Chronic Aroclor 1260 Exposure Alters the Mouse Liver Proteome, Selenoproteins, and Metals in Steatotic Liver Disease

Full text

Environmental Toxicology and Pharmacology 107 (2024) 104430
Available online 27 March 2024
1382-6689/© 2024 Elsevier B.V. All rights reserved.
Chronic Aroclor 1260 exposure alters the mouse liver proteome,
selenoproteins, and metals in steatotic liver disease
Kellianne M. Piell
a
, Belinda J. Petri
a
,
b
, Jason Xu
c
, Lu Cai
c
,
d
,
e
, Shesh N. Rai
f
, Ming Li
g
,
Daniel W. Wilkey
h
, Michael L. Merchant
e
,
g
,
h
, Matthew C. Cave
e
,
h
,
i
,
j
, Carolyn M. Klinge
a
,
e
,
*
a
Department of Biochemistry & Molecular Genetics, University of Louisville School of Medicine, Louisville, KY 40292, USA
b
Kentucky IDeA Networks of Biomedical Research Excellence (KY INBRE) Bioinformatics Core, University of Louisville, Louisville, KY 40202, USA
c
Pediatric Research Institute, Department of Pediatrics, University of Louisville School of Medicine, Louisville, KY 40292, USA
d
Departments of Radiation Oncology, Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA
e
University of Louisville Center for Integrative Environmental Health Sciences (CIEHS), University of Louisville, Louisville, KY 40292, USA
f
Division of Biostatistics and Bioinformatics, Department of Environmental and Public Health Sciences, College of Medicine, University of Cincinnati, Cincinnati, OH
45267, USA
g
Division of Nephrology & Hypertension, Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40202, USA
h
University of Louisville Hepatobiology and Toxicology Center; University of Louisville School of Medicine, Louisville, KY 40202, USA
i
Division of Gastroenterology, Hepatology & Nutrition, Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40292, USA
j
The University of Louisville Superfund Research Center, University of Louisville School of Medicine, Louisville, KY 40292, USA
ARTICLE INFO
Edited by Dr. M.D. Coleman
Keywords:
PCBs
Diet
Liver
RNA modications
TRNA
Selenoproteins
Proteome
ABSTRACT
The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) continues to increase due in
part to the obesity epidemic and to environmental exposures to metabolism disrupting chemicals. A single
gavage exposure of male mice to Aroclor 1260 (Ar1260), an environmentally relevant mixture of non-dioxin-like
polychlorinated biphenyls (PCBs), resulted in steatohepatitis and altered RNA modications in selenocysteine
tRNA 34 weeks post-exposure. Unbiased approaches identied the liver proteome, selenoproteins, and levels of
25 metals. Ar1260 altered the abundance of 128 proteins. Enrichment analysis of the liver Ar1260 proteome
included glutathione metabolism and translation of selenoproteins. Hepatic glutathione peroxidase 4 (GPX4) and
Selenoprotein O (SELENOO) were increased and Selenoprotein F (SELENOF), Selenoprotein S (SELENOS), Se-
lenium binding protein 2 (SELENBP2) were decreased with Ar1260 exposure. Increased copper, selenium (Se),
and zinc and reduced iron levels were detected. These data demonstrate that Ar1260 exposure alters the (seleno)
proteome, Se, and metals in MASLD-associated pathways.
1. Introduction
Polychlorinated biphenyls (PCBs) are metabolism disrupting chem-
icals (MDC) that persist in the environment and promote metabolic
disease including metabolic dysfunction-associated steatotic liver dis-
ease (MASLD) (reviewed in (Wahlang et al., 2019)). MASLD may
progress from steatosis to steatohepatitis (metabolic
dysfunction-associated steatohepatitis (MASH)), in which liver injury
and inammation are detected with or without brosis, cirrhosis, and
hepatocellular carcinoma (HCC) (Cave et al., 2016). Although PCB
manufacture was banned in the U.S. in 1979, exposure to PCBs is
considered “ubiquitous” for the U.S. population (Curtis et al., 2021).
Human PCB exposure is primarily by dietary intake from contaminated
food, e.g., sh and dairy products (reviewed in (Montano et al., 2022)).
The biological effects of PCBs are considered to be mediated by
receptor-based modes of action. Coplanar, or “dioxin-like” (DL) PCBs
activate the aryl hydrocarbon receptor (AHR), while the non-coplanar,
or “non-dioxin-like” (NDL) PCBs activate nuclear receptors, e.g.,
constitutive androstane receptor (CAR) and pregnane X receptor (PXR)
(Safe et al., 1985) and inhibit epidermal growth factor receptor (EGFR)
signaling (Hardesty et al., 2021). PCB mixtures were sold under the
trade name Aroclor followed by four digits in which the rst two indi-
cate that the mixture is composed of chlorinated biphenyls and the last
two digits indicate the percent chlorine by weight (Master et al., 2002).
Aroclor1260 (Ar1260) is a mixture of highly chlorinated, NDL PCBs. Due
to the high thermodynamic stability of these congeners, Ar1260 reects
* Correspondence to: Department of Biochemistry & Molecular Genetics University of Louisville School of Medicine, Louisville, KY 40292, USA.
E-mail address: carolyn.klinge@louisville.edu (C.M. Klinge).
Contents lists available at ScienceDirect
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https://doi.org/10.1016/j.etap.2024.104430
Received 9 January 2024; Received in revised form 19 March 2024; Accepted 23 March 2024

Environmental Toxicology and Pharmacology 107 (2024) 104430
2
the PCB bioaccumulation patterns by mass in human tissue (Wahlang
et al., 2014a).
We recently reported that male C57Bl/6 J mice receiving a single
oral dose of 20 mg/kg Ar1260 developed, at the 34th week (wks.) post-
Ar1260, liver steatosis and brosis with increased serum AST and liver
Col1a1 (collagen type 1 alpha 1 chain) transcript abundance (Piell et al.,
2023). The human environmental relevance of the 20 mg/kg Ar1260
dose was conrmed by exposure assessment in mouse liver (Hardesty
et al., 2019; Wahlang et al., 2014b) to model the upper end of observed
human exposure in our translational human cohort, the Anniston
Community Health Survey (ACHS) (Cave et al., 2022; Clair et al., 2018),
based on PCB partitioning ratios observed in NTP studies (ANON, 2006).
We reported 12 RNA epitranscriptomic changes in the livers of mice at
the 34th wks. post-Ar1260 exposure compared to control mice (Piell
et al., 2023). Using a comprehensive bioinformatics approach, we
identied cell pathway networks related to MAFLD that were associated
with the epitranscriptome changes and observed a reduction in the
levels of the hub protein NRF2 in the Ar1260-exposed livers, which we
suggested would impair hepatic detoxication and anti-oxidative stress
responses.
Among the tRNA modications we reported, i6A (N6-isopentenyl-
adenosine) was increased and mcm5U (5-methoxy-carbon-
ylmethyluridine) was decreased (Piell et al., 2023). mcm5U and i6A are
specically located in the anti-codon loop of selenocysteine (Sec, the
21st amino acid) transfer RNA (Sec-tRNA, also called tRNA
[Ser]Sec
)
(Moustafa Mohamed et al., 2001). The essential trace element selenium
(Se) is ingested in food as various organic Se species (Marschall et al.,
2016). The liver is the central organ for Se regulation (Burk and Hill,
2015). Se is co-translationally incorporated into Sec which is part of the
active site in selenoprotein antioxidant enzymes (Lai et al., 2011). There
are 24- and 25- Se-dependent proteins (selenoproteins) in rodents and
humans, respectively (Zhang et al., 2020b). Selenoprotein synthesis
uniquely depend on Sec-tRNA. Although there is only one gene encoding
Sec-tRNA (n-TUtca2 in mouse), there are two Sec-tRNAs isoforms with
either mcm5U or mcm5Um at position 34 (Sengupta et al., 2008), and
then invariably i6A at position 37 in the anticodon loop, Y at 55, and
m1A at 58 (Carlson et al., 2018). i6A is required for Sec insertion into
selenoproteins at UGA, a process requiring a special elongation factor
eEFsec (Eukaryotic Elongation Factor, Selenocysteine-TRNA Specic,
Eefsec in mouse) and SBP2 (Secisbp2, SECIS binding protein 2 in mouse)
(Moustafa Mohamed et al., 2001; Peng et al., 2021).
PCB exposures do not affect hepatic total glutathione concentrations
in rodents (Twaroski et al., 2001). However, hepatic Se levels in rats
were reduced two wks. after a single i.p. injection of 100
μ
mol/kg of the
DL PCB77 (Twaroski et al., 2001) or 5
μ
mol/kg of the DL PCB126
(Klaren et al., 2015). Dietary Se supplementation prevented a PCB126
exposure-dependent decrease in the activities of selenoproteins gluta-
thione peroxidase (GPX) and thioredoxin (TRX) reductase in rat liver
(Lai et al., 2011). High Se (0.15 or 0.60 ppm) for seven wks directly after
weaning increased GPX and TRX activity and plasma triglycerides in
mice (Speckmann et al., 2017). To our knowledge, no one has evaluated
the impact of Ar1260 exposures on liver Se levels or selenoprotein
expression. To address this gap, we used an untargeted proteomics
approach to identify protein changes in the livers of male mice treated
with the same experimental procedure as previous study (9), i.e., a single
gavage of Ar1260 at 20 mg/kg and examination at the 34th wks.
post-exposure. Pathway enrichment analysis of the proteome revealed
glutathione metabolism as the top pathway in the livers of the
Ar1260-exposed mice. In addition, we evaluated the levels of 25 metals
and identied signicant increases in Se, zinc (Zn), and copper (Cu) and
reduced iron (Fe) with Ar1260 exposure that are associated with path-
ways and transcription factors regulating the hepatic pathways altered
by Ar1260 exposure.
2. Materials and methods
2.1. Materials
Aroclor 1260 (Ar1260) was purchased from AccuStandard (New
Haven, CT, USA). Ar1260 was a commercial heavily chlorinated PCB
mixture containing ~ 99 % non-coplanar, di-ortho substituted PCBs that
have 5, 6, 7, or 8 chlorines (see Supplementary Table 1 in (Wahlang
et al., 2014a)).
2.2. Animal studies
The experimental design is modeled in Supplementary Figure 1 as
reported in (Piell et al., 2023). The animal protocol was ratied by the
University of Louisville Institutional Animal Care and Use Committee
(Piell et al., 2023). Adult male C57BL/6 mice (8 wks. old) were pur-
chased from Jackson Laboratory and randomized into two groups
(n =10 control and n =30 Ar1260 exposure) (Piell et al., 2023). All mice
were fed ad libitum a normal, control diet with 20 %, 69.8 %, and 10.2 %
of total calories from protein, carbohydrate, and fat, respectively
(TekLad 06416, Envigo, Indianapolis, IN, USA) throughout the study
(Piell et al., 2023). At 10 wks. of age, the mice in each group were given
either corn oil or Aroclor1260 (20 mg/kg) via a one-time oral gavage as
reported in our previous study (Piell et al., 2023). This concentration
was based on previous studies showing that Ar1260 acts as a ‘second hit’
in high-fat diet (HFD)-fed mice to induce steatohepatitis in a chronic (12
wks.) mouse diet +PCB exposure model of human MASLD (Jin et al.,
2020; Shi et al., 2019). Although Ar1260 contains the PCB congeners
that are reective of human adipose bioaccumulation patterns (Wahlang
et al., 2014a), it did not activate mouse AHR at environmentally relevant
doses (Wahlang et al., 2014b). The dose of Ar1260 in this study is the
same as in our previous studies (Jin et al., 2020; Piell et al., 2023; Shi
et al., 2019; Wahlang et al., 2014b). The total experimental time was 36
wks. and post-exposure time of single gavage with Ar1260 was 34 wks
(Supplementary Figure 1). At the end of 36th wks., the mice were fasted
for ~ 6 h prior to euthanasia and liver samples were harvested as
described (Piell et al., 2023; Shi et al., 2019).
2.3. RNA isolation
Mouse liver was preserved in RNAlater Stabilization Solution
(ThermoFisher, Waltham, MA) immediately following excision. Total
RNA was isolated as previously described (Klinge et al., 2021; Piell et al.,
2023). In summary, liver tissue was washed in PBS, and total RNA was
isolated using the Qiagen (Germantown, MD) RNeasy Mini Kit according
to manufacturer’s protocol. Total RNA concentration and quality were
measured using a NanoDrop spectrophotometer and a Qubit Fluorom-
eter (ThermoFisher, Rockford, IL, USA).
2.4. LC-MS/MS analysis of RNA modications and data processing
All liver RNA samples (n =10 control +30 Ar1260-exposed) were
analyzed in random order on a Thermo Orbitrap Fusion Lumos Tribrid
mass spectrometer coupled with a Thermo Vanquish™ HPLC system
(Thermo Fisher) as previously described (Piell et al., 2023). Xcalibur
software (v4.5, Thermo Fisher) was used to identify and quantify the
LC-MS data. Quan Browser, a build-in component in Xcalibur was used
to quantify the nucleosides using the peak area of daughter ion.
Nucleoside identication was achieved by matching the experimental
value of retention time, parent ion m/z, and the m/z of the most abun-
dant daughter ion to the corresponding data of the standard (Piell et al.,
2023). The threshold for the retention time difference and m/z variation
window were set as ≤3 s and ≤5 ppm, respectively.
K.M. Piell et al.

Environmental Toxicology and Pharmacology 107 (2024) 104430
3
2.5. Sample preparation for proteomics analysis
Proteins were extracted from liver tissues in RIPA buffer supple-
mented with protease and phosphatase inhibitors using a bead homog-
enizer and protein amounts were quantitated by BioRad DC protein
assay (Bio-Rad Laboratories, Inc, Hercules, CA.) (Piell et al., 2023).
Protein lysates (200
μ
g) were diluted to 4 % SDS plus 1X HALT™ pro-
tease/phosphatase inhibitors. Samples (100 µg) were trypsinized for
proteomic analyses with an S-Trap™ mini column (Proti, Fairport, NY,
USA) according to the manufacturer’s recommendations. After drying
the S-Trap eluate, each sample was dissolved in LC-MS grade water and a
NanoDrop 2000 (ThermoFisher Scientic, Waltham, MA, USA) was used
to estimate peptide concentration by absorbance at 205 nm (extinction
coefcient 31(mL⋅cm)/mg).
2.6. Liquid chromatography
The samples were resuspended in 2 % acetonitrile/0.1 % formic acid
(0.1 µg/µL) and 250 ng injected onto a 300 µm x 5 mm, 5 µm PepMap100
C18 trap cartridge heated at 30◦C (ThermoFisher Scientic) before
elution onto a 75 µm x 15 cm, 3 µm, 100 Å PepMapTM RSLC C18 EASY-
spray separating column heated at 40◦C. Peptides were separated using
a Dionex Ultimate3000 RSLCnano system (ThermoFisher) at 200 nL/
min with an 90 min 5–35 % acetonitrile (0.1 % v/v formic acid)
gradient. An EASY-spray source (ThermoFisher) was used to position the
emitter of the separating column 1 mm from the ion transfer capillary of
the mass spectrometer. The ion transfer capillary temperature and spray
voltage of the mass spectrometer were set at 320◦C and 1.8 kV,
respectively.
2.7. Mass spectrometry
An Orbitrap Exploris480 mass spectrometer (ThermoFisher) was
used to collect data from the LC eluate. A Full MS – ddMS2 method with
a 3 sec cycle time was created in Xcalibur v4.5.445.18 (ThermoFisher)
operating in positive polarity. Scan event one obtained an MS1 scan
(60,000 resolution, Normalized AGC target of 100 %, scan range
350–1400 m/z). Scan event two obtained dd-MS2 scans (7500 resolu-
tion, Normalized AGC target of 50 %) on ions with charge states from 2
to 6 and a minimum intensity of 8000 until the cycle time was complete.
2.8. Proteome data analysis
Proteome Discoverer v2.5.0.400 (ThermoFisher) was used to analyze
the data collected by the mass spectrometer. In the processing step, the
database used in SequestHT was the 1/24/2023 version of the Uni-
protKB canonical Mus musculus sequences (Proteome ID
UP000000589). Trypsin (KR|P) digestion with up to two missed cleav-
ages was assumed with the dynamic modications Oxidation (M), Acetyl
(Protein N-term), Met-los (Protein N-term), and Met-loss+Acetyl (Pro-
tein N-term); and the static modication Carbamidomethyl (C). Pre-
cursor and fragment mass tolerances were 10 ppm and 0.02 Da,
respectively.
In the consensus step, proteins were quantied from the summed
abundances of all high condence unique and razor precursor ion in-
tensities. Samples were normalized to total peptide amount and scaled
to 100 on all average. Proteins were grouped by the strict parsimony
principle. Peptides and proteins were accepted at 1 % FDR for high
condence or 5 % for medium condence based on the q-value. A pro-
teins text le was exported from the consensus workow result of Pro-
teome Discoverer for curation in Microsoft Excel (Supplementary
Methods). Primary proteomic data will be shared through the Mass
Spectrometry Interactive Virtual Environment (MassIVE, https://mass
ive.ucsd.edu/ProteoSAFe/static/massive.jsp) hosted by the Center for
Computational Mass Spectrometry at the University of California, San
Diego. Data will be shared from MassIVE into the ProteomeXchange
Consortium data sharing site (http://www.proteomexchange.org/). The
data will be made public upon manuscript publication.
Hepatic proteins identied in minimally three of the ve individual
livers examined and that had signicance abundance were imported
into MetaCore software (Clarivate Analytics, Philadelphia, PA) for the
following analyses: enrichment by pathway maps, enrichment by pro-
cess networks, gene ontology (GO) process, enrichment by protein
function (EPF), and interaction by protein function (IPF), and tran-
scription factor associated function.
2.9. Western blots
Liver tissues were homogenized, sonicated, and supernatants
collected after 21,000 x g centrifugation. Protein concentrations deter-
mined (BioRad DC protein assay, Bio-Rad Laboratories) and samples
stored as previously described (Petri et al., 2023b). 50 µg of protein were
separated on 10–15 % SDS-PAGE gels and transferred to PVDF mem-
branes (Bio-Rad Laboratories). Membranes were blocked and incubated
overnight at 4◦C with primary antibodies: GPX4 (#67763–1-Ig, Protein
Tech, Rosemont, IL, USA); Selenoprotein S (SELENOS, SELS, #
15591-1-AP, Protein Tech); and
α
-tubulin (Fisher Scientic #
MS581P1). Membranes were washed with TBS-Tween followed by in-
cubation with anti-mouse (#7076 S) or anti-rabbit (#7074 S) (Cell
Signaling Technology, Danvers, MA, USA) secondary antibodies. Mem-
branes were incubated with Clarity Western ECL (Bio-Rad) and imaged
on a Bio-Rad ChemiDoc™ XRS+System with Image Lab™ Software
(Bio-Rad). Blots were stained with Ponceau S and Amido Black for
additional quantication.
2.10. Metals analysis
50–100 mg of each mouse liver sample were homogenized in RIPA
buffer supplemented with protease and phosphatase inhibitors and
centrifuged at 21,000 x g for 10 min. at 4 ◦C. Protein concentrations in
the supernatant were determined using the Bio-Rad DC protein-assay
(Bio-Rad Laboratories Inc., Hercules, CA). 5 mg protein was brought
to a nal volume of 200
μ
l with nuclease-free H
2
O. Each liver protein
sample was digested in 800
μ
l 70 % nitric acid (trace metal grade, Fisher
Scientic Cat# A509-P500) and 200
μ
l H
2
O
2
(Sigma Cat# 95321) at
85 ◦C for 4 hours. Digested samples were transferred to a chemical hood,
allowed to cool to room temperature, and diluted to a nal 5 % nitric
acid with deionized water (Milli Q system). Each sample was ltered by
45 µm lter.
To measure the metal content in liver samples, Agilent 7800 Induc-
tively Coupled Plasma Quadrupole Mass Spectrometer (ICP-MS) (Agi-
lent Technologies, Japan) was used. The Agilent 7800 ICP-MS was
optimized by performance checks with 1 ppb tuning solution and the
assay program was auto tuned by 10 ppb tuning solution (Agilent
Cat#5188–6564). The autosampler SPS 4 was used for sample intro-
duction. A 25 metals calibration standard was purchased from Inorganic
Ventures (Cat# IV-STOCK-50) and serial metal standard dilutions were
made with same acid matrix of samples. Internal standards were pur-
chased from Agilent (Cat# 5188–6525). During sample injection, in-
ternal standards including bismuth, indium, lithium, scandium, terbium
and yttrium were mixed with each sample for drift correction and ac-
curacy improvement. The assay program was run by Agilent MassHunter
software with He mode and each sample was read three times for nal
mean value. Data were analyzed quantile normalization to normalize
the intensity data after logarithm transformation with base 2. Then, the
LIMMA/Moderated t-test was used to compare the vehicle control to
Ar1260 exposure. The p value is the adjustment for the raw p-value
using Benjamini-Hochberg method for multiple testing of chemicals
(Benjamini and Hochberg, 1995).
K.M. Piell et al.

Environmental Toxicology and Pharmacology 107 (2024) 104430
4
3. Results
3.1. Effects of Ar1260 exposure on the hepatic proteome
We recently reported that the top pathway from MetaCore enrich-
ment by pathway maps in our analysis of epitranscriptome and mRNA
changes in the livers from control diet-fed male mice 34 wks. after a
single oral gavage of a mixture of NDL PCBs Ar1260 (20 mg/kg) was
“Response to hypoxia and oxidative stress” (Piell et al., 2023). Inte-
grated network analysis of epitranscriptomic modications identied a
NRF2 (gene Nfe2l2) pathway and we reported that NRF2 protein was
reduced in the mouse livers after chronic Ar1260 exposure (Piell et al.,
2023). We also identied changes in the abundance of RNA modica-
tions mcm5U and i6A (Piell et al., 2023), located specically in the
anticodon loop of Sec-tRNA which is required for the synthesis of sele-
noprotein synthesis (Moustafa Mohamed et al., 2001).
To evaluate the impact of these Ar1260 exposure-mediated alter-
ations in transcript abundance and RNA chemical modications, we
used an unbiased approach to identify proteins altered after the long-
term exposure Ar1260 using mass spectrometry. Of the 2156 proteins
detected in the liver samples (Supplementary Table 1), the abundance of
128 proteins was signicantly different between control and Ar1260-
exposed mice (Fig. 1A and B, Supplementary Table 2). MetaCore
enrichment analysis was performed to identify pathways and networks
specic to these liver protein changes with chronic Ar1260 exposure
(Supplementary Tables 3 and 4). The top signicant pathway was
“Glutathione metabolism” and the fth signicant pathway was
“Translation_(L)-selenoaminoacids incorporation in proteins during
translation” (Supplementary Table 3). We identied seven selenopro-
teins and three Sec synthesis proteins in the liver proteomes of both
control and Ar1260-exposed mice (Table 1, Supplementary Table 5)
with three signicantly different proteins with Ar1260 exposure
(Table 1, Fig. 1C). Glutathione peroxidase 4 (GPX4) and Selenoprotein O
(SELENOO) were increased and Selenoprotein F (SELENOF) was
decreased with Ar1260 exposure (Fig. 1C). A western blot conrmed
higher GPX4 protein levels in the Ar1260- exposed liver samples (Fig. 2).
We also observed a reduction in Selenium binding protein 2 (SELENBP2)
with Ar1260 exposure (Fig. 1B). We identied nine proteins uniquely
expressed in the livers of the Ar1260-exposed mice (Table 2) and 15
proteins uniquely identied in the livers of the control, but not Ar1260
exposed mice (Table 3).
As seen previously in our integrated analysis of transcript (gene,
mRNA-seq) and global epitranscriptomic changes with chronic Ar1260
exposure (Piell et al., 2023), the top signicant network identied in this
proteome analysis was “Response to hypoxia and oxidative stress”
(Supplementary Table 4). The top two GO (gene ontology) processes
were “small molecule metabolic process” and “cellular detoxication”
(Supplementary Table 6). Enrichment by Protein Function (EFP) anal-
ysis was performed and revealed that 24 of the 128 Ar1260-regulated
proteins were enzymes (Supplementary Table 7).
Interaction by protein function (IFP) analysis (Vazquez et al., 2003)
was performed in MetaCore for the 128 proteins regulated by Ar1260
exposure. This analysis identied 68 transcription factors, 19 enzymes,
11 kinases, 7 ligands, and 5 receptors, and 42 ‘other’ proteins over-
connected with Ar1260 exposure (Supplementary Table 8). The
over-connected interactions by z-score are shown in Fig. 3. Thyroid
hormone receptor beta (THRβ) showed the highest overconnection
among the transcription factors. Reduced local hepatic thyroid hormone
activity and THRβ expression have been reported in human MASH
(Krause et al., 2018). Resmetirom, an oral, once-daily, liver-targeted
THR-β selective agonist was reported to be safe and well-tolerated in a
phase 3 clinical trial demonstrating reduced LDL-C, apoB, hepatic fat,
and liver stiffness in adults with presumed MASH (Harrison et al., 2023)
and was recently (3/14/2024) approved by the FDA for treatment of
NASH (MASH) patients who have progressed to brosis. Three tran-
scription factors: Glucocorticoid receptor (GR), SRY-Box Transcription
Factor 17 (SOX17), and ETS Proto-Oncogene 1 (ETS1) (Fig. 3 and Sup-
plementary Table 8) and one enzyme, protein arginine methyltransfer-
ase 1 (PRMT1), were in common with our previous HFD-fed Ar1260
liver proteome analysis (Jin et al., 2020). None of the other enzymes,
ligands, kinases, or proteases in the current analysis of
theAr1260-exposed livers were identied in the IFP analysis of the
HFD-fed, Ar1260-exposed mouse liver proteome (Jin et al., 2020). The
low overlap in IFP suggests that diet and time after initial oral gavage of
Ar1260 impacts hepatic proteome response to Ar1260 exposure.
Methionine adenosyltransferase 1A (MAT1A) had the top z-score
among enzymes. MAT1A is exclusively expressed in the liver and is the
terminal enzyme in the synthesis of S-adenosylmethionine (SAM), the
methyl donor for DNA, RNA, and proteins as well as glutathione syn-
thesis, that is reduced in chronic liver disease (Mato et al., 2013).
Immunoglobulin heavy constant gamma 3 (IGHG3) was the top z-score
in ligands (Fig. 3) and is produced by B lymphocytes which are recruited
to the liver, activated, and release pro-inammatory IL-6 and TNF
α
, that
promote inammation and brogenesis in MAFLD progression to MASH
(reviewed in (Barrow et al., 2021)). IGHG3 (Immunoglobulin heavy
constant gamma 3) transcript was identied in differentiated plasma
cells in human liver by single cell RNA sequencing (MacParland et al.,
2018). IGHG3 was more highly expressed in extracellular vesicles iso-
lated from blood serum of patients with alcoholic hepatitis (AH)
compared to those with MASLD (Nguyen et al., 2021). Dual-Specicity
Tyrosine Phosphorylation–Regulated Kinase 3 (DYRK3) was the top
z-score in kinases (Fig. 3). DYRK3 was reduced in HCC and acts as a
Fig. 1. Long term Ar1260 exposure alters the hepatic proteome. A) Volcano plot of all 2715 proteins in the mouse liver plots showing signicance (y-axis) versus log2
fold change in protein abundance between Ar1260-exposed vs. control livers. B) Signicant alterations in 128 hepatic proteins after Ar1260 exposure were graphed
in a volcano plot. C) Volcano plot of the 7 seleonoproteins and 4 Sec synthesis machinery proteins identied in the proteome analysis. The dotted line separates the
signicantly altered proteins (red dots) with log2FC >2 or <2. Black circles are unaltered proteins.
K.M. Piell et al.

Environmental Toxicology and Pharmacology 107 (2024) 104430
5
tumor suppressor by inhibiting the de novo purine synthesis (Ma et al.,
2019)
Using a cut-off of detection of a protein in minimally three of the ve
individual liver samples/exposure group, nine proteins were uniquely
detected in Ar1260-exposed livers and 15 proteins were uniquely
detected in the control group livers (Tables 1 and 2). For the nine hepatic
proteins uniquely detected in the Ar1260-exposed mice, the top
pathway map was “Androstenedione and testosterone biosynthesis and
metabolism” for UGT1A9 (UDP Glucuronosyltransferase Family 1
Member A9, Supplementary Table 9) in the data and the top process
network was “Response to hypoxia and oxidative stress” for GSTM4
(Glutathione S-Transferase Mu 4, Supplementary Table 10). UGT1A9
protein was reduced in human MASH (Hardwick et al., 2013). GSTM4
transcript was reduced in the livers of patients with steatosis alone or
steatosis +inammation versus obese controls (Younossi et al., 2005).
For the fteen liver proteins detected only in the control diet-fed, vehicle
control mice, the top pathway map was “Development_Epigenetic and
transcriptional regulation of oligodendrocyte precursor cell differenti-
ation and myelination” for QKI (Quaking Homolog, KH Domain RNA
Binding, Supplementary Table 11) and the top process network was
“Protein folding_ER and cytoplasm” for SCO2 (Synthesis Of Cytochrome
C Oxidase 2, Supplementary Table 12). These data suggest that Ar1260
exposure in mice altered the liver proteome in pathways related to
human MASH.
None of the changes in the 128 signicantly altered proteins in the
Ar1260 exposed versus control liver proteome were observed at the
transcript level in the mRNA seq transcriptome (Piell et al., 2023) from
the same liver samples. However, since we observed a decrease in mouse
Selenos (Selenoprotein S) transcript abundance in livers from
Ar1260-exposed mice, although no Selenoprotein S (SELS) in the mouse
proteome, we performed western blots to examine Selenoprotein S.
Western blot conrmed reduced SELENOS liver protein with Ar1260
exposure (Fig. 4). None of the protein changes in the livers of the control
(normal) diet-fed, Ar1260 exposed mice identied here were detected in
proteome data from the livers of HFD-fed, Ar1260 (12 wks., same
dose)-exposed mice (Jin et al., 2020). These data suggest that diet before
and after Ar1260 exposure regulates the liver proteome.
3.2. Effects of Ar1260 exposure on liver metal levels
GO analysis of the Ar1260-altered proteome using the web-based g:
Proler tool (https://biit.cs. ut.ee/gproler/) (Raudvere et al., 2019)
identied “catalytic function” and “selenium binding” as the top GO
molecular function process (Supplementary Figure 2). Ten metals are
considered “essential” for liver homeostasis including Fe, Cu, and Zn
functioning in redox reactions enzymes, metabolism, and in transcrip-
tion (Jomova et al., 2022; Kardos et al., 2018). To our knowledge, no one
has examined the impact of Ar1260 exposure on hepatic metals. The
levels of three metals: Cu, Fe, and Zn and the microelement Se (Table 4)
showed signicant differences in liver samples between mice with and
without Ar1260-exposure with Fe, reduced and Se, Cu, and Zn
increased.
4. Discussion
Although PCBs are ‘legacy contaminants’, they persist in the envi-
ronment and are consumed in foods including sh and dairy products
(reviewed in (Montano et al., 2022)). Human PCB exposures interact
with diet and are associated with metabolic dysfunction and MASLD
(Cave et al., 2022; Clair et al., 2018; Pavuk et al., 2023, 2019; Petriello
et al., 2022). However, the precise mechanisms by which diet and PCB
exposures result in steatohepatitis remain unclear. We recently reported
specic diet and PCB exposure-induced changes in the global tran-
scriptome of mouse liver (Klinge et al., 2021; Piell et al., 2023). For mice
on a normal diet and exposed to Ar1260, we found that the abundance of
specic RNA modications, Am and m6A, were associated with reduced
NRF2 (Nfe2l2) and NFATC4 (Nfatc4) proteins which would be expected
to impair anti-oxidant stress responses and increase fatty acid accumu-
lation in the liver (Piell et al., 2023). In addition, we reported that i6A
was increased and mcm5U was decreased (Petri et al., 2023c), because
Table 1
Seven Selenoproteins and four Sec synthesis proteins were identied in the livers of the normal fed control and Ar1260 exposed mice. These proteins were detected in
at least three of the ve individual livers in each group examined (Supplementary Table 5).
Accession Description # Unique Peptides Gene Symbol Abundance Ratio Adj. P-Value * P <0.05 Log2 fold-
change
P97364 Selenide, water dikinase 2 15 Sephs2 0.864 -0.242
Q9ERR7 Selenoprotein F 2 Selenof 0.014 * -0.894
A4FUU9 Selenoprotein O (Fragment) 5 Selenoo 0.048 * 0.196
O70325 Phospholipid hydroperoxide glutathione
peroxidase
8 Gpx4 0.033 * 0.872
P11352 Glutathione peroxidase 1 21 Gpx1 0.732 0.217
A0A0M3HEQ0 thioredoxin-disulde reductase 5 Txnrd2 0.804 -0.676
Q9JMH6 Thioredoxin reductase 1, cytoplasmic 9 Txnrd1 0.888 -0.125
Q6P6M7 O-phosphoseryl-tRNA(Sec) selenium transferase 2 Sepsecs 0.915 0.135
Q3UGH6 Tr-type G domain-containing protein 6 Eefsec 0.920 0.055
Q8BH69 Selenide, water dikinase 1 2 Sephs1 0.925 0.103
A0A0R4J069 Selenocysteine lyase 7 Scly 0.969 -0.302
Fig. 2. GPX4 was increased in Ar1260-exposed mouse livers. Liver homogenates were prepared from the liver samples from control or Ar1260-exposed mice. GPX4
was quantied relative to Ponceau S staining and normalized to the values in the ve control liver samples. A two-tailed t-test was performed ** p <0.01.
K.M. Piell et al.

Environmental Toxicology and Pharmacology 107 (2024) 104430
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Table 2
Nine proteins uniquely identied in livers of mice 34 wks. after a single oral
gavage of Ar1260. These proteins were detected in at least three of the ve in-
dividual Ar1260-exposed mouse livers examined and in none of the control
mouse livers.
Accession Description and role(s) in liver and
liver diseases
# Unique
Peptides
Gene
Symbol
P62838 Ubiquitin-conjugating enzyme E2 D2 –
ubiquitin-mediated proteolysis; UBE2D2
was increased in human NASH liver
samples (Dahlhoff et al., 2014).
1 Ube2d2
E9QA46 Peroxisomal assembly protein PEX3-
essential for peroxisome membrane
assembly and mutations, frameshifts, or
deletions cause severe metabolic disease
(Fujiki et al., 2012). PEX3 is
phosphorylated by CaMMK2
(calcium/calmodulin-dependent protein
kinase kinase 2) resulting in larger lipid
droplets accumulating in steatosis (Stork
et al., 2022)
1 Pex3
Q8JZL3 Thiamine-triphosphatase
Thtpa transcript abundance was
increased in the livers of HFD-fed male
C7Bl/6 J mice (Soltis et al., 2017)
1 Thtpa
Q3URM1 Caseinolytic peptidase B protein
homolog – acts as tissue-specic
mammalian mitochondrial chaperone
that may play a role in mitochondrial
protein homeostasis (Santagata et al.,
1999)
1 Clpb
Q8R5I6 Glutathione S-transferase Mu 4 – an
oxidative stress-related gene
Induced by 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD) and Aroclor1254 co-
exposure in ApoE KO mice (Shan et al.,
2015). Liver transcript levels were
up-regulated by the herbicides
Glyphosate and/or 2,4-D in male
C57Bl/6 J mice (Romualdo et al., 2023).
2 Gstm4
A0A0R4J1R7 4a-hydroxytetrahydrobiopterin
dehydratase
PCBD2 interacts directly with
hepatocyte nuclear factor 1β (HNF1β)
shuttling between the cytoplasm and the
nucleus, acting as a coactivator to
regulate the ability of HNF1β to regulate
transcription (Tholen et al., 2021).
2 Pcbd2
Q62452 UDP-glucuronosyltransferase 1A9
Induced by TCDD and PCB126 in male
C57Bl/6 mouse liver (Buckley and
Klaassen, 2009).
3 Ugt1a9
Q9EQI8 39 S ribosomal protein L46,
mitochondrial
Is considered a ‘house-keeping gene’ for
mouse liver (Mazin et al., 2022;
Yarushkin et al., 2018).
3 Mrpl46
Q8BTZ7 Mannose-1-phosphate guanyltransferase
beta is a key enzyme in the glycosylation
pathway, which catalyzes the synthesis
of GDP-mannose from mannose-1-
phosphate and guanosine triphosphate (
Franzka et al., 2022). More than 50
mutations in the GMPPB gene have been
identied in patients with congenital
disorders of glycosylation affecting
multiple tissues especially the nervous
system, muscles, and intestines (Liu
et al., 2021). Increased in mouse liver
after 90 consecutive days of intragastric
administration with titanium dioxide
nanoparticles (TiO
2
NPs) (Liu et al.,
2021).
5 Gmppb
Table 3
Fifteen proteins uniquely identied in livers of control mice that were not
detected in the livers of the Ar1260-exposed mice. These proteins were detected
in at least three of the ve individual livers examined.
Accession Description and role(s) in liver and
liver diseases
# Unique
Peptides
Gene
Symbol
H7BXC3 Triosephosphate isomerase: catalyzes
the interconversion of
dihydroxyacetone phosphate (DHAP)
and d-glyceraldehyde-3-phosphate
(G3P) during glycolysis and
gluconeogenesis
TPI1 is reduced in HCC and its
overexpression in HCC cell lines
reduced cell proliferation, invasion,
and migration and induced cell cycle
arrest (Jiang et al., 2017).
1 Tpi1
Q80UT3 Tsfm protein (Fragment): TSFM
activates mitochondrial elongation
factor Tu. Mutation in TSFM results in
severe infantile liver failure (Vedrenne
et al., 2012).
1 Tsfm
A0A077S9N1 Lysozyme f3 – Lysozyme P, a glycosyl
hydrolase; Lyz1 transcript is a maker
for Kupffer cells and monocytes/
macrophages and is elevated in
MAFLD mouse liver after high fructose
and HFD (Su et al., 2021).
1 Lyz1
Q9CWW6 Peptidyl-prolyl cis-trans isomerase
NIMA-interacting 4
PIN4 protein was decreased in the
livers of APOE3 (steatosis and brosis)
versus APOE4 mice fed a HFD +0.2 %
cholesterol (Huebbe et al., 2023).
1 Pin4
Q8BUR8 Steroid 5-alpha reductase C-terminal
domain-containing protein – converts
testosterone to dihydrotestosterone or
3
α
-androstenediol.
Srd5a1 ko mice have reduced
clearance of corticosterone without
primary histological abnormality in
the adrenal gland (Livingstone et al.,
2014). Srd5a1 ko mice show no change
in liver weight (males or females) (
Windahl et al., 2011).
1 Srd5a1
Q9QYS9 KH domain-containing RNA-binding
protein QKI is an RNA reader protein
that binds specic RNA and is involved
in pre-mRNA splicing, circRNA
formation, and in mRNA export,
stability and translation (Neumann
et al., 2022). QKI is upregulated in
HCC (Han et al., 2019). QKI serves as a
coactivator for the PPARβ-RXR
α
complex, which controls the
transcription of lipid-metabolism
genes in oligodendrocytes (Zhou et al.,
2020). Adipocyte-specic deletion of
QKI in mice prevented HFD-induced
liver steatosis and reduced hepatic TG
content (Lu et al., 2020).
1 Qki
O35457 C-C chemokine receptor-like 2 - is a
non-functional G-protein coupled
receptor for the adipokine chemerin
but does not transduce any signals to
the cell. It may dampen inammation
by binding chemokines and reducing
signaling (Del Prete et al., 2013).
Hepatic CCRL2 transcript levels are
positively associated with MASH (
Zimny et al., 2017).
1 Ccrl2
Q3V250 Protein-serine/threonine kinase –
located in the mitochondrial matrix
Pdk3 is expressed in hepatocytes and
was activated by liver overload and
suppressed the activity of AKT,
mTORC1, and mTORC2 thus curtailing
1 Pdk3
(continued on next page)
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Environmental Toxicology and Pharmacology 107 (2024) 104430
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these modications are specically located in the anti-codon loop of
Sec-tRNA (Moustafa Mohamed et al., 2001), we hypothesized that liver
selenoprotein abundance may be altered in response to Ar1260 expo-
sure. Here we used an unbiased approach to evaluate the liver proteomic
prole of these same mice. We report that the abundance of 128 pro-
teins, including seven selenoproteins and four Sec synthesis proteins,
was altered by Ar1260 exposure.The pathways identied from the
altered proteome analysis include “Glutathione Metabolism” and
“Translation (L)-selenoaminoacids incorporation in proteins during
translation”. Based on these terms and GO terms “catalytic function and
“selenium binding, we used metallomics analysis and observed increases
in Se, Cu, Zn and a decrease in Fe in the livers of the Ar1260-exposed
mice.
The decrease in hepatic mcm5U abundance in RNA isolated from the
Ar1260-exposed mice is in agreement with a study in mice showing a
reciprocal relationship between i6A and mcm5U in tRNA iso-
pentenyltransferase 1 (Trit1)-hepatocyte specic knockout mice (Fra-
dejas-Villar et al., 2021). However, Ar1260 exposure did not affect
hepatic Trit1 transcript or protein abundance. TRIT1 is the writer for the
i6A mark; however, i6A abundance was not rate-limiting for seleno-
protein expression, although translation of Sephs2 (Selenophosphate
Synthetase 2), Selenop (Selenoprotein P), and Txnrd1 (Thioredoxin
reductase 1) were reduced in the livers of the Trit1-hepatocyte specic
knockout mice (Fradejas-Villar et al., 2021). This study noted that a
deciency of selenoproteins induced NRF2 (Fradejas-Villar et al., 2021)
and earlier studies showed that knockout of nuclear-encoded tRNA
selenocysteine 2 (gene n-TUtca2 in mouse, protein TRSP) increased liver
phase II detoxifying enzyme gene expression in mice (Sengupta et al.,
2008). Although NRF2 (mouse gene Nfe2l2) was not detected in this
proteome analysis, we recently reported that Ar1260 exposure reduced
Table 3 (continued )
Accession Description and role(s) in liver and
liver diseases
# Unique
Peptides
Gene
Symbol
insulin signaling, lipid synthesis, and
lipid accumulation in liver (Mayer
et al., 2019).
G3UW40 Mutated in colorectal cancers 1 Mcc
Q9QWR8 Alpha-N-acetylgalactosaminidase -
removes terminal alpha-N-
acetylgalactosamine residues from
glycolipids and glycopeptides
2 Naga
P58044 Isopentenyl-diphosphate Delta-
isomerase 1
Idi1 transcript levels were lower in rat
liver regeneration than control livers (
Xu et al., 2008). Idi1 was
down-regulated at the hepatic
transcript level by a 1-wk. high
cholesterol (0.5 %) diet in male and
female C57Bl/6 mice (Maxwell et al.,
2003).
2 Idi1
Q8CAS9 Protein mono-ADP-ribosyltransferase
PARP9
The activity of PARP enzymes is
regulated by lipid molecules including
oxidized cholesterol derivatives,
steroid hormones or bile acids and
PARPs regulate lipid homeostasis (
Sz´
ant´
o et al., 2021). PARP9 and
PARP14 regulate the expression of the
LDL receptor and apolipoproteins in
macrophages (Sz´
ant´
o et al., 2021).
PARP9 promotes macrophage
responses to IFNγ (Iwata et al., 2016).
2 Parp9
E9Q616 AHNAK nucleoprotein (desmoyokin)
Identied as a phosphoprotein in
female C57Bl/6 mouse liver (Jin et al.,
2004). Ahnak KO mice are resistant to
HFD-induced obesity and hepatic
steatosis because FGF21 and PPAR
α
are upregulated in the absence of
Ahnak expression (Kim et al., 2021).
3 Ahnak
Q8VCL2 Protein SCO2 homolog, mitochondrial
SCO2 is copper chaperone required for
Cytochrome c oxidase (COX) activity
in Complex IV of the mitochondrial
respiratory chain (Hill et al., 2017).
Sco2 knockout mice show increased
hepatic steatosis, elevated serum and
liver TG and serum cholesterol (Hill
et al., 2017).
4 Sco2
Fig. 3. Effect of Ar1260 on liver protein function. Interaction by protein
function (IFP) analysis was performed in MetaCore for the 128 liver proteins
differentially expressed in Ar1260-exposed mice with the top proteins shown
with the corresponding z-scores shown in the heatmap.
Fig. 4. Selenoprotein S (SELENOS) was decreased in Ar1260-exposed mouse
livers. Liver homogenates were prepared from the liver samples from control or
Ar1260-exposed mice. SELS was quantied relative to Amido Black staining
and normalized to the values in the ve control liver samples. A two-tailed
Mann-Whitney was performed. * p =0.0159.
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Environmental Toxicology and Pharmacology 107 (2024) 104430
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NRF2 liver protein levels in mice exposed to Ar1260 (Piell et al., 2023)
and mice exposed to HFD (Hardesty et al., 2019).
The liver is the central organ for Se regulation and the synthesis of
selenoproteins requires unique RNA elements and proteins. We found
that Ar1260 exposure increased liver Se in mice. The Sec insertion
sequence (SECIS) element in the 3
′
-untranslated region (3
′
-UTR) is
required for recruitment of Sec-tRNA (N-TUtca2), in a complex with
Eefsec (eukaryotic elongation factor, selenocysteine-tRNA-specic,
EFSEC or SELB) and SECIS binding protein 2 (Secisbps, SBP2) to insert
Sec in response to a UGA codon (Burk and Hill, 2015; Labunskyy et al.,
2014). There was no change in n-TUtca2, Eefsec, or Secisbp2 transcript
abundance with Ar1260 exposure (Piell et al., 2023). An increase in
Secisbp2l (SECIS binding protein 2-like, protein SBP2L) transcript in
Ar1260-exposed mouse liver (Piell et al., 2023). SBP2L binds SECIS el-
ements in Sec-protein mRNAs (The UniProt, 2021). SBP2L was not
detected in our mouse proteome analysis.
Of the 24 mouse selenoprotein genes, only glutathione peroxidase
(Gpx3) transcript abundance was increased in Ar1260-exposed livers
and Selenoprotein S (Selenos, protein SELS) transcript was decreased in
Ar1260-exposed livers (Piell et al., 2023). In the present study, GPX4
and SELENOO (SELO) were increased and SELENOF was decreased. The
increases in GPX4 and SELENOO were associated with the “Glutathione
Metabolism” and “Translation_(L)-selenoaminoacids incorporation in
proteins during translation” enrichment pathways identied. Increased
GPX4 protein was conrmed by western blot. GPX4 protects cells
against lipid peroxidation and inhibits ferroptosis (Wu et al., 2021; Yang
et al., 2014).
Here, reduced SELENOF protein was observed in the Ar1260-
exposed livers, despite not identifying Selenof in the mouse tran-
scriptome (Piell et al., 2023). Selenof KO mice (C57Bl/6) showed
enhanced HFD (45 % fat)-induced hepatic steatosis and reduced
SELENBP2 (Zheng et al., 2020), as seen in the current study. Previously,
we reported a decrease in mouse Selenos hepatic transcript abundance
in Ar1260-exposed mice (Piell et al., 2023) and here we demonstrated a
reduction in SELENOS (SELS) protein in the same livers. In contrast,
Selenof KO mice showed increased SELENOS (SELS) protein in liver (Li
et al., 2022). We note that MASH livers have lower SELENOS transcript
expression compared to healthy controls (Day et al., 2021). Thus, the
potential interplay among selenoproteins in liver in response to Ar1260
exposure requires further study.
We detected a decrease in SELENBP2 protein, previously known as
acetaminophen-binding protein (AP56), a cytosolic xenobiotic receptor
(Giometti et al., 2000; Mattow et al., 2006). We did not detect a dif-
ference in Selenbp2 transcript abundance in the Ar1260 exposed liver
relative to controls (Piell et al., 2023), suggesting that Ar1260 regulation
of SELENBP2 protein may be post-transcriptional. Since miRNAs
post-transcriptionally regulate protein abundance, we examined which
miRs regulate SELENBP2 with one result: miR-24–3p repressed
SELENBP2 translation in mouse liver (Zhang et al., 2020a). However,
miR-24–3p levels were not affected by Ar1260-exposure in HFD-fed
mouse liver (Petri et al., 2022) and miR-24–3p not identied among
altered miRNAs found in our RNA-seq analysis of the control or
Ar1260-exposed livers used here, although miRNA-seq was not per-
formed (Piell et al., 2023). Future studies will examine the mechanism
by which Ar1260 exposure reduces hepatic SELENBP2 levels.
To our knowledge, this is the rst examination of metals in the livers
of rodents exposed to Ar1260, a predominantly NDL PCB mixture. We
report increased Se, Cu, and Zn whereas Fe was decreased in the livers of
Ar1260-exposed mice. Previously, a single i.p. injection of male
Sprague-Dawley (SD) rats with a 1
μ
mol/kg dose of DL PCB126 resulted
a decrease in Se and Zn and an increase in Cu after 2 wks. (Lai et al.,
2010). Similarly, a single i.p. injection of male SD rats with a 5
μ
mol/kg
(1.63 mg/kg) dose of DL PCB126 resulted in a time-dependent increase
in hepatic Cu between days 3 – 12, increases in hepatic Zn at 9 hr.
post-injection and again day 3, and an increase in hepatic Se at 9 hr., but
decrease at day 12 (Klaren et al., 2015). The study also reported a
non-signicant decrease in hepatic Fe after days 6 – 12 (Klaren et al.,
2015). In another study, female SD rats received multi i.p. injections of
100
μ
mol/kg DL PCB77, which was not detected in Ar1260 (Wahlang
et al., 2014a), twice weekly for 1, 2 or 3 wks. (Twaroski et al., 2001). The
authors reported a time- (accumulated dose) dependent decrease in
hepatic Se levels to almost half of control levels by week 3, while male
SD rats showed a signicant decrease in hepatic Se (Twaroski et al.,
2001). Although these limited studies did not demonstrate specic
patterns of DL PCBs exposures on hepatic essential metals due to the
differences in exposure doses, frequency (single vs multi exposure), and
sexes, increased hepatic Cu and decreased hepatic Se levels showed
apparent similarity among the three studies. Here we demonstrated that
a single oral exposure of mice to Ar1260 also resulted in increased he-
patic Cu, consistent with the ndings in SD rats exposed to DL PCBs
(Klaren et al., 2015; Qian et al., 2015; Twaroski et al., 2001).
Few studies in mice have investigated the effects of exposure to PCBs
on liver Fe metabolism. A single i.p. injection of PCB126 (20 or 70 mg/
kg) signicantly reduced hepatic Fe and increased serum Fe in female
C57Bl/6 mice at 48 hr. (Qian et al., 2015). In contrast, four i.p. in-
jections of PCB126 (1 or 5 mg/kg at 2, 3, 4, and 5 wks.), which resulted
in steatohepatitis, increased hepatic Fe and reduced serum Fe at the 6th
wk. in male C57Bl/6 mice (Kim et al., 2022). Another study adminis-
tered Ar1260 (10 or 20 mg/kg) i.p. at 2, 3, 4, and 5 wks., which induced
hepatic steatosis and inammation, but Fe was not measured (52).
Therefore, no model similar to that used in our current study is available
to directly compare the effects of Ar1260 and DL PCBs on mouse liver
metals. Here, decreased hepatic Fe levels in Ar1260 exposed mice were
similar to the ndings of a small decrease in hepatic Fe in female
C57Bl/6 mice 48 hr after i.p. injection of PCB126 (Qian et al., 2015).
Taken together, we suggest that exposure of rats and mice to DL PCBs
and NDL PCBs caused signicant changes of hepatic essential metals
(Cu, Fe, Zn, and Se). Further examination of dose-, time-, sex-, species-
dependent responses to PCBs on essential metal homeostasis remains
further explored.
In human MASH livers, altered levels of essential elements including
Se, Cu, Zn, and Fe have been reported (reviewed in (Day et al., 2021; Ma
et al., 2022)) and dyshomeostasis of essential metals, particularly
increased Cu or the ratio of Cu to Zn, has been extensively discussed as
important mechanisms for the liver diseases and cancer development
(Lin et al., 2006; Liu et al., 2023; Tamai et al., 2020). For example,
patients with Wilson disease, caused by inactivation of the Cu trans-
porter ATP7B that results in increased liver Cu liver, have steatosis,
inammation, brosis, and liver failure (Gottlieb et al., 2022). It is
difcult to directly compare changes in essential metals and elements by
PCB exposures in this mouse model of MASLD with human studies.
There are few studies examining metals and elements in livers from
human MASLD or MASH patients. Livers from patients with MASLD
showed low Cu (Aigner et al., 2008) and high Fe (Younossi et al., 1999),
ndings opposite of the current study in Ar1260-exposed mice. Most
human studies examine blood, serum, or plasma elements in MASLD
patients. High blood Se was associated with MASLD while MASH was
Table 4
Metal analysis in liver samples by ICP-MS. Five liver samples from normal diet-
fed, vehicle control mice and ten liver samples from Ar1260-exposed mice were
analyzed. Data were analyzed quantile normalization to normalize the intensity
data after logarithm transformation with base 2. Then, the LIMMA/Moderated t-
test was used to compare the vehicle control to Ar1260 exposure. The p value is
the adjustment for the raw p-value using BH method for multiple testing of
chemicals (Benjamini and Hochberg, 1995). Positive and negative values mean
increased and decreased levels, respectively.
Metal Estimate logFC adj. p-value
Cu 0.0152 0.0000
Fe -0.1238 0.0001
Se 0.2170 0.0000
Zn 0.0456 0.0000
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Environmental Toxicology and Pharmacology 107 (2024) 104430
9
associated with low blood Se (Liu et al., 2022). Patients with MASLD
showed lower serum Zn (Ito et al., 2020).
In the present study, hepatic Fe levels were reduced along with
increased Se and GPX4, suggesting that the livers of the mice exposed to
Ar1260 for 34 wks. demonstrated ferroptosis resistance. We also
observed increased hepatic Cu. Exogenous Cu was reported to increase
GPX4 protein levels along with ubiquitination and aggregation, pro-
moting ferroptosis under certain conditions (Xue et al., 2023). The in-
crease in GPX4 protein agrees with other reports: GPX4 protein was
increased ~ 15–25 % in C57Bl/6 mouse liver with MCD (methionine
and choline decient) diet-induced steatosis for 8–12 wks. (Lu et al.,
2021) and GPX4 is elevated in MASH (Day et al., 2021). SELENOO lo-
calizes to mitochondria and participates in redox reactions (Han et al.,
2014). SELENOO transcript abundance was lower in MASH compared to
healthy controls (Day et al., 2021) and lower in HepG2 and Huh7 human
hepatoma cells compared to normal human hepatocytes (Guariniello
et al., 2015). In contrast with these ndings, we demonstrated the
increased expression of SELENOO in the liver of mice at 34 wks. after a
single exposure to Ar1260. We suggest that increased GPX4 and SELE-
NOO with Ar1260 exposure are likely hepatic protective measures.
In the present study, the biological role of the increase in hepatic Zn
after Ar1260 exposure in male mice observed here is unknown. In SD
rats, Zn status and dietary supplementation did not affect PCB126 he-
patic toxicity (Klaren et al., 2016). In the present study, we speculate
that the Ar1260-exposed mouse liver may be ferroptosis-resistant due to
increased expression of GPX4 and a lack of free iron and apoptotic
resistance due to the increased Zn and Zn-metallothionein. However, the
chronic pathogenic metabolism and carcinogenic processes caused by
PCB exposures and increased reactive metals such as Cu, which causes
liver cancer development as reported in human studies (Deen et al.,
2022; Donato et al., 2021; Ludewig and Robertson, 2013), was seen in
some mice in our recent long-term Ar1260 study (Head et al., 2023).
5. Limitations
Despite the strengths of the current study, e.g., high sample numbers
and unbiased analysis, the identication of 128 proteins differentially
expressed in Ar1260-exposed mice 34 wks. after a single oral gavage of
20 mg/kg is a small change in the liver proteome. We eliminated pro-
teins from analysis in which less than three liver samples in each of the
two groups (control or Ar1260-exposed) showed values. Further, as re-
ported for our study in HFD-fed mice exposed to Ar1260 (Klinge et al.,
2021; Petri et al., 2023b, 2022, 2023c), and in other models of steatotic
liver disease (Herranz et al., 2023), the transcript abundance does not
reect protein abundance likely due to multiple mechanisms and anal-
ysis issues (Fortelny et al., 2017; Poverennaya et al., 2017), including
epitranscriptomic and epigenetic pathways regulating
post-transcriptional processing and protein synthesis (Kan et al., 2022;
Petri et al., 2023a).
6. Conclusions
In summary, this study identied 128 liver proteins, including
changes in seven selenoproteins and four Sec synthesis proteins, regu-
lated by exposure of mice to environmentally relevant PCB mixture,
Ar1260. Major ndings were the regulation of selenoproteins and
SELENBP2 and the increase in liver Se, Cu, and Zn and the reduction in
Fe in these mice. The lack of concordance between transcript and pro-
tein abundance in this Ar1260-exposure-mediated model of MAFLD in
mice on a normal diet suggest that epigenetic and epitranscriptomic
regulation of proteins in pathways associated with liver homeostasis and
pathology require further investigation.
Funding
This work was supported, in part by NIH R21ES031510, ES031510-
01S1, P30ES030283, R35ES028373, R01ES032189, T32ES011564,
P42ES023716, P20GM113226; P50AA024337, P20GM103436, the
Kentucky Council on Postsecondary Education (PON2 415
1900002934). The authors acknowledge the following CIEHS cores for
their assistance with this project: the Integrative Health Science Facility
Core (IHSFC); the Integrated Toxicomics & Environment Measurement
Facility Core (ITEMFC); and an ITEMFC Research Voucher Award.
CRediT authorship contribution statement
Kellianne M. Piell: Investigation. Carolyn M. Klinge: Writing –
review & editing, Conceptualization, Data curation, Formal analysis,
Funding acquisition, Project administration, Resources, Supervision,
Writing – original draft. Shesh N. Rai: Formal analysis. Lu Cai: Writing
– review & editing. Jason Xu: Investigation, Formal analysis. Belinda J.
Petri: Writing – review & editing, Investigation, Formal analysis.
Matthew C. Cave: Writing – review & editing, Supervision, Resources,
Funding acquisition. Michael L. Merchant: Writing – review & editing,
Formal analysis, Data curation. Daniel W. Wilkey: Funding acquisition.
Ming Li: Formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
Primary proteomic data will be shared in https://massive.ucsd.edu/
ProteoSAFe/static/massive.jsp and from MassIVE into the Proteo-
meXchange Consortium (http://www.proteomexchange.org/) upon
publication
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.etap.2024.104430.
References
Aigner, E., Theurl, I., Haufe, H., Seifert, M., Hohla, F., Scharinger, L., Stickel, F.,
Mourlane, F., Weiss, G., Datz, C., 2008. Copper Availability Contributes to Iron
Perturbations in Human Nonalcoholic Fatty Liver Disease. Gastroenterology 135,
680–688 e681.
ANON, 2006. Toxicology and carcinogenesis studies of a binary mixture of 3,3
′
,4,4
′
,5-
pentachlorobiphenyl (PCB 126) (Cas No. 57465-28-8) and 2,3
′
,4,4
′
,5-
pentachlorobiphenyl (PCB 118) (Cas No. 31508-00-6) in female Harlan Sprague-
Dawley rats (gavage studies). Natl. Toxicol. Program Tech. Rep. Ser. 1–218, 2007/
03/08 ed.
Barrow, F., Khan, S., Wang, H., Revelo, X.S., 2021. The Emerging Role of B Cells in the
Pathogenesis of NAFLD. Hepatology(Baltim. Md.) 74, 2277–2286.
Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and
powerful approach to multiple testing. J. Roy. Stat. Soc. Ser. B 57, 289–300.
Buckley, D., B., Klaassen, C., D., 2009. Induction of mouse UDP-glucuronosyltransferase
mRNA expression in liver and intestine by activators of aryl-hydrocarbon receptor,
constitutive androstane receptor, pregnane x receptor, peroxisome proliferator-
activated receptor
α
, and nuclear factor erythroid 2-related factor 2. Drug Metab.
Dispos. 37, 847.
Burk, R.F., Hill, K.E., 2015. Regulation of selenium metabolism and transport. Annu. Rev.
Nutr. 35, 109–134.
Carlson, B.A., Lee, B.J., Tsuji, P.A., Copeland, P.R., Schweizer, U., Gladyshev, V.N.,
Hateld, D.L., 2018. Selenocysteine tRNA([Ser]Sec), the Central Component of
Selenoprotein Biosynthesis: Isolation, Identication, Modication, and Sequencing.
Methods Mol. Biol. (Clifton, N. J.) 1661, 43–60.
Cave, M., C., Pinkston, C., M., Rai, S.N., Wahlang, B., Pavuk, M., Head, K., Z.,
Carswell, G., K., Nelson, G., M., Klinge, C., M., Bell, D., A., Birnbaum, L., S.,
Chorley, B., N., 2022. Circulating MicroRNAs, polychlorinated biphenyls, and
environmental liver disease in the anniston community health survey. Environ.
Health Perspect. 130, 017003.
Cave, M.C., Clair, H.B., Hardesty, J.E., Falkner, K.C., Feng, W., Clark, B.J., Sidey, J.,
Shi, H., Aqel, B.A., McClain, C.J., Prough, R.A., 2016. Nuclear receptors and
K.M. Piell et al.

Environmental Toxicology and Pharmacology 107 (2024) 104430
10
nonalcoholic fatty liver disease. Biochim. Et. Biophys. Acta (BBA) - Gene Regul.
Mech. 1859, 1083–1099.
Clair, H.B., Prough, R.A., Cave, M.C., Brock, G.N., Pinkston, C.M., Rai, S.N., McClain, C.
J., Falkner, K.C., Pavuk, M., Dutton, N.D., 2018. Liver disease in a residential cohort
with elevated polychlorinated biphenyl exposures. Toxicol. Sci. 164, 39–49.
Curtis, S.W., Cobb, D.O., Kilaru, V., Terrell, M.L., Marder, M.E., Barr, D.B., Marsit, C.J.,
Marcus, M., Conneely, K.N., Smith, A.K., 2021. Genome-wide DNA methylation
differences and polychlorinated biphenyl (PCB) exposure in a US population.
Epigenetics 16, 338–352.
Dahlhoff, M., Pster, S., Blutke, A., Rozman, J., Klingenspor, M., Deutsch, M.J.,
Rathkolb, B., Fink, B., Gimp, M., Hrabˇ
e de Angelis, M., Roscher, A.A., Wolf, E.,
Ensenauer, R., 2014. Peri-conceptional obesogenic exposure induces sex-specic
programming of disease susceptibilities in adult mouse offspring. Biochim. Et.
Biophys. Acta 1842, 304–317.
Day, K., Seale, L.A., Graham, R.M., Cardoso, B.R., 2021. Selenotranscriptome Network in
Non-alcoholic Fatty Liver Disease. Front. Nutr. 8.
Deen, L., Hougaard Karin, S., Clark, A., Meyer Harald, W., Frederiksen, M.,
Gunnarsen, L., Andersen Helle, V., Hougaard, T., Petersen Kajsa Kirstine, U., Ebbehøj
Niels, E., Bonde Jens, P., Tøttenborg Sandra, S., 2022. Cancer risk following
residential exposure to airborne polychlorinated biphenyls: a danish register-based
cohort study. Environ. Health Perspect. 130, 107003.
Del Prete, A., Bonecchi, R., Vecchi, A., Mantovani, A., Sozzani, S., 2013. CCRL2, a fringe
member of the atypical chemoattractant receptor family. Eur. J. Immunol. 43,
1418–1422.
Donato, F., Moneda, M., Portolani, N., Rossini, A., Molno, S., Ministrini, S., Contessi, G.
B., Pesenti, S., De Palma, G., Gaia, A., Zanardini, E., Sileo, C.V., Magoni, M., 2021.
Polychlorinated biphenyls and risk of hepatocellular carcinoma in the population
living in a highly polluted area in Italy. Sci. Rep. 11, 3064.
Fortelny, N., Overall, C.M., Pavlidis, P., Freue, G.V.C., 2017. Can we predict protein from
mRNA levels? Nature 547, E19–E20.
Fradejas-Villar, N., Bohleber, S., Zhao, W., Reuter, U., Kotter, A., Helm, M., Knoll, R.,
McFarland, R., Taylor, R.W., Mo, Y., Miyauchi, K., Sakaguchi, Y., Suzuki, T.,
Schweizer, U., 2021. The effect of tRNA([Ser]Sec) isopentenylation on selenoprotein
expression. Int. J. Mol. Sci. 22.
Franzka, P., Turecki, G., Cubillos, S., Kentache, T., Steiner, J., Walter, M., Hübner, C.A.,
Engmann, O., 2022. Altered mannose metabolism in chronic stress and depression is
rapidly reversed by vitamin B12. Front. Nutr. 9.
Fujiki, Y., Yagita, Y., Matsuzaki, T., 2012. Peroxisome biogenesis disorders: Molecular
basis for impaired peroxisomal membrane assembly: In metabolic functions and
biogenesis of peroxisomes in health and disease. Biochim. Et. Biophys. Acta (BBA) -
Mol. Basis Dis. 1822, 1337–1342.
Giometti, C.S., Liang, X., Tollaksen, S.L., Wall, D.B., Lubman, D.M., Subbarao, V., Rao, M.
S., 2000. Mouse liver selenium-binding protein decreased in abundance by
peroxisome proliferators. Electrophoresis 21, 2162–2169.
Gottlieb, A., Dev, S., DeVine, L., Gabrielson, K.L., Cole, R.N., Hamilton, J.P., Lutsenko, S.,
2022. Hepatic Steatosis in the Mouse Model of Wilson Disease Coincides with a
Muted Inammatory Response. Am. J. Pathol. 192, 146–159.
Guariniello, S., Di Bernardo, G., Colonna, G., Cammarota, M., Castello, G., Costantini, S.,
2015. Evaluation of the selenotranscriptome expression in two hepatocellular
carcinoma cell lines. Anal. Cell. Pathol. (Amst.) 2015, 419561.
Han, J., Meng, J., Chen, S., Wang, X., Yin, S., Zhang, Q., Liu, H., Qin, R., Li, Z., Zhong, W.,
Zhang, C., Zhang, H., Tang, Y., Lin, T., Gao, W., Zhang, X., Yang, L., Liu, Y., Zhou, H.-
g, Sun, T., Yang, C., 2019. YY1 complex promotes quaking expression via super-
enhancer binding during EMT of hepatocellular carcinoma. Cancer Res. 79,
1451–1464.
Han, S.-J., Lee, B.C., Yim, S.H., Gladyshev, V.N., Lee, S.-R., 2014. Characterization of
mammalian selenoprotein O: a redox-active mitochondrial protein. PLoS One 9,
e95518.
Hardesty, J., E., Wahlang, B., Prough Russell, A., Head Kim, Z., Wilkey, D., Merchant, M.,
Shi, H., Jin, J., Cave, M., C., 2021. Effect of epidermal growth factor treatment and
polychlorinated biphenyl exposure in a dietary-exposure mouse model of
steatohepatitis. Environ. Health Perspect. 129, 037010.
Hardesty, J.E., Wahlang, B., Falkner, K.C., Shi, H., Jin, J., Zhou, Y., Wilkey, D.W.,
Merchant, M.L., Watson, C.T., Feng, W., Morris, A.J., Hennig, B., Prough, R.A.,
Cave, M.C., 2019. Proteomic Analysis Reveals Novel Mechanisms by Which
Polychlorinated Biphenyls Compromise the Liver Promoting Diet-Induced
Steatohepatitis. J. Proteome Res. 18, 1582–1594.
Hardwick, R.N., Ferreira, D.W., More, V.R., Lake, A.D., Lu, Z., Manautou, J.E., Slitt, A.L.,
Cherrington, N.J., 2013. Altered UDP-glucuronosyltransferase and sulfotransferase
expression and function during progressive stages of human nonalcoholic fatty liver
disease. Drug Metab. Dispos.: Biol. Fate Chem. 41, 554–561.
Harrison, S.A., Taub, R., Neff, G.W., Lucas, K.J., Labriola, D., Moussa, S.E., Alkhouri, N.,
Bashir, M.R., 2023. Resmetirom for nonalcoholic fatty liver disease: a randomized,
double-blind, placebo-controlled phase 3 trial. Nat. Med. 29, 2919–2928.
Head, K.Z., Bolatimi, O.E., Gripshover, T.C., Tan, M., Li, Y., Audam, T.N., Jones, S.P.,
Klinge, C.M., Cave, M.C., Wahlang, B., 2023. Investigating the effects of long-term
Aroclor 1260 exposure on fatty liver disease in a diet-induced obesity mouse model.
Front. Gastroenterol. 2.
Herranz, J.M., L´
opez-Pascual, A., Clavería-Cabello, A., Uriarte, I., Latasa, M.U., Irigaray-
Miramon, A., Ad´
an-Villaescusa, E., Castell´
o-Uribe, B., Sangro, B., Arechederra, M.,
Berasain, C., Avila, M.A., Fern´
andez-Barrena, M.G., 2023. Comprehensive analysis of
epigenetic and epitranscriptomic genes’ expression in human NAFLD. J. Physiol.
Biochem. 79, 901–924.
Hill, S., Deepa, S.S., Sataranatarajan, K., Premkumar, P., Pulliam, D., Liu, Y., Soto, V.Y.,
Fischer, K.E., Van Remmen, H., 2017. Sco2 decient mice develop increased
adiposity and insulin resistance. Mol. Cell. Endocrinol. 455, 103–114.
Huebbe, P., Bilke, S., Rueter, J., Schloesser, A., Campbel, G., Glüer, C.C., Lucius, R.,
R¨
ocken, C., Tholey, A., Rimbach, G., 2023. Human APOE4 Protects High-Fat and
High-Sucrose Diet Fed Targeted Replacement Mice against Fatty Liver Disease
Compared to APOE3. Aging and disease.
Ito, T., Ishigami, M., Ishizu, Y., Kuzuya, T., Honda, T., Ishikawa, T., Toyoda, H.,
Kumada, T., Fujishiro, M., 2020. Correlation of serum zinc levels with pathological
and laboratory ndings in patients with nonalcoholic fatty liver disease. Eur. J.
Gastroenterol. Hepatol. 32.
Iwata, H., Goettsch, C., Sharma, A., Ricchiuto, P., Goh, W.W., Halu, A., Yamada, I.,
Yoshida, H., Hara, T., Wei, M., Inoue, N., Fukuda, D., Mojcher, A., Mattson, P.C.,
Barab´
asi, A.L., Boothby, M., Aikawa, E., Singh, S.A., Aikawa, M., 2016. PARP9 and
PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation. Nat.
Commun. 7, 12849.
Jiang, H., Ma, N., Shang, Y., Zhou, W., Chen, T., Guan, D., Li, J., Wang, J., Zhang, E.,
Feng, Y., Yin, F., Yuan, Y., Fang, Y., Qiu, L., Xie, D., Wei, D., 2017. Triosephosphate
isomerase 1 suppresses growth, migration and invasion of hepatocellular carcinoma
cells. Biochem. Biophys. Res. Commun. 482, 1048–1053.
Jin, J., Wahlang, B., Shi, H., Hardesty, J.E., Falkner, K.C., Head, K.Z., Srivastava, S.,
Merchant, M.L., Rai, S.N., Cave, M.C., Prough, R.A., 2020. Dioxin-like and non-
dioxin-like PCBs differentially regulate the hepatic proteome and modify diet-
induced nonalcoholic fatty liver disease severity. Med. Chem. Res. 29, 1247–1263.
Jin, W.-H., Dai, J., Zhou, H., Xia, Q.-C., Zou, H.-F., Zeng, R., 2004. Phosphoproteome
analysis of mouse liver using immobilized metal afnity purication and linear ion
trap mass spectrometry. Rapid Commun. Mass Spectrom. 18, 2169–2176.
Jomova, K., Makova, M., Alomar, S.Y., Alwasel, S.H., Nepovimova, E., Kuca, K.,
Rhodes, C.J., Valko, M., 2022. Essential metals in health and disease. Chem. -Biol.
Interact. 367, 110173.
Kan, R.L., Chen, J., Sallam, T., 2022. Crosstalk between epitranscriptomic and epigenetic
mechanisms in gene regulation. Trends Genet. 38, 182–193.
Kardos, J., H´
eja, L., Simon, ´
A., Jablonkai, I., Kov´
acs, R., Jemnitz, K., 2018. Copper
signalling: causes and consequences. Cell Commun. Signal.: CCS 16, 71.
Kim, H.Y., Park, C.H., Park, J.B., Ko, K., Lee, M.H., Chung, J., Yoo, Y.H., 2022. Hepatic
STAMP2 alleviates polychlorinated biphenyl-induced steatosis and hepatic iron
overload in NAFLD models. Environ. Toxicol. 37, 2223–2234.
Kim, Y.N., Shin, J.H., Kyeong, D.S., Cho, S.Y., Kim, M.-Y., Lim, H.J., Jimenez, M.R.R.,
Kim, I.Y., Lee, M.-O., Bae, Y.S., Seong, J.K., 2021. Ahnak deciency attenuates high-
fat diet-induced fatty liver in mice through FGF21 induction. Exp. Mol. Med. 53,
468–482.
Klaren, W.D., Gadupudi, G.S., Wels, B., Simmons, D.L., Olivier, A.K., Robertson, L.W.,
2015. Progression of micronutrient alteration and hepatotoxicity following acute
PCB126 exposure. Toxicology 338, 1–7.
Klaren, W.D., Gibson-Corley, K.N., Wels, B., Simmons, D.L., McCormick, M.L., Spitz, D.
R., Robertson, L.W., 2016. Assessment of the mitigative capacity of dietary zinc on
PCB126 hepatotoxicity and the contribution of zinc to toxicity. Chem. Res. Toxicol.
29, 851–859.
Klinge, C.M., Piell, K.M., Petri, B.J., He, L., Zhang, X., Pan, J., Rai, S.N., Andreeva, K.,
Rouchka, E.C., Wahlang, B., Beier, J.I., Cave, M.C., 2021. Combined exposure to
polychlorinated biphenyls and high-fat diet modies the global epitranscriptomic
landscape in mouse liver. Environ. Epigenet. 7, dvab008.
Krause, C., Grohs, M., El Gammal, A.T., Wolter, S., Lehnert, H., Mann, O., Mittag, J.,
Kirchner, H., 2018. Reduced expression of thyroid hormone receptor β in human
nonalcoholic steatohepatitis. Endocr. Connect. 7, 1448–1456.
Labunskyy, V.M., Hateld, D.L., Gladyshev, V.N., 2014. Selenoproteins: molecular
pathways and physiological roles. Physiol. Rev. 94, 739–777.
Lai, I., Chai, Y., Simmons, D., Luthe, G., Coleman, M.C., Spitz, D., Haschek, W.M.,
Ludewig, G., Robertson, L.W., 2010. Acute toxicity of 3,3
′
,4,4
′
,5-
pentachlorobiphenyl (PCB 126) in male Sprague–Dawley rats: effects on hepatic
oxidative stress, glutathione and metals status. Environ. Int. 36, 918–923.
Lai, I.K., Chai, Y., Simmons, D., Watson, W.H., Tan, R., Haschek, W.M., Wang, K.,
Wang, B., Ludewig, G., Robertson, L.W., 2011. Dietary selenium as a modulator of
PCB 126-induced hepatotoxicity in male Sprague-Dawley rats. Toxicol. Sci. 124,
202–214.
Li, M., Zhang, Y., Zhou, J., Liu, H., 2022. Selenoprotein F knockout caused glucose
metabolism disorder in young mice by disrupting redox homeostasis. Antioxidants
11, 2105.
Lin, C.-C., Huang, J.-F., Tsai, L.-Y., Huang, Y.-L., 2006. Selenium, iron, copper, and zinc
levels and copper-to-zinc ratios in serum of patients at different stages of viral
hepatic diseases. Biol. Trace Elem. Res. 109, 15–23.
Liu, J., Tan, L., Liu, Z., Shi, R., 2022. The association between non-alcoholic fatty liver
disease (NAFLD) and advanced brosis with blood selenium level based on the
NHANES 2017-2018. Ann. Med. 54, 2258–2267.
Liu, X., Zhang, Y., Yishake, D., Luo, Y., Liu, Z., Chen, Y., Zhu, H., Fang, A., 2023. Dietary
intake and serum levels of copper and zinc and risk of hepatocellular carcinoma: a
matched case-control study. Chin. Med. J.
Liu, Z., Wang, Y., Yang, F., Yang, Q., Mo, X., Burstein, E., Jia, D., Cai, X.-t, Tu, Y., 2021.
GMPPB-congenital disorders of glycosylation associate with decreased enzymatic
activity of GMPPB. Mol. Biomed. 2, 13.
Livingstone, D.E., Di Rollo, E.M., Yang, C., Codrington, L.E., Mathews, J.A., Kara, M.,
Hughes, K.A., Kenyon, C.J., Walker, B.R., Andrew, R., 2014. Relative adrenal
insufciency in mice decient in 5
α
-reductase 1. J. Endocrinol. 222, 257–266.
Lu, D., Xia, Q., Yang, Z., Gao, S., Sun, S., Luo, X., Li, Z., Zhang, X., Han, S., Li, X., Cao, M.,
2021. ENO3 promoted the progression of NASH by negatively regulating ferroptosis
via elevation of GPX4 expression and lipid accumulation. Ann. Transl. Med 9, 661.
Lu, H., Ye, Z., Zhai, Y., Wang, L., Liu, Y., Wang, J., Zhang, W., Luo, W., Lu, Z., Chen, J.,
2020. QKI regulates adipose tissue metabolism by acting as a brake on
thermogenesis and promoting obesity. EMBO Rep. 21, e47929.
K.M. Piell et al.

Environmental Toxicology and Pharmacology 107 (2024) 104430
11
Ludewig, G., Robertson, L.W., 2013. Polychlorinated biphenyls (PCBs) as initiating
agents in hepatocellular carcinoma. Cancer Lett. 334, 46–55.
Ma, C., Han, L., Zhu, Z., Heng Pang, C., Pan, G., 2022. Mineral metabolism and
ferroptosis in non-alcoholic fatty liver diseases. Biochem. Pharmacol. 205, 115242.
Ma, F., Zhu, Y., Liu, X., Zhou, Q., Hong, X., Qu, C., Feng, X., Zhang, Y., Ding, Q., Zhao, J.,
Hou, J., Zhong, M., Zhuo, H., Zhong, L., Ye, Z., Xie, W., Liu, Y., Xiong, Y., Chen, H.,
Piao, D., Sun, B., Gao, Z., Li, Q., Zhang, Z., Qiu, X., Zhang, Z., 2019. Dual-specicity
tyrosine phosphorylation–regulated kinase 3 loss activates purine metabolism and
promotes hepatocellular carcinoma progression. Hepatology 70.
MacParland, S.A., Liu, J.C., Ma, X.-Z., Innes, B.T., Bartczak, A.M., Gage, B.K., Manuel, J.,
Khuu, N., Echeverri, J., Linares, I., Gupta, R., Cheng, M.L., Liu, L.Y., Camat, D.,
Chung, S.W., Seliga, R.K., Shao, Z., Lee, E., Ogawa, S., Ogawa, M., Wilson, M.D.,
Fish, J.E., Selzner, M., Ghanekar, A., Grant, D., Greig, P., Sapisochin, G., Selzner, N.,
Winegarden, N., Adeyi, O., Keller, G., Bader, G.D., McGilvray, I.D., 2018. Single cell
RNA sequencing of human liver reveals distinct intrahepatic macrophage
populations. Nat. Commun. 9, 4383.
Marschall, T.A., Bornhorst, J., Kuehnelt, D., Schwerdtle, T., 2016. Differing cytotoxicity
and bioavailability of selenite, methylselenocysteine, selenomethionine, selenosugar
1 and trimethylselenonium ion and their underlying metabolic transformations in
human cells. Mol. Nutr. Food Res. 60, 2622–2632.
Master, E.R., Lai, V.W.M., Kuipers, B., Cullen, W.R., Mohn, W.W., 2002. Sequential
Anaerobic−Aerobic Treatment of Soil Contaminated with Weathered Aroclor 1260.
Environ. Sci. Technol. 36, 100–103.
Mato, J.M., Martínez-Chantar, M.L., Lu, S.C., 2013. S-adenosylmethionine metabolism
and liver disease. Ann. Hepatol. 12, 183–189.
Mattow, J., Demuth, I., Haeselbarth, G., Jungblut, P.R., Klose, J., 2006. Selenium-
binding protein 2, the major hepatic target for acetaminophen, shows sex differences
in protein abundance. Electrophoresis 27, 1683–1691.
Maxwell, K.N., Soccio, R.E., Duncan, E.M., Sehayek, E., Breslow, J.L., 2003. Novel
putative SREBP and LXR target genes identied by microarray analysis in liver of
cholesterol-fed mice. J. Lipid Res. 44, 2109–2119.
Mayer, A.E., L¨
ofer, M.C., Loza Vald´
es, A.E., Schmitz, W., El-Merahbi, R., Viera, J.T.,
Erk, M., Zhang, T., Braun, U., Heikenwalder, M., Leitges, M., Schulze, A., Sumara, G.,
2019. The kinase PKD3 provides negative feedback on cholesterol and triglyceride
synthesis by suppressing insulin signaling. Sci. Signal. 12, eaav9150.
Mazin, M.E., Yarushkin, A.A., Pustylnyak, Y.A., Prokopyeva, E.A., Pustylnyak, V.O.,
2022. Promotion of NR1I3-mediated liver growth is accompanied by STAT3
activation. Mol. Biol. Rep. 49, 4089–4093.
Montano, L., Pironti, C., Pinto, G., Ricciardi, M., Buono, A., Brogna, C., Venier, M.,
Piscopo, M., Amoresano, A., Motta, O., 2022. Polychlorinated Biphenyls (PCBs) in
the environment: occupational and exposure events, effects on human health and
fertility. Toxics 10, 365.
Moustafa Mohamed, E., Carlson Bradley, A., El-Saadani Muhammad, A., Kryukov
Gregory, V., Sun, Q.-A., Harney John, W., Hill Kristina, E., Combs Gerald, F.,
Feigenbaum, L., Mansur David, B., Burk Raymond, F., Berry Marla, J., Diamond
Alan, M., Lee Byeong, J., Gladyshev Vadim, N., Hateld Dolph, L., 2001. Selective
Inhibition of Selenocysteine tRNA Maturation and Selenoprotein Synthesis in
Transgenic Mice Expressing Isopentenyladenosine-Decient Selenocysteine tRNA.
Mol. Cell. Biol. 21, 3840–3852.
Neumann, D.P., Goodall, G.J., Gregory, P.A., 2022. The Quaking RNA-binding proteins
as regulators of cell differentiation. WIREs RNA 13, e1724.
Nguyen, H.-Q., Lee, D., Kim, Y., Bang, G., Cho, K., Lee, Y.-S., Yeon, J.E., Lubman, D.M.,
Kim, J., 2021. Label-free quantitative proteomic analysis of serum extracellular
vesicles differentiating patients of alcoholic and nonalcoholic fatty liver diseases.
J. Proteom. 245, 104278.
Pavuk, M., Serio, T.C., Cusack, C., Cave, M., Rosenbaum, P.F., Birnbaum, L.S., 2019.
Hypertension in relation to dioxins and polychlorinated biphenyls from the anniston
community health survey follow-up. Environ. Health Perspect. 127, 127007.
Pavuk, M., Rosenbaum, P.F., Lewin, M.D., Serio, T.C., Rago, P., Cave, M.C., Birnbaum, L.
S., 2023. Polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins,
polychlorinated dibenzofurans, pesticides, and diabetes in the Anniston Community
Health Survey follow-up (ACHS II): single exposure and mixture analysis
approaches. Sci. Total Environ. 877, 162920.
Peng, J.J., Yue, S.Y., Fang, Y.H., Liu, X.L., Wang, C.H., 2021. Mechanisms affecting the
biosynthesis and incorporation rate of selenocysteine. Molecules 26.
Petri, B.J., Piell, K.M., Wahlang, B., Head, K.Z., Andreeva, K., Rouchka, E.C., Pan, J.,
Rai, S.N., Cave, M.C., Klinge, C.M., 2022. Multiomics analysis of the impact of
polychlorinated biphenyls on environmental liver disease in a mouse model.
Environ. Toxicol. Pharmacol. 94, 103928.
Petri, B.J., Piell, K.M., Wahlang, B., Head, K.Z., Andreeva, K., Rouchka, E.C., Cave, M.C.,
Klinge, C.M., 2023b. Polychlorinated biphenyls alter hepatic m6A mRNA
methylation in a mouse model of environmental liver disease. Environ. Res. 216,
114686.
Petri, B.J., Piell, K.M., Wahlang, B., Head, K.Z., Rouchka, E.C., Park, J.W., Hwang, J.Y.,
Banerjee, M., Cave, M.C., Klinge, C.M., 2023c. Altered splicing factor and alternative
splicing events in a mouse model of diet- and polychlorinated biphenyl-induced liver
disease. Environ. Toxicol. Pharmacol. 103, 104260.
Petri, B.J., Cave, M.C., Klinge, C.M., 2023a. Changes in m6A in Steatotic Liver Disease.
Genes 14, 1653.
Petriello, M.C., Mottaleb, M.A., Serio, T.C., Balyan, B., Cave, M.C., Pavuk, M.,
Birnbaum, L.S., Morris, A.J., 2022. Serum concentrations of legacy and emerging
per- and polyuoroalkyl substances in the Anniston Community Health Surveys
(ACHS I and ACHS II). Environ. Int. 158, 106907.
Piell, K.M., Petri, B.J., Head, K.Z., Wahlang, B., Xu, R., Zhang, X., Pan, J., Rai, S.N., de
Silva, K., Chariker, J.H., Rouchka, E.C., Tan, M., Li, Y., Cave, M.C., Klinge, C.M.,
2023. Disruption of the mouse liver epitranscriptome by long-term aroclor 1260
exposure. Environ. Toxicol. Pharmacol. 100, 104138.
Poverennaya, E.V., Ilgisonis, E.V., Ponomarenko, E.A., Kopylov, A.T., Zgoda, V.G.,
Radko, S.P., Lisitsa, A.V., Archakov, A.I., 2017. Why are the correlations between
mRNA and protein levels so low among the 275 predicted protein-coding genes on
human chromosome 18? J. Proteome Res. 16, 4311–4318.
Qian, Y., Zhang, S., Guo, W., Ma, J., Chen, Y., Wang, L., Zhao, M., Liu, S., 2015.
Polychlorinated Biphenyls (PCBs) inhibit hepcidin expression through an estrogen-
like effect associated with disordered systemic iron homeostasis. Chem. Res. Toxicol.
28, 629–640.
Raudvere, U., Kolberg, L., Kuzmin, I., Arak, T., Adler, P., Peterson, H., Vilo, J., 2019. g:
Proler: a web server for functional enrichment analysis and conversions of gene
lists (2019 update). Nucleic Acids Res. 47, W191–W198.
Romualdo, G.R., Valente, L.C., de Souza, J.L.H., Rodrigues, J., Barbisan, L.F., 2023.
Modifying effects of 2,4-D and Glyphosate exposures on gut-liver-adipose tissue axis
of diet-induced non-alcoholic fatty liver disease in mice. Ecotoxicol. Environ. Saf.
268, 115688.
Safe, S., Bandiera, S., Sawyer, T., Robertson, L., Safe, L., Parkinson, A., Thomas, P.E.,
Ryan, D.E., Reik, L.M., Levin, W., et al., 1985. PCBs: structure-function relationships
and mechanism of action. Environ. Health Perspect. 60, 47–56.
Santagata, S., Bhattacharyya, D., Wang, F.-H., Singha, N., Hodtsev, A., Spanopoulou, E.,
1999. Molecular cloning and characterization of a mouse homolog of bacterial ClpX,
a novel mammalian class II member of the Hsp100/Clp chaperone family. J. Biol.
Chem. 274, 16311–16319.
Sengupta, A., Carlson, Bradley A., Weaver, James A., Novoselov, Sergey V.,
Fomenko, Dmitri E., Gladyshev, Vadim N., Hateld, Dolph L., 2008. A functional
link between housekeeping selenoproteins and phase II enzymes. Biochem. J. 413,
151–161.
Shan, Q., Huang, F., Wang, J., Du, Y., 2015. Effects of co-exposure to 2,3,7,8-tetra-
chlorodibenzo-p-dioxin and polychlorinated biphenyls on nonalcoholic fatty liver
disease in mice. Environ. Toxicol. 30, 1364–1374.
Shi, H., Jan, J., Hardesty, J.E., Falkner, K.C., Prough, R.A., Balamurugan, A.N.,
Mokshagundam, S.P., Chari, S.T., Cave, M.C., 2019. Polychlorinated biphenyl
exposures differentially regulate hepatic metabolism and pancreatic function:
Implications for nonalcoholic steatohepatitis and diabetes. Toxicol. Appl. Pharmacol.
363, 22–33.
Soltis, A.R., Kennedy, N.J., Xin, X., Zhou, F., Ficarro, S.B., Yap, Y.S., Matthews, B.J.,
Lauffenburger, D.A., White, F.M., Marto, J.A., Davis, R.J., Fraenkel, E., 2017.
Hepatic dysfunction caused by consumption of a high-fat diet. Cell Rep. 21,
3317–3328.
Speckmann, B., Schulz, S., Hiller, F., Hesse, D., Schumacher, F., Kleuser, B., Geisel, J.,
Obeid, R., Grune, T., Kipp, A.P., 2017. Selenium increases hepatic DNA methylation
and modulates one-carbon metabolism in the liver of mice. J. Nutr. Biochem. 48,
112–119.
Stork, B.A., Dean, A., Ortiz, A.R., Saha, P., Putluri, N., Planas-Silva, M.D., Mahmud, I.,
Rajapakshe, K., Coarfa, C., Knapp, S., Lorenzi, P.L., Kemp, B.E., Turk, B.E., Scott, J.
W., Means, A.R., York, B., 2022. Calcium/calmodulin-dependent protein kinase
kinase 2 regulates hepatic fuel metabolism. Mol. Metab. 62, 101513.
Su, Q., Kim, S.Y., Adewale, F., Zhou, Y., Aldler, C., Ni, M., Wei, Y., Burczynski, M.E.,
Atwal, G.S., Sleeman, M.W., Murphy, A.J., Xin, Y., Cheng, X., 2021. Single-cell RNA
transcriptome landscape of hepatocytes and non-parenchymal cells in healthy and
NAFLD mouse liver. iScience 24, 103233.
Sz´
ant´
o, M., Gupte, R., Kraus, W.L., Pacher, P., Bai, P., 2021. PARPs in lipid metabolism
and related diseases. Prog. Lipid Res. 84, 101117.
Tamai, Y., Iwasa, M., Eguchi, A., Shigefuku, R., Sugimoto, K., Hasegawa, H., Takei, Y.,
2020. Serum copper, zinc and metallothionein serve as potential biomarkers for
hepatocellular carcinoma. PLoS One 15, e0237370.
The UniProt, C., 2021. UniProt: the universal protein knowledgebase in 2021. Nucleic
Acids Res. 49, D480–D489.
Tholen, L.E., Bos, C., Jansen, P., Venselaar, H., Vermeulen, M., Hoenderop, J.G.J., de
Baaij, J.H.F., 2021. Bifunctional protein PCBD2 operates as a co-factor for
hepatocyte nuclear factor 1β and modulates gene transcription. FASEB J.: Off. Publ.
Fed. Am. Soc. Exp. Biol. 35, e21366.
Twaroski, T.P., O’Brien, M.L., Robertson, L.W., 2001. Effects of selected polychlorinated
biphenyl (PCB) congeners on hepatic glutathione, glutathione-related enzymes, and
selenium status: implications for oxidative stress. Biochem. Pharmacol. 62, 273–281.
Vazquez, A., Flammini, A., Maritan, A., Vespignani, A., 2003. Global protein function
prediction from protein-protein interaction networks. Nat. Biotechnol. 21, 697–700.
Vedrenne, V., Galmiche, L., Chretien, D., de Lonlay, P., Munnich, A., R¨
otig, A., 2012.
Mutation in the mitochondrial translation elongation factor EFTs results in severe
infantile liver failure. J. Hepatol. 56, 294–297.
Wahlang, B., Song, M., Beier, J.I., Cameron Falkner, K., Al-Eryani, L., Clair, H.B.,
Prough, R.A., Osborne, T.S., Malarkey, D.E., Christopher States, J., Cave, M.C.,
2014b. Evaluation of Aroclor 1260 exposure in a mouse model of diet-induced
obesity and non-alcoholic fatty liver disease. Toxicol. Appl. Pharmacol. 279,
380–390.
Wahlang, B., Falkner, K.C., Clair, H.B., Al-Eryani, L., Prough, R.A., States, J.C., Coslo, D.
M., Omiecinski, C.J., Cave, M.C., 2014a. Human receptor activation by aroclor 1260,
a polychlorinated biphenyl mixture. Toxicol. Sci. 140, 283–297.
Wahlang, B., Jin, J., Beier, J.I., Hardesty, J.E., Daly, E.F., Schnegelberger, R.D.,
Falkner, K.C., Prough, R.A., Kirpich, I.A., Cave, M.C., 2019. Mechanisms of
environmental contributions to fatty liver disease. Curr. Environ. Health Rep. 6,
80–94.
Windahl, S.H., Andersson, N., B¨
orjesson, A.E., Swanson, C., Svensson, J., Mov´
erare-
Skrtic, S., Sj¨
ogren, K., Shao, R., Lagerquist, M.K., Ohlsson, C., 2011. Reduced bone
K.M. Piell et al.

Environmental Toxicology and Pharmacology 107 (2024) 104430
12
mass and muscle strength in male 5
α
-reductase Type 1 inactivated mice. PLoS One 6,
e21402.
Wu, J., Wang, Y., Jiang, R., Xue, R., Yin, X., Wu, M., Meng, Q., 2021. Ferroptosis in liver
disease: new insights into disease mechanisms. Cell Death Discov. 7, 276.
Xu, C., Lin, F., Qin, S., 2008. Relevance between lipid metabolism-associated genes and
rat liver regeneration. Hepatol. Res. 38, 825–837.
Xue, Q., Yan, D., Chen, X., Li, X., Kang, R., Klionsky, D.J., Kroemer, G., Chen, X.,
Tang, D., Liu, J., 2023. Copper-dependent autophagic degradation of GPX4 drives
ferroptosis. Autophagy 19, 1982–1996.
Yang, Wan S., SriRamaratnam, R., Welsch, Matthew E., Shimada, K., Skouta, R.,
Viswanathan, Vasanthi S., Cheah, Jaime H., Clemons, Paul A., Shamji, Alykhan F.,
Clish, Clary B., Brown, Lewis M., Girotti, Albert W., Cornish, Virginia W.,
Schreiber, Stuart L., Stockwell, Brent R., 2014. Regulation of ferroptotic cancer cell
death by GPX4. Cell 156, 317–331.
Yarushkin, A.A., Mazin, M.E., Yunusova, A.Y., Korchagina, K.V., Pustylnyak, Y.A.,
Prokopyeva, E.A., Pustylnyak, V.O., 2018. CAR-mediated repression of Cdkn1a(p21)
is accompanied by the Akt activation. Biochem. Biophys. Res. Commun. 504,
361–366.
Younossi, Z.M., Gramlich, T., Bacon, B.R., Matteoni, C.A., Boparai, N., O’Neill, R.,
McCullough, A.J., 1999. Hepatic iron and nonalcoholic fatty liver disease.
Hepatology 30, 847–850.
Younossi, Z.M., Baranova, A., Ziegler, K., Del Giacco, L., Schlauch, K., Born, T.L.,
Elariny, H., Gorreta, F., VanMeter, A., Younoszai, A., Ong, J.P., Goodman, Z.,
Chandhoke, V., 2005. A genomic and proteomic study of the spectrum of
nonalcoholic fatty liver disease. Hepatology 42, 665–674.
Zhang, L., Wu, K., Bo, T., Zhou, L., Gao, L., Zhou, X., Chen, W., 2020a. Integrated
microRNA and proteome analysis reveal a regulatory module in hepatic lipid
metabolism disorders in mice with subclinical hypothyroidism. Exp. Ther. Med. 19,
897–906.
Zhang, X., Xiong, W., Chen, L.-L., Huang, J.-Q., Lei, X.G., 2020b. Selenoprotein V
protects against endoplasmic reticulum stress and oxidative injury induced by pro-
oxidants. Free Radic. Biol. Med. 160, 670–679.
Zheng, X., Ren, B., Li, X., Yan, H., Xie, Q., Liu, H., Zhou, J., Tian, J., Huang, K., 2020.
Selenoprotein F knockout leads to glucose and lipid metabolism disorders in mice.
JBIC J. Biol. Inorg. Chem. 25, 1009–1022.
Zhou, X., He, C., Ren, J., Dai, C., Stevens, S.R., Wang, Q., Zamler, D., Shingu, T., Yuan, L.,
Chandregowda, C.R., Wang, Y., Ravikumar, V., Rao, A.U., Zhou, F., Zheng, H.,
Rasband, M.N., Chen, Y., Lan, F., Heimberger, A.B., Segal, B.M., Hu, J., 2020. Mature
myelin maintenance requires Qki to coactivate PPARβ-RXR
α
-mediated lipid
metabolism. J. Clin. Invest 130, 2220–2236.
Zimny, S., Pohl, R., Rein-Fischboeck, L., Haberl, E.M., Krautbauer, S., Weiss, T.S.,
Buechler, C., 2017. Chemokine (CC-motif) receptor-like 2 mRNA is expressed in
hepatic stellate cells and is positively associated with characteristics of non-alcoholic
steatohepatitis in mice and men. Exp. Mol. Pathol. 103, 1–8.
K.M. Piell et al.