The flame retardant triphenyl phosphate alters the epigenome of embryonic cells in an aquatic in vitro model

Triphenyl phosphate (TPhP) is an organophosphate flame retardant and plasticizer that is added to a wide variety of consumer and industrial products. It is also a ubiquitous environmental pollutant. Exposure to TPhP has been shown to alter gene expression in metabolic and estrogenic signaling pathways in in vitro and in vivo models of a variety of species, and as such, is considered to be an endocrine disrupting chemical. Exposure to endocrine disrupting chemicals is increasingly being associated with changes to the epigenome, especially during embryonic development. The aim of this study was to evaluate whether TPhP exposure in aquatic ecosystems has the ability to alter the epigenome in two immortal cell lines derived from trout (Oncorhynchus mykiss). This study assessed whether 24 h exposure to TPhP resulted in changes to histone modification and DNA methylation profiles in steelhead trout embryonic cells and rainbow trout gill epithelial cells. Results show that several epigenetic modifications on histone H3 and DNA methylation are altered in the embryonic cells following TPhP exposure, but not in the gill epithelial cells. Specifically, histone H3 acetylation, histone H3 mono‐methylation and global DNA methylation were found to be reduced. The alterations of these epigenetic modification profiles in the embryonic cells suggest that exposure to TPhP during fetal development may alter gene expression in the developing embryo, likely in metabolic and estrogenic pathways. The impacts to the epigenome determined in this study may even carry multigenerational detrimental effects on human and ecosystem health, which requires further investigation.

plastics, foam, lubricant and insulation (van der Veen & de Boer, 2012).Flame retardants are omnipresent in global ecosystems and can be long-range transported via water and atmospheric transport to widespread locations such as Arctic ecosystems (Fu et al., 2021;Möller et al., 2012).Brominated flame retardants, specifically polybrominated diphenyl ethers (PBDEs), were the dominant flame retardants on the market for several decades given their low cost and effective fire prevention.It is now known that PBDEs are extremely persistent in the environment and undergo limited metabolism within organisms (Allchin et al., 1999;Covaci et al., 2003;Rahman et al., 2001).PBDEs are now classified as a Schedule 1 substance on the Government of Canada's Toxic Substances List and their use was extremely restricted in Canada beginning in 2004 (Environment and Climate Change Canada [ECCC], 2013).In the wake of this ban, alternative flame retardants gained popularity on the market.
Presently, organophosphate-based flame retardants are a widely used class, and up to 1,000,000 kg have been reported to be imported into Canada within the past decade (Environment and Climate Change Canada, Health Canada., 2021).Triphenyl phosphate (TPhP) is a component of the popular Firemaster 550 mix, which is typically added to products in the form of a powder and therefore has a tendency to leach into surrounding environments (Carlsson et al., 2000).TPhP has been detected in many settings including in water systems (Bollmann et al., 2012), indoor dust (Stapleton et al., 2009) and in soil (Dong et al., 2022), including in remote Arctic regions (Fu et al., 2021).
TPhP is highly pervasive in water systems, with aquatic exposure being as high as 180 μg/kg lipid weight (Sundkvist et al., 2010).TPhP is not considered to be acutely toxic at the concentrations that organisms are typically exposed to, with the exception of some aquatic organisms (Environment and Climate Change Canada, Health Canada., 2021), based on a relatively low 96 h LC 50 of 0.36 mg/l in rainbow trout (Palawski et al., 1983).
Beyond potential acute toxicity to aquatic organisms, TPhP may have more subtle adverse effects to both humans and the broader ecosystem as organisms are chronically exposed to low concentrations.TPhP has been shown to cause gene expression changes in a wide variety of nuclear receptor networks and act as an endocrine disrupting chemical (EDC).TPhP has been shown to alter sex hormone balance through altered estrogen metabolism in zebrafish, specifically altering cyp19a expression, which encodes the aromatase enzyme (Liu et al., 2012).TPhP also acted agonistically on the estrogen receptor alpha (ERα) and antagonistically on the androgen receptor in an in vitro model and in zebrafish embryos (Kojima et al., 2013;Liu et al., 2013).Perhaps due to estrogenic effects, TPhP has also been shown to decrease sperm concentration in human semen, therefore reducing reproductive capabilities (Meeker & Stapleton, 2010).Further, TPhP altered mRNA expression of the glucocorticoid receptor, thyroid hormone receptor alpha and the mineralocorticoid receptor in zebrafish embryos (Liu et al., 2013;Liu, Cai, et al., 2019).Mice exposed to TPhP in utero experienced increased gene expression of insulin-like growth factors and insulin receptor substrate indicating that TPhP may interfere with normal development and metabolism (Philbrook et al., 2018).
The developmental origins of health and disease hypothesis states that environmental factors during development may result in disease states later in life (Barker, 2007).Such environmental factors include maternal diet, stressed states and exposure to xenobiotics.Specifically, it is thought that exposure to EDCs have the potential to cause transgenerational alterations in gene expression in offspring through epigenetic mechanisms (Anway et al., 2005;Anway & Skinner, 2006;Skinner, 2008;Skinner et al., 2011).Exposures during embryonic development pose a particular concern, as alterations to the epigenome during this period can predispose organisms to dysfunction transgenerationally (Anway et al., 2005;Anway & Skinner, 2006;Skinner, 2008;Skinner et al., 2011).An example of this occurrence is a study that found that exposure to the fungicide vinclozolin (an antiandrogenic compound) in male rats resulted in decreased sperm quality in the F 1 , F 2 , F 3 and even F 4 generations (Anway et al., 2005).This decrease in sperm quality correlated with altered DNA methylation patterns, a type of epigenetic modification.Other flame retardants have also been shown to alter the epigenome.Tris (1,3-dichloro-2-propyl) phosphate, another organophosphate flame retardant, alters DNA methylation profiles of zebrafish embryos resulting in developmental toxicities (Kupsco et al., 2017;McGee et al., 2012).More recently Shafique et al., 2023, demonstrated that gestational exposure to TPhP in mice resulted in decreased fetal global DNA methylation.Although the epigenetic effects of many EDCs have been documented, the exact mechanism by which they alter epigenetic patterns is not yet completely understood.It is thought that one mechanism is that EDCs can interfere with the activity of enzymes that regulate epigenetic patterns (Rattan & Flaws, 2019).
Commonly studied epigenetic modifications include DNA methylation and post-translational histone modifications, including acetylation and methylation.DNA methylation involves the addition of a methyl group to cytosine residues by DNA methyltransferase enzymes (DNMTs) (Jin et al., 2011), which typically results in gene expression silencing (Rattan & Flaws, 2019).Another mechanism that changes gene expression is alterations to the N-terminals of histone tails.Histone acetylation is increased by histone acetyltransferase enzymes (HATs), which add acetyl groups and decreased by histone deacetylase enzymes (HDACs), which remove acetyl groups.It is known that global acetylation of histones H3 and H4 can result in transcriptional activation (Alavian-Ghavanini & Rüegg, 2018).Histone methylation is more complex as depending on where the methylation occurs, it can have different effects on chromatin compaction (Alavian-Ghavanini & Rüegg, 2018).Methylation at histone 3 at lysine 9 (H3K9) is known to cause transcriptional silencing and is increasingly being considered a marker for more permanent DNA methylation to occur, as well as involved with X-chromosome inactivation and alternative splicing patterns (Alavian-Ghavanini & Rüegg, 2018;Hyun et al., 2017).
Currently, there are very limited regulations on the use of TPhP in Canada (Environment and Climate Change Canada, Health Canada., 2021).Chemicals used in Canada, similar to in the EU and in other countries, must undergo toxicological studies.These studies, however, are limited to classical toxicological endpoints such as LD 50 , genotoxicity, carcinogenicity, skin and respiratory sensitivity, and reproductive toxicity (ECCC, 2016;Krivoshiev et al., 2016).Only recently has the endocrine disrupting capability of chemicals been included in the toxicological assessment of their safety.There is a lack of screening for the ability of chemicals in Canada and globally to modify the epigenome of organisms, and thus have the potential to cause transgenerational harm.Given the pervasive presence of TPhP in the environment, it is critical to further understand the broader and more nuanced toxicological consequences of exposure to this compound.Exposure to EDCs during development can result in alterations to the epigenome (Skinner et al., 2011).Furthermore, alterations to the epigenome have been shown to be associated with transgenerational disease states, such as increased occurrence of cancers, metabolic disorders and reproductive disorders (Alavian-Ghavanini & Rüegg, 2018;Nilsson & Skinner, 2015;Villeneuve et al., 2008).It is now known that TPhP is an EDC and that exposure to TPhP can alter gene expression in critical signaling pathways in exposed generations, such as estrogenic and metabolic genes.Due to the widespread presence of TPhP in the environment, the purpose of this study was to investigate the potential for epigenetic changes following exposure to TPhP in an aquatic in vitro model using embryonic and gill epithelial cells derived from trout.These two cell types were chosen to broadly evaluate the potential for changes to the epigenome in a developmentally inert and a developmentally active cell type.
Here, we examine global DNA methylation as a marker for gene expression silencing, global histone acetylation as a marker for gene expression activation and histone methylation at lysine 9 as a marker for gene expression silencing, in association with TPhP concentration to determine the impact of TPhP on the epigenome in an aquatic in vitro model.

| Cell culture and cell exposure model
This study used two immortalized cell lines as models for aquatic organisms.RTgill-W1 is derived from rainbow trout (Onchorhynchus mykiss) gill epithelial tissue and STE-137 is derived from pooled steelhead trout (Onchorhynchus mykiss irideus) embryonic tissue.RTgill-W1 (ATCC CRL-2523) cells were purchased from the American Type Culture Collection.STE-137 cells were kindly supplied by the United States Geological Survey Western Fisheries Research Centre (Seattle, WA, USA).The protocol for the growth and maintenance of RTgill-W1 and STE-137 cells was adapted from previously described protocols (Bols et al., 2017;Lannan et al., 1984).Both cells lines were grown in a supplemented growth media containing 88% Leibovitz's L-15 media (Sigma-Aldrich, MO, USA), 10% fetal bovine serum from Gibco (Grand Island, New York), 1% L-glutamine (Sigma-Aldrich, MO, USA), and 2% penicillin-streptomycin solution (Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada) in free gas exchange with air at 18 C. Experiments were carried out when cell populations were at 80-90% confluency.

| Preparation of test chemical solution
Triphenyl phosphate (TPhP)  was purchased from Sigma-Aldrich (MO, USA).A working stock solution of 100 mM TPhP in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, MO, USA) was prepared and was stored in the dark at room temperature.This working stock solution was diluted into the supplemented growth media to achieve the desired concentrations.The final concentration of solvent in the supplemented growth media of all exposure groups in this study, including in the vehicle control group, was 0.08% (v/v).

| Cell proliferation and viability assay
Cell viability was assessed as the proportion of alive cells within a population and is an indicator for acute cell death.Cell viability was measured using the trypan blue exclusion method (Louis & Siegel, 2011) in biological triplicates and technical duplicates.In brief, both cell lines were seeded at a density of 250,000 cells/mL into 24-well plates for 24 h in biological triplicates.Following this attachment period, cells were exposed to a range of TPhP concentrations (0-3000 μM) for 24 h.Following exposure, the cells were harvested, and the cell suspension was mixed with an equal part of 0.4% trypan blue dye from Sigma-Aldrich (MO, USA).Following 5-10 min of incubation, the cell mixture was pipetted into a hemocytometer and the ratio of alive to dead cells was determined under microscope.Cell proliferation refers to the growth in the number of cells over time and is an indicator for the health of a cell population.To assess cell proliferation, both cell lines were seeded at a density of 150,000-200,000 cells/mL into 24-well plates for 24 h in biological triplicates.The 24 h adhesion rates differ between the two cell lines, so this was taken into account by performing a 0 h cell count pre-exposure in both cell lines for the proliferation assay.Following this attachment period, cells were exposed to either 0, 40 or 80 μM of TPhP.Cell density was counted via hemocytometer in technical duplicates following 0, 24, 48 or 72 h of exposure.

| Histone extractions
The exposure groups for histone modifications were as follows: 0 μM as a vehicle control, 40 and 80 μM of TPhP in both cell lines.These

| Western blotting
Histone aliquots were thawed from À70 C and 2.5 μg of histone protein was loaded in duplicate on a 15% polyacrylamide gel and separated at 100 V for 1 h 40 min.Proteins were then transferred to 0.45 μm pore size low fluorescence polyvinylidene fluoride membrane (Bio-Rad, Mississauga, ON, Canada) using a wet transfer at 100 V for 1 h at 4 C. Following transfer, membranes were incubated for 1 h at room temperature in 5% bovine serum albumin (BSA) and probed overnight at 4 C with two primary antibodiesone antibody for the loading control and the other antibody for the protein of interest, diluted in accordance their respective product sheet and found to be within the linear range of detection.The following morning, membranes were incubated with the respective secondary antibody for 1 h at room temperature with gentle rocking.All antibody information can be found in Table S1.Membranes with STE-137 samples were imaged in an Azure Biosystems c600 in the fluorescent setting.The membranes with RTgill-W1 samples were imaged in a LI-COR Odyssey CLx Imaging machine in the 700 nm and 800 nm channels.Densitometry was performed using ImageJ to quantify the relative optical densities of the bands.

| DNA methylation
The exposure groups for histone modifications were as follows: 0 μM

| Statistical analysis
Cell viability at each tested TPhP concentration was normalized to a vehicle control, transformed to a logarithmic scale and fit using a fourparameter logistic (4PL) non-linear regression model.Cell proliferation was compared between concentration groups and time points using a two-way ANOVA with Tukey's multiple comparisons test post-hoc.
For immunoblotting, the densitometry values of the proteins of interest were normalized to the densitometry values of the respective loading controls and expressed as a ratio relative to the normalized control value, as previously described by Shafique et al., 2023.The ratios were then compared between groups using a one-way ANOVA and Tukey's multiple comparisons test post-hoc.For DNA methylation, the percent of methylated DNA (5-mc) out of the known total input DNA was calculated as per the manufacturer's instructions and was compared between groups using a one-way ANOVA and Tukey's multiple comparisons test post-hoc.All analyses were performed on GraphPad Prism9 software and the ROUT outlier test (Q = 10%) was applied to all data.Error bars represent standard deviation and statistical significance is defined as a p-value <0.05.

| Cell viability and proliferation
In this study, we determined a sub-lethal TPhP exposure regimen for epigenetic investigations in STE-137 and RTgill-W1 cells to the flame retardant triphenyl phosphate.We found that the 24 h LC 50 for TPhP was 307 μM in STE-137 and 107 μM in RTgill-W1 cells (Figure 1A,B).
We determined that at 40 and 80 μM of TPhP, there was no significant difference in cell proliferation at 24 or 48 h post-exposure compared with control (Figure 2A,B).However, following 72 h of exposure, 80 μM of TPhP caused a significant reduction in cell proliferation in RTgill-W1 cells (Figure 2B).No significant differences in cell proliferation were found at any concentration at any time point compared with control (Figure 2A).

| Acetylated H3 and H4
Here, we assessed whether in vitro exposure to TPhP caused changes to chromatin conformation by analyzing levels of acetylated histone H3 and H4.Histone acetylation is known to cause loosening of DNA structure and increased gene transcription.Following exposure to 40 and 80 μM of TPhP in STE-137 cells, histone H3 acetylation was significantly reduced in a concentration-dependant manner (Figure 3A).No significant differences to H3 acetylation were seen at any concentration point compared with control in RTgill-W1 cells (Figure 3B).The levels of histone H4 acetylation were unchanged at all concentration points compared wit control levels in both the STE-137 cells and the RTgill-W1 cells (Figure 4A,B).

| Mono-and tri-methylated H3K9
Methylation on histone H3 at lysine 9 is associated with transcriptional repression and reduced gene expression.We found that following exposure to both 40 and 80 μM of TPhP, there was a significant and concentration-dependent reduction in levels of H3K9 mono-methylation in STE-137 cells (Figure 5A).However, there were no changes to H3K9 mono-methylation at any concentration point in RTgill-W1 cells, when compared with control (Figure 5B).We also assessed tri-methylated H3K9 and found that there were no changes to this epigenetic marker in either the STE-137 or RTgill-W1 cells at any concentration point (Figure 6A,B).

| DNA methylation
DNA methylation impedes active transcription and results in gene expression silencing.Here, we determined that exposure to 80 μM of TPhP resulted in significantly reduced global DNA methylation in STE-137 cells (Figure 7A).Contrastingly, we again observed that there were no changes to DNA methylation at any concentration point compared with control in the RTgill-W1 cells (Figure 7B).

| DISCUSSION
Given the environmental persistence and evidence of endocrine disruption for flame retardants in both in vitro and in vivo models (Krivoshiev et al., 2016), the present study evaluated potential epigenetic impacts of the popular flame retardant TPhP using two aquatic cell lines that are commonly used for aquatic toxicological assessments of environmental contaminants.Specifically, we evaluated changes to well-established epigenetic modifications involved with gene expression in aquatic embryonic cells following TPhP exposure at concentrations that did not outright cause cell death or reduce cell proliferation.Our overall results are summarized in Table 1 and  Though the mechanism by which these modifications were reduced is unknown, it is possible that TPhP is altering the activity of epigenetic modifying enzymes.It is known that some steroid receptors are cofactors to histone modifying enzyme activity (Casati et al., 2015), so the endocrine disrupting actions of TPhP on endogenous steroid hormones may have downstream implications on enzymes involved in the maintenance of H3 acetylation, H3 methylation and DNA methylation.None of these epigenetic modification profiles were altered in the RTgill-W1 epithelial cells.We chose to evaluate the potential for epigenetic implications of TPhP exposures in developmentally active embryonic cells and developmentally inert gill epithelial cells, as this difference in cell type may contribute to differences in susceptibility to epigenetic disruption.Development is an especially sensitive period for the epigenome, as this is when many marks are installed by epigenetic modifying enzymes (Prusinski et al., 2016).Additionally, the epithelial cells were also of interest, as the gills are the primary site of uptake for xenobiotics in fish and would be the first point of contact to TPhP for fish (Hayton & Barron, 1990).
DNA methylation is a commonly studied epigenetic modification.
It involves the addition of a methyl group to cytosine residues by DNA methyltransferase enzymes (Jin et al., 2011).This methylation commonly occurs in CpG islands, which are cytosine and guanine dinucleotides that are commonly found in promoter regions of transcriptional start sites (Lim et al., 2019).Methylation of these sites creates a physical barrier that impedes transcription factor or replication machinery binding, and thus causes gene expression silencing (Rattan & Flaws, 2019).This study chose to investigate global DNA methylation rather than any promoter-specific site of DNA methylation, as it was previously unknown whether TPhP had the capability of disrupting this process during embryogenesis.Given that global DNA methylation was found to be reduced following TPhP exposure in our study, we would expect to see increased gene expression of genes that were epigenetically controlled via this mechanism.This would be consistent with a previous study from our laboratory, which found increased gene expression of the insulin-like growth factor 1 (Igf1) in the liver of mice who were exposed in utero to TPhP (Philbrook et al., 2018).Histone proteins contain an N-terminal domain that protrudes from the core and can be chemically modified by acetylation, methylation, phosphorylation or ubiquitination (Shahid et al., 2022).These modifications change the affinity of the histone protein to the DNA backbone, which changes the relative compactness of the chromatin and thus the accessibility of the DNA to transcription factors and replication machinery.The end result is a change in gene expression (Shahid et al., 2022).Histone acetylation at lysine residues is known to cause an opening of chromatin and typically results in increased gene expression.It is known that acetylation of histones H3 (at K9 and K18) and of H4 (at K5, K8 and K16) can result in transcriptional activation (Alavian-Ghavanini & Rüegg, 2018).We chose to investigate global histone acetylation rather than site-specific acetylation patterns, as acetylation at any of these sites will cause transcriptional activation and it was previously unknown whether TPhP had the capability of altering this specific epigenetic modification during embryogenesis.Our finding that global histone H3 acetylation was decreased following TPhP exposure suggests there would be decreased expression of genes that may be epigenetically controlled via this mechanism.Our results for histone H4 acetylation demonstrate considerable variability at the 80 μM concentration point.Many endocrine disrupting chemicals exhibit non-linear and non-monotonic dose responses for cell viability, proliferation, and gene expression (Vandenberg et al., 2012).It is possible that at 80 μM, there is a differ- concentrations above 80 μM would need to be conducted in order to conclude whether a non-linear, non-monotonic effect is occurring to histone H4 acetylation.
Histone methylation is more complex as depending on the site of methylation, it can have different effects on chromatin.We chose to specifically investigate methylation of H3K9 because it is one of the histone methylation patterns considered to be a hallmark of transcriptional repression, is involved in the maintenance of stable heterochromatin and X chromosome inactivation, is a signal for DNA methylation establishment and also thought to be involved in alternative splicing patterns (Hyun et al., 2017).Furthermore, histone methylation has been implicated in processes that are susceptible to endocrine disruption such as steroidogenesis (Zhu et al., 2021), spermatogenesis (Liu et al., 2019) and embryonic development (Hublitz et al., 2009).Interestingly, histone methylation at H3K9, amongst other sites, is increasingly being considered as a prerequisite for the more permanent modification of DNA methylation (Cedar & Bergman, 2009).This is consistent with our findings that both H3K9 mono-methylation and DNA methylation are reduced following TPhP exposure.Furthermore, our findings that H3 acetylation and H3K9 mono-methylation are reduced is consistent with a previously conducted study in our laboratory, which found sexspecific changes to both of these same modifications in mice exposed in utero to TPhP (Shafique et al., 2023).Specifically, it was found that these modifications were significantly decreased in male mice compared with female mice.This decrease in histone acetylation and methylation would likely have opposite effects on genes that are epigenetically controlled via this mechanism (Alavian-Ghavanini & Rüegg, 2018).Previous studies have demonstrated that estrogenrelated genes such as the ERα are epigenetically controlled by histone modifications on histone H3 (Dumasia et al., 2017).For example, demethylation of H3K9 is thought to be required for the expression of many genes controlled by ERα-mediated transcription (Dumasia et al., 2017).However, the specific gene expression implications of our findings are currently unknown.
A limitation of this study is that no sex-specific analysis of the impacts of TPhP on the epigenome was possible as both cell lines come from pooled tissue derived from multiple organisms of the same species.Therefore, the biological sex of the cell lines is unknown and is likely a mixture.Additionally, the concentrations used in this study are higher than are typically found in the environment (Environment and Climate Change Canada, Health Canada., 2021), and the determined in vitro 24 h LC 50 concentrations for the cell lines are about two orders of magnitude greater than the 96 h LC 50 in vivo for rainbow trout (Palawski et al., 1983).The concentrations in this study were chosen based on having no significant effect on cell viability and proliferation for both the STE-137 and RTgill-W1 cells following 24 h exposure (Figures 1 and 2).Future studies using environmentally relevant low dose exposures would be of interest, as it is well-established that the endogenous hormones exert their effects at extremely low serum concentrations and can exhibit non-monotonic dose-response relationships.For example, the plasticizer Bisphenol A is an environmental EDC, which exhibits estrogenic endocrine disruption in the nanomolar and picomolar range (Vandenberg et al., 2009(Vandenberg et al., , 2012;;Wozniak et al., 2005).Due to a limited variety of cell types used in this study and species-specific susceptibilities to environmental compounds, conclusions about the implications to the health of aquatic organisms cannot yet be drawn.However, teleost fish such as zebrafish and rainbow trout have been highlighted for their predictive RTgill-W1 (epithelial) ns ns ns potential of epigenetics in aquatic toxicology and as a model for human disease due to the highly methylated nature of their DNA, responsiveness to their environment and highly conserved metabolic and developmental pathways (Best et al., 2018;Kamstra, Aleström, Kooter & Legler, 2015).
In conclusion, the present study demonstrates that TPhP exposure has the capability to alter the epigenome of aquatic embryonic cells at sub-lethal concentrations.Specifically, global DNA methylation, histone H3 acetylation and histone H3 mono-methylation at lysine 9 was reduced in exposed cells when compared with control populations.These findings validate the need for future investigations into the downstream gene expression consequences of these changes to the epigenome and broader epigenetic alterations in aquatic ecosystems.Epigenetic control of gene expression is a highly regulated system, and to which perturbations during critical developmental windows such as embryogenesis have been associated with many disease states.TPhP has been shown to be associated with disease states such as obesity (Wang et al., 2019), diabetes (Cano-Sancho et al., 2017;Yue et al., 2023), endometrial cancer (Kwon et al., 2022) and reduced fertility (Shi et al., 2023) in a variety of in vivo and in vitro models.Flame retardants such as TPhP are produced with the intention of increasing human safety, specifically by protecting humans from fire hazards.However, these chemicals also have inherent hazards to ecosystems, including causing developmental toxicity to a wide range of species.Thus, understanding the toxicological risk and their molecular mechanisms associated with TPhP usage is critical for maintaining ecosystem health.
concentrations and time point were chosen based on results from the cell proliferation and viability experiments that demonstrated they would cause no reductions to cell viability or proliferation.Populations of cells were exposed in T25 flasks to one of the three test concentrations for 24 h for each biological replicate.Following exposure, cells were harvested, and the histones were acid extracted using a protocol described by Abcam (Cambridge, MA, USA).Briefly, cells were lysed in freshly prepared nuclear extraction buffer.The histones were then extracted overnight in 0.2 N HCl with gentle rocking.Following extraction, the histone solution was neutralized, and the protein concentrations measured via Bradford assay.Histone protein aliquots were stored at À70 C until further use.
as a vehicle control, 40 and 80 μM of TPhP in both cell lines.Following exposure, the cells were harvested, and the DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Mississauga, ON, Canada) as per the manufacturer's instructions.Following extraction, the DNA quality and quantity was measured using the NanoDrop 2000 Spectrophotometer (ThermoFischer Scientific, Wilmingdon, DE, USA) and assessed for an appropriate 260 nm/280 nm absorbance ratio (>1.5) to indicate purity.DNA methylation was assessed using the MethylFlash Global DNA Methylation ELISA Easy Kit (Colorimetric) from Epigentek (CedarLane, Burlington, ON, Canada) with no deviations from the manufacturer's protocol.The DNA input was 100 ng, as outlined by the manufacturer.The optical density of samples was measured using a SpectraMax iD3 microplate reader (Molecular Devices, California, USA) at 450 nm.The amount of methylated DNA is proportional to the optical density measured and is based on the generation of a standard curve.

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I G U R E 2 Cell proliferation following 0-72 h of exposure to either 0 (vehicle control), 40 or 80 μM of TPhP expressed as number of cells/mL.(A) Cell proliferation in STE-137 cells.(B) Cell proliferation in RTgill-W1 cells (n = 3, *p < 0.05).
show that TPhP exposure downregulated several epigenetic modifications in the STE-137 embryonic cells.DNA methylation was significantly decreased following 80 μM of TPhP and two histone modifications on histone H3 were downregulated following 40 and 80 μM of TPhP.

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I G U R E 3 Levels of acetylated H3 following 24 h exposure to either 0 (vehicle control), 40 or 80 μM of TPhP.Fluorescent signal of acetylated H3 was normalized to the fluorescent signal of total H3 and expressed relative to the control within each western blot.(A) Acetylated H3 in STE-137 cells.(B) Acetylated H3 in RTgill-W1 cells.(C) Representative western blot for acetylated H3 and total H3 in STE-137 and RTgill-W1 cells (n = 5, *p < 0.05).

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I G U R E 4 Levels of acetylated H4 following 24 h exposure to either 0 (vehicle control), 40 or 80 μM of TPhP.Fluorescent signal of acetylated H4 was normalized to the fluorescent signal of total H4 and expressed relative to the control within each western blot.(A) Acetylated H4 in STE-137 cells.(B) Acetylated H4 in RTgill-W1 cells.(C) Representative western blot for acetylated H4 and total H4 in STE-137 and RTgill-W1 cells (n = 5).
ential response occurring due to being at the cusp of a change in the slope of histone H4 acetylation.Further investigation into F I G U R E 5 Levels of monomethylated H3K9 following 24 h exposure to either 0 (vehicle control), 40 or 80 μM of TPhP.Fluorescent signal of monomethylated H3K9 was normalized to the fluorescent signal of total H3 and expressed relative to the control within each western blot.(A) Monomethylated H3K9 in STE-137 cells.(B) Monomethylated H3K9 in RTgill-W1 cells.(C) Representative western blot for monomethylated H3K9 and total H3 in STE-137 and RTgill-W1 cells (n = 5-6, *p < 0.05).

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I G U R E 6 Levels of trimethylated H3K9 following 24 h exposure to either 0 (vehicle control), 40 or 80 μM of TPhP.Fluorescent signal of trimethylated H3K9 was normalized to the fluorescent signal of total H3 and expressed relative to the control within each western blot.(A) Trimethylated H3K9 in STE-137 cells.(B) Trimethylated H3K9 in RTgill-W1 cells.(C) Representative western blot for trimethylated H3K9 and total H3 in STE-137 and RTgill-W1 cells (n = 5-6).