE/
Fish/Form
(CT)
No Yes Yes
F2TCDD Yes No No
F2TCDD/Fish Yes Yes No
F2TCDD/Form Yes No Yes
F2TCDD/Fish/Form Yes Yes Yes
“CT” represents “control” groups that do not have a history of TCDD exposure.
F2
None
and F2
None
/Form pups displayed healthy lungs with no visible signs of
hemorrhaging (Figure 1B). The gross lung anatomy of F2
None
/Fish pups was also com-
parable to F2
None
pups (Figure 1C) and was unaffected by formula supplementation
(F2None/Fish/Form pups) (Figure 1D).
In contrast to F2
None
offspring, abnormal gross anatomy of the lungs was observed
in approximately 35% of F2
TCDD
pups. Additionally, F2
TCDD
pups also exhibited hemor-
rhaging, noted by the coagulation of blood on the surface of the lung (Figure 1E). Lung
hemorrhaging was most severe among F2
TCDD
/Form pups and affected about 45% of
Mar. Drugs 2023,21, 161 4 of 15
these animals (Figure 1F). However, paternal fish oil supplementation improved these
outcomes resulting in 100% of F2
TCDD
/Fish pups exhibiting normal lung anatomy with
little-to-no hemorrhaging and was comparable to F2
None
pups (Figure 1G). Minimal lung
hemorrhaging was also noted in F2TCDD/Fish/Form pups (Figure 1H).
Next, we measured lung edema in all groups by assessing the mean wet-to-dry lung
weight ratio. Among all groups, there were no significant differences when considering
wet lung weight or dry lung weight individually (Supplemental Figures S1 and S2). F2
None
pups, independent of paternal and neonatal diet, did not exhibit lung edema as charac-
terized by a wet-to-dry lung weight ratio of 5 or greater. However, F2
TCDD
(
p= 0.0168
)
and F2
TCDD
/Form (p= 0.0246) pups displayed a significant increase in wet-to-dry lung
weight compared to F2
None
pups. Additionally, 100% (4/4) of F2
TCDD
pups exhibited a
wet-to-dry lung weight ratio of 5 or greater. Interestingly, 60% (3/5) of F2
TCDD
/Form
pups exhibited a wet-to-dry lung weight ratio of 5 or greater. None of the F2
TCDD
/Fish
(
N= 4
) and F2
TCDD
/Fish/Form pups (N= 5) exhibited a wet-to-dry lung weight ratio of 5
or greater (Figure 2).
Figure 2.
A paternal fish oil diet preconception reduced the risk of toxicant-driven edema in F2
TCDD
pups. Average wet-to-dry lung weights (* p< 0.05).
Mar. Drugs 2023,21, 161 5 of 15
2.2. A Paternal Fish Oil Diet Preconception Reduced Pulmonary Expression of Toxicant and
Formula Driven Pro-Inflammatory Mediators in F2TCDD Pups
Cxcr2, a CXC chemokine receptor, is commonly overexpressed in pulmonary diseases
such as acute lung disease, chronic obstructive pulmonary disease, and new
BPD [21–23]
.
Additionally, TCDD exposure can elicit immune responses in a Cxcr2-dependent man-
ner [
24
]. Herein, we evaluated whether a paternal history of TCDD exposure was associ-
ated with increased pulmonary expression of Cxcr2 in neonatal offspring. We additionally
assessed the impact of both the paternal diet (standard or fish oil) and neonatal diet (with
or without formula supplementation) on this outcome
.
We measured the mRNA transcript
level of Cxcr2 in the lungs of all F2 neonates. Our studies revealed that F2
TCDD
(p= 0.0007)
and F2
TCDD
/Form pups (p= 0.0077) exhibited significant increases in the normalized rel-
ative transcript expression of Cxcr2 when compared to F2
None
pups. Intervening with a
paternal fish oil diet preconception normalized the relative transcript expression of Cxcr2
in F2
TCDD
/Fish and F2
TCDD
/Fish/Form pups to F2
None
pups (Figure 3). It is interest-
ing to note that formula, in the absence of paternal TCDD exposure, had little impact
on pulmonary Cxcr2 gene expression. In contrast, expression of this gene was highest
in F2
TCDD
/Form pups suggesting a potential synergistic effect of paternal exposure and
neonatal diet.
Figure 3.
A paternal fish oil diet preconception reduced toxicant-driven pulmonary Cxcr2 gene
expression in F2
TCDD
pups. Lung RNA was isolated from all groups and subjected to qRT-PCR to
measure Cxcr2 mRNA expression. For this set of experiments, each exposure and treatment group
consisted of 3 pups from different litters. (** p< 0.01, *** p< 0.001).
Mar. Drugs 2023,21, 161 6 of 15
Activation of the Il-1 family of cytokines is downstream of Cxcr2 activation and
aberrant expression of the Il-1 family has been implicated in pulmonary diseases such as
new BPD [
25
,
26
]. Therefore, we assessed the relative mRNA transcript expression of Il-1
alpha in F2
None
and F2
TCDD
groups. We found that Il-1 alpha expression in lungs of F2 pups
was not impacted by formula alone (F2
None
/Form) or paternal toxicant exposure alone
(F2
TCDD
). In contrast, Il-1 alpha expression was significantly increased in F2
TCDD
/Form
pups (p< 0.0001), strongly suggesting a synergistic effect of these treatments. Intervening
with a paternal fish oil diet preconception reduced the relative expression level of Il-1 alpha
among F2TCDD/Fish/Form pups when compared to F2TCDD/Form pups (Figure 4).
Figure 4.
A paternal fish oil diet preconception reduced pulmonary gene expression of toxicant and
formula-driven Il-1 alpha in F2TCDD pups. Lung RNA was isolated from all groups and subjected to
qRT-PCR for Il-1 alpha. For this set of experiments, each exposure and treatment group consisted of
3 pups from multiple litters (**** p< 0.0001).
We previously reported that, in addition to new BPD, F2
TCDD
pups are susceptible
to developing necrotizing enterocolitis (NEC) [
9
,
10
]. NEC is a life-threatening inflam-
matory disease of the intestine and is thought to promote development of new BPD as
a consequence of systemic inflammation and activation of pulmonary Tlr4.Tlr4 is a
proinflammatory cytokine that is commonly upregulated in infants with new BPD [
27
,
28
].
Herein, we performed an immunohistochemical analysis to observe the localization of Tlr4
expression in the lungs of F2
NONE
and F2
TCDD
pups. We report that maternal milk-fed
F2
TCDD
pups exhibited interalveolar Tlr4 expression, which was heightened when pups
received a supplemental formula diet. Formula supplementation among F2
NONE
pups
also led to an increase in interalveolar Tlr4 expression when compared to their maternal
milk-fed counterparts. However, after intervening with a paternal fish oil diet precon-
ception, we found that interalveolar Tlr4 expression was eliminated in maternal milk-fed
and formula-supplemented F2
TCDD
pups, as well as formula-supplemented F2
NONE
pups
(Figure 5A–H).
Mar. Drugs 2023,21, 161 7 of 15
Figure 5.
A paternal fish oil diet normalized pulmonary Tlr4 gene and protein expression in F2
TCDD
pups. Representative images from multiple lungs (N= 3 or more /group) of Tlr4-stained per-
fused lung tissue on postnatal day 11. Original magnification 200
×
(
A
–
H
);
F2NONE (A)
, F2
NONE
/
Form (B)
, F2
NONE
/Fish (
C
), F2
NONE
/Fish/Form (
D
), F2
TCDD
(
E
), F2
TCDD
/Form (
F
), F2
TCDD
/
Fish (G)
, F2
TCDD
/Fish/Form (
H
). Lung RNA was isolated from all groups and subjected to qRT-
PCR for
Tlr4 (I)
. TLR4 protein expression was determined by computer-assisted quantification of
DAB substrate from lung immunohistochemistry experiments (
J
). For this set of experiments, each
exposure and treatment group consisted of 3 pups from multiple litters (* p< 0.05).
We also measured pulmonary Tlr4 relative mRNA transcript and protein expression
in all F2 pups. We found that F2
TCDD
/Form pups exhibited a significant increase in Tlr4
mRNA expression compared to F2
None
pups (p= 0.0415) while Tlr4 protein was significantly
increased in both F2
TCDD
and F2
TCDD
/Form pups (p= 0.0232; p= 0.0149, respectively)
(Figure 5). Notably, formula supplementation of pups in the absence of paternal TCDD
exposure did not have a significant impact on Tlr4 mRNA or protein expression.
Mar. Drugs 2023,21, 161 8 of 15
2.3. Impact of Paternal and Neonatal Diet on Pulmonary Fibrosis in F2 Neonates
Finally, we performed Masson’s Trichrome staining to assess the incidence of pul-
monary fibrosis in all F2 groups (Figure 6). Although it is normal for collagen to be
deposited around pulmonary vessels [
29
], collagen deposition surrounding alveoli (iden-
tified by the overlap of blue and red staining) is not normal and indicates fibrosis. We
found that F2
NONE
pups displayed little-to-no signs of pulmonary fibrosis (Figure 6A,E),
but collagen deposition was commonly observed in control pups receiving supplemental
formula (F2
NONE
/Form pups) (Figure 6B,F). However, pulmonary fibrosis was reduced in
formula-fed control pups whose fathers were provided fish oil (F2
None
/Fish/Form pups)
(Figure 6D,H). F2
TCDD
and F2
TCDD
/Form pups also demonstrated signs of pulmonary
fibrosis (Figure 6I,M). However, only F2
TCDD
/Form pups exhibited interalveolar red blood
cell infiltration (Figure 6J,N), consistent with our previous observations [
9
]. F2
TCDD
/Fish
pups displayed a reduction in fibrosis and improvement in alveolarization (Figure 6K,O).
Although F2
TCDD
/Fish/Form pups also exhibited improved alveolarization and their risk
of interalveolar red blood cell infiltration was eliminated, pulmonary fibrosis was still
apparent in approximately 50% of these pups (Figure 6L,P).
Figure 6.
Impact of paternal and neonatal exposures and risk of pulmonary fibrosis. Repre-
sentative images from multiple lungs (N= 3/group) of Masson’s Trichrome-stained perfused
lung tissue on postnatal day 11. Original magnification 200
×
(
A
–
D
,
I
–
L
) and 400
×
(
E
–
H
,
M
–
P
).
F2NONE (A,E)
, F2
NONE
/Form (
B
,
F
), F2
NONE
/Fish (
C
,
G
), F2
NONE
/Fish/Form (
D
,
H
), F2
TCDD
(
I
,
M
),
F2TCDD/ Form (J,N), F2TCDD /Fish ( K,O), F2TCDD/Fish/Form (L,P).
Mar. Drugs 2023,21, 161 9 of 15
3. Discussion
New BPD, characterized by reduced alveolarization, most commonly occurs in pre-
mature infants and places them at increased risk of developing chronic lung disease [
30
].
Inflammation plays a major role in both the development and progression of new BPD and
thus post-natal anti-inflammatory agents (e.g., corticosteroids) are often used therapeuti-
cally. Unfortunately, for poorly understood reasons, not all patients at risk for BPD respond
to this treatment [
30
]. Thus, developing a better understanding of events associated with
disease initiation is needed in order to identify more effective treatment or prevention
strategies.
Several studies indicate that new BPD is initiated in the prenatal environment [
31
], sug-
gesting to us that paternal factors capable of impeding placental function would contribute
to its development. We have previously reported that, compared to control pregnancies,
placentae arising in control female mice mated to TCDD exposed males exhibit reduced
gene and protein expression of the progesterone receptor and insulin-like growth factor 2
as well has enhanced expression of Tlr4 [
6
]. These placental changes were associated with
pups being born preterm and with IUGR [
6
,
7
], conditions which are known risk factors
for BPD in human infants. Indeed, using this same model, we previously reported that
a paternal history of TCDD exposure was associated with an increased risk of new BPD
in his offspring [
10
]. However, intervening with a paternal fish oil diet preconception
eliminated the risk of PTB and IUGR [
8
] and significantly reduced the risk of new BPD in
these pups [
9
]. The goal of the current study was to determine if the reduced risk of new
BPD in offspring of fish oil supplemented fathers was also associated with attenuation of
neonatal lung inflammation.
Evidence from the literature suggests that hemorrhaging and edema are both as-
sociated with inflammation in BPD and other pulmonary diseases [
11
,
32
] and further
compromises lung capacity [
11
,
33
]. We report here that independent of neonatal diet,
F2
TCDD
pups exhibited both lung hemorrhaging and edema denoted by gross anatomy and
an increased ratio of wet-to-dry lung weight (Figures 1and 2). Our findings in mice born
to toxicant-exposed fathers mirror the data of studies examining human infants with new
BPD [34,35].
In the current study, we also found that intervening with a paternal fish oil diet prior to
conception reduced lung hemorrhaging and edema in F2
TCDD
/Fish and F2
TCDD
/Fish/Form
pups (Figures 1and 2). As stated above, both lung edema and hemorrhaging are associated
with pulmonary inflammation, most likely as a consequence of pro-inflammatory mediators
within alveolar interstitial fluid [
36
–
38
]. Therefore, we next measured pro-inflammatory
mediator expression in neonatal lungs in concert with the assessment of gross morphology
(hemorrhaging and edema).
Our studies revealed that compared to all F2
none
groups, a paternal history of TCDD
exposure was associated with heightened pulmonary expression of Cxcr2 and Il-1 alpha
transcripts in neonatal mice. However, gene expression was normalized in pups whose
fathers were provided the fish oil diet. Interestingly, formula supplementation alone had
virtually no impact on Cxcr2 or Il-1 alpha expression (Figures 3and 4). These findings
suggest that indirect fish oil supplementation can reduce the expression of Cxcr2 and its
downstream mediator, Il-1 alpha. These data also support other studies that suggest the
antagonism of Cxcr2 is a potential therapeutic approach for new BPD [21].
We next measured Tlr4 protein and mRNA expression since studies from others have
suggested that antagonism of this receptor can protect against lung inflammation and
associated diseases [
39
,
40
]. We report that, compared to control pups, Tlr4 mRNA and
protein levels were increased within the alveolar space of lungs of F2
TCDD
pups arising
from standard diet fathers. However, F2
TCDD
pups whose fathers were provided a fish
oil supplemented diet exhibited Tlr4 levels similar to that of control pups. Unexpectedly,
formula feeding of pups did not have a significant impact on Tlr4 expression unless the
paternal parent had a history of TCDD exposure. The lungs also appeared to contain
interstitial fluid (Figure 5). This finding supports the work of others suggesting that
Mar. Drugs 2023,21, 161 10 of 15
infants with new BPD are at risk of developing pulmonary edema due to proinflammatory
interstitial fluid within the alveolar space [41,42].
Lastly, we report that control pups provided formula (F2
none
/Form), and F2
TCDD
pups with and without formula all exhibited signs of lung fibrosis; however, abnormal
collagen deposition was not observed in F2
None
/Fish/Form and F2
TCDD
/Fish pups. Sur-
prisingly, paternal fish oil supplementation did not reduce the risk of lung fibrosis in
F2
TCDD
/Fish/Form pups despite the absence of interalveolar red blood cell infiltration
(Figure 6). These results suggest that historical TCDD exposure and postnatal formula
supplementation act as independent risk factors for pulmonary fibrosis. Our results also in-
dicate that intervening with a paternal fish oil diet can reduce formula-driven lung fibrosis
in the absence of historical TCDD exposure, but not in the presence of this variable.
The omega-3 fatty acids are known for their anti-inflammatory effects [
43
,
44
] and
supplementation with fish oil has been found to lower the risk of internal bleeding [
45
] and
edema [
46
]. Overall, our data also indicate that fish oil can reduce pulmonary inflammation,
as well as associated hemorrhaging, edema, and fibrosis [
7
,
35
]. However, our studies are
unique in that the fish oil intervention was provided to the paternal parent prior to conception.
Although F2
TCDD
and F2
TCDD
/Form pups frequently exhibited new BPD, paternal fish oil
supplementation attenuated the expression of pulmonary inflammation (Cxcr2,Il-1 alpha,
and Tlr4) and significantly reduced the incidence of lung disease in offspring. As stated
above, antagonism of Cxcr2 has been suggested as a potential treatment for new BPD [
21
].
Our studies revealed that preconception fish oil was effective in reducing pulmonary Cxcr2
expression in offspring and thus may have value as a preventive measure. Since fish oil
was not provided to the F2 generation directly, this intervention likely improved lung
development and health outcomes by reducing placental inflammation during pregnancy
which also reduces the risk of PTB and IUGR [
8
]. Our studies not only provide strong
evidence that new BPD can begin in utero, but that effective treatment for this devastating
disease can be initiated prior to pregnancy.
Development of new BPD remains a major problem facing premature infants; thus,
identifying effective preventative measures is a paramount concern. Our studies in mice
demonstrating that the toxicant exposure and dietary history of the father can significantly
impact the development of this disease may also shed light on factors that influence BPD
in human infants. Due to the potential importance of our findings using a small cohort of
animals, future studies will need to be conducted with a larger sample size as well as in
additional experimental models of new BPD.
4. Materials and Methods
4.1. Animals
Adult (10–12 weeks) and neonatal C57BL/6 mice were used in this study. Adult mice
were obtained from Envigo (Indianapolis, IN, USA) or born in-house. All neonatal mice
were born in-house. Animals were housed in the Barrier Animal Care Facility at Vanderbilt
University Medical Center, which is free of common mouse pathogens. Adult mice were
provided food and water ad libitum. Animal room temperatures were maintained between
22–24
◦
C and relative humidity of 40–50% on a 12-h light: dark schedule. Experiments
described in this study were approved by Vanderbilt University’s Institutional Animal
Care and Use Committee (IACUC) per the Animal Welfare Act protocol #M2000098.
4.2. Chemicals
Esbilac Puppy Milk Replacer Powder was purchased from Pet-Ag, Inc (Hampshire, IL,
USA). All other chemicals were obtained from Sigma-Aldrich unless otherwise stated.
4.3. Exposure, Mating and Diet Scheme
Virgin 10 to 12-week-old C57BL/6 females were mated with intact males of similar
age. Females were weighed daily and monitored for the presence of a vaginal semen plug;
denoting copulation had occurred. The morning a vaginal plug was identified, the dam
Mar. Drugs 2023,21, 161 11 of 15
was considered pregnant [denoted as embryonic day 0.5 (E0.5)] and moved to a new cage.
Following confirmation of pregnancy, dams were exposed to TCDD (10
µ
g/kg) in corn oil
or corn oil vehicle alone by gavage on E15.5 at 1100 h CST. Dams provided vehicle only
were used as unexposed controls while dams receiving TCDD were designated F0 mice
(the founding generation).
Although the selected dose of TCDD is higher than typical human exposures, this dose
reflects the more rapid clearance of this toxicant in mice compared to humans. This dose is
well below the LD50 for adult C57BL/6 mice (230
µ
g/kg) [
47
] and is not overtly teratogenic
or abortogenic. In our hands, parturition typically occurs on E20 for both control and F0
pregnancies. Finally, since the half-life of TCDD is 11 days in C57BL/6 mice, offspring of
F0 dams (F1 pups) are directly exposed to TCDD in utero and during lactation [
47
]. Germ
cells present within F1 feti are also directly exposed to TCDD; these cells have the potential
to become the F2 generation.
4.4. Diet and Mating Scheme for the F1 Generation
Purina Mills (TestDiet division) provided the 5% Menhaden fish oil diet, which also
contained 1.5% corn oil to prevent depletion of omega-6 fatty acids. Menhaden fish oil,
(OmegaProtein, Houston, TX, USA), has an established fatty acid profile (~40% omega-3
fatty acids) and was processed to remove dioxins and polychlorinated biphenyls. The fish
oil diet is a modification of Purina’s low phytoestrogen rodent chow, 5VR5, which was
used as the control (standard) diet. Protein, total fat, and energy content are similar across
diets. The fish oil diet was maintained in vacuum-sealed bags at
−
20
◦
C until use and once
provided to mice, replaced every 3 days.
After weaning, F1 males were maintained on a standard or fish oil diet for 7-weeks
(one full cycle of spermatogenesis) and mated at 10–12 weeks of age with age-matched
unexposed C57BL/6 females. Once a vaginal semen plug was identified, dams were singly
housed until parturition.
4.5. Formula Feeding
Beginning on postnatal day 7 (PND7), pups were weighed, and randomized to a strict
maternal milk diet or a supplemental formula diet. Pups were bottle-fed 30
µ
L of formula
three times a day over the course of four days using a small nipple attached to a 1ml syringe
(Miracle Nipple Mini for Pets and Wildlife). Each 30
µ
L dose was provided in two aliquots
of 15
µ
L, 10 min apart. All pups (independent of neonatal diet) remained with dams for the
duration of the study and formula-supplemented pups were allowed to nurse ad libitum.
Pup nomenclature is described in Table 1.
4.6. Euthanasia and Sample Collection
On PND11 at 1100 h local time, pups were weighed, then euthanized by decapitation
performed under deep anesthesia per AAALAC guidelines. Following euthanasia, the rib
cavity was opened, and the lungs were identified. The lungs were weighed, and some were
dried to obtain the dry weight or stored at −80 ◦C until further use.
4.7. qRT-PCR
Lung tissue was lysed using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) and
total RNA was purified from tissue lysates using the RNeasy Mini Kit (Qiagen, Valencia,
CA, USA). RNA quality was verified using Nanodrop and RNA with a 260/280 ratio of ~2.0
were used for qRT-PCR. An iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) was
used to generate cDNA from 1
µ
g of total RNA using random decamer primers as described
by the manufacturers. The same thermal cycling program was applied Cxcr2,Il-1 alpha and
Tlr4: 95
◦
C for 30 s, 40 cycles of 95
◦
C for 5 s, 60
◦
C for 5 s using a Bio-Rad CFX96 Real-
time thermocycler. The melt curve was analyzed to confirm product purity. All reactions
were performed in triplicate. Ribosomal 18s transcript was used as a housekeeping gene to
normalize transcript levels of Cxcr2,Il-1 alpha, and Tlr4 (Sino Biological, Chesterbrook, PA,
Mar. Drugs 2023,21, 161 12 of 15
USA) for all samples (primer sequences were not disclosed by the company). Results were
evaluated using the delta-delta Ct method as previously described [9].
4.8. Masson’s Trichrome Stain
At the time of necropsy, the whole lung tissue from a subset F2 mice was perfused with
phosphate-buffered saline (PBS), embedded in paraffin and sectioned at 5
µ
m as previously
described [
9
]. Lung slides were deparaffinized in xylene, then rehydrated in increasing
concentrations of ethanol, followed by dH2O. Slides were then incubated overnight at
room temperature in Bouin’s solution. The next day, slides were washed and incubated
with Wiegert’s iron hematoxylin solution for 10 min, then washed. Next, slides were
stained with Biebrich scarlet-acid fuchsin solution for 15 min then washed. Slides were
then differentiated by incubating in phosphomolybdic- phosphotungstic acid solution for
20 min. Without washing, aniline blue solution was added, and the slides incubated for an
additional 15 min. Slides were then washed briefly and differentiated by incubating in a
1% acetic acid solution for 3 min. Slides were then washed and dehydrated in increasing
concentrations of ethanol and xylene. Slides were coverslipped and viewed using an
Olympus B071 microscope. Collagen is denoted by blue staining; nuclei are denoted by
black staining and muscle/cytoplasm/keratin are denoted by red staining.
4.9. Lung Hemorrhaging and Edema Analysis
At the time of necropsy the whole lung was quickly removed from the chest cavity
and immediately photographed. This photograph was used to assess lung hemorrhaging
via gross analyses (e.g., coagulation of blood at the surface of the lung or discoloration) as
previously described [
48
,
49
]. Next, the right lobe of the lung was excised from the whole
lung and weighed. The right lobe was then placed in an oven set at 20
◦
C for 5 days. On
the 6th day, the lung lobes were reweighed. The wet lung weight was then divided by
the dry lung weight and an increased ratio was a sign of edema. Herein, a ratio of 5 or
greater was classified as edema and a ratio of less than 5 suggested no edema as previously
described [50,51].
4.10. Immunohistochemistry
Immunohistochemistry: At the time of necropsy, the whole lung tissue from some F2
mice was perfused as previously described [
3
]. Slides were deparaffinized in xylene, then
rehydrated in increasing concentrations of ethanol, followed by dH
2
O. Antigen retrieval
was performed by placing slides in citrate buffer within a warm rice cooker. After a dH2O
wash, endogenous peroxidase activity was blocked by incubating slides in 3% hydrogen
peroxide in methanol. Slides were washed in phosphate buffered saline (PBS) and blocked
for non-specific binding using powerblock reagent (BioGenex, Freemont, California; Cat#
HK085-5K). Slides were washed in PBS and incubated overnight with rabbit Toll like 4
Receptor Primary antibodies (Abcam, Boston, MA, USA; Cat# ab13556) diluted 1:200 in PBS
containing 0.5% triton x-100 (PBST) in a humidifying chamber. After a PBST wash, slides
were incubated with a premade goat anti-rabbit secondary antibody (Abcam, Waltham,
Massachusetts; Cat# ab64256) for 20 min at room temperature. After a PBST wash, slides
were incubated with streptavidin peroxidase (Thermo Scientific, Waltham, MA, USA; Cat#
TS-125-HR) for 20 min at room temperature. After a PBS wash, color was developed
using the Vector DAB peroxidase chromogen kit (Vector Laboratories, Newark, CA, USA;
Cat# ZG0306) as described by the manufacturer. After washing in dH20, slides were
counterstained with Hematoxylin (Ricca, Arlington, TX, USA; Cat# 3537-32). Next, slides
were placed under warm running water for a bluing effect, then dehydrated in increasing
concentrations of ethanol and xylene. Slides were coverslipped and viewed using an
Olympus B071 microscope. DAB was quantified using the DAB quantification Image J
plugin.
Mar. Drugs 2023,21, 161 13 of 15
4.11. Statistics
All data were analyzed using GraphPad Prism’s one-way ANOVA and the Tukey
post-hoc test. For all experiments, three to six non-littermates were used to obtain the
average for each group. The presented images are representative of each group. The data
are represented as the mean
±
standard deviation. Values of p< 0.05 were considered
significant. Significance was determined by comparing each treatment group to Control
(CT)/F2
NONE
pups. All experiments were repeated twice using different non-littermates.
In each group, approximately half of the pups were male, and the other half were female.
The majority of the pups were male in groups with uneven samples sizes.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/md21030161/s1, Figure S1: Wet lung weights; Figure S2: Dry
lung weights.
Author Contributions:
Conceptualization, J.T.R. and K.L.B.-T.; methodology, J.T.R.; validation, J.T.R.
and K.L.B.-T.; formal analysis, J.T.R.; investigation, J.T.R., V.R.S., S.A. and L.K.B.; resources, K.L.B.-T.
and K.G.O.; data curation, J.T.R. and K.L.B.-T.; writing—original draft preparation, J.T.R.; writing—
review and editing, J.T.R., K.L.B.-T., K.J.R. and P.N.N.; visualization, J.T.R. and K.J.R.; supervision,
K.L.B.-T.; project administration, K.L.B.-T.; funding acquisition, K.L.B.-T. and K.G.O. All authors have
read and agreed to the published version of the manuscript.
Funding:
This research was funded by the National Institute of Environmental Health Sciences,
Grant/Award Number TOX T32 ES007028; the National Institute of General Medical Sciences of
the National Institutes of Health, Award Numbers T32GM007628, 1SC1AI127352, 5R25GM059994,
1F31AI67579, and U54MD007586; the Veterans Administration, Grant/Award Number I01BX002583;
and the Bill & Melinda Gates Foundation INV-03510.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors would like to acknowledge Paula Austin, Lou Ann Brown, Phillip
Gaines, Kamiya Bridges and Tianbing Ding for their contributions.
Conflicts of Interest: The authors report no conflict of interest.
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