Barrier disruptions were attributed to the toxic effects of PFOS on f-actin, microtubule, and gap junction organization. Specifically, dose dependent PFOS exposure disassembled tight and gap junctions responsible for barrier function of blood-testis barrier, increased blood-testis barrier permeability, and disrupted spermatogenesis. PFAS chemicals have also demonstrated tight junction opening in brain endothelial cells, a main component of the blood-brain barrier, and increases in reactive oxygen species that increases endothelial permeability. PFOS was found to induce the remodeling of actin filaments through ROS production in human endothelial cells and increased gaps/breaks between endothelial cells in monolayers which resulted in increased permeability. Authors observed formation of central cell actin stress fibers and the formation of lamellipodia and filopodia structures at cell periphery. In alignment with the previous findings summarized, we observed the shift in f-actin expression toward the center of N/TERT-1 cells which could indicate formation of central stress fibers. The representative images shown display increased fiber formation near the center of the cells . We also observed the formation of filopodia structures at cell edges which were observed due to ROS production by PFOS by Qian et al.
Although we did not measure ROS production,greenhouse snap clamps is likely that these cell models are being affected in the same way; PFOS may increase ROS production by the host cell and in turn dysregulate the cytoskeleton. Importantly, previous literature showed that the disruptions caused to the cytoskeleton by PFAS increase permeability of crucial barriers such as the blood-brain barrier and the blood-testis barrier. Skin keratinocytes are responsible for formation of the skin barrier, but instead of working in a monolayer, keratinocytes differentiate into 4 different epidermal layers with the top corneal layer acting as the barrier . N/TERT-1 cells are skin keratinocytes and are what were used for epidermal monolayer cultures in this work. We used these cells as a general model to study PFAS effects, but the implications of PFAS toxicity on epidermal skin cells are important in understanding PFAS absorption through the skin and the dangers it might cause to human health. The study of PFAS effects on skin tissue and cells is lacking with only a few investigations completed thus far using human tissue engineered skin and animal models. PFAS was previously deemed to be not well absorbed through the skin by the US Environmental Protection Agency in 2002, but since then, it has been demonstrated that after topical exposure, PFOA permeates human tissue engineered skin, human and rat skin explants , and mouse skin. In the mouse studies completed by Franko et al., it was demonstrated that there were dose-responsive increases in serum PFOA after dermal exposure and concluded that PFOA is dermally absorbed. Han et al. showed that after 6 days of dermal exposure to PFOA , there was decreased skin thickness and portions of degeneration in the epidermal layers and necrotic fibroblasts in human tissue engineered skin but did not observe decreases in cellular viability.
They also demonstrated that after 2 weeks of dermal exposure in rats with another type of PFAS, short-chain perfluoroalkyl carboxylic acids rather than long, there were adverse effects on kidney, liver, testes, and skin that resulted in death via ulcerative dermatitis at application sites with high doses . Our findings of PFAS toxicity and cytoskeletal dysregulation on N/TERT1 cells support the need for more research on dermal absorption of PFAS. Previous investigations have concluded that tubulin acetylation plays a role in cell migration and cell development and it has been concluded that alpha tubulin acetylationhelps to protect microtubules against mechanical stress and enhance microtubule flexibility. Acetylation of alpha tubulin is a common post-translational modification of alpha tubulin that happens on stable microtubules. Disrupted acetylation process can contribute to negative effects in cell polarization, cell division, adhesion, motility and have been linked to neurodegenerative disorders and tumor metastasis. To our knowledge, no studies have investigated tubulin acetylation specifically in regards to PFAS toxicity. The work presented here demonstrates dysregulation of acetylated tubulin by differences in localization within N/TERT1 cells and further implicates PFAS as a cytoskeletal disrupter. These underlying roles of tubulin acetylation and f-actin and the consequences of dysregulation may be involved in the developmental changes of children and metabolic/reproductive toxicity changes in adults Many studies have focused on YAP localization and Hippo signaling regarding organ size and development.
YAP and its upstream regulators have been investigated in liver, pancreas, intestine, kidney, lung, and bone but regulation through the Hippo pathway is different in each system. Disruption of YAP localization can cause several developmental problems in many organs. Notably, deletion of YAP in the lung epithelium of mice and humans was shown to cause defective lung development; correct YAP localization in the cytoplasm is required for proximal airway maturation while nuclear YAP is required for progenitor specification. In the mouse kidney, deletion of YAP led to hypoplastic kidneys, fewer glomeruli, and defect formations in distal tubules and loop of Henle YAP is essential for nephron development and establishing kidney morphology and function. YAP knockout has also been found to decrease bone formation and increases bone marrow fat331. The pancreas and liver have been a focus of Hippo signaling pathway and organ development research. Rather than YAP knockout and upregulation, the Hippo pathway was altered via deletion/knockout of Mst1/2 in the pancreas324 and deletion of Mst1/2 and Lats1/2 in the liver. Changes in the levels of Hippo pathway regulators MST and LATS and their effect on organ size poses other mechanisms of organ development dysregulation; in which Hippo is inactivated and nuclear YAP is upregulated. These too could be causes of changes in organ mass and body weight decrease during infancy and in mice have been shown to be a cause of decrease in pancreatic mass resulting in body weight differences. Although the current study did not investigate knockout of YAP and PFAS effects, these summarized studies suggest that perturbation of YAP localization and Hippo signaling pathway activity by PFAS can lead to organ developmental changes and possible decreased body weight due to changes in organ size/mass tied to developmental issues. To investigate the possibility that PFAS acts directly on the Hippo pathway effector, YAP, we studied localization of YAP in the cytosol and nuclei under PFAS exposure in the N/TERT-1 and ASC52telo cell lines. Through this work, data show that in N/TERT1 cells at the 72 h time point, expression of nuclear YAP increased for PFOS doses of 30 and 40 µM and PFOA doses of 100 and 125 µM. These data indicate that these PFAS chemicals regulate the Hippo pathway in N/TERTs through YAP/TAZ regulation. Because of the crosstalk between Hippo pathway and Rho/ROCK cytoskeletal perturbation, it is plausible that PFAS regulates Hippo through mechanosensing and extracellular matrix/focal adhesion changes that propagate through the Hippo mediator YAP/TAZ . At the 24 h time point in N/TERT-1 cells and at 48 h in ASC52telo cells, there were no differences in YAP localization.General mesenchymal stem cell polarization toward adipogenesis and osteogenesis is dependent on Hippo signaling and particularly related to YAP localization . Reduced expression of upstream regulators of YAP such as Lats1/2 inhibit Hippo signaling and promote in vitro adipogenic and osteogenic differentiation, proliferation, and migration of bone marrow derived mesenchymal stem cells . With increased nuclear localization of YAP, there is increased osteogenic differentiation. Conversely, with decreased nuclear localization of YAP there is increased adipogenic differentiation. Though we did not directly quantify YAP in mature adipocyte cells under PFAS exposure, to aid in the understanding of adiposity changes,snap clamps for greenhouse we explored PFAS’ impact on mesenchymal stem cell differentiation toward adipogenesis.
We found that some doses of PFOA and PFOS upregulated adipogenesis as measured by lipid droplet presence . qPCR results further support the finding that PFAS disrupts fat homeostasis, but instead of showing upregulation of fat markers, data shows down regulation of mRNA expressions of the adipokine leptin and no changes in Adiponectin . PFOA and PFOS upregulated adipogenesis indicated by lipid droplet analysis but the mechanism of action still requires more investigation especially in understanding the non-supporting mRNA expression results. One avenue that may elucidate changes in the adipogenic process is specifically investigating cytoskeleton changes that may occur when PFAS is present during adipogenesis. Here, we investigated cytoskeleton in pre-adipogenic ASC52telo cells under PFAS, but not changes during or after exposure through 19 days of differentiation. Cytoskeletal components are involved in the maturation and homeostasis of fat cells. Our findings that PFAS chemicals disrupt cytoskeletal stability in skin and pre-adipose cells lend support to previous literature and suggest that the upregulation of adipogenesis could be due to the cytoskeletal perturbations. Palanivel et al. found, in rat cardiomyocytes, that an increase in adiponectin induces cytoskeletal remodeling and increases membrane microvillar like protrustions and actin polymerization. It is clear that PFAS is effecting pre-adipose and adipose cells, but it is unclear whether or not PFAS upregulates adipokines in mature fat cells which then disrupt the cytoskeleton through rho/rock and/or if PFAS acts directly on the cytoskeleton or not at all. In their undifferentiated state, ASC52telos did exhibit cytoskeletal changes including increase of protrustions at the cell boarder, shifts in cytoskeletal component intensities and location, and dysregulation of fibers .This dysregulation may be contributing to the PFAS associated increased adolescent obesity. Further investigation on lipid profiles and hormonal changes due to increased adipogenesis should be explored to help in understanding prior observations regarding increased childhood adiposity and higher PFAS serum concentrations. The goal of this work was to better understand the mechanistic actions of PFAS on human cells specifically of their effect in altering the cell cytoskeleton and the Hippo signaling pathway. We investigated PFAS chemicals, PFOA and PFOS, for their effects on an adipose derived mesenchymal stem cell line and a keratinocyte skin cell line . In conclusion, these data support previous findings that PFAS chemicals affect cytoskeletal integrity of the f-actin and microtubules and have demonstrated this in the human skin keratinocyte cell line, N/TERT-1 and the adipose derived mesenchymal stem cell line, ASC52telo. We also investigated if PFOA and PFOS directly effect the Hippo signaling pathway modulator, YAP. Our results support a direct modification of YAP localization by PFOA and PFOS chemicals. Finally, to gain an understanding into the negative effects of PFAS chemicals on infant and adolescent body weight, we examined the adipogenic process under PFAS dosing. PFAS indeed upregulates the adipogenic process as quantified by presence of lipid droplets through ORO assays and qPCR. Key adipokines were dysregulated in dose-dependent exposure to PFOA and PFOS. Although more investigation is required in understanding how PFAS specifically acts to upregulate adipogenesis, we speculate that the cytoskeleton changes involved in adipogenesis are being dysregulated and aiding in dysregulation of fat maturation. These findings reinforce the need for more in depth detection and more stringent limitations of PFAS exposure to the public, particularly pregnant mothers and children. In the 1960s, the state of Punjab led in the adoption of new high-yielding varieties of wheat and rice. Production of these new varieties required innovations in the use of fertilizer and water, which occurred in a complementary manner to the innovation in seed choices. Mechanization of several aspects of farming also became a supporting innovation. Agricultural extension services based in Punjab’s public universities guided farmers in their transition to the new modes of production. Furthermore, an infrastructure of local roads and market towns had been developed by the state government: these, along with central government procurement guarantees, gave farmers access and security in earning income from their produce. In the private sector, new providers of seeds and fertilizer, as well as farm equipment and equipment maintenance services also arose. All of these conditions together created what has been known as the Green Revolution economy. With the Green Revolution, Punjab quickly became the state with the highest per capita income. This ranking persisted into the 1990s, but underlying conditions became less favorable well before then. Gains in agricultural yields and productivity slowed, due to diminishing returns. While India began to grow faster after trade and industrial policy liberalization of 1991 and subsequent creeping reforms in other sectors, agriculture remained locked into the old policies, and Punjab mostly into the old equilibrium.