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Citation: Bhatia, S.; Bodenstein, D.;
Cheng, A.P.; Wells, P.G. Altered
Epigenetic Marks and Gene
Expression in Fetal Brain, and
Postnatal Behavioural Disorders,
Following Prenatal Exposure of Ogg1
Knockout Mice to Saline or Ethanol.
Cells 2023,12, 2308. https://
doi.org/10.3390/cells12182308
Academic Editor: Mojgan Rastegar
Received: 30 June 2023
Revised: 12 September 2023
Accepted: 13 September 2023
Published: 19 September 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/).
cells
Article
Altered Epigenetic Marks and Gene Expression in Fetal Brain,
and Postnatal Behavioural Disorders, Following Prenatal
Exposure of Ogg1 Knockout Mice to Saline or Ethanol
Shama Bhatia 1,2, David Bodenstein 3, †, Ashley P. Cheng 1,2,† and Peter G. Wells 1,2,3,*
1Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto,
Toronto, ON M5S 3M2, Canada; shama.bhatia@mail.utoronto.ca (S.B.); ashley.cheng@mail.utoronto.ca (A.P.C.)
2Centre for Pharmaceutical Oncology, Faculty of Pharmacy, University of Toronto,
Toronto, ON M5S 3M2, Canada
3Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto,
Toronto, ON M5S 1A8, Canada; d.bodenstein@mail.utoronto.ca
*Correspondence: pg.wells@utoronto.ca
†These authors contributed equally to this work.
Abstract:
Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species
(ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an
epigenetic modifier. We have previously shown that saline-exposed Ogg1
−
/
−
knockout progeny
exhibited learning and memory deficits, which were enhanced by in utero exposure to a single low
dose of ethanol (EtOH) in both Ogg1 +/+ and
−
/
−
progeny, but more so in Ogg1
−
/
−
progeny.
Herein, OGG1-deficient progeny exposed in utero to a single low dose of EtOH or its saline vehicle
exhibited OGG1- and/or EtOH-dependent alterations in global histone methylation and acetylation,
DNA methylation and gene expression (Tet1 (Tet Methylcytosine Dioxygenase 1), Nlgn3 (Neuroligin
3), Hdac2 (Histone Deacetylase 2), Reln (Reelin) and Esr1 (Estrogen Receptor 1)) in fetal brains, and
behavioural changes in open field activity, social interaction and ultrasonic vocalization, but not
prepulse inhibition. OGG1- and EtOH-dependent changes in Esr1 and Esr2 mRNA and protein levels
were sex-dependent, as was the association of Esr1 gene expression with gene activation mark histone
H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27 trimethylation
(H3K27me3) measured via ChIP-qPCR. The OGG1-dependent changes in global epigenetic marks and
gene/protein expression in fetal brains, and postnatal behavioural changes, observed in both saline-
and EtOH-exposed progeny, suggest the involvement of epigenetic mechanisms in developmental
disorders mediated by 8-oxoG and/or OGG1. Epigenetic effects of OGG1 may be involved in
ESR1-mediated gene regulation, which may be altered by physiological and EtOH-enhanced levels
of ROS formation, possibly contributing to sex-dependent developmental disorders observed in
Ogg1 knockout mice. The OGG1- and EtOH-dependent associations provide a basis for more
comprehensive mechanistic studies to determine the causal involvement of oxidative DNA damage
and epigenetic changes in ROS-mediated neurodevelopmental disorders.
Keywords:
alcohol (ethanol, EtOH); DNA damage and repair; DNA methylation; epigenetic changes;
fetal alcohol spectrum disorders (FASDs); histone methylation and acetylation; neurodevelopmental
disorders; oxoguanine glycosylase 1 (OGG1); reactive oxygen species (ROS)
1. Introduction
Oxoguanine glycosylase 1 (OGG1) repairs 8-oxoguanine (8-oxoG), the major DNA
lesion caused by reactive oxygen species (ROS) [
1
,
2
]. 8-OxoG is a mutagenic lesion po-
tentially involved in carcinogenesis, and more recently has been shown to contribute to
developmental disorders, likely via epigenetic (non-mutagenic) mechanisms [
3
–
5
]. ROS
Cells 2023,12, 2308. https://doi.org/10.3390/cells12182308 https://www.mdpi.com/journal/cells
Cells 2023,12, 2308 2 of 24
are involved in many processes that are essential for life, yet even normal levels cause mea-
surable oxidative DNA damage including 8-oxoG formation [
6
], which is further increased
by ROS-enhancing drugs like ethanol (EtOH) [
3
–
5
]. In humans, exposure of the embryo or
fetus to EtOH during pregnancy results in an array of morphological and functional fetal al-
cohol spectrum disorders (FASDs) via multiple mechanisms likely including ROS-initiated
oxidative stress and oxidative DNA damage [
3
,
4
]. Using Ogg1 knockout mice, our lab has
previously found that Ogg1
−
/
−
progeny exposed in utero to saline exhibited learning and
memory deficits, which were further enhanced by in utero exposure to a single low dose of
EtOH on gestational day (GD) 17 in both Ogg1 +/+ and
−
/
−
littermates, with Ogg1
−
/
−
progeny being most severely affected [
7
]. This OGG1-dependent susceptibility to altered
learning and memory caused by both physiological and EtOH-enhanced levels of ROS
formation suggests a role for oxidative DNA damage in the pathogenic mechanism. Aside
from the role of OGG1 in DNA repair, recent studies have revealed multiple epigenetic and
other non-mutational mechanisms by which 8-oxoG and OGG1 may regulate gene tran-
scription and signal transduction [
8
,
9
]. We recently found that untreated OGG1-deficient
progeny exhibit a sex-dependent enhancement in DNA strand breaks, decreased DNA
methylation levels and disorders of brain function compared to Ogg1 +/+ littermates [
10
].
Over 3% of Canadian children aged 5–14 are estimated to have a neurodevelopmental
disorder [
11
], while the incidence of FASDs in Canada and the USA is at least 1%, but may
be as high as 3.1–9.8% in some regions [
12
,
13
]. EtOH causes several epigenetic modifications
including changes in histone acetylation and methylation as well as DNA methylation,
which have been associated with its embryopathic mechanism [
14
–
16
]. To determine if ROS-
initiated DNA damage may cause neurodevelopmental disorders in part via epigenetic
mechanisms, we measured OGG1- and EtOH-dependent (1) changes in epigenetic histone
protein and DNA modifications, (2) altered expression of representative genes involved
in epigenetic regulation and neurodevelopment, (3) involvement of candidate locus Esr1
and (4) neurodevelopmental disorders using a battery of behavioural tests. We were
particularly interested in estrogen receptors 1 and 2 (ESR1, ESR2), as one study has reported
increased expression of ESR1 target genes in brains of untreated Ogg1
−
/
−
young mice [
17
],
and we have found sex-dependent differences in epigenetic changes and behaviour in
OGG1-deficient mice [
10
]. These two studies together suggest a potential role for OGG1-
dependent, ESR-mediated gene regulation in brain function development. Thus, in fetal
brains exposed in utero to EtOH, both gene expression and protein levels of ESR1 and
ESR2 were analyzed. In addition, the association of both gene activation mark histone
H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27
trimethylation (H3K27me3) [
18
] within various regions of the Esr1 gene were analyzed
via chromatin immunoprecipitation followed by quantitative polymerase chain reaction
analysis (ChIP-qPCR) [
19
]. In companion studies, OGG1-deficient progeny exposed in
utero to the same single low dose of EtOH were analyzed for behavioural abnormalities
using tests for open field activity, social interaction, ultrasonic vocalizations and prepulse
inhibition. The OGG1-dependent changes in global epigenetic marks and gene/protein
expression in fetal brains, and behavioural changes, observed in both saline- and EtOH-
exposed progeny suggest the involvement of epigenetic mechanisms in developmental
disorders mediated by 8-oxoG and/or OGG1.
2. Methods
2.1. Animals and Diet
All animal protocols were approved by the institutional animal care committee in
conformance with the guidelines established by the Canadian Council on Animal Care.
Ogg1 knockout mice on a 129SV/C57BL/6J background strain were originally generated
by Klungland and coworkers [
2
] and were generously provided by Dr. Tomas Lindahl
(Imperial Cancer Research Fund, UK) through Dr. Christi A. Walter at the University of
Texas Health Science Center at San Antonio. Based on single nucleotide polymorphism
analysis (SNP) (Jackson Laboratory) that identifies ~150 SNPs and covers 19 autosomes
Cells 2023,12, 2308 3 of 24
and the X chromosome, this Ogg1 mouse strain was identified as 58% C57BL/6 and 42%
129. Mice were housed in vented plastic cages (Allentown Inc., Allentown, NJ, USA)
with ground corncob bedding (Bed-O’Cobs Laboratory Animal Bedding; The Andersons
Industrial Products Group, Maumee, OH, USA). Mouse cages were maintained in a room
with controlled light (14 h light–10 h dark cycle) and climate (21–23
◦
C, with approximately
40–50% room humidity). Mice were provided with rodent chow (Harlan Labs: 2018 Harlan
Teklad, Montreal, QC, Canada) and water (U.V. sterilized reverse osmosis water acidified
to a pH of 3 using HCl to minimize pathogen dissemination) ad libitum. Breeder pairs
were set up by placing one sexually naïve Ogg1 +/
−
male with one sexually naïve Ogg1
+/
−
female per cage, which were left for their entire lifetime to generate progeny of all
three genotypes (+/+, +/
−
and
−
/
−
) within the same litter. The number of animals used
in each experiment is shown in each figure in parentheses or its legend.
2.2. Treatment Regimen
One Ogg1 heterozygous (+/
−
) knockout female mouse was mated overnight with
one Ogg1 +/
−
male, and the presence of vaginal plug in the morning was designated as
gestation day (GD) 1. GD 17 pregnant dams were given an intraperitoneal (i.p.) injection
of a single dose of 2 g/kg EtOH (25% solution (v/v) in saline) or its saline vehicle. GD
17 lies within the sensitive time window for the development of brain function in mice,
and we have shown in several mouse strains that in utero exposure on this day to a
range of ROS-enhancing drugs reproducibly causes neurodevelopmental disorders similar
to those observed in humans [
3
,
4
,
7
,
20
]. For EtOH, the 2 g/kg dose in Ogg1 females
produces blood alcohol concentrations equivalent in humans to 4–6 drinks (based on
women weighing
100–130 pounds
) (Cheng et al., unpublished data), and in Ogg1 mice
causes neurodevelopmental disorders similar to those observed in human FASDs without
maternal toxicity or fetal morphological abnormalities [
7
,
21
]. Fetuses were sacrificed 1, 6
or 24 h post-EtOH exposure (depending on the measure), and fetal brains were removed,
snap frozen and stored at
−
80
◦
C for biochemical assessments. For behavioural tests, the
dams were allowed to deliver spontaneously, and the progeny were assessed after weaning
as described below. For both types of assessment, progeny were obtained from at least
three litters to minimize potential litter effects.
2.3. DNA Extraction and Determination of Ogg1 Genotype and Sex
DNA was isolated from a 1–2 mm tail snip of either fetuses or weaned progeny by
heating the samples at 62
◦
C in 200
µ
L lysis solution (0.5% SDS, 0.1 M NaCl, 50 mM Tris
HCl, pH 8.0, and 0.5
µ
M EDTA) containing 80
µ
g of proteinase K (8
µ
L of 10 mg/mL)
for 3 h. After the entire tissue was dissolved, 37
µ
L of 8 M potassium acetate solution
was added to each sample, followed by freezing samples for >15 min (or overnight) at
−
20
◦
C. Samples were spun for 5 min at 15,700
×
gin a microcentrifuge. The supernatant
or aqueous top phase was transferred to a new tube and 500
µ
L of 100% ethanol was added
to the supernatant of each sample to precipitate DNA. The samples were spun at 15,700
×
g
for 3 min, and the precipitate was washed with 500
µ
L of 70% ethanol, followed by another
centrifugation at 15,700
×
gfor 2 min. The supernatant was discarded, and the samples
were air dried for 15–20 min. The pellets were resuspended with 100
µ
L ddH
2
O and were
incubated at 4 ◦C overnight to solubilize DNA.
DNA was genotyped for the presence or absence of Ogg1 using a PCR-based assay.
Primers (Sigma-Aldrich, St. Louis, MO, USA) used to amplify the 500-base pair (bp) band
for the Ogg1 gene were Ogg1-sense (5
0
-ACTGCATCTGCTTAATGGCC-3
0
) (forward primer)
and Ogg1-antisense (5
0
-CGAAGGTCAGCACTGAACAG-3
0
) (reverse primer). Primers
used to amplify the 300 bp band for the neo-cassette responsible for disruption of the Ogg1
gene in the Ogg1 knockout mice were neo-sense (5
0
-CTGAATGAACTGCAGGACGA-3
0
)
(forward primer) and neo-antisense (5
0
-CTCTTCGTCCAGATCATCCT-3
0
) (reverse primer).
PCR reaction conditions were 2
µ
L genomic DNA, 2
µ
L per sample of 10
×
DreamTaq Green
Buffer (Thermo Scientific, Burlington, ON, Canada, Cat. EP0713), 0.4
µ
L per sample of
Cells 2023,12, 2308 4 of 24
10 mM deoxyribonucleotides (dNTP) (Thermo Scientific, Cat. R0192), 0.3
µ
L per sample of
each of the four 20
µ
M primers, 0.2
µ
L of DreamTaq DNA Polymerase (Thermo Scientific,
Cat. EP0713) and 14.2
µ
L per sample of ddH
2
O for a final volume of 20
µ
L. Cycling
conditions were 95
◦
C for 5 min and 35 cycles of 94
◦
C for 1 min, 55
◦
C for 1.5 min, 72
◦
C
for 2 min and a final extension at 72 ◦C for 10 min.
2.4. Sex Genotyping
DNA extracted from the tail snips of fetuses (as described above) was used to assess
the sex of the fetus using a published protocol [
22
], with ~280 bp product amplified from
male DNA and ~685 bp product (along with ~480 and 660 bp products) amplified from
female DNA [
22
]. The PCR products were separated on a 1.5% (w/v) agarose gel in 1
×
TAE
buffer (40 mM Tris, pH 8.3–8.5, 40 mM glacial acetic acid, 1 mM EDTA, BioShop, Burlington,
ON, Canada) and 8
µ
g ethidium bromide. The agarose gel was run at 130 V for 40 min and
then visualized and photographed under an ultraviolet light.
2.5. Histone Modifications
Histones were isolated from fetal brain samples using a protocol adapted from Kim
and Shukla 2006 [
23
]. Briefly, fetal brains were homogenized and nuclei were isolated by
sucrose density gradient centrifugation. Histone proteins were then acid-extracted from the
nuclei, and about 5
µ
g of protein was loaded onto an SDS-PAGE gel, which was transferred
to a nitrocellulose membrane for the assessment of histone acetylation and methylation
changes by western blot. Activation modifications include acetylation of histone H3 at
lysine 9 (H3K9ac, Cat. 06-942, Millipore Sigma, Burlington, MA, USA) and trimethylation
of histone H3 at lysine 4 (H3K4me3, Cat. ab8580, Abcam, Cambridge, UK), and repressive
modifications include trimethylation of histone H3 at lysine 9 and lysine 27 (H3K9me3, Cat.
07-442, and H3K27me3, Cat. 07-449, Millipore Sigma, respectively).
2.6. 5-Methylcytosine (5-mC) and 5-Hydroxymethylcytosine (5-hmC) Levels in GD 17 Fetal Brains
DNA from the fetal brains was extracted using a DNA extraction kit (FitAmp General
Tissue Section DNA Isolation Kit, Cat. P-1003, EpiGentek, Farmingdale, NY, USA), with
minor modifications. Briefly, brains were homogenized using 2 mL of lysing buffer con-
taining 320 mM sucrose, 5 mM MgCl
2
, 10 mM Tris, 0.1 mM deferoxamine and 1% Triton
X-100, pH 7.5. The entire homogenate was centrifuged at 1000
×
gfor 15 min at 4
◦
C, and
DNA was extracted from the nuclear pellets following the procedure in the kit. A 100-ng
aliquot of DNA was used to measure the levels of 5-mC using an ELISA-based kit (Cat.
P-1034, EpiGentek), while 150 ng of DNA was used to measure the 5-hmC levels using an
ELISA-based kit (Cat. P-1036, EpiGentek). 5-mC and 5-hmC levels were measured in the
same samples. The levels were quantified as suggested in the kit.
2.7. Gene Expression via Reverse Transcriptase Followed by Quantitative Polymerase Chain
Reaction (RT-qPCR)
A published protocol was adapted [
24
] with minor modifications. Fetal brains were
homogenized using 1 mL of TRIzol
™
LS Reagent (Cat. 10296-010, Invitrogen, Waltham,
MA, USA) and incubated at room temperature for 5 min, followed by addition of 200
µ
L of
chloroform and incubation at room temperature for 15 min. Samples were then centrifuged
at 12,000
×
gfor 15 min at 4
◦
C. In a new tube, isopropyl alcohol was added in a 1:1 ratio of
isopropyl alcohol:RNA. Samples were incubated at room temperature for 10 min followed
by centrifugation at 12,000
×
gfor 10 min at 4
◦
C. The pellet was washed with 1 mL of
75% ethanol (in diethylpyrocarbonate (DEPC) water) followed by centrifugation at
7600×g
for 5 min at 4
◦
C. The pellet was dissolved in 70
µ
L of DEPC water and its concentration
was measured to be between 500 and 1000 ng/µL. The purity of RNA was determined by
ensuring that the A260/A280 ratio was ~1.8–2.0 for pure RNA and the A260/A230 ratio
was ~2.0. Before conversion to cDNA, samples containing 2 µg of RNA were treated with
DNAse (4.2 mM MgCl
2
, 1.28 U of DNAse I, total volume 20
µ
L) at 37
◦
C for 30 min, followed
by inactivation of enzyme at 75
◦
C for 10 min. RNA was then converted to cDNA using
Cells 2023,12, 2308 5 of 24
a High-Capacity cDNA Reverse Transcription Kit (Cat. 4368814, ThermoFisher Scientific,
Waltham, MA, USA) and a 40
µ
L reaction was run under the following conditions: 25
◦
C
for 10 min, 37
◦
C for 120 min and 85
◦
C for 5 min. Samples were then diluted to 10 ng/
µ
L.
About 12.5 ng of cDNA was used to run the RT-qPCR reaction. See Supplementary Figure
S8 for primer information.
2.8. Protein Levels
Fetal brains were homogenized using radioimmunoprecipitation assay (RIPA) buffer
(150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH
8.0) containing protease inhibitor cocktail (Roche, Basel, Switzerland, Cat. No: 11836170001).
The protein concentrations of the supernatants were determined with bicinchoninic acid
(BCA) assay. About 75
µ
g of protein homogenate was loaded onto a 12.5% SDS PAGE
gel. The gel was run at constant 150 V for 1 h in electrophoresis buffer (25 mM Tris base,
192 mM glycine and 0.1% sodium dodecyl sulfate, pH 8.3). The protein was transferred
onto a nitrocellulose membrane using 1
×
Transfer Buffer (25 mM Tris base, 192 mM glycine
and 20% methanol) at 100 V for 1 h 30 min. Membranes were then washed three times in
Tris-buffered saline, 0.1% Tween-20 (TBST), followed by blocking using 5% non-fat milk
blocking solution in TBST for 1 h at room temperature. Membranes were then washed again
three times with TBST. The membranes were cut into three pieces to be able to incubate the
blots with estrogen receptor 1 (1:2000, Millipore Sigma, Cat. 06-935) and estrogen receptor
2 (1:2000, Abcam, Cat. ab3576) in 5% TBST-milk and Gapdh (1:35,000, Millipore SIGMA,
Cat. G9295) in 3% bovine serum albumin in TBST overnight in a cold room. The next
day, membranes were incubated with secondary antibody for both ESR1 and ESR2 (Goat
anti-rabbit horseradish peroxidase, 1:30,000, Millipore Sigma, Cat. A0505) in TBST for 1 h
at room temperature. All membranes (including Gapdh) were then washed three times
for 5 min each with TBST, followed by a 10-min single wash with TBST. Membranes were
then incubated with enhanced chemiluminescence stain (Pierce ECL Plus Western Blotting
Substrate, Thermo Scientific, Burlington, ON, Canada, Cat. 32132) for 5 min and analyzed
with a FluorChem8800 imager.
2.9. Chromatin Immunoprecipitation and qPCR (ChIP-qPCR)
Chromatin was immunoprecipitated as previously described [
19
]. Briefly, fetal brains
were homogenized in 1% formaldehyde in phosphate buffered saline (PBS) for 10 min.
Cross-linking was quenched by adding 125 mM glycine for 5 min at room temperature,
and the nuclear pellet was washed with PBS, pelleted and resuspended in 800
µ
L of
lysis buffer (20 mM Tris.HCl, pH 8.0, 1% Triton X-100, 150 mM NaCl, 0.1% w/vSDS,
2 mM EDTA) containing protease inhibitors (Roche, Cat. 11836170001) and sonicated at
high intensity in 30 s on, 30 s off cycles for 15 min twice using a Bioruptor (model UCD-
200, Diagenode, Denville, NJ, USA). An aliquot of sonicated chromatin was set aside to
represent input fraction and to verify for sonication efficiency and stored at
−
80
◦
C. The
sonicated chromatin was incubated with 30
µ
L Protein A agarose/salmon sperm DNA
slurry (50% slurry, Cat. 16-157, Sigma Millipore), under gentle rotation on a tube rotator for
1 h at 4
◦
C, after which the sample was centrifuged to separate the beads. The supernatant
was transferred to a new low DNA-binding tube (150
µ
L/immunoprecipitation). 4
µ
g
antibody and 20
µ
L BSA was added to each immunoprecipitation tube (IgG, H3K9ac,
H3K27me3 and H3) and the samples were incubated overnight at 4
◦
C. The next morning,
30
µ
L Protein A agarose/salmon sperm DNA slurry was added and samples were incubated
at 4
◦
C for 2 h. The pellets were successively washed three times for 10 min each in 1 mL
of Tris.Saline.EDTA (TSE I) buffer (20 mM Tris.HCl, pH 8.0, 1% Triton X-100, 150 mM
NaCl, 0.1% w/vSDS, 2 mM EDTA.Na
2
), twice in 1 mL of TSE II (20 mM Tris.HCl, pH
8.0, 1% Triton X-100, 500 mM NaCl, 0.1% w/vSDS, 2 mM EDTA.Na
2
), twice in 1 mL of
LiCL (20 mM Tris.HCl, pH 8.0, 1 mM EDTA.Na
2
, 250 mM LiCl, 1% v/vNP-40, 1% w/v
Na-deoxycholate) and three times in 1 mL of Tris.EDTA (TE) buffer (10 mM Tris.HCl pH
8.0, 1 mM EDTA.Na
2
). Protein–DNA complexes were eluted in 110
µ
L elution buffer
Cells 2023,12, 2308 6 of 24
(1% SDS/TE) for 30 min, and the cross-links were reversed by overnight incubation at
65
◦
C (5
µ
L of 5 M NaCl and 1
µ
L of RNase A). DNA was purified using the Biobasic PCR
Purification kit (Cat. BS664-250) and eluted in 40
µ
L TE buffer. CHIP DNA (5
µ
L) was
amplified by PCR using primers listed in Supplementary Figure S9.
2.10. Behavioural Tests
After weaning at 3 weeks of age, pups were handled for three consecutive days to ac-
custom them to human exposure and reduce stress and anxiety. The following behavioural
studies were completed in a single mouse cohort at the specified times: interaction-induced
ultrasonic vocalization (USV) (3–4 weeks), open field activity (6 weeks), social interaction
(7–8 weeks old), female-induced USV (4–5 months) and prepulse inhibition (~5 months)
(Figure 1). At least three litters were tested for each treatment group, and the number of
mice tested is indicated in parentheses.
Cells 2023, 12, x FOR PEER REVIEW 6 of 25
mM NaCl, 0.1% w/v SDS, 2 mM EDTA.Na
2
), twice in 1 mL of TSE II (20 mM Tris.HCl, pH
8.0, 1% Triton X-100, 500 mM NaCl, 0.1% w/v SDS, 2 mM EDTA.Na
2
), twice in 1 mL of
LiCL (20 mM Tris.HCl, pH 8.0, 1 mM EDTA.Na
2
, 250 mM LiCl, 1% v/v NP-40, 1% w/v Na-
deoxycholate) and three times in 1 mL of Tris.EDTA (TE) buffer (10 mM Tris.HCl pH 8.0,
1 mM EDTA.Na
2
). Protein–DNA complexes were eluted in 110 µL elution buffer (1%
SDS/TE) for 30 min, and the cross-links were reversed by overnight incubation at 65 °C (5
µL of 5 M NaCl and 1 µL of RNase A). DNA was purified using the Biobasic PCR Purifi-
cation kit (Cat. BS664-250) and eluted in 40 µL TE buffer. CHIP DNA (5 µL) was amplified
by PCR using primers listed in Supplementary Figure S9.
2.10. Behavioural Tests
After weaning at 3 weeks of age, pups were handled for three consecutive days to
accustom them to human exposure and reduce stress and anxiety. The following behav-
ioural studies were completed in a single mouse cohort at the specified times: interaction-
induced ultrasonic vocalization (USV) (3–4 weeks), open field activity (6 weeks), social
interaction (7–8 weeks old), female-induced USV (4–5 months) and prepulse inhibition (~5
months) (Figure 1). At least three liers were tested for each treatment group, and the
number of mice tested is indicated in parentheses.
Figure 1. Dosing and behavioural testing timeline. Ogg1 heterozygous mice were mated, and on
gestational day (GD) 17, the dams received a single dose of EtOH (2 g/kg i.p.) or its saline vehicle.
The pups were delivered spontaneously, weaned after 3 weeks and subjected to a series of behav-
ioural tests including interaction-induced ultrasonic vocalization (USV) at 3–4 weeks of age, open
field activity at 6 weeks of age, social interaction with a novel mouse at 7–8 weeks of age, female-
induced USV at 4–5 months of age and prepulse inhibition at approximately 5 months of age.
2.11. Interaction-Induced Ultrasonic Vocalizations (USV)
Interaction-induced USV was performed to assess social interactive behaviour at 3–4
weeks of age using lighting conditions of ~15–20 lux to minimize anxiety in the mice [25–
27]. The test was performed by placing two mice in an empty mouse cage for 5 min with
a microphone placed directly above the centre. The number of USVs emied were rec-
orded and manually counted, with USVs defined as vocalizations between 50 and 100
kHz. Mice were matched by age, sex, Ogg1 genotype and treatment group.
Figure 1. Dosing and behavioural testing timeline
.Ogg1 heterozygous mice were mated, and on
gestational day (
GD
) 17, the dams received a single dose of EtOH (2 g/kg i.p.) or its saline vehicle.
The pups were delivered spontaneously, weaned after 3 weeks and subjected to a series of behavioural
tests including interaction-induced ultrasonic vocalization (USV) at
3–4 weeks
of age, open field
activity at 6 weeks of age, social interaction with a novel mouse at 7–8 weeks of age, female-induced
USV at 4–5 months of age and prepulse inhibition at approximately 5 months of age.
2.11. Interaction-Induced Ultrasonic Vocalizations (USV)
Interaction-induced USV was performed to assess social interactive behaviour at
3–4 weeks
of age using lighting conditions of ~15–20 lux to minimize anxiety in the
mice [25–27]
. The test was performed by placing two mice in an empty mouse cage for
5 min with a microphone placed directly above the centre. The number of USVs emitted
were recorded and manually counted, with USVs defined as vocalizations between 50 and
100 kHz. Mice were matched by age, sex, Ogg1 genotype and treatment group.
2.12. Open Field Activity
Locomotor activity and anxiety were measured using an open field activity box at
~130 lux for 1 h at 6 weeks of age [
20
,
28
–
30
]. During the test, the mouse was placed in a
16-inch by 16-inch arena with sensory infrared beams that span the whole area. Breaks
in these beams detect movement. The mouse was allowed to explore the arena for 1 h.
Various activity parameters were recorded and analyzed.
Cells 2023,12, 2308 7 of 24
2.13. Social Interaction Test
Alterations in social behaviour were assessed at 7–8 weeks of age at ~6–8 lux [
25
,
26
].
The test involved placing a mouse in a white walled arena of 62
×
40.5
×
23 cm with a
novel social mouse placed inside one of the two inverted wire cups (defined as the social
zone), with the other cup placed empty (defined as the nonsocial zone). The test mouse
was monitored for 10 min using video tracking software. An Ogg1 wild-type untreated
mouse was used as a “novel mouse”, was age and sex-matched and was used up to about
four times throughout the experiment. The novel mouse was placed first in the arena and
was allowed to habituate for 2 min prior to the addition of the test mouse. A 20
×
20 cm
zone was defined around each of the inverted wire cup leaving a 5 cm gap in which the
whole body of the test mouse must enter to interact with the novel mouse.
2.14. Female-Induced Ultrasonic Vocalization (USV)
This test was performed to analyze female-induced USV in male mice [
25
,
26
]. The
test was performed at ~15–20 lux to minimize anxiety in mice. The test was performed
at
4–5 months
of age by placing one male and one female mouse together for 5 min in an
empty mouse cage with a microphone placed directly above the centre. Mice were matched
by age, Ogg1 genotype and treatment group.
2.15. Prepulse Inhibition
A prepulse inhibition test was performed to measure abnormalities in sensorimotor
gating or startle response in OGG1 mice (~5 months old) [
31
]. The prepulse inhibition test
was conducted using SR-LAB equipment and SR-LAB software from San Diego Instruments.
Accelerometers were calibrated to 700
±
5 mV and output voltages were amplified and
analyzed for voltage changes using SR Analysis (San Diego Instruments, San Diego, CA,
USA). Each mouse was tested for 30 min. The background white noise was maintained
at 65 dB, and each mouse was subjected to 80 randomized trials of pulse alone (100 dB
above background), prepulse alone (4, 8 or 16 dB above background), prepulse plus pulse
and no pulse, with five pulse-alone trials performed at the start and end of the 80 trials.
Time intervals between trials were randomized from 5 s to 20 s, with a delay of 100 ms
between the prepulse and the pulse. Prepulse inhibition was thus measured as a decrease
in the amplitude of startle response to a 100 dB (above background) acoustic startle pulse
following each prepulse (4, 8 and 16dB above background).
3. Results
3.1. Altered Histone and DNA Modifications in Fetal Brains of Ogg1
−
/
−
Mice Exposed In Utero
to EtOH
Global histone protein activation marks (H3K9ac and H3K4me3) and repressive marks
(H3K9me3 and H3K27me3) were assessed via western blot in fetal brains exposed in
utero to saline or EtOH (Figure 2A, Supplementary Figure S1). EtOH exposure enhanced
histone acetylation (H3K9ac) levels at 6 h in Ogg1 +/+(p< 0.01) but not −/−fetal brains,
which had returned to baseline at 24 h. However, at 24 h, H3K9ac levels were elevated in
saline-exposed
−
/
−
vs. +/+ (p< 0.05) but not EtOH-exposed Ogg1
−
/
−
vs. +/+ brains.
H3K9me3 levels were increased by EtOH exposure in
−
/
−
but not +/+ brains at both 6
and 24 h (p< 0.01), whereas H3K27me3 levels were increased in only saline-exposed
−
/
−
vs. +/+ brains at only 24 h (p< 0.05).
Cells 2023,12, 2308 8 of 24
Cells 2023, 12, x FOR PEER REVIEW 8 of 25
For DNA modifications (Figure 2B), at 24 h, an OGG1-dependent increase in 5-mC
levels was observed only in saline-exposed −/− vs. +/+ brains (p < 0.05), with no effect of
EtOH observed. No differences were observed in 5-mC and 5-hmC levels at 1 or 6 h, and
there were no OGG1- or EtOH-dependent changes in 5-hmC levels. The significance for
both histone and DNA modifications is based on two-way ANOVA and a post hoc Tukey’s
test.
Figure 2. Altered histone and DNA modifications in fetal brains of Ogg1 −/− mice exposed in utero
to EtOH. GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle
were extracted 1, 6 and 24 h later from Ogg1 +/+ and −/− littermates and were assessed for histone and
DNA modifications. Fetal brains from at least three litters were used to minimize potential litter effects,
and the number of fetal brains for each group is shown in parentheses. (A). The following histone
modifications were analyzed: H3K9ac (activation mark), H3K9me3 and H3K27me3 (repressive
marks). See Supplementary Figure S1 for 1 h results for H3K9ac and H3K9me3 (no differences ob-
served) and for 6 and 24 h results for H3K4me3 (activation mark, no differences observed). (B). Increase
in 5-methylcytosine (5-mC) levels in saline-exposed Ogg1 −/− vs. +/+ fetal brains at 24 h, with a similar
trend in EtOH-exposed fetal brains. The same DNA sample was used for both 5-mC and 5-hmC meas-
urements. The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s
test. Abbreviations: 5-mC: 5-methylcytosine; 5-hmC: 5-hydroxymethylcytosine; H3K4me3: histone 3
lysine 9 trimethylation; H3K9ac: histone 3 lysine 9 acetylation; H3K9me3: histone 3 lysine 9 trimethyl-
ation; H3K27me3: histone 3 lysine 27 trimethylation.
Figure 2. Altered histone and DNA modifications in fetal brains of Ogg1 −/−mice exposed in
utero to EtOH.
GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its
saline vehicle were extracted 1, 6 and 24 h later from Ogg1 +/+ and
−
/
−
littermates and were
assessed for histone and DNA modifications. Fetal brains from at least three litters were used
to minimize potential litter effects, and the number of fetal brains for each group is shown in
parentheses. (
A
). The following histone modifications were analyzed: H3K9ac (activation mark),
H3K9me3 and H3K27me3 (repressive marks). See Supplementary Figure S1 for 1 h results for H3K9ac
and H3K9me3 (no differences observed) and for 6 and 24 h results for H3K4me3 (activation mark,
no differences observed). (
B
). Increase in 5-methylcytosine (5-mC) levels in saline-exposed Ogg1
−
/
−
vs. +/+ fetal brains at 24 h, with a similar trend in EtOH-exposed fetal brains. The same
DNA sample was used for both 5-mC and 5-hmC measurements. The significance of differences was
determined by two-way ANOVA and a post hoc Tukey’s test. Abbreviations: 5-mC: 5-methylcytosine;
5-hmC: 5-hydroxymethylcytosine; H3K4me3: histone 3 lysine 9 trimethylation; H3K9ac: histone
3 lysine 9 acetylation; H3K9me3: histone 3 lysine 9 trimethylation; H3K27me3: histone 3 lysine
27 trimethylation.
Cells 2023,12, 2308 9 of 24
For DNA modifications (Figure 2B), at 24 h, an OGG1-dependent increase in 5-mC
levels was observed only in saline-exposed
−
/
−
vs. +/+ brains (p< 0.05), with no effect
of EtOH observed. No differences were observed in 5-mC and 5-hmC levels at 1 or 6 h,
and there were no OGG1- or EtOH-dependent changes in 5-hmC levels. The significance
for both histone and DNA modifications is based on two-way ANOVA and a post hoc
Tukey’s test.
3.2. Altered Gene Expression in OGG1 Fetal Brains Exposed In Utero to EtOH
Expression levels of representative genes involved in learning and memory were mea-
sured via RT-qPCR. In Ogg1
−
/
−
fetal brains, EtOH increased Tet1 expression (24 h, p< 0.05)
and decreased Nlgn3 expression (6 h, p< 0.05) compared to saline (Figure 3). In contrast, in
Ogg1 +/+ fetal brains, EtOH increased Hdac2 and Reln expression (p< 0.05) compared to
saline, neither of which were altered in Ogg1
−
/
−
progeny. See
Supplementary Figure S2
for a list of other genes with no OGG1- or EtOH-dependent differences. The significance is
based on two-way ANOVA and a post hoc Tukey’s test.
Cells 2023, 12, x FOR PEER REVIEW 9 of 25
3.2. Altered Gene Expression in OGG1 Fetal Brains Exposed In Utero to EtOH
Expression levels of representative genes involved in learning and memory were
measured via RT-qPCR. In Ogg1 −/− fetal brains, EtOH increased Tet 1 expression (24 h, p
< 0.05) and decreased Nlgn3 expression (6 h, p < 0.05) compared to saline (Figure 3). In
contrast, in Ogg1 +/+ fetal brains, EtOH increased Hdac2 and Reln expression (p < 0.05)
compared to saline, neither of which were altered in Ogg1 −/− progeny. See Supplemen-
tary Figure S2 for a list of other genes with no OGG1- or EtOH-dependent differences. The
significance is based on two-way ANOVA and a post hoc Tukey’s test.
Figure 3. Altered gene expression in Ogg1 −/− fetal brains exposed in utero to EtOH. GD 17 fetal
brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle were extracted 6
and 24 h later from Ogg1 +/+ and −/− liermates. Fetal brains from at least three liers were used to
minimize potential lier effects, and the number of fetal brains for each group is shown in paren-
theses. Fetal brains were homogenized, RNA was extracted using the TRIzol method and mRNA
expression levels were measured via RT-qPCR. Gapdh was used as a control. Expression levels of
various learning and memory candidate genes were measured (see Supplementary Figure S2).
Above are the results for OGG1- and EtOH-dependent differences in mRNA levels in fetal brains.
The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test.
Abbreviations: Hdac2: histone deacetylase 2; Nlgn3: neuroligin 3; Reln: Reelin; Tet 1: Ten-eleven trans-
location methylcytosine dioxygenase 1.
3.3. OGG1-and Sex-Dependent Differences in Esr1 and Esr2 mRNA Levels and Their Ratios in
Fetal Brains Exposed In Utero to EtOH
Since one study has reported increased expression of ESR1-regulated genes (i.e.,
ESR1 target genes) in brains of untreated Ogg1 −/− young mice [17], we analyzed the Esr1
gene expression separated by sex (Figure 4). In the combined sex data, at 6 h, no differ-
ences were observed in expression levels for Esr1, Esr2 or the Esr1/2 ratio, whereas at 24
h, EtOH-exposed Ogg1 −/− fetal brains showed a small but significant increase in Esr1 ex-
pression (p < 0.05) in comparison to saline-exposed Ogg1 −/− fetal brains, and among
EtOH-exposed progeny, Esr1 expression was marginally increased in Ogg1 −/− fetal brains
(p = 0.06) compared to Ogg1 +/+ liermates. When the data were separated by sex, Esr1
expression at 6 h was decreased in EtOH- vs. saline-exposed Ogg1 +/+ male fetal brains (p
< 0.05), but not in Ogg1 −/− male brains nor in female fetal brains. At 24 h, although not
Figure 3. Altered gene expression in Ogg1 −/−fetal brains exposed in utero to EtOH.
GD 17 fetal
brains exposed in utero to a single dose of EtOH (2 g/kg i.p.) or its saline vehicle were extracted
6 and 24 h later from Ogg1 +/+ and
−
/
−
littermates. Fetal brains from at least three litters were
used to minimize potential litter effects, and the number of fetal brains for each group is shown
in parentheses. Fetal brains were homogenized, RNA was extracted using the TRIzol method and
mRNA expression levels were measured via RT-qPCR. Gapdh was used as a control. Expression levels
of various learning and memory candidate genes were measured (see Supplementary Figure S2).
Above are the results for OGG1- and EtOH-dependent differences in mRNA levels in fetal brains.
The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test.
Abbreviations: Hdac2: histone deacetylase 2; Nlgn3: neuroligin 3; Reln: Reelin; Tet1: Ten-eleven
translocation methylcytosine dioxygenase 1.
3.3. OGG1-and Sex-Dependent Differences in Esr1 and Esr2 mRNA Levels and Their Ratios in
Fetal Brains Exposed In Utero to EtOH
Since one study has reported increased expression of ESR1-regulated genes (i.e., ESR1
target genes) in brains of untreated Ogg1
−
/
−
young mice [
17
], we analyzed the Esr1 gene
expression separated by sex (Figure 4). In the combined sex data, at 6 h, no differences
Cells 2023,12, 2308 10 of 24
were observed in expression levels for Esr1,Esr2 or the Esr1/2ratio, whereas at 24 h,
EtOH-exposed Ogg1
−
/
−
fetal brains showed a small but significant increase in Esr1
expression (p< 0.05) in comparison to saline-exposed Ogg1
−
/
−
fetal brains, and among
EtOH-exposed progeny, Esr1 expression was marginally increased in Ogg1
−
/
−
fetal brains
(p= 0.06) compared to Ogg1 +/+ littermates. When the data were separated by sex, Esr1
expression at 6 h was decreased in EtOH- vs. saline-exposed Ogg1 +/+ male fetal brains
(p< 0.05)
, but not in Ogg1
−
/
−
male brains nor in female fetal brains. At 24 h, although
not significant, a similar trend for decreased mRNA was seen in male fetal brains. Esr2
gene expression at 6 h was decreased in EtOH vs. saline-exposed Ogg1
−
/
−
(p< 0.05) but
not in +/+ male brains nor in female fetal brains. No significant differences were observed
in Esr2 levels at 24 h. When analyzed for Esr1/2ratio, there was a decrease in its ratio at
6 h in EtOH-exposed Ogg1 +/+ male fetal brains but not
−
/
−
fetal brains (p< 0.05), with
no differences observed in the females. The significance is based on two-way ANOVA and
a post hoc Tukey’s test.
Cells 2023, 12, x FOR PEER REVIEW 10 of 25
significant, a similar trend for decreased mRNA was seen in male fetal brains. Esr2 gene
expression at 6 h was decreased in EtOH vs. saline-exposed Ogg1 −/− (p < 0.05) but not in
+/+ male brains nor in female fetal brains. No significant differences were observed in Esr2
levels at 24 h. When analyzed for Esr1/2 ratio, there was a decrease in its ratio at 6 h in
EtOH-exposed Ogg1 +/+ male fetal brains but not −/− fetal brains (p < 0.05), with no differ-
ences observed in the females. The significance is based on two-way ANOVA and a post
hoc Tukey’s test.
Figure 4. OGG1- and sex-dependent differences in Esr1 and Esr2 mRNA levels and their ratios in
fetal brains exposed in utero to EtOH. GD 17 fetal brains exposed in utero to a single dose of EtOH
(2 g/kg i.p.) or its saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and −/− liermates.
Fetal brains from at least three liers were used to minimize potential lier effects, and the number
of fetal brains for each group is shown in parentheses. Fetal brains were homogenized, RNA was
extracted using the TRIzol method and mRNA expression levels were measured via RT-qPCR.
Gapdh was used as a control. Above are the results for Esr1 and Esr2 mRNA levels as well as their
ratios in fetal brains. The significance of differences was determined by two-way ANOVA and a post
hoc Tukey’s test. Abbreviations: Esr1: estrogen receptor 1; Esr2: estrogen receptor 2.
3.4. OGG1-and Sex-Dependent Differences in ESR1 and ESR2 Protein Levels and Their Ratios
in Fetal Brains Exposed In Utero to EtOH
Protein levels were measured at 24 h post maternal treatment (Figure 5). No differ-
ences were seen in ESR1 protein levels; however, ESR2 protein levels were decreased in
EtOH- vs. saline-exposed Ogg1 −/− male fetal brains (p < 0.05), but not in Ogg1 +/+ male
brains nor in female fetal brains. No differences in ESR1/ESR2 protein level ratios were
seen. The significance is based on two-way ANOVA and a post hoc Tukey’s test.
Figure 4. OGG1- and sex-dependent differences in Esr1 and Esr2 mRNA levels and their ratios in
fetal brains exposed in utero to EtOH
. GD 17 fetal brains exposed in utero to a single dose of EtOH
(2 g/kg i.p.) or its saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and
−
/
−
littermates.
Fetal brains from at least three litters were used to minimize potential litter effects, and the number
of fetal brains for each group is shown in parentheses. Fetal brains were homogenized, RNA was
extracted using the TRIzol method and mRNA expression levels were measured via RT-qPCR. Gapdh
was used as a control. Above are the results for Esr1 and Esr2 mRNA levels as well as their ratios
in fetal brains. The significance of differences was determined by two-way ANOVA and a post hoc
Tukey’s test. Abbreviations: Esr1: estrogen receptor 1; Esr2: estrogen receptor 2.
Cells 2023,12, 2308 11 of 24
3.4. OGG1-and Sex-Dependent Differences in ESR1 and ESR2 Protein Levels and Their Ratios in
Fetal Brains Exposed In Utero to EtOH
Protein levels were measured at 24 h post maternal treatment (Figure 5). No differences
were seen in ESR1 protein levels; however, ESR2 protein levels were decreased in EtOH-
vs. saline-exposed Ogg1
−
/
−
male fetal brains (p< 0.05), but not in Ogg1 +/+ male brains
nor in female fetal brains. No differences in ESR1/ESR2 protein level ratios were seen. The
significance is based on two-way ANOVA and a post hoc Tukey’s test.
Cells 2023, 12, x FOR PEER REVIEW 11 of 25
Figure 5. OGG1- and sex-dependent differences in ESR1 and ESR2 protein levels and their ratios
in fetal brains exposed in utero to EtOH. GD 17 fetal brains exposed in utero to a single dose of
EtOH (2 g/kg i.p.) or its saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and −/− lier-
mates. Fetal brains from at least three liers were used to minimize potential lier effects, and the
number of fetal brains for each group is shown in parentheses. Fetal brains were homogenized, and
protein levels were quantified via western blot. GAPDH was used as a loading control. Above are
the results for ESR1 and ESR2 proteins levels as well as their ratios in fetal brains. The significance
of differences for each sex was determined by two-way ANOVA and a post hoc Tukey’s test. Abbre-
viations: ESR1: estrogen receptor 1; ESR2: estrogen receptor 2.
3.5. EtOH-Mediated Increased Association of H3K27me3 with Esr1 Gene in Ogg1 +/+ but Not
−/− Fetal Brains
The association of histone modifications H3K27me3 (repression mark) and H3K4me3
(activation mark) within various regions of the Esr1 gene were quantified via chromatin im-
munoprecipitation followed by quantitative PCR (Figure 6A). We chose these marks based
on the Ensembl database, which showed a high association of activation mark H4K3me3
around the Esr1 promoter, and to complement this activation mark, we chose to also meas-
ure the repression mark H3K27me3. At 6 h, there was an increased association of H3K27me3
in EtOH- vs. saline-exposed Ogg1 +/+ but not −/− progeny at various regions of the Esr1 gene
(relative to transcription start site (+ 1 bp): −34 to 31 bp (p < 0.01), + 538 bp (p < 0.01), + 2.3
kbp (p < 0.01) and + 100.3 kbp (p < 0.05)) (Figure 6B). No OGG1- or EtOH-dependent
Figure 5. OGG1- and sex-dependent differences in ESR1 and ESR2 protein levels and their ratios
in fetal brains exposed in utero to EtOH
. GD 17 fetal brains exposed in utero to a single dose of EtOH
(2 g/kg i.p.) or its saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and
−
/
−
littermates.
Fetal brains from at least three litters were used to minimize potential litter effects, and the number
of fetal brains for each group is shown in parentheses. Fetal brains were homogenized, and protein
levels were quantified via western blot. GAPDH was used as a loading control. Above are the results
for ESR1 and ESR2 proteins levels as well as their ratios in fetal brains. The significance of differences
for each sex was determined by two-way ANOVA and a post hoc Tukey’s test. Abbreviations: ESR1:
estrogen receptor 1; ESR2: estrogen receptor 2.
3.5. EtOH-Mediated Increased Association of H3K27me3 with Esr1 Gene in Ogg1 +/+ but
Not −/−Fetal Brains
The association of histone modifications H3K27me3 (repression mark) and H3K4me3
(activation mark) within various regions of the Esr1 gene were quantified via chromatin
immunoprecipitation followed by quantitative PCR (Figure 6A). We chose these marks
based on the Ensembl database, which showed a high association of activation mark
H4K3me3 around the Esr1 promoter, and to complement this activation mark, we chose to
also measure the repression mark H3K27me3. At 6 h, there was an increased association of
Cells 2023,12, 2308 12 of 24
H3K27me3 in EtOH- vs. saline-exposed Ogg1 +/+ but not
−
/
−
progeny at various regions
of the Esr1 gene (relative to transcription start site (+ 1 bp):
−
34 to 31 bp (p< 0.01), + 538 bp
(p< 0.01), + 2.3 kbp (p< 0.01) and + 100.3 kbp (p< 0.05)) (Figure 6B). No OGG1- or EtOH-
dependent differences in H3K4me3 levels within the Esr1 gene were observed (Figure 6c).
See Supplementary Figure S3 for controls and Figures S4 and S5 for sex-separated data
with low n. The significance is based on two-way ANOVA and a post hoc Tukey’s test.
Cells 2023, 12, x FOR PEER REVIEW 12 of 25
differences in H3K4me3 levels within the Esr1 gene were observed (Figure 6c). See Supple-
mentary Figure S3 for controls and Figures S4 and S5 for sex-separated data with low n. The
significance is based on two-way ANOVA and a post hoc Tukey’s test.
Figure 6. EtOH-mediated increased association of H3K27me3 with Esr1 gene expression in Ogg1
+/+ but not −/− fetal brains. GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg i.p.)
or its saline vehicle were extracted 6 h later from Ogg1 +/+ and −/− liermates. Fetal brains from at
least three liers were used to minimize potential lier effects, and the number of fetal brains for
each group is shown in parentheses. (A). ChIP was performed using fetal brains, and extracted DNA
was used to perform quantitative PCR using five different sets of primers directed against various
regions of the Esr1 gene. The Esr1 gene has two promoter regions, marked with arrows, which can
generate four transcript variants via gene splicing. The locations of the exons and introns on chro-
mosome 10 are marked. The primer locations are relative to the first transcription start site (TSS).
Regions 1–5 were chosen based on the Ensembl database reporting their association with activation
or repressive epigenetic marks. Each of the regions was amplified after chromatin was immunopre-
cipitated using antibodies against H3K4me3 (active promoter) and H3K27me3 (inactive promoter)
and histone H3 (control). This figure is not drawn to scale. (B). The association of the H3K27me3:H3
ratio was normalized to 1% input in various regions of Esr1 of fetal brains exposed in utero to saline
or EtOH. (C). The association of the H3K4me3:H3 ratio was normalized to 1% input in various re-
gions of Esr1 of fetal brains exposed in utero to saline or EtOH. The significance of differences was
Figure 6. EtOH-mediated increased association of H3K27me3 with Esr1 gene expression in Ogg1
+/+ but not −/−fetal brains.
GD 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg
i.p.) or its saline vehicle were extracted 6 h later from Ogg1 +/+ and
−
/
−
littermates. Fetal brains
from at least three litters were used to minimize potential litter effects, and the number of fetal brains
for each group is shown in parentheses. (
A
). ChIP was performed using fetal brains, and extracted
DNA was used to perform quantitative PCR using five different sets of primers directed against
various regions of the Esr1 gene. The Esr1 gene has two promoter regions, marked with arrows,
which can generate four transcript variants via gene splicing. The locations of the exons and introns
on chromosome 10 are marked. The primer locations are relative to the first transcription start site
(
TSS
). Regions 1–5 were chosen based on the Ensembl database reporting their association with
activation or repressive epigenetic marks. Each of the regions was amplified after chromatin was
immunoprecipitated using antibodies against H3K4me3 (active promoter) and H3K27me3 (inactive
promoter) and histone H3 (control). This figure is not drawn to scale. (
B
). The association of the
H3K27me3:H3 ratio was normalized to 1% input in various regions of Esr1 of fetal brains exposed in
utero to saline or EtOH. (
C
). The association of the H3K4me3:H3 ratio was normalized to 1% input
in various regions of Esr1 of fetal brains exposed in utero to saline or EtOH. The significance of
differences was determined by two-way ANOVA and a post hoc Tukey’s test. See Supplementary
Figure S3 for controls and Figures S4 and S5 for sex-separated data.
Cells 2023,12, 2308 13 of 24
3.6. Increased Hyperactivity in Saline- but Not EtOH-Exposed Ogg1 −/−vs. +/+ Females and
Ogg1- and Sex-Dependent Changes in Centre Time Spent in Saline- and EtOH-Exposed Progeny
Increased hyperactivity was observed in saline-exposed Ogg1
−
/
−
vs. +/+ females
(p< 0.05) but not males when averaged over 1 h (Figure 7). When analyzed for the last
half hour, there was increased hyperactivity in saline-exposed Ogg1
−
/
−
and +/
−
vs. +/+
females (p< 0.05). Although similar trends were seen with in utero EtOH exposure, they
were not significant. Analysis of centre zone activity revealed that saline-exposed Ogg1
+/
−
vs. +/+ male progeny showed greater time spent in the centre zone, but there was
no Ogg1-dependent effect in saline-exposed females. In contrast, in utero EtOH exposure
decreased the total time spent in the centre zone by Ogg1 +/
−
female progeny compared
to +/
−
saline controls (p< 0.05), but had no effect on males. EtOH exposure also increased
total time spent in the centre zone by Ogg1
−
/
−
female progeny compared to both EtOH-
exposed +/
−
and +/+ female littermates (p< 0.05). Interestingly, the increased time spent
by EtOH-exposed Ogg1
−
/
−
females was similar to that observed in saline-exposed female
progeny of all Ogg1 genotypes. The significance is based on two-way ANOVA and a post
hoc Tukey’s test.
Cells 2023, 12, x FOR PEER REVIEW 14 of 25
Figure 7. Increased hyperactivity for saline- but not EtOH-exposed Ogg1 −/− vs. +/+ females, and
decreased centre time for EtOH-exposed Ogg1 +/+ but not −/− female mice. For all behavioural stud-
ies, pregnant dams were treated with single dose of EtOH (2 g/kg i.p.) or its saline vehicle on GD
17, as shown in Figure 1, and progeny were delivered spontaneously. Fetal brains from at least three
liers were used to minimize potential lier effects, and the number of mice tested for each group
is shown in parentheses. Results show total distance travelled during the entire test (1 h), total dis-
tance travelled during the last 30 min of the test and total time spent in the centre zone (10 × 10
inches). The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s
test. See Supplementary Figure S6a for data for the nonsocial zone.
3.7. OGG1- and Sex-Dependent Effect on Social Interaction and Interaction-Induced Ultrasonic
Vocalizations but Not Startle Response
No OGG1-dependent or EtOH-dependent differences were observed in social inter-
action, including total duration in social zone, visits or time/visit (Figure 8A). No sex-de-
pendent differences were observed, so the data were combined. Interestingly, EtOH- vs.
saline-exposed Ogg1 +/− progeny exhibited decreased velocity and track length in the so-
cial zone, with no differences in Ogg1 +/+ and −/− mice (see Supplementary Figure S6a for
data for the nonsocial zone).
Figure 7.
Increased hyperactivity for saline- but not EtOH-exposed Ogg1
−
/
−
vs. +/+ females, and
decreased centre time for EtOH-exposed Ogg1 +/+ but not
−
/
−
female mice. For all behavioural
studies, pregnant dams were treated with single dose of EtOH (2 g/kg i.p.) or its saline vehicle on GD
17, as shown in Figure 1, and progeny were delivered spontaneously. Fetal brains from at least three
litters were used to minimize potential litter effects, and the number of mice tested for each group is
shown in parentheses. Results show total distance travelled during the entire test (1 h), total distance
travelled during the last 30 min of the test and total time spent in the centre zone (
10 ×10 inches
).
The significance of differences was determined by two-way ANOVA and a post hoc Tukey’s test. See
Supplementary Figure S6a for data for the nonsocial zone.
Cells 2023,12, 2308 14 of 24
3.7. OGG1- and Sex-Dependent Effect on Social Interaction and Interaction-Induced Ultrasonic
Vocalizations but Not Startle Response
No OGG1-dependent or EtOH-dependent differences were observed in social in-
teraction, including total duration in social zone, visits or time/visit (Figure 8A). No
sex-dependent differences were observed, so the data were combined. Interestingly, EtOH-
vs. saline-exposed Ogg1 +/
−
progeny exhibited decreased velocity and track length in the
social zone, with no differences in Ogg1 +/+ and
−
/
−
mice (see
Supplementary Figure S6a
for data for the nonsocial zone).
Cells 2023, 12, x FOR PEER REVIEW 15 of 25
EtOH-exposed Ogg1 +/− progeny showed increased interaction-induced ultrasonic
vocalizations (USVs) compared to EtOH-exposed Ogg1 +/+ liermates (p < 0.05), with a
similar trend observed in saline-exposed Ogg1 +/− mice (Figure 8B). Unexpectedly, Ogg1
−/− mice were similar to Ogg1 +/+ mice (Figure 8B). No differences were observed in fe-
male-induced USVs (see Supplementary Figure S6b). The significance is based on two-
way ANOVA and a post hoc Tukey’s test.
Figure 8. OGG1- and sex-dependent effect on social interaction and interaction-induced ultra-
sonic vocalizations. For all behavioural studies, pregnant dams were treated with a single dose of
EtOH (2 g/kg i.p.) or its saline vehicle on GD 17, as described in Figure 1, and progeny were deliv-
ered spontaneously. Fetal brains from at least three liers were used to minimize potential lier
effects, and the number of mice tested for each group is shown in parentheses. (Panel A). For social
interaction, EtOH vs. saline decreased velocity and track length in Ogg1 +/− progeny, but not in +/+
or −/− liermates, although the laer exhibited a similar non-significant trend. (Panel B). As with
social interaction, EtOH vs. saline increased interaction-induced ultrasonic vocalizations in Ogg1
+/− progeny, but not in +/+ or −/− liermates. The significance of differences was determined by two-
way ANOVA and a post hoc Tukey’s test.
3.8. Prepulse Inhibition
No differences in startle response measured via prepulse inhibition were observed
due to Ogg1 genotype, sex or treatment (see Supplementary Figure S7).
Figure 8. OGG1- and sex-dependent effect on social interaction and interaction-induced ultra-
sonic vocalizations.
For all behavioural studies, pregnant dams were treated with a single dose
of EtOH (2 g/kg i.p.) or its saline vehicle on GD 17, as described in Figure 1, and progeny were
delivered spontaneously. Fetal brains from at least three litters were used to minimize potential litter
effects, and the number of mice tested for each group is shown in parentheses. (Panel
A
). For social
interaction, EtOH vs. saline decreased velocity and track length in Ogg1 +/
−
progeny, but not in +/+
or
−
/
−
littermates, although the latter exhibited a similar non-significant trend. (Panel
B
). As with
social interaction, EtOH vs. saline increased interaction-induced ultrasonic vocalizations in Ogg1
+/
−
progeny, but not in +/+ or
−
/
−
littermates. The significance of differences was determined by
two-way ANOVA and a post hoc Tukey’s test.
Cells 2023,12, 2308 15 of 24
EtOH-exposed Ogg1 +/
−
progeny showed increased interaction-induced ultrasonic
vocalizations (USVs) compared to EtOH-exposed Ogg1 +/+ littermates (p< 0.05), with
a similar trend observed in saline-exposed Ogg1 +/
−
mice (Figure 8B). Unexpectedly,
Ogg1
−
/
−
mice were similar to Ogg1 +/+ mice (Figure 8B). No differences were observed
in female-induced USVs (see Supplementary Figure S6b). The significance is based on
two-way ANOVA and a post hoc Tukey’s test.
3.8. Prepulse Inhibition
No differences in startle response measured via prepulse inhibition were observed
due to Ogg1 genotype, sex or treatment (see Supplementary Figure S7).
4. Discussion
4.1. Overview
OGG1- and/or EtOH-dependent changes were observed in most biochemical out-
comes in whole fetal brains and postnatal behavioural disorders, which in some cases were
sex-dependent, as summarized in Table 1. The OGG1-dependent biochemical changes
in fetal brains exposed to saline potentially reflect the role of oxidative DNA damage in
DNA repair-deficient progeny due to physiological levels of ROS formation (Figure 9). This
oxidative DNA damage may contribute to developmental disorders not dependent upon
xenobiotic exposures, such as some components of attention-deficit hyperactivity disorders,
obsessive-compulsive disorders and autism spectrum disorders. The changes in represen-
tative epigenetic histone/DNA marks and behavioural disorders are consistent with our
hypothesis (Figure 9) and provide a rationale for more comprehensive mechanistic studies
to determine causal relationships. The absence of changes in gene expression with saline
exposure may in part reflect the limited selection of representative genes assessed herein,
and/or the use of whole fetal brains rather than brain regions or brain cell types. Also,
the direction and time course of epigenetic changes and gene expression may vary within
different brain regions and brain cell types, and for different genes and their associated
histone proteins. Finally, although the limited behavioural tests were consistent with our
hypothesis, and provided a proof of concept, a more comprehensive battery of tests would
be necessary to determine the full breadth of brain functions affected by physiological
levels of ROS formation in DNA repair-deficient progeny.
Table 1.
Summary of OGG1-dependent changes in saline- and EtOH-exposed
−
/
−
or +/
−
Ogg1
DNA repair-deficient fetal brains compared to +/+ control brains.
Gene/Protein Time Post-Exposure (h) Saline-Exposed
EtOH-Exposed
(Compared to Saline-Exposed Progeny of the Same Ogg1
Genotype, Unless Otherwise Stated)
Epigenetic Marks (Figure 2)
H3K9ac 6 - ↑Ogg1 +/+
↓Ogg1 −/−(vs. EtOH-exposed +/+)
24 ↑Ogg1 −/−-
H3K9me3 6 - ↑Ogg1 −/−
24 - ↑Ogg1 −/−
H3K27me3 6 - -
24 ↑Ogg1 −/−-
5-mC 24 ↑Ogg1 −/−-
5-hmC 24 - -
Cells 2023,12, 2308 16 of 24
Table 1. Cont.
Gene/Protein Time Post-Exposure (h) Saline-Exposed
EtOH-Exposed
(Compared to Saline-Exposed Progeny of the Same Ogg1
Genotype, Unless Otherwise Stated)
Gene expression (Figure 3)
Tet1 6 - -
24 - ↑Ogg1 −/−
Hdac2 6 - -
24 - ↑Ogg1 +/+
Nlgn3 6 - ↓Ogg1 −/−
24 - -
Reln 6 - -
24 - ↑Ogg1 +/+
Estrogen mRNA levels (Figure 4)
Esr1 6 - ↓Ogg1 +/+ (M)
24 - ↑Ogg1 −/−(combined sexes)
↑Ogg1 −/−(combined sexes, vs. EtOH-exposed +/+) 1
Esr2 6 - ↓Ogg1−/−(M)
24 - -
Esr1/2 ratio 6 - ↓Ogg1 +/+ (M)
24 - ↑Ogg1 −/−(combined sexes)
↑Ogg1 −/−(F) (vs. EtOH-exposed +/+) 1
Estrogen Protein levels 2(Figure 5)
ESR1 24 - -
ESR2 24 - ↓Ogg1 −/−(M)
↓Ogg1 −/−(M) (vs. EtOH-exposed +/+) 1
ESR1/2 ratio 24 - -
Esr1 loci select epigenetic marks (Figure 6)
H3K27me3 6 - ↑Ogg1 +/+ (regions 2, 3, 4, 5)
H3K4me3 6 - -
Behaviour (Figures 7and 8)
Open field activity
(total distance) 6 weeks ↑Ogg1 +/−(F)
↑Ogg1 −/−(F)
Open field activity
(centre zone time) 6 weeks ↑Ogg1 +/−(M) ↑Ogg1 −/−(F) (vs. EtOH-exposed +/+ and +/−)
↓Ogg1 +/−(F)
Social interaction
(velocity) 8 weeks - ↓Ogg1 +/−(combined sexes)
Social interaction
(track length) 8 weeks - ↓Ogg1 +/−(combined sexes)
Female-induced USV 4–5 months - ↑Ogg1 +/−(compared to EtOH-exposed +/+)
Abbreviations: - = no change; M = males; F = females; USV = ultrasonic vocalization.
1
Significance level with this
superscript: 0.05 < p< 0.1.
2
All other changes, at least p< 0.05 (see figures for specific levels). Protein levels were
not determined at 6 h post treatment.
Prenatal exposure to the ROS-enhancing drug EtOH was similarly associated with
OGG1-dependent changes in epigenetic marks in the fetal brain, but with different patterns
than with saline and more extensive behavioural disorders (Table 1). In addition, unlike
saline, EtOH altered the expression of all the representative genes, and a specific Esr1 gene
histone inactivation mark and ESR2 protein levels, in an OGG1-dependent fashion. In
certain cases, consistent changes were not observed among epigenetic changes, estrogen
receptor gene/protein levels and behaviour. These inconsistencies may have been due
in part to our use of whole fetal brains rather than brain regions or specific cell types,
limited sampling times, and the limited number and particular selection of representative
genes. The data for prenatal EtOH exposure provide an even more compelling proof of
concept consistent with our hypothesis for a role of oxidative DNA damage in epigenetic
mechanisms of neurodevelopmental disorders relevant to FASD (Figure 9) and a rationale
for more comprehensive mechanistic studies including whole-genome and proteomic
approaches to determine causal relationships.
Cells 2023,12, 2308 17 of 24
Cells 2023, 12, x FOR PEER REVIEW 16 of 25
4. Discussion
4.1. Overview
OGG1- and/or EtOH-dependent changes were observed in most biochemical out-
comes in whole fetal brains and postnatal behavioural disorders, which in some cases
were sex-dependent, as summarized in Table 1. The OGG1-dependent biochemical
changes in fetal brains exposed to saline potentially reflect the role of oxidative DNA dam-
age in DNA repair-deficient progeny due to physiological levels of ROS formation (Figure
9). This oxidative DNA damage may contribute to developmental disorders not depend-
ent upon xenobiotic exposures, such as some components of aention-deficit hyperactiv-
ity disorders, obsessive-compulsive disorders and autism spectrum disorders. The
changes in representative epigenetic histone/DNA marks and behavioural disorders are
consistent with our hypothesis (Figure 9) and provide a rationale for more comprehensive
mechanistic studies to determine causal relationships. The absence of changes in gene ex-
pression with saline exposure may in part reflect the limited selection of representative
genes assessed herein, and/or the use of whole fetal brains rather than brain regions or
brain cell types. Also, the direction and time course of epigenetic changes and gene ex-
pression may vary within different brain regions and brain cell types, and for different
genes and their associated histone proteins. Finally, although the limited behavioural tests
were consistent with our hypothesis, and provided a proof of concept, a more compre-
hensive baery of tests would be necessary to determine the full breadth of brain functions
affected by physiological levels of ROS formation in DNA repair-deficient progeny.
Figure 9. Postulated epigenetic role of oxidative DNA damage initiated by reactive oxygen spe-
cies (ROS) in neurodevelopmental disorders caused by physiological or ethanol-enhanced levels
of ROS formation. ROS, which are naturally produced in the body and essential for normal devel-
opment and life, can oxidatively damage DNA, resulting in multiple types of lesions. The most
prevalent DNA lesion, 8-oxoguanine (8-oxoG), is developmentally pathogenic, and is repaired by
Figure 9. Postulated epigenetic role of oxidative DNA damage initiated by reactive oxygen
species (ROS) in neurodevelopmental disorders caused by physiological or ethanol-enhanced
levels of ROS formation
. ROS, which are naturally produced in the body and essential for normal
development and life, can oxidatively damage DNA, resulting in multiple types of lesions. The
most prevalent DNA lesion, 8-oxoguanine (8-oxoG), is developmentally pathogenic, and is repaired
by oxoguanine glycosylase 1 (OGG1). Heterozygous (+/
−
) and particularly homozygous (
−
/
−
)
Ogg1 knockout progeny have decreased DNA repair activity compared to their wild-type (+/+)
littermates, and can accumulate oxidative DNA damage due to even physiological levels of ROS
formation, leading to epigenetic changes in DNA methylation and modifications to histone proteins,
exemplified by histone methylation and acetylation. These epigenetic changes can alter the expression
of developmentally important genes, leading to neurodevelopmental disorders. Prenatal exposure
to ROS-enhancing drugs like alcohol (ethanol, EtOH) can further enhance both the complexity and
magnitude of epigenetic changes initiated by oxidative DNA damage, increasing the spectrum and
severity of neurodevelopmental disorders.
4.2. Ethanol-Initiated Alterations in Histone Modifications and Gene Expression in Fetal Brains
EtOH causes several epigenetic modifications, which have been associated with its
embryopathic mechanism [
14
–
16
]. The effect of EtOH on histone acetylation patterns
depends on the EtOH treatment paradigm, timing of exposure and brain regions examined,
with varying results even within a region [16,32,33].
Herein, an EtOH-initiated increase in global H3K9ac indicated early gene activation in
Ogg1 +/+ but not
−
/
−
fetal brains, which returned to baseline at a later time point, likely
due to increased HDAC2 activity, as an increase in Hdac2 mRNA levels was observed in
EtOH-exposed Ogg1 +/+ progeny at 24 h. The initial increase in H3K9ac in EtOH-exposed
Ogg1 +/+ brains may play a role in regulating gene transcription to maintain the redox
balance, as previously proposed [
8
]. Under oxidative stress conditions, recognition of
8-oxoG by OGG1 results in the oxidation of a cysteine sulfhydryl group in OGG1, which in
Cells 2023,12, 2308 18 of 24
its oxidized state can bind but not repair 8-oxoG. Subsequent assembly of transcriptional
machinery and regulation of gene transcription restores the redox state of the cell. This
in turn results in reduced OGG1, thereby excising 8-oxoG, suggesting a role for OGG1 in
gene transcription regulation [
8
]. This is consistent with the observed increase in H3K9ac
in EtOH- vs. saline-exposed Ogg1 +/+but not Ogg1
−
/
−
fetal brains. EtOH exposure also
resulted in an increase in H3K9me3, indicating gene suppression, in Ogg1
−
/
−
but not
+/+ fetal brains, suggesting OGG1 dependence. These results suggest that EtOH causes
OGG1-dependent alterations in histone marks that alter gene expression. Although the
above levels were measured globally, it is possible that EtOH may increase or decrease
these marks in an Ogg1 gene-dependent manner, as 8-oxoG formation has recently been
shown to be gene-selective [34,35].
EtOH-initiated, OGG1-dependent alterations in gene expression were found for only
a few of representative genes tested (see Supplementary Figure S2). Aside from the limited
number of representative genes examined herein, this may be in part because the gene
expression changes in our study were measured earlier in life (i.e., in the fetal brain),
whereas some gene expression changes may not be seen until later in life (postnatally),
exemplified by OGG1-dependent differences in gene expression in Ogg1
−
/
−
vs. +/+
untreated adult hippocampi around 6 months of age [
17
]. In addition, the use of a single low
dose of EtOH may result in negligible or minor differences in gene expression, and could
thus limit the quantitative discrimination of small differences in mRNA level observed via
RT-qPCR when only one housekeeping gene is used as a control [
36
], as was the case herein
(i.e., Gapdh).
In this study, the EtOH-mediated increase in Ten-eleven translocation methylcytosine
dioxygenase 1 (Tet1) gene expression in Ogg1
−
/
−
but not Ogg1 +/+ fetal brains may
have contributed to altered methylation levels at the later time point, as TET proteins are
involved in active demethylation of 5-mC to cytosine [
37
]. Reelin (RELN), along with its
essential adapter protein, Disabled-1 (Dab1), regulates neuronal plasticity and subsequently
memory formation [
38
]. A deficiency in Reelin can result in behavioural dysfunction and
may contribute to schizophrenia [
39
] or autism [
40
]. One study suggests that exposure
to EtOH initially activates but later silences the Reelin–Dab1 signaling pathway via brief
activation and subsequently inactivation of Src-family kinases both
in vitro
and
in vivo
[
41
].
Interestingly, we saw an EtOH-dependent increase in Reln mRNA levels in Ogg1 +/+ but
not Ogg1
−
/
−
fetal brains, possibly because baseline Reln levels seemed to be already
higher in saline-exposed Ogg1
−
/
−
fetal brains, resulting in no further increase with
EtOH exposure. EtOH appeared to transiently decrease neuroligin 3 (Nlgn3) regardless
of Ogg1 genotype, although the decrease was significant only in Ogg1
−
/
−
fetal brains.
Neuroligin-3 (NLGN3) is an X-linked neuronal cell surface protein and a postsynaptic
cell adhesion molecule involved in the formation and remodeling of the central nervous
systems [
42
]. One study reported decreased methylation levels in the Nlgn3 gene with
EtOH exposure in embryo culture without altering its expression [
43
]. A gain of function of
Nlgn3 resulting in inhibition of synaptic transmission is associated with a model for autism
spectrum disorders [44].
4.3. Alterations in Histone Modifications, DNA Methylation and Gene Expression in Fetal Brains
Exposed to Saline
For some measures, OGG1-dependent changes were seen only in saline-exposed Ogg1
−
/
−
fetal brains. The increase in repression marks H3K27me3 and 5-mC in saline-exposed
Ogg1
−
/
−
vs. +/+ fetal brains at 24 h suggests that OGG1 can regulate gene methylation
levels. A role for OGG1 in oxidative stress-induced DNA demethylation via recruiting
TET1 to the 8-oxoG lesion has been reported [
45
]. This is consistent with our results
showing an increase in repression marks only in Ogg1
−
/
−
fetal brains. This suggests
OGG1 may be important in maintaining normal levels of repressive marks by facilitating
the demethylation process. In addition, there was also an OGG1-dependent increase in
H3K9ac in saline-exposed Ogg1
−
/
−
vs. +/+ brains at 24 h, which could be involved
Cells 2023,12, 2308 19 of 24
in the activation of genes facilitating recovery after the stress of a single injection and/or
suggest a role for OGG1 in regulating histone acetylation marks similar to its regulation of
DNA demethylation.
4.4. OGG1- and Sex-Dependent Differences in mRNA and Protein Levels of ESR1 and ESR2 and
Their Ratios in Fetal Brains, and the Association of H3K27me3 and H3K4me3 with the Esr1 Gene
Previous evidence suggests that OGG1 may be involved in estrogen receptor (ESR)-
mediated gene transcription [
17
,
46
]. For example, during estrogen-bound ESR-mediated
gene transcription, a lysine-specific demethylase 1 (LSD1) demethylates histone H3 lysine
9 dimethylation marks (H3K9me2), and in the process produces localized ROS and
8-oxoG
formation in discrete foci including at the promoters of various ESR1 target genes. Localized
8-oxoG recruits OGG1, which nicks the DNA, resulting in conformational changes of
chromatin necessary for estrogen-dependent transcription [46].
ESRs are nuclear hormone receptors and transcription factors [
47
] that serve various
functions, including playing an important neuroprotective role in memory formation and
maintenance [
47
]. ESR1 inhibition is important in the formation of long-term memory
in the mouse hippocampus [
48
], and a role for OGG1 in repressing ESR1 to modulate
learning and memory formation has been postulated because hippocampal regions of
Ogg1 KO mice exhibited upregulation of Esr1 target genes [
17
]. We previously saw OGG1-
and sex-dependent enhanced DNA strand breaks, decreased DNA methylation levels and
behaviour deficits including alterations in spatial and recognition memory in untreated
OGG1-deficient young mice (~2–3 months) [
10
]. The presence of sex-dependent differences
in young OGG1-deficient mice suggests that estrogen may play an important role in regu-
lating gene expression in an OGG1-dependent manner, particularly during development,
so Esr1 and Esr2 mRNA levels and protein levels were quantified in fetal brains.
Only limited OGG1- and treatment-dependent differences were seen in Esr1 gene
expression. A marginal increase in Esr1 expression in EtOH-exposed Ogg1
−
/
−
vs. +/+
fetal brain (sexes combined) was associated with a consistent trend for a decrease in gene-
specific H3K27me3 marks measured via ChIP-qPCR in EtOH-exposed Ogg1
−
/
−
vs. +/+
fetal brains across various regions of the Esr1 gene
(
Figure 6), suggesting that EtOH can
alter the ESR1 signaling pathway in an OGG1- and sex-dependent manner. Furthermore,
this increase in Esr1 expression in EtOH-exposed Ogg1
−
/
−
vs. +/+ fetal brains seemed to
be due to an EtOH-dependent decrease in Esr1 expression and the Esr1/Esr2 ratio in EtOH-
vs. saline-exposed Ogg1 +/+ but not Ogg1
−
/
−
male fetal brains. This was reflected by
ChIP-qPCR results, which showed an increased association of H3K27me3 (repression mark)
in EtOH- vs. saline-exposed Ogg1 +/+ but not Ogg1
−
/
−
brains. Analysis of ChIP-qPCR
data by sex suggested that, although a trend for EtOH-mediated increase in H3K27me3
in Ogg1 +/+ fetal brains was observed in both males and females (Supplementary Figure
S4), a similar trend for an EtOH-mediated increase in H3K4me3 (activation mark) in Ogg1
+/+ brains seemed to occur only in females (Supplementary Figure S5). An increase in
both H3K27me3 (repressor) and H3K4me3 (activator) in females may cancel each other,
resulting in no difference in Esr1 gene expression in EtOH-exposed Ogg1 +/+ females,
whereas an increase only in H3K27me3 but not H3K4me3 in males may cause decreased
Esr1 gene expression in EtOH- vs. saline-exposed Ogg1 +/+ males.
A decrease in Esr1 gene expression in EtOH- vs. saline-exposed Ogg1 +/+ but not
−
/
−
progeny suggests a role for OGG1 and possibly 8-oxoG in regulating Esr1 gene expression.
Mapping of
8-oxoG
in the genome of Ogg1
−
/
−
vs. +/+ mouse embryonic fibroblasts
revealed a three-fold enrichment of 8-oxoG peaks in the Esr1 gene in Ogg1 +/+ but not
Ogg1
−
/
−
cells [
34
]. A decrease in 8-oxoG peaks in the Esr1 gene in Ogg1
−
/
−
fetal brains
may alter Esr1 gene expression in Ogg1
−
/
−
progeny, as the presence of 8-oxoG may either
increase or decrease gene expression depending on the site oxidized [
8
]. Interestingly, the
EtOH-mediated increase in association of H3K27me3 marks with various regions of the Esr1
gene and decreased Esr1 gene expression in Ogg1 +/+ male mice but not
−
/
−
male mice
further suggests a role for EtOH in altering the ESR1 signaling pathway in both an OGG1-
Cells 2023,12, 2308 20 of 24
and sex-dependent manner. We hypothesize that EtOH, being a ROS inducer, may further
increase
8-oxoG
levels in the Esr1 gene in both Ogg1 +/+ and
−
/
−
fetal brains. However,
in Ogg1 +/+ brains, 8-oxoG may be recognized by OGG1, resulting in the recruitment of
epigenetic modifiers and hence increased H3K27me3 levels and gene repression, whereas
in Ogg1
−
/
−
mice lacking OGG1, 8-oxoG cannot be recognized by OGG1 and thus the
repressive complex may not be recruited as efficiently as in Ogg1 +/+ brains, resulting
in no changes in H3K27me3 levels or gene repression. In a related example, OGG1 can
recruit chromodomain helicase DNA-binding protein 4 (CHD4) to interact with 8-oxoG
sites in genes, and CHD4 recruits repressive epigenetic modifiers complexes including
DNA methyltransferases and histone H3K27 methyltransferases (such as EZH2 and G9a)
to DNA damage sites, resulting in silencing of genes [
49
]. Thus, EtOH-enhanced ROS
and 8-oxoG levels specifically in the Esr1 gene may act similarly to repress Esr1 gene
transcription mediated by OGG1, as previously postulated [17].
The absence of differences in ESR1 protein levels may have been due in part to limited
differences in Esr1 mRNA levels and/or the time point analyzed. The protein levels were
only measured using fetal brains extracted 24 h after maternal treatment due to loss of
samples extracted 6 h later. However, the EtOH-mediated decrease in Esr2 mRNA and
ESR2 protein levels in Ogg1
−
/
−
(but not +/+) males (but not females) suggests that OGG1
may also sex-dependently regulate ESR2-mediated gene transcription, which has not been
previously reported and merits further investigation.
4.5. OGG1- and EtOH-Dependent Effects on Behavioural Abnormalities
Previous studies that measured open field activity in untreated OGG1-deficient mice
did not find differences at 3 months of age (sexes combined and tested at 20 lux) [
50
] or
in 4-month-old male Ogg1
−
/
−
mice (lighting conditions not provided) [
17
]. However a
decreased performance in an open field box test (measured via number of crossed lines,
number of rears, mean speed and time immobile) was observed in aging (26 months) Ogg1
−
/
−
mice (sexes combined and tested at 20 lux) [
50
]. Our own data using 3-month-old
untreated OGG1-deficient mice showed no differences in open field activity except for a
trend for increased time spent in the centre zone by Ogg1
−
/
−
females when tested at
~50 lux [10].
Herein, sex-dependent differences were observed in the saline-exposed group in our
studies. This could be due to several factors that varied across studies [
51
], including age
(6 weeks in our study), testing time, size of the apparatus, centre zone size and lighting
conditions (~130 lux in our study), as well as the effect of a single saline injection during
pregnancy. Altered time spent in the centre zone without differences in the total distance
travelled have been reported in mouse models reflecting altered anxiety-related behaviour
without any effect on ambulatory ability [
28
]. In our study, saline-exposed OGG1-deficient
males spent more time in the centre without any effect on the total distance travelled,
suggesting reduced fear and anxiety in OGG1-deficient males, which was not significantly
altered in EtOH-exposed Ogg1
−
/
−
males. Unlike males, saline-exposed OGG1-deficient
females travelled a greater distance compared to Ogg1 +/+ females, without exhibiting
any differences in time spent in the centre zone, suggesting increased ambulatory activity
without any effect on fear and anxiety in saline-exposed OGG1-deficient females. In
contrast, with in utero EtOH exposure, although the total distance travelled in Ogg1 +/+
and
−
/
−
females was unaffected, the time spent in the centre zone was decreased in Ogg1
+/+ but not
−
/
−
females, suggesting that the EtOH-mediated increased fear and anxiety
was OGG1-dependent.
Prenatal exposure to EtOH has been shown to induce anxiety-like behaviour in rats
and mice [
52
,
53
]. ESR knockout mice exhibit altered open field activity at 10–12 weeks
of age in mice, confirming a role for ESRs in altered open field activity [
54
]. Our results
suggest that anxiety-like behaviour may be both an OGG1- and sex-dependent effect of
EtOH, and may involve mechanisms such as estrogen receptors and the interaction between
OGG1- and ESR-mediated gene regulation.
Cells 2023,12, 2308 21 of 24
Although no differences were observed in social interaction in saline-exposed Ogg1
mice, EtOH decreased velocity and track length in Ogg1 +/
−
mice, with a similar non-
significant trend in Ogg1
−
/
−
mice, but no effect in Ogg1 +/+ mice. In the USV test,
both the saline- and EtOH-exposed Ogg1 +/
−
progeny exhibited similarly increased
interaction-induced USVs compared to Ogg1 +/+ mice, which was significant for EtOH,
suggesting that the increase was due to a heterozygous loss of OGG1 rather than EtOH.
The mechanism may involve ESR-mediated gene regulation, as one study reported that
Esr1/2knockout male mice exhibited an abolition of sexual behaviour and decreased
USVs in the presence of females, and a role for ESRs in regulating aggressive behaviour in
male mice has been reported [
55
]. In addition, mice lacking either ESR1 or ESR2 exhibit
decreased social behaviour [
56
,
57
], whereas ovariectomized female rats exposed to estrogen
subcutaneously exhibited decreased USVs when introduced to a familiar cage mate after a
week of separation, which may be due to estrogen-induced increased social memory [
58
].
These studies suggest a role for estrogen receptors in interaction-induced USV, which our
results suggest may be OGG1-dependent.
The absence of a significant effect of EtOH or Ogg1 genotype on startle response in the
prepulse inhibition test may be due to the relatively low dose of EtOH, given there was a
non-significant trend of an EtOH-dependent decrease in prepulse inhibition in Ogg1
−
/
−
but not +/+ males and a decrease in Ogg1 +/+ but not −/−females.
5. Conclusions
This study demonstrated OGG1-dependent effects of in utero exposure of the fetal
brain to physiological ROS levels, or to a single low dose of EtOH on epigenetic marks
and gene expression in fetal brains, and postnatal behaviour, potentially relevant to neu-
rodevelopmental disorders including FASD. In particular, OGG1 alone or in association
with 8-oxoG may play a role in estrogen receptor-mediated gene regulation, which may
be further altered by the ROS-enhancing effect of in utero EtOH exposure, possibly con-
tributing to sex-dependent differences in some behavioural disorders observed in OGG1
mice. The relatively modest effects of EtOH observed herein suggest that follow-up studies
using a higher dose of EtOH will be necessary to fully understand the effect of EtOH
on behavioural abnormalities in OGG1-deficient mice and the role of OGG1 in estrogen
receptor-mediated gene regulation, among other pathways, at higher levels of oxidative
stress, where OGG1 may work differently to regulate estrogen receptor-mediated gene
expression. Given the OGG1-dependent alterations in some epigenetic marks and be-
havioural outcomes in saline-exposed progeny, it is likely that OGG1 epigenetically alters
the regulation of additional genes contributing to developmental disorders, including
disorders not involving xenobiotic exposure.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/cells12182308/s1, Figure S1: Histone modifications with no OGG1-
or EtOH-dependent differences. Gestational Day (GD) 17 fetal brains exposed in utero to a single
dose of EtOH (2 g/kg, i.p.) or its saline vehicle were extracted 1, 6 and 24 h later from Ogg1
+/+ and -/- littermates and were assessed for histone modifications. No differences observed in
H3K9ac (activation mark) and H3K9me3 (inactivation mark) at 1h and in H3K4me3 at 6 and 24 h
(1 h not tested). The differences observed were determined using two-way ANOVA and a post-hoc
Tukey’s test; Figure S2: List of genes with no OGG1- or EtOH-dependent differences in expression.
Gestational Day (GD) 17 fetal brains exposed in utero to a single dose of EtOH (2 g/kg, i.p.) or its
saline vehicle were extracted 6 and 24 h later from Ogg1 +/+ and -/- littermates and were assessed
gene expression. No differences were observed in mRNA levels of the listed genes using the primers
shown in Figure S8. The differences observed were determined using two-way ANOVA and a
post-hoc Tukey’s test. Figure S3: Controls to validate the ChIP-qPCR technique. Positive controls
are
β
-actin and Gapdh, and the negative control is Hbby (hemoglobin subunit beta y). Associations
of histone H3 (total histone), histone H3 lysine 9 acetylation (H3K9ac, activation mark), histone H3
lysine 27 trimethylation (H3K27me3, repressive mark), and RNA polymerase II (Pol II, activation
mark) with Esr1 gene were measured via ChIP-qPCR. The positive controls appropriately show
Cells 2023,12, 2308 22 of 24
enrichment for H3K9ac and Pol II (activation mark) and low levels for H3K27me3 (inactivation mark),
as they are constitutively active. Conversely, the negative control appropriately shows a low or
absent signal for H3K9ac and Pol II with some enrichment for repressive mark H3K27me3, as Hbby
is not transcribed in the brain. Association of H3K27me3 and Pol II with
β
-actin, Gapdh, and the
negative control Hbby were not measured in Ogg1 -/- fetal brain (listed as N/A or not applicable).
IgG represents the negative control isotype IgG. Above data represent 1 sample. Figure S5: EtOH-
mediated trend for increased association of H3K4me3 with the Esr1 gene in female Ogg1 +/+ but
not -/- fetal brains. Quantitative PCR was performed using five different sets of primers directed
against various regions of the Esr1 gene (Figure S9), and each of the regions were amplified after
chromatin was immunoprecipitated using antibodies against H3K4me3 (active promoter) and histone
H3 (control). The association of H3K4me3:H3 ratio was normalized to 1 % input in various regions of
Esr1 of fetal brains exposed in utero to saline or EtOH. The significance of differences was determined
using two-way ANOVA and a post hoc Tukey’s test. Figure S7: OGG1- and EtOH-dependent effects
on the startle response measured via prepulse inhibition. No differences were observed aside from the
expected decibel dose-response relationship. Significance was determined using two-way ANOVA
and a post hoc Tukey’s test. Figure S8: Primer sequences used to analyze mRNA levels of genes via
RT-qPCR. Figure S9: Primer sequences used to analyze the association of histone modifications on
various sites of the Esr1 gene and control genes via ChIP-qPCR.
Author Contributions:
Conceptualization: S.B. and P.G.W.; Methodology: S.B. and A.P.C.; Formal
analysis: S.B. and D.B.; Investigation: S.B. and D.B.; Data curation: S.B. and D.B.; Writing—original
draft: S.B. and P.G.W.; Writing—review and editing: A.P.C. and P.G.W. All authors have read and
agreed to the published version of the manuscript.
Funding:
These studies were supported by grants from the Canadian Institutes of Health Research
(CIHR) (PJT-156023, MOP-115108) and the Faculty of Pharmacy at University of Toronto. SB and
AC were supported in part by fellowship awards from the Centre for Pharmaceutical Oncology at
University of Toronto. We thank Dr. Jason Matthews for his help in the epigenetic studies.
Institutional Review Board Statement:
The animal study protocol was approved by the Institutional
Review Board of University of Toronto (protocol code 20011157, 6 August 2018).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available in this paper and the
Supplementary Material.
Conflicts of Interest: The authors declare no conflict of interest.
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