Naphthenic acid fraction components from oil sands process‐affected water from the Athabasca Oil Sands Region impair murine osteoblast differentiation and function

Abstract The extraction of bitumen from surface mining in the Athabasca Oil Sands Region (AOSR) produces large quantities of oil sands process‐affected water (OSPW) that needs to be stored in settling basins near extraction sites. Chemical constituents of OSPW are known to impair bone health in some organisms, which can lead to increased fracture risk and lower reproductive fitness. Naphthenic acid fraction components (NAFCs) are thought to be among the most toxic class of compounds in OSPW; however, the effect of NAFCs on osteoblast development is largely unknown. In this study, we demonstrate that NAFCs from OSPW inhibit osteoblast differentiation and deposition of extracellular matrix, which is required for bone formation. Extracellular matrix deposition was inhibited in osteoblasts exposed to 12.5–125 mg/L of NAFC for 21 days. We also show that components within NAFCs inhibit the expression of gene markers of osteoblast differentiation and function, namely, alkaline phosphatase (Alp), osteocalcin, and collagen type 1 alpha 1 (Col1a1). These effects were partially mediated by the induction of glucocorticoid receptor (GR) activity; NAFC induces the expression of the GR activity marker genes Sgk1 (12.5 mg/L) and p85a (125 mg/L) and inhibits GR protein (125 mg/L) and Opg RNA (12.5 mg/L) expression. This study provides evidence that NAFC concentrations of 12.5 mg/L and above can directly act on osteoblasts to inhibit bone formation and suggests that NAFCs contain components that can act as GR agonists, which may have further endocrine disrupting effects on exposed wildlife.


| INTRODUCTION
The Athabasca Oil Sands Region (AOSR) is one of the world's largest deposits of extractable bitumen (Alberta Energy Regulator, 2015). The process of bitumen extraction uses and recycles water, which becomes oil sands process-affected water (OSPW); this OSPW is then stored in tailings ponds and cannot be discharged into receiving waters after use (Wu et al., 2019). There is concern that chemicals from bitumen extraction may be affecting development and reproductive health of animals in areas impacted by mining activity; many of these concerns are centered around the toxicity of OSPW (Li et al., 2017). There is further concern that components of OSPW are able to migrate beyond tailings ponds containment structures into nearby waterbodies and wetlands (Hewitt et al., 2020).
OSPW contains a variety of compounds including naphthenic acid fraction components (NAFCs) which are a large and diverse group of organic acids that are natural components of bitumen (Clemente & Fedorak, 2005). NAFCs are generally considered to be the most toxic class of organic compound in OSPW (Li et al., 2017). As a result, the toxicity of NAFCs has been widely studied. Indeed, there is evidence demonstrating the toxicity of OSPW on invertebrates (Bartlett et al., 2017) and fish (Mahaffey & Dubé, 2016;Marentette et al., 2015Marentette et al., , 2017. Although few studies have investigated the effects that chronic exposures to NAFCs have on major organ systems in mammals (Clemente & Fedorak, 2005;Li et al., 2017), there is evidence that OSPW and NAFCs can act as endocrine disrupting compounds. Endocrine effects of NAFCs that have been observed include alterations in sex hormone and glucocorticoid production (Knag et al., 2013;Lister et al., 2008), impaired development and reproductive success, and reduced secondary sexual characteristics in animals exposed to NAFCs (Kavanagh et al., 2013;Li et al., 2019;Philibert et al., 2019;Rogers et al., 2002).
More recently, in a study investigating the baculum strength of animals in the AOSR, lower baculum strength was observed in river otters (Lontra canadensis) collected in areas with high levels of mining activity compared with those collected in areas far from mining activity (Thomas et al., 2021). A decrease in baculum bone material properties can reduce the reproductive fitness of male mammals, thereby increasing pressures on local river otter populations (Brassey et al., 2018;Sonne et al., 2015;Stockley et al., 2013). Similar to Thomas et al. (2021), previous studies in other free-ranging mammals have also shown that exposure to endocrine disrupting chemicals can lower baculum bone mineral density (BMD) (Sonne et al., 2015).
Although there is limited information available regarding the effects of OSPW or NAFCs on mammalian bone, a study in fathead minnow larvae reported that both a commercial technical naphthenic acid mixture and NAFCs of tailings water from the AOSR induced skeletal deformities and altered gene networks related to chondrocyte development and endochondral ossification (Marentette et al., 2015). These data suggest that it is biologically plausible that NAFCs may also impact mammalian bone development.
Bone strength is maintained throughout life by the process of remodeling, which allows bone to adapt and repair microdamage through the resorption of old bone and deposition of new bone matrix (Seeman, 2003). This process is regulated by endocrine factors and paracrine signaling between osteocytes, osteoblasts, and osteoclasts (Hauge et al., 2001). Osteoblasts synthesize new bone matrix on bone-forming surfaces as they differentiate from osteoblast progenitor cells during the formation phase of the remodeling cycle (Komori, 2016). The primary endocrine pathways regulating BMD are parathyroid hormone (PTH) (Silva & Bilezikian, 2015;Stein et al., 2013), insulin-like growth factor 1 (IGF-1) (McClung et al., 2014;Plotkin & Bellido, 2016), and glucocorticoids (Rauch et al., 2010;Weinstein et al., 1998). Glucocorticoid receptor (GR) activation in osteoblasts inhibits differentiation and production of extracellular matrix (ECM) (Rauch et al., 2010). Further, the use of synthetic glucocorticoids like dexamethasone is also the most common cause of secondary osteoporosis (Briot & Roux, 2015). Exposure to OSPW has been found to increase plasma cortisol levels in fish (Lister et al., 2008), but studies have yet to investigate whether chemicals in OSPW act as agonists for PTH, IGF-1, or glucocorticoid receptors.
The goals of this study were (1) to investigate the effects of the NAFC extracted from OSPW from an active mining operation within the AOSR (Frank et al., 2006) on the osteogenic potential of mouse preosteoblastic (MC3T3-E1) cells and (2) to determine if the underlying mechanism is glucocorticoid receptor dependent.

| NAFC preparation
Naphthenic acid extracts were isolated from tailings pond water collected from Syncrude Canada Ltd. West In-pit settling basin in Fort McMurray, Alberta, Canada, in 2009, using a procedure described in (Frank et al., 2006). An NAFC stock solution was prepared and stored in glass vessels in darkness and preserved in 0.05 M NaOH with a final concentration of 2504 mg/L determined via liquid chromatography/quadrupole mass spectrometry with time-of-flight detection (LC/QTofF) as previously described in Bartlett et al. (2017) and Marentette et al. (2015Marentette et al. ( , 2017.
To investigate whether NAFCs inhibits osteoblastogenesis, cells were treated beginning on day 0 with 125 mg/L, 25 mg/L, 12.5 mg/L, and 1.25 mg/L of the NAFC mixture, which was diluted in differentiation media (described above). These concentrations encompass the range of NAFCs, which have been reported in surface waters, wetlands, and tailings ponds in the AOSR (Vander Meulen et al., 2021).
Dexamethasone (1 μM, Sigma), a glucocorticoid receptor agonist, was used as a positive control to assess the effects of GR activation during differentiation (Rauch et al., 2010). The pH of the media was adjusted to be similar across all groups using NaOH or HCl after the addition of NAFCs. Cells were treated for 7, 14, or 21 days after the initiation of differentiation; media was changed every 4 days.

| Cell viability
Cells were seeded and treated in 96-well plates following the protocols described above. After 7 and 14 days of treatment, 10% ala-marBlue reagent (BioRad Laboratories, Hercules, CA, USA) was added to fresh media and cells were incubated for 2 h. Absorbance was then read at 570 nm in a plate reader (Synergy H1 microplate reader, Norgen BioTek, Thorold, ON, Canada) with a reference read at 600 nm. Treatments were considered to be cytotoxic if the average absorbance for the group was below 80% of the control group. Levels of gene expression were generated using the 2(ÀΔΔC(T)) method (Livak & Schmittgen, 2001) and normalized using the geometric means of two reference genes: tyrosine 3-monooxygenase/ tryptophan 5-monooxygnase activation protein zeta (Ywhaz) and peptidylprolyl isomerase A (Ppia). Gene targets for osteoblast differentiation: alkaline phosphatase (Alp), collagen type 1 alpha 1 chain (Col1a1),

| Real-time quantitative PCR
Osteocalcin, and runt-related transcription factor 2 (Runx2), glucocorticoid receptor activity: serum/glucocorticoid-inducible kinase 1 (Sgk1), receptor activator of nuclear factor kappa-B ligand (Rankl), osteoprotegerin (Opg), and phosphatidylinositol 3-kinase regulatory subunit alpha (p85a) were measured. Primer sequences are presented in Table 1. Briefly, cells were harvested with a cell lysis solution, a p-nitrophenyl phosphate (pNPP) phosphatase substrate was applied to the samples, and absorbance was read at 405 nm using a microplate reader (Norgen BioTek). Absorbance data were corrected to background and compared with control.

| Alizarin Red S staining of MC3T3-E1 osteoblasts
To measure calcium deposition, after 21 days of treatment, cells were washed with Dulbecco's phosphate-buffered saline (DPBS), then fixed with 10% formalin and stained with 2% Alizarin Red S (ARS) solution (EMD) for 15 min. The ARS solution was removed, and the cells were then washed five times with water. Representative images were taken using phase-contrast microscopy (Motic Microscopes). The dye was extracted using 10% acetic acid, cells were separated, and supernatant was transferred to a 96-well plate and absorbance was read at 405 nm in a microplate reader (Norgen BioTek). Absorbance data were corrected for background and compared with control.

| Immunoblotting
After 21 days of treatment, cells were isolated in lysis buffer (50 mM HEPES, 150 mM NaCl, 100 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol, and 1 mM sodium orthovanadate, with 1% Triton X and one tablet of cOmplete Protease Inhibitor Cocktail [Roche, Basil, Switzerland] per 50 ml) then stored at À80 C until analysis. Protein concentration was determined with the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Lysates were then diluted with Laemmli buffer and run on a 10% polyacrylamide gel (Bio-Rad Laboratories) to separate proteins based on size. Samples were then transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 5% BSA for 1 h at room temperature (20-25 C).
Membranes were incubated with primary antibody (1:1000) overnight at 4 C. Appropriate secondary antibodies were used at a concentration of 1:10,000. Bound antibodies were detected using Clarity Western ECL Substrate (BioRad Laboratories). For primary antibody information, see Table 2.

| Statistical analysis
All statistical analyses were carried out using GraphPad Prism 9.2 (GraphPad Software Inc, San Diego, CA, USA). One-way analysis of variance (ANOVA) was performed to detect significant interactions between experimental groups and negative control groups. Multiple comparisons were analyzed following Dunnett's multiple comparisons post hoc tests. Positive control groups (dexamethasone) were compared with negative control groups using a Student's t test. Any p value lower than 0.05 was considered significant. All results are presented as means ± SEM.   NAFCs induced significant reductions in the expression of Alp, osteocalcin, and Col1a1 genes at the 14-and 21-day time points relative to control treated cells ( Figure 3B Figure 3C). Accordingly, we observed significantly lower osteocalcin protein expression in cells exposed to 125 mg/L of NAFCs than control cells ( Figure 3E). Col1a1 expression was unchanged after 7 days of NAFCs treatment, but after 14 days, cells treated with ≥12.5 mg/L of NAFCs exhibited significantly lower Col1a1 expression than control cells, which then persisted to 21 days in cells treated with 125 mg/L of NAFCs ( Figure 3D).

| NAFCs inhibition of osteoblastogenesis is mediated by glucocorticoid receptor activity
Given that GR activity has a common and strong inhibitory effect on osteoblastogenesis, we sought to determine if GR was implicated in the mechanism by which NAFCs inhibits osteoblastogenesis. We mea- The order of the lanes is consistent, though the alignment of the blots may vary. Each value is the mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, treated groups compared with the differentiation control group. Each value is the mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, treated groups compared with the differentiation control group.
( Figure 4F,G). We then measured protein markers of GR activity after 21 days of treatment with NAFCs and found that there was no change in OPG or RANKL protein expression with NAFCs or the 1 μM dexamethasone GR agonist positive control, but there was a significant decrease in GR protein expression in cells treated with both 125 mg/L of NAFCs and dexamethasone ( Figure 4H-L). These results are consistent with an induction of GR activity by NAFCs functioning as GR agonists.

| DISCUSSION
Although there are numerous studies which have investigated the toxicological effects of environmental contaminants derived from oil and gas production (Li et al., 2017;Rodríguez-Estival & Smits, 2016), few of these have focused on bone health. Three studies have found skeletal deformities caused by OSPW or light oil fraction on bone in fish (Danion et al., 2011;Loughery et al., 2019;Marentette et al., 2015), and another found that river otters living closer to AOSR mining projects had generally poorer baculum bone material properties than otters farther from mining activity (Thomas et al., 2021). In the present study, we found that treatment with NAFCs derived from the AOSR are sufficient to inhibit osteoblast differentiation, determined by measuring calcified ECM, ALP activity, and gene and protein markers of osteoblastogenesis. We also found that NAFCs exposure at concentrations as low as 12.5 mg/L alters the expression of components of GR signaling similar to what is seen with a known GR agonist dexamethasone.
In the current study, we found that treatment with NAFCs inhibited the differentiation of osteoblast precursor cells into mature osteoblasts. Treatment with 12.5-125 mg/L of NAFCs for 21 days significantly decreased the deposition of extracellular matrix in differentiating osteoblasts ( Figure 2D), indicating an inhibition in the differentiation and primary function of these cells. ALP enzyme activity, a major marker of osteogenic differentiation (Lee et al., 2017), was reduced with 125 mg/L of NAFCs treatment for 14 days ( Figure 2B), and we observed corresponding reductions in Alp gene expression at doses of 12.5 mg/L and higher at 14 and 21 days ( Figure 3B). We also observed lower expression of osteocalcin, a marker of osteoblast differentiation that is specifically expressed by mature osteoblasts (Huang et al., 2007)  In this study, we also show that NAFCs can activate GR in a manner similar to dexamethasone. Glucocorticoids like dexamethasone inhibit osteoblast mineral deposition and ALP activity through inducing GR activity (Rauch et al., 2010). NAFCs and dexamethasone treatment resulted in similar changes in the expression of GR protein ( Figure 4K) suggesting NAFCs is also acting as a GR agonist (Kuo et al., 2012;Xu et al., 2019). We also observed increases in the expression of gene markers of GR activity p85a and Sgk1 after 21 days of treatment with NAFCs treatment ( Figure 4D-G). Sgk1 plays an important role in calcification of vascular muscle cells (Poetsch et al., 2020) and is required for the late stages of osteoblastogenesis (Kim et al., 2022). Other markers, RANKL and OPG are osteoblast-derived proteins that are important for regulating bone mass and are disrupted with GR activity (Humphrey et al., 2006). We  (Chen et al., 2018;Li et al., 2015). Due to the fact that the NAFCs is a mixture of several thousand organic acids, various components of the NAFCs mixture could be functioning through multiple pathways that contribute to the totality of responses observed with the whole mixture. It is likely that some components of the NAFCs may be inhibiting osteoblast differentiation through other mechanisms, in addition to those acting via the GR pathway. NAFCs from other sources have previously been shown to alter other pathways that can affect BMD, including altered sex hormone function (Knag et al., 2013;Lister et al., 2008) and increased inflammatory cytokine expression (Garcia-Garcia et al., 2011); the effects of our NAFC mixture on these pathways remains to be determined. Our data suggest that NAFC is acting through GR. However, NAFCs are complex mixtures that could also act through other pathways to inhibit osteoblastogenesis such as PTH, wingless-related integration site (Wnt), or peroxisome proliferator-activated receptor gamma (PPARγ) among others (Bateman et al., 2017;Silva & Bilezikian, 2015).
Although our findings are consistent with the hypothesis that NAFCs exposure has the potential to inhibit bone formation, this study was conducted in osteoblasts, which only represent one bone cell lineage. Moreover, using this model, we cannot assess if the effects of NAFCs on bone are reversible. Another major limitation of studies investigating the toxicological effects of NAFCs is the challenge of measuring the tissue concentrations and compositions of NAFCs in wildlife (Bartlett et al., 2017;Marentette et al., 2015Marentette et al., , 2017. Although our findings utilized concentrations of NAFCs that have been reported in surface water, industrially affected wetlands, and tailings ponds in the AOSR (Vander Meulen et al., 2021), the concentration at the tissue level is uncertain. However, the concentrations of NAFCs used in this study are similar to those used in other toxicological studies. Despite the limitations of this study, our finding that osteoblast differentiation is inhibited with exposure to NAFCs may have important implications for the bone strength of exposed animals and warrants the study of the effects of NAFCs exposures in vivo.