It is possible that PFAS effects YAP/TAZ localization via cytoskeleton integrity. These connections between adipose tissue maturation and cytoskeletal remodeling are a potential avenue for PFAS perturbation and how it effects lipid profiles and fat development. In obesity, adipocytes are hypertrophic and the expression of the adipokine leptin increases and inflammatory cytokines are increased while adiponectin and lipoprotein lipase are decreased. There are also increases in angiogenesis, immune cell infiltration, and adipose inflammation. These changes are characteristics of insulin resistance, hyperglycemia, dyslipidemia, hypertension, and obesity/adiposity and could be common ground for PFAS associations with obesity in humans. Based on these observations, we hypothesize that PFAS may be mechanistically perturbing the cell cytoskeleton and working through the Hippo pathway resulting in abnormal developmental outcomes in children. In the current study, we have evaluated PFAS’ effects on skin epithelial and adipose derived stem cell lines. We performed in vitro experiments to determine if the cytoskeleton is modulated by PFAS in two types of cells and investigated dysregulation of f-actin and acetylated tubulin. Functional effects on cell survival of both cell types and differentiation of adipose was evaluated in response to PFAS exposure. To investigate if the Hippo signaling effector, YAP, is modulated by PFAS, we quantified YAP expression and localization. Further, we have shown that damaged phenotypes due to PFAS effect the wound healing processes of keratinocyte skin cells and adipose derived mesenchymal stem cells and that some doses of PFAS chemicals induce higher amounts of adipogenesis as determined by lipid droplet staining and qPCR. PFAS effect on the differentiation process of adipose derived mesenchymal stem cells into adipogenic phenotype was also investigated.
Adipogenic differentiation was induced via media,greenhouse ABS snap clamp as completed previously. Adipogenic induction media was DMEM supplemented with 10% FBS and 1% P/S, 500 µM 3-isobutyl-1-methylxanthine , 1 µM Dexamethasone , 10 µg/mL Insulin , and 10 µg/mL indomethacin . To examine how PFAS effected these processes, ASC52telo cells were plated at a density of 25,000 cells/coverslip in 24 well plates on 12 mm glass slides and cultured in adipogenic conditions. After initial seeding in routine culture media, at 48 hours PFOA [100 and 125 µM ] and PFOS [20, 30, 40 µM] were added to well plates. Control media was Adipogenic Induction media. Cultures were carried out for 19 days, at endpoint, all wells were fixed with 4% PFA for 15 minutes then washed x 3 with PBS . Plates were either stored at 4 °C or were immediately exposed to oil-red-o staining for adipogenesis assessment. To assess adipogenesis, lipid content was assayed using Oil Red O staining which marks lipids secreted by adipocytes. ORO staining was completed similar to previous studies. Briefly, a 0.5% lab stock was made up by dissolving 0.2 g Oil Red O in 40 mL Isopropanol . After dissolving overnight, a working stock was made up by diluting the lab stock 2:3 with culture grade water to yield a 0.2% solution in 40% Isopropanol. The 0.2% solution was made up immediately before exposure then filtered before use with a 0.2 µm sterile syringe filter. Each 24 well was filled with 0.5 mL ORO and incubated for 30 minutes at room temperature. After exposure, coverslips were washed 5x with autoclaved distilled water. Phase Contrast imaging at 10x was performed to evaluate adipogenesis and oil-red-o accumulation. For oil-red-o analysis, 3 images were taken per coverslip for each replicate set. For RNA isolations, ASC52telo cells were plated at a density of 500,000 cells/60 mm tissue culture plate then grown up for 48 hours in their routine culture media. At 48 hours, adipogenesis was induced via media and cells were cultured in adipogenic conditions for 19 days with experimental chemical doses. At day 19, mRNA was isolated using the Thermo GeneJet RNA Purification Kit .
All RNA isolations were carried out as outlined by the manufacturer. Assays were completed to assess for adiponectin, leptin, PPARγ, CEBP, and CTGF, using the housekeeping gene GAPdh. Primer sequences given in supplementary data Table 5.1 Primer Sequences used for qPCR. . Quantitative polymerase chain reaction was performed via SensiFAST™ SYBR No-ROX One-Step mastermix from Bioline. All reactions were performed in triplicate. qPCR reactions were completed using a Mic Real Time PCR Cycler at 10 minutes at 45 °C then 2 minutes at 95 °C then 40 3-step cycles of 95 °C for 5 seconds, 60 °C for 20 seconds, and 72 °C for 10 seconds and a melt from 72 °C to 95 °C at 0.3 °C/s. For quantification of mRNA, n = 4. Following culture, coverslips were fixed and permeabilized in place for 20 minutes via 4% paraformaldehyde and 0.25% triton x 100. After fixation, coverslips were washed with PBS x 3 and either stored at 4 °C in PBS or blocked and stained immediately. Samples were first blocked for one hour then stain solution of primary antibodies was added . All stain solutions were made up in blocking buffer except DAPI which was made up in 1 x PBS. Primary antibodies and chemical stains used include: YAP-1, Acetylated Tubulin, Ki67, Phalloidin, DAPI ; antibody specifications given in supplemental data Table 5.2. Coverslips were exposed overnight at 4 °C to primary stain solution then removed, washed x3 with 1x PBS, then exposed overnight to the secondary solution . DAPI was used to mark nuclei and coverslips were exposed for 20-30 minutes at room temperature in 1x PBS, after initial staining was complete. All coverslips were mounted to glass slides using gelvitol allowed to dry, then cleaned with 70% ethanol prior to imaging. Confocal imaging was performed using a Leica TCS SPEII confocal, sCMOS camera attachment. Imaging parameters remained the same for each coverslip in order to compare intensities; each coverslip was imaged using a 20-25 position tilescan and a 1 micron voxel size. Confocal volumes were assessed via custom algorithms designed in MATLAB . Briefly, maximum projections of each tile-position per sample was filtered and segmented which enabled antibody intensity quantification and cell colony health evaluations.
YAP intensity at nuclei and cytosol was quantified in N/TERT-1 and ASC52telo and the number of nuclei per sample was also quantified as an indication of cellcolony health after PFAS exposure. For ASC52telo cells, Ki67 was quantified at nuclei as an indication of proliferation. To evaluate dysregulation of the cytoskeleton of N/TERT-1 cells, expression of phalloidin and acetylated tubulin were quantified using concentric annular rings. Cells were identified using the phalloidin stain then two regions were indicated. A peri-nuclear region and a peripheral nuclear region was identified for each cell and the intensities of phalloidin and acetylated tubulin were quantified within the segments. For all analysis, each experimental condition quantification was based on at least 20 positions per coverslip, intensities were averaged per position per coverslip. For all analysis of confocal images, n = 3 for N/TERT-1 cells for each timepoint and n = 4 for all ASC52telo cells for each time point. For quantification of adipogenesis,snap clamps ABS pvc pipe clip custom MATLAB algorithms were written to assess pixel area of ORO present in each control and dosed coverslips. Three phase contrast images were taken per well; cell coverage and ORO content was determined via image segmentation and averaged for each sample then ORO content was normalized to both the cell coverage of the sample and to the control of each experiment group . PFAS chemicals have stable and bio-accumulative properties which makes them particularly dangerous to biological membrane structures and to the environment in general. Previous work has shown that higher maternal serum concentration of PFAS are associated with decreased birthweight but increased adiposity in infants and in pediatric/young adults, but the mechanism of these changes and toxicity of PFAS chemicals are still largely unknown. Work in animal models has shown that PFAS chemicals have toxic effects and that in some cases these follow nonmonotonic responses. Generally, there is a discrepancy in research doses of PFAS and environmental exposure and detection of PFAS. The U.S Environmental Protection Agency set federal drinking water guideline limits for PFAS at 70 ng/L but, as of 2019 only seven states developed guidelines varying from 13-1000 ng/L. Several states do not have any guidelines at all or only have guidelines for one of many PFAS chemicals. Although the PFAS doses administered in this work are higher than the limitations set , the bio-accumulative properties of PFAS are an important consideration. Guidelines set by the EPA cannot account for bioaccumulation and only apply to drinking water even though humans are exposed to PFAS through many avenues including high amounts from contaminated aquatic species. Thus, there is a mismatch between what guidelines say are safe to consume and what humans are actually consuming or being exposed to. Further, bio-accumulative properties of PFAS chemicals are difficult to model in vitro and hard to discern in vivo because of environmental and body variability.
In the experiments completed here, samples were given the same doses for each time point and were only dosed a single time. For example, the 72 h time point experiments did not get exposed to more PFAS toxicant than the 24 hr time point, but the cells were exposed longer. For the adipogenesis assays, cells were exposed to PFAS at every media feeding, and therefore may have modeled bio-accumulation in a different way than the N/TERT-1s and undifferentiated ASC52telos. To help understand underlying mechanisms of PFAS toxicity in human cells, we studied PFAS effects on cell monolayers. The data presented here show that PFAS effects cell and colony health, as determined by nuclear counts . Interestingly, ASC52telo cell monolayers that were exposed to PFAS remained stable 48 h after exposure and there was not a significant difference in cell number. To understand if PFAS had detrimental effects on proliferation of ASC52telos, we quantified the proliferative marker, Ki67 but concluded that these doses of PFOS and PFOA do not have significant effects on proliferative capacity of ASCs either. However, in agreement with past literature that investigated PFAS effects in the African clawed frog, we show that PFOS and PFOA treatments induce significant declines in cell number of N/TERT-1 cells, and that these declines are greater at longer time points . Additionally, we similarly show that higher PFOS doses produce the greatest cell decreases but our results from PFOA exposure do not follow nonmonotonic relationships as previously shown in the African clawed frog cell monolayers. Importantly, the high dose of PFOS used here is similar to the frog study where the high PFOA and PFOS dose was 10 µM. However, the low dose used for both chemicals from the frog study is not similar to our PFOA doses . More investigation is required to understand if low doses of PFOA produce nonmonotonic effects in human cells. PFAS chemicals have been found to disrupt f-actin, microtubules, and gap junctions of the cell cytoskeleton. We have demonstrated that PFOA and PFOS disrupt the cytoskeletal components, acetylated tubulin and f-actin, in two human cell lines. Increased intensities and shifts in expression of acetylated tubulin and f-actin support cytoskeletal disruption by PFAS that has been reported in other studies. As seen through f-actin fibers , there were also differences in N/TERT-1 cell edges with increased presence of filipodia and prominent protrusions. It could be argued that these cytoskeletal changes are largely due to cell death and colony disruption due to PFAS toxicity, but we have demonstrated that there are cytoskeletal disruptions in ASC52telo cell lines as well. ASC cell lines were resistant to the cytotoxic effects of PFAS exposure and the number of cells and their proliferative capacities did not change after 48 h of exposure. Additionally, the cell death at 24 h seen with the high dose of PFOS did not correspond with radial distribution changes of cytoskeletal components. It is likely that cell death does play a part in the cytoskeletal disruption since colonies themselves are disrupted, but it does not seem to be the only mechanism here. PFAS likely perturbs the cytoskeleton independently of cell death, but this mechanism of action still requires more investigation.Disruption has been demonstrated through actin filament remodeling, central actin stress fiber formation, microtubule and gap junction disorganization, and formation of lamellipodia and filipodia structures at cell periphery. PFOS has been explicitly linked to disruption of blood testi barriers established by Sertoli cells in human and animal studies and thus, accumulation of testicular PFOS.